**Part 7**

## **Research and New Challenges in Osteoporosis**

664 Osteoporosis

Mathis JM. Percutaneous vertebroplasty:complication avoidance and technique

Naganathan V, Jones G , Nash P, Nicholson G, Eisman J & Sambrook PN. Vertebral

Nolla JM, Gomez-Vaquesro C, Romera M, Roig-Vilaseca D . Osteoporotic vertebral fracture

O'Neill T. W., Felsenberg D., Varlow J., Cooper C., Kanis J. A. & Silman A. J. The prevalence

Oleksik A, Lips P, Dawson A, Minshall ME, Shen W, Cooper C & Kanis J. Health-related

Ortiz OO, Deramond H. Spine anatomy.In :*Percutaneous Vertebroplasty*. JM Mathis, H

Prather H, Watson JO & Gilula LA. Nonoperative management of osteoporotic vertebral compression fractures. *Injury .* 2007; 38 Suppl 3:S40-8 (ISSN: 0020-1383) Rousing R, Hansen KL , Andersen MO, Jespersen SM, Thomsen K & Lauritsen JM. Twelve-

Schindler OS ,Watura R & Cobby M . Sacral insufficiency fractures. *Journal of Orthopaedic* 

Sclaich C, Minne HW, Bruckner T, Wagner G et al. Reduced pulmonary function in patients

Scroop R, Eskridge J & Britz GW. Paradoxical cerebral arterial embolization of cement

Smit RS, van der Velde D & Hegeman JH . Augmented pin fixation with Cortoss® for an

Sun K, Mendel E, Rhines L, Burton A & Liebschner M. Disperse cement filling in

Taylor RS, Fritzell P & Taylor RJ. Ballon kyphoplasty in the management of vertebral

Trials of Vertebroplasty for Vertebral Fractures. *N Engl J Med* 2009; Dec 24, 361, 26:2097-2100 US Department of Health and Human Services. Bone Health and Osteoporosis: a report of

Wehrli FW, Ford JC & Haddad JG. Osteoporosis: clinical assessment with quantitative MR

Weinstein JN. Balancing Science and Informed Choice in Decisions about Vertebroplasty. *N* 

prevalent vertebral fractures. *J Bone Miner Res* 2000; 15:1384-1392.

Deramond, and SM Belkoff (eds).New York Springer, 2001:7-24

clinical randomized study. *Spine*, 2010; vol. 35, no. 5, pp. 478–482,.

with spinal osteoporotic fractures. *Osteoporosis Int* 1998;8, 3:261-267

*Arch Orthop Trauma Surg* 2008;Sep 128, 9: 989-993

imaging in diagnosis. *Radiology* 1995;Sep 196, 3:631–641

*Engl J Med* 2009; Dec 24, 361, 26:619-621

Fracture Risk With Long-term Corticosteroid Therapy. *Arch Intern Med*. 2000;Vol

in clinical practice:669 patients diagnosed over a 10 year period. *J. Rheumatol.*

of vertebral deformity in European men and women: The european vertebral osteoporosis study. Journal of Bone and Mineral Research.1996,July Vol 11, Issue 7:

quality of life (HRQOL) in postmenopausal women with low BMD with or without

months follow-up in forty-nine patients with acute/semiacute osteoporotic vertebral fractures treated conservatively or with percutaneous vertebroplasty: a

during intraoperative vertebroplasty: case report. *AJNR Am J Neuroradiol* 2002; May

unstable AOA3 type distal radius fracture in a patient with a manifest osteoporosis.

vertebroplasty may reduce risk of secondary tissue damage. *Eur Cell Mater* 2006;11:

compression fractures: an updated systematic review and meta-analysis. Eur Spine

the surgeon general. Rockville, MD:*US Department of Health and Human* 

optimization. *AJNR Am J Neuroradiol* 2003;Sep 24, 8:1697-1706

160:2917-2922

pp 1010–1018

23, 5:868–870

12 (abstr).

*Services*,2004

J 2007;16:1085-1100

2001;Oct 28, 10:2289-2293

*Surgery* 2007;Vol. 15, no. 3:339-46

**33** 

**Osteoporosis: A Look at the Future** 

Anelia Dimitrova2, Cyril Popov3 and Margarita D. Apostolova1 *1Medical and Biological Research Lab, Institute of Molecular Biology,* 

Osteoporosis – the commonest age-related skeletal chronic disorder– is characterized by loss of bone mass, alterations of bone micro-architecture, and increased fracture risk. It has, however, received much less attention than most chronic diseases. Osteoporotic fractures are expensive to treat, and cause significant mortality, morbidity, and loss of independence in an ever broader population of patients. As we have said, osteoporosis is a disease in which the mineral density of the bone (BMD) is reduced, its microarchitecture disrupted, and the expression profile of non-collagenous proteins altered. All these factors predispose bones to fractures, particularly the hip, spine and wrist, and are a major cause of disability, severe back pain and deformity. The World Health Organization estimates that approximately 70 million people worldwide have osteoporosis (Penrod J. et al., 2008). The total cost of osteoporosis is difficult to calculate because it includes in-patient and outpatient medical care, loss of working days, chronic nursing-home costs, and medication. The direct costs of osteoporosis arise mainly from the management of patients with hip fractures. Hip fractures also account for 40% of all deaths from trauma in patients over 75, with 68% of all patients not returning to their former level of activity following an osteoporotic hip fractures. The annual worldwide incidence of hip fracture is 1.5 million, a number projected to grow to 2.6 million by 2025 and to 4.5 million by 2050 (Penrod J. et al., 2008). For these reasons, it is necessary to pay a special attention to these health problems

Both disease prevention and innovation in therapy are critically dependent on identifying the factors that predispose to the development of osteoporosis (Marini & Brandi, 2010). Several studies that have investigated the influence of genetic factors on the development of osteoporosis have found a weaker contribution than to peak bone mass where up to 80% of

**1. Introduction** 

which disturb the quality of life.

**1.1 Evidence for genetic variation influencing fractures** 

Iliyan Kolev1, Lyudmila Ivanova1, Leni Markova1,

*Bulgarian Academy of Sciences, Sofia,* 

*Medical University, Pleven,* 

*University of Kassel,* 

*1,2Bulgaria 3Germany* 

*2Department of Physiology and Pathophysiology,* 

*3Institute of Nanostructure Technologies and Analytics,* 

## **Osteoporosis: A Look at the Future**

Iliyan Kolev1, Lyudmila Ivanova1, Leni Markova1, Anelia Dimitrova2, Cyril Popov3 and Margarita D. Apostolova1 *1Medical and Biological Research Lab, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, 2Department of Physiology and Pathophysiology, Medical University, Pleven, 3Institute of Nanostructure Technologies and Analytics, University of Kassel, 1,2Bulgaria 3Germany* 

### **1. Introduction**

Osteoporosis – the commonest age-related skeletal chronic disorder– is characterized by loss of bone mass, alterations of bone micro-architecture, and increased fracture risk. It has, however, received much less attention than most chronic diseases. Osteoporotic fractures are expensive to treat, and cause significant mortality, morbidity, and loss of independence in an ever broader population of patients. As we have said, osteoporosis is a disease in which the mineral density of the bone (BMD) is reduced, its microarchitecture disrupted, and the expression profile of non-collagenous proteins altered. All these factors predispose bones to fractures, particularly the hip, spine and wrist, and are a major cause of disability, severe back pain and deformity. The World Health Organization estimates that approximately 70 million people worldwide have osteoporosis (Penrod J. et al., 2008). The total cost of osteoporosis is difficult to calculate because it includes in-patient and outpatient medical care, loss of working days, chronic nursing-home costs, and medication. The direct costs of osteoporosis arise mainly from the management of patients with hip fractures. Hip fractures also account for 40% of all deaths from trauma in patients over 75, with 68% of all patients not returning to their former level of activity following an osteoporotic hip fractures. The annual worldwide incidence of hip fracture is 1.5 million, a number projected to grow to 2.6 million by 2025 and to 4.5 million by 2050 (Penrod J. et al., 2008). For these reasons, it is necessary to pay a special attention to these health problems which disturb the quality of life.

#### **1.1 Evidence for genetic variation influencing fractures**

Both disease prevention and innovation in therapy are critically dependent on identifying the factors that predispose to the development of osteoporosis (Marini & Brandi, 2010). Several studies that have investigated the influence of genetic factors on the development of osteoporosis have found a weaker contribution than to peak bone mass where up to 80% of

Osteoporosis: A Look at the Future 669

Fig. 1. Overview of signal-transduction pathways associated with candidate genes

protective role associated with stronger bones and reducing the risk of osteoporotic fractures. The extent to which genes cause alteration in combination with different risk factors for fracture might explain the incidence of fractures in the population, and will depend on strength of association between the risk factor and fracture risk, and on proportion of the population at different levels of allele frequencies (Hopper, 2000). Therefore, although a variation in a risk factor may be strongly genetically determined, it may have little consequence for the disease in terms of explaining different patient's clinical

Bone fracture healing is a complicated, multistage process, influenced by cells events and regulated by local and systematic factors. Recent data concerning the regulatory factors correlated with fracture healing suggest that several chemical compounds can be used to stimulate bone growth, and enhance callus formation and maturation. The pharmacological approach towards osteoporosis prevention aims to increase the bone mass by decreasing of osteoclastic bone resorption with the aid of estrogens, bisphosphonates, calcitonin, calcium plus, cholecalciferol, calcitriol and selective estrogen receptor modulators (Grabo &

Moving away from these conventional approaches, investigators have recently used lovastatin loaded nanoparticles (Garrett et al., 2007) or Ossein – Hydroxyapatit compounds (OHC) for osteoporosis treatment. One of the most important pleiotropic actions of statins is their effect on bone metabolism (Horiuchi & Maeda, 2006). Statins form a class of hypolipidemic agents, known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A) reductase inhibitors, commonly used as therapy to lower the cholesterol levels in people with/or at risk of cardiovascular disease. Statins exhibit a pleiotropic effect in preclinical models, accelerating fracture healing and increasing angiogenesis when the fracture area is

underlying susceptibility to osteoporosis.

outcomes.

**2. State-of-the-art** 

Longyhore, 2008).

the variance can be explained by genetics alone in both sexes (Ralston & de Crombrugghe, 2006).

High-throughput technologies facilitate the identification of genetic, genomic, proteomic, and metabolomic markers of osteoporosis risk that may find a place in clinical prediction algorithms. The operation of intricate networks of genes, environmental factors, and geneby-environmental interactions further complicate our understanding of the genetic components of the osteoporosis. Until recently, single nucleotide polymorphisms (SNPs) were thought to be the predominant form of genomic variation and to account for phenotypic variation in patients with osteoporotic disorders and those without. However, with the advent and application of array-based comparative genomic hybridization (aCGH), which allows analysis of the genome with a significantly higher resolution than previously possible, scientists have demonstrated that humans are much more genetically variable than previously thought. In two different publications in 2004 (Iafrate et al., 2004; Sebat et al., 2004) hundreds of genomic regions that varied significantly with respect to the number of copies (CNVs) have been reported. Since then, older observations (Iafrate et al., 2004; Sebat et al., 2004) have been replicated and expanded (Conrad et al., 2006; deVries et al., 2005; Repping et al., 2006; Schoumans et al., 2005; Sharp et al., 2005; Tuzun et al., 2005). CNVs vary greatly in size, with insertions or deletions ranging from below 1 kb to several Mb in length (Feuk et al., 2006). As with other types of genetic variation, some gene CNVs have been associated with susceptibility or resistance to disease (Aitman et al., 2006; Cappuzzo et al., 2005; Feuk et al., 2006; Gonzalez et al., 2005; Redon et al., 2006; Sebat et al., 2007; Deng et al., 2010). In humans, CNVs encompass more DNA than SNPs and may be responsible for a substantial part of human phenotypic variability and disease susceptibility (Freeman et al., 2006; Redon et al., 2006). In spite of revolutionary technologies, the major genes determining the bone density and the fracture risk in humans remain uncertain. Approximately 150 candidate genes that might influence the BMD have been identified (Richards et al., 2009; Zhang et al., 2010). Confirmation analyses have revealed that only 30 of these SNPs are somehow connected with the development of osteoporosis.

Preliminary Pathway analysis (Molecular INTeraction database) of the differentially regulated genes/proteins in patients with osteoporosis (Richards et al., 2009) has revealed significance for the highlighted nodes (Mothers against decapentaplegic homolog (SMAD) 2, 3, 4 and 7, Tumor necrosis factor superfamily (TNFRSF), Integrin beta-3 (ITGB3), Bone morphogenetic proteins (BMPs), Transforming growth factor beta-receptors (TGFBRs), Calmodulin 3 (CALM3), TANK protein, Cystic fibrosis transmembrane conductance regulator (CFTR), Pro-neuropeptide Y (NPY)), suggesting that these pathways might play a concomitant role in the pathogenesis of osteoporosis (Fig. 1). New findings notwithstanding, we have to remember that the formation of bone and its repair is a complex process including skeletal patterning, remodeling, and bone growth. The rate of bone formation is dependent on the commitment and replication of several cell types, including osteoprogenitor cells. Their differentiation into functional osteoblasts and the life span of mature osteoblasts is very important. Although a few signaling pathways and patterns of gene expression have been identified in the process of osteoblast differentiation, the exact molecular mechanisms are poorly understood.

A recent genome wide association study (GWAS) has identified approximately 9000 CNVs in patients with lower BMD at spine, hip, and femoral neck (Deng et al., 2010). This study showed that only one low copy number variant of VPS13B gene had some possible

the variance can be explained by genetics alone in both sexes (Ralston & de Crombrugghe,

High-throughput technologies facilitate the identification of genetic, genomic, proteomic, and metabolomic markers of osteoporosis risk that may find a place in clinical prediction algorithms. The operation of intricate networks of genes, environmental factors, and geneby-environmental interactions further complicate our understanding of the genetic components of the osteoporosis. Until recently, single nucleotide polymorphisms (SNPs) were thought to be the predominant form of genomic variation and to account for phenotypic variation in patients with osteoporotic disorders and those without. However, with the advent and application of array-based comparative genomic hybridization (aCGH), which allows analysis of the genome with a significantly higher resolution than previously possible, scientists have demonstrated that humans are much more genetically variable than previously thought. In two different publications in 2004 (Iafrate et al., 2004; Sebat et al., 2004) hundreds of genomic regions that varied significantly with respect to the number of copies (CNVs) have been reported. Since then, older observations (Iafrate et al., 2004; Sebat et al., 2004) have been replicated and expanded (Conrad et al., 2006; deVries et al., 2005; Repping et al., 2006; Schoumans et al., 2005; Sharp et al., 2005; Tuzun et al., 2005). CNVs vary greatly in size, with insertions or deletions ranging from below 1 kb to several Mb in length (Feuk et al., 2006). As with other types of genetic variation, some gene CNVs have been associated with susceptibility or resistance to disease (Aitman et al., 2006; Cappuzzo et al., 2005; Feuk et al., 2006; Gonzalez et al., 2005; Redon et al., 2006; Sebat et al., 2007; Deng et al., 2010). In humans, CNVs encompass more DNA than SNPs and may be responsible for a substantial part of human phenotypic variability and disease susceptibility (Freeman et al., 2006; Redon et al., 2006). In spite of revolutionary technologies, the major genes determining the bone density and the fracture risk in humans remain uncertain. Approximately 150 candidate genes that might influence the BMD have been identified (Richards et al., 2009; Zhang et al., 2010). Confirmation analyses have revealed that only 30 of these SNPs are

Preliminary Pathway analysis (Molecular INTeraction database) of the differentially regulated genes/proteins in patients with osteoporosis (Richards et al., 2009) has revealed significance for the highlighted nodes (Mothers against decapentaplegic homolog (SMAD) 2, 3, 4 and 7, Tumor necrosis factor superfamily (TNFRSF), Integrin beta-3 (ITGB3), Bone morphogenetic proteins (BMPs), Transforming growth factor beta-receptors (TGFBRs), Calmodulin 3 (CALM3), TANK protein, Cystic fibrosis transmembrane conductance regulator (CFTR), Pro-neuropeptide Y (NPY)), suggesting that these pathways might play a concomitant role in the pathogenesis of osteoporosis (Fig. 1). New findings notwithstanding, we have to remember that the formation of bone and its repair is a complex process including skeletal patterning, remodeling, and bone growth. The rate of bone formation is dependent on the commitment and replication of several cell types, including osteoprogenitor cells. Their differentiation into functional osteoblasts and the life span of mature osteoblasts is very important. Although a few signaling pathways and patterns of gene expression have been identified in the process of osteoblast differentiation, the exact

A recent genome wide association study (GWAS) has identified approximately 9000 CNVs in patients with lower BMD at spine, hip, and femoral neck (Deng et al., 2010). This study showed that only one low copy number variant of VPS13B gene had some possible

somehow connected with the development of osteoporosis.

molecular mechanisms are poorly understood.

2006).

Fig. 1. Overview of signal-transduction pathways associated with candidate genes underlying susceptibility to osteoporosis.

protective role associated with stronger bones and reducing the risk of osteoporotic fractures. The extent to which genes cause alteration in combination with different risk factors for fracture might explain the incidence of fractures in the population, and will depend on strength of association between the risk factor and fracture risk, and on proportion of the population at different levels of allele frequencies (Hopper, 2000). Therefore, although a variation in a risk factor may be strongly genetically determined, it may have little consequence for the disease in terms of explaining different patient's clinical outcomes.

### **2. State-of-the-art**

Bone fracture healing is a complicated, multistage process, influenced by cells events and regulated by local and systematic factors. Recent data concerning the regulatory factors correlated with fracture healing suggest that several chemical compounds can be used to stimulate bone growth, and enhance callus formation and maturation. The pharmacological approach towards osteoporosis prevention aims to increase the bone mass by decreasing of osteoclastic bone resorption with the aid of estrogens, bisphosphonates, calcitonin, calcium plus, cholecalciferol, calcitriol and selective estrogen receptor modulators (Grabo & Longyhore, 2008).

Moving away from these conventional approaches, investigators have recently used lovastatin loaded nanoparticles (Garrett et al., 2007) or Ossein – Hydroxyapatit compounds (OHC) for osteoporosis treatment. One of the most important pleiotropic actions of statins is their effect on bone metabolism (Horiuchi & Maeda, 2006). Statins form a class of hypolipidemic agents, known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG Co-A) reductase inhibitors, commonly used as therapy to lower the cholesterol levels in people with/or at risk of cardiovascular disease. Statins exhibit a pleiotropic effect in preclinical models, accelerating fracture healing and increasing angiogenesis when the fracture area is

Osteoporosis: A Look at the Future 671

developed in last years, their application in the treatment of osteoporotic bones is limited

A significant number of osteoporotic bone fractures are treated with implants which are generally fixed with bone cement. However, this fixation requires good quality cancellous bone, generally absent in the osteoporotic population. The incidence of implant loosening and revision surgery is high when osteoporosis is present. For years, attempts have been made to use tissue engineering to develop functional substitutes for damaged or diseased tissues through complex constructs with living cells, bioactive molecules and threedimensional scaffolds, which can support cell attachment, proliferation and differentiation. By integrating of nanotechnology with cellular and molecular biology we aimed to develop injectable, bioresorbable polymers, in acellular and autologous cell-seeded forms, to enhance the bone fracture fixation and healing, and to promote regeneration of the natural tissue.





Strategies that allow efficient scaffold injectability are critical for the realization of successful

The synthesis of block copolymers with a general structure PLGA-PEG-PLGA was accomplished as follows: A mixture of polyethylene glycol (PEG), lactide and glycolide with a predetermined molar ratio (Table 1) was placed into a polymerization flask and dried by azeotropic distillation using anhydrous toluene as an entraining agent. After heating at 80 °C under stirring, 0.5 mL of 0.06 M Sn(Oct)2 was rapidly injected through a septum into the polymerization mixture. Then the reaction temperature was elevated to 140 °C and maintained constant for 24 hours. After that, the polymerization flask was cooled down to room temperature and the reaction mixture was dissolved in a small amount of CH2Cl2. The copolymers were collected by precipitation in cooled diethyl ether and dried at 40 °C in vacuum for few minutes. The obtained copolymers were dissolved in cold water and then

**3. New concepts and strategies for the treatment of osteoporosis** 




because of their weak angiogenic properties.

New strategies involve:

**4. Methods** 

• Preparation of injectable scaffolds


bone tissue and material

nanocoating technology.

treatment for osteoporosis with minimally invasive surgery.

**4.1 Synthesis of PLGA-PEG-PLGA triblock copolymers** 

systemically or locally treated. OHCs also have osteogenic and chondrogenic properties *in vitro* and accelerate fracture healing *in vivo* (Annefeld et al., 1986).

Some patients respond poorly to these therapies, including the integration of implants such as hip replacements. They develop post-surgical complications, caused mainly by weakening of the implant-bone bond due to bone resorption at the interface. This is caused by necrosis of the surrounding host tissue due to surgery, heat released during cement polymerization and micromotion caused by poor fixation. To improve the integration of implants with weak osteoporotic bone, fixation augmentation techniques have been recently proposed such as superficial coatings of polymethylmethacrylate, calcium phosphate ceramics or hydroxyapatite (HA) onto the metallic surfaces (Annefeld et al., 1986; Moroni et al., 2001; Moroni et al., 2005a; Moroni et al., 2005b). Metal surfaces coated with nanohydroxyapatite or biomimetic calcium phosphate (CaP) have been used to improve the fracture fixation stability, which has been proposed as a strategy for porous bones. However, the conventional method for CaP-coating requires high temperature treatment, which prevents the addition of bioactive pharmaceuticals. Post-treatment loading with drugs generally results in their uncontrolled, high kinetic release. A biomimetic nano-CaP coating technology which retains all the features of plasma spraying, including controllable porosity and 3D morphology was recently developed. This technology results in the deposition of amorphous CaP coprecipitated with bioactive substances through the full thickness of the coating, allowing gradual release. The influence of resorbable CaP particles and paste on bone healing was investigated by Bloemers et al. (Bloemers et al., 2003). Twelve weeks after the defect reconstruction, radiological, biomechanical, and histological analyses were performed. Biomechanical tests showed a significantly higher torsional stiffness for the resorbable CaP paste group when compared with the autologous bone.

Several other investigations (de Melo et al., 2006; Niemeyer et al., 2010) published recently utilized the transplantation of xenogenic human and autologous ovine mesenchymal progenitor cells in an ovine critical-size defect or study whether blood-derived endothelial progenitor cells promote the bone regeneration once transplanted into an ovine and tibial defect in animal models (Rozen et al., 2009). Both studies showed significant bone regenerative processes, but the overall results are inconclusive.

Nanoscale materials currently investigated for bone tissue engineering applications can be placed in the following categories: ceramics, metals, and composites. Each type has distinct properties that can be advantageous for specific bone repairing applications. For example, HA, an inorganic compound of bone can be made synthetically. Ceramics are not mechanically tough enough to be used in bulk for large scale bone fractures. However, for a long time they have found applications as bioactive coatings, owing to their ionic bonding mechanisms that are favorable for osteoblast functions (Hench, 1982; Hench, 2004). Unlike ceramic materials, metals are not present in the body as bulk materials. However, because of their mechanical strength and relative inactivity with biological substances, metals and alloys (Ti, Ti6Al4V, CoCrMo, etc.) have been the materials of choice for large bone fractures. Composites of the above mentioned materials can be synthesized to provide a wide range of material properties and also to increase the bone implant performance (Nikolovski & Mooney, 2000). It was found that detonation-generated nanodiamond (DND) inclusions can stimulate the biological performance of the composite layer (Pramatarova et al., 2007).

Although novel bioactive and organic materials with good bioavailability and osteogenic activity such as new nano CaP and sulfate cements, bioactive glass and polymers, have been

systemically or locally treated. OHCs also have osteogenic and chondrogenic properties *in* 

Some patients respond poorly to these therapies, including the integration of implants such as hip replacements. They develop post-surgical complications, caused mainly by weakening of the implant-bone bond due to bone resorption at the interface. This is caused by necrosis of the surrounding host tissue due to surgery, heat released during cement polymerization and micromotion caused by poor fixation. To improve the integration of implants with weak osteoporotic bone, fixation augmentation techniques have been recently proposed such as superficial coatings of polymethylmethacrylate, calcium phosphate ceramics or hydroxyapatite (HA) onto the metallic surfaces (Annefeld et al., 1986; Moroni et al., 2001; Moroni et al., 2005a; Moroni et al., 2005b). Metal surfaces coated with nanohydroxyapatite or biomimetic calcium phosphate (CaP) have been used to improve the fracture fixation stability, which has been proposed as a strategy for porous bones. However, the conventional method for CaP-coating requires high temperature treatment, which prevents the addition of bioactive pharmaceuticals. Post-treatment loading with drugs generally results in their uncontrolled, high kinetic release. A biomimetic nano-CaP coating technology which retains all the features of plasma spraying, including controllable porosity and 3D morphology was recently developed. This technology results in the deposition of amorphous CaP coprecipitated with bioactive substances through the full thickness of the coating, allowing gradual release. The influence of resorbable CaP particles and paste on bone healing was investigated by Bloemers et al. (Bloemers et al., 2003). Twelve weeks after the defect reconstruction, radiological, biomechanical, and histological analyses were performed. Biomechanical tests showed a significantly higher torsional stiffness for the resorbable CaP paste group when compared with the autologous bone. Several other investigations (de Melo et al., 2006; Niemeyer et al., 2010) published recently utilized the transplantation of xenogenic human and autologous ovine mesenchymal progenitor cells in an ovine critical-size defect or study whether blood-derived endothelial progenitor cells promote the bone regeneration once transplanted into an ovine and tibial defect in animal models (Rozen et al., 2009). Both studies showed significant bone

*vitro* and accelerate fracture healing *in vivo* (Annefeld et al., 1986).

regenerative processes, but the overall results are inconclusive.

Nanoscale materials currently investigated for bone tissue engineering applications can be placed in the following categories: ceramics, metals, and composites. Each type has distinct properties that can be advantageous for specific bone repairing applications. For example, HA, an inorganic compound of bone can be made synthetically. Ceramics are not mechanically tough enough to be used in bulk for large scale bone fractures. However, for a long time they have found applications as bioactive coatings, owing to their ionic bonding mechanisms that are favorable for osteoblast functions (Hench, 1982; Hench, 2004). Unlike ceramic materials, metals are not present in the body as bulk materials. However, because of their mechanical strength and relative inactivity with biological substances, metals and alloys (Ti, Ti6Al4V, CoCrMo, etc.) have been the materials of choice for large bone fractures. Composites of the above mentioned materials can be synthesized to provide a wide range of material properties and also to increase the bone implant performance (Nikolovski & Mooney, 2000). It was found that detonation-generated nanodiamond (DND) inclusions can stimulate the biological performance of the composite layer (Pramatarova et al., 2007). Although novel bioactive and organic materials with good bioavailability and osteogenic activity such as new nano CaP and sulfate cements, bioactive glass and polymers, have been developed in last years, their application in the treatment of osteoporotic bones is limited because of their weak angiogenic properties.

### **3. New concepts and strategies for the treatment of osteoporosis**

A significant number of osteoporotic bone fractures are treated with implants which are generally fixed with bone cement. However, this fixation requires good quality cancellous bone, generally absent in the osteoporotic population. The incidence of implant loosening and revision surgery is high when osteoporosis is present. For years, attempts have been made to use tissue engineering to develop functional substitutes for damaged or diseased tissues through complex constructs with living cells, bioactive molecules and threedimensional scaffolds, which can support cell attachment, proliferation and differentiation. By integrating of nanotechnology with cellular and molecular biology we aimed to develop injectable, bioresorbable polymers, in acellular and autologous cell-seeded forms, to enhance the bone fracture fixation and healing, and to promote regeneration of the natural tissue. New strategies involve:

	- Incorporation of angiogenic/osteogenic factors within the scaffolds (if necessary)
	- Homogeneous distribution within a cavity of any shape
	- Easy integration of biologically active substances and nanomaterials for improvement of the pro-angiogenetic properties of bone macroporous scaffolds
	- The ability to deliver progenitor cells as autologous transplant
	- Manipulation of cell function
	- Avoidance of foreign body reactions
	- Transplant assimilation and remodelling
	- Enhancing minimally invasive application and securing better apposition between bone tissue and material
	- Deposition of angiogenic extracellular matrixes on the surfaces
	- Preparation of new implants with increased biocompatibility by specific nanocoating technology.

Strategies that allow efficient scaffold injectability are critical for the realization of successful treatment for osteoporosis with minimally invasive surgery.

### **4. Methods**

### **4.1 Synthesis of PLGA-PEG-PLGA triblock copolymers**

The synthesis of block copolymers with a general structure PLGA-PEG-PLGA was accomplished as follows: A mixture of polyethylene glycol (PEG), lactide and glycolide with a predetermined molar ratio (Table 1) was placed into a polymerization flask and dried by azeotropic distillation using anhydrous toluene as an entraining agent. After heating at 80 °C under stirring, 0.5 mL of 0.06 M Sn(Oct)2 was rapidly injected through a septum into the polymerization mixture. Then the reaction temperature was elevated to 140 °C and maintained constant for 24 hours. After that, the polymerization flask was cooled down to room temperature and the reaction mixture was dissolved in a small amount of CH2Cl2. The copolymers were collected by precipitation in cooled diethyl ether and dried at 40 °C in vacuum for few minutes. The obtained copolymers were dissolved in cold water and then

Osteoporosis: A Look at the Future 673

the samples at a temperature of 4.0 °C for 48 h. The gel-sol transition behavior of the block copolymer solutions was investigated following the procedure described by Lee et al. (Lee et al., 2006) using a WB-4MS water bath-thermostat (Biosan) and also was studied by rheological analysis (Yu et al., 2010). The introduction of NCDs in the polymer hydrogels was made by preparation of sterile aqueous NCD suspensions with different concentrations, used in order to get a final concentration of 0.5 mg/ml in the hydrogels. The sterilization of the NCDs was achieved through stepwise washing with 99% and 45% ethanol and autoclaved deionized water. Lovastatin loaded hydrogels were obtained by dissolution of lovastatin (powder) in the

The degradation of the polymeric hydrogels (166 µl of 25% (w/w) PLGA1000-PEG1000- PLGA1000 or PLGA900-PEG1000-PLGA900) was determined by their incubation in the presence of 5.0 ml EPCs cell culture media (see below) in 24 well plates, in a humidified 5.0% CO2 incubator at 37 °C for 10, 20, and 30 days. Following the incubation periods, the cell culture media was removed and the remaining hydrogels were washed with distilled H2O several times and dried under vacuum up to a constant weight. The obtained dried polymer residuals were dissolved in CDCl3 and characterized by 1H NMR spectroscopy. The hydrogels degradation was also followed by phase contrast microscopy (Carl Zeiss Teraval 3) equipped with a DCM 300 digital camera (Hangzhou Huaxin IC Technology Inc., China).

already prepared hydrogels. The final concentration of lovastatin was 825 µM.

**4.5 Preparation and surface modification of nanodiamond films for implants** 

several references (Koch et al., 2011; Kulisch et al., 2010; Popov et al., 2008b).

**4.6 Isolation and characterization of endothelial progenitor cells (EPCs)** 

Endothelial progenitor cells were isolated and cultured with minor modifications of the protocols described in earlier works (Fuchs et al., 2006a; Fuchs et al., 2006b). In brief, mononuclear cells were harvested from human peripheral blood buffy coats using Ficoll

The ultrananocrystalline diamond (UNCD) coatings discussed in this chapter were grown by microwave plasma chemical vapour deposition (MWCVD) from gas mixtures containing 17% CH4 in N2. The substrate temperature was kept 600 °C, the working pressure 2.2 kPa, the microwave power 800 W, the deposition time 360 min. Detailed description of the MWCVD set-up is given in (Popov et al., 2006b). Monocrystalline silicon wafers were used as substrates which were ultrasonically pretreated in a suspension of diamond powders with different fractions in n-pentane prior the deposition in order to enhance the diamond nucleation on the surface. Such coatings were prepared also on a number of other materials of biomedical interest, for example, Ti-alloy used for implants (Kulisch & Popov, 2006). In order to tailor the surface properties of the UNCD films with respect to their wettability, surface conductivity, etc., they were subjected to a number of surface modification processes as follows: i) H2 microwave plasma at 400 °C in the deposition set-up (in the following: *HP*); ii) O2 microwave plasma at room temperature (*OP*); iii) CHF3 plasma in a 13.56 MHz parallel plate reactor also at room temperature (*FP*); iv) NH3/N2 plasma in a 13.56 MHz plasma enhanced chemical vapour deposition set-up at room temperature (*NP*); v) UV/O3 photochemical treatment at room temperature (*UV*) and vi) chemical treatment with aqua regia (HCl/HNO3 with a ratio of 3:1) at room temperature (*AR*). Further experimental details can be found in

**4.4** *In vitro* **degradation of the polymeric hydrogels** 

**functionalization** 


heated to 80 ºC to precipitate, and remove the water-soluble impurities and unreacted polyethylene oxide. The purification process was repeated three times.

Table 1. The starting monomer and oligomer amounts for the synthesized block copolymers and their yields.

The polymers were characterized by 1H NMR (250 MHz) spectroscopy recorded with a Bruker Avance DRX 250 apparatus, Fourier transforms infrared (FTIR) spectroscopy, and Size exclusion chromatography (GPC). GPC was performed on Waters chromatographic system, equipped with a double detection- differential refractometer M410 and M484 UV detector. Data collection and processing were done using Clarity software. The analyses were performed on Ultrastyragel Linear**,** Styragel 100 Å**,** and Styragel 500 Å columns (Waters) calibrated with polyethylene oxide (PEO) standards. Tetrahydrofuran was used as mobile phase with a flow rate of 1.0 ml/min at 45 °C. FTIR spectra (KBr pallets) were recorded on Bruker-Vector 22 FTIR spectrometer at a resolution of 1-2 cm-1 accumulating 50 scans.

#### **4.2 Nanocrystalline diamonds production and characterization**

Shock-wave synthesis was used to produce nanocrystalline diamonds **(**NCDs) by explosive conversion of a trinitrotoluene/hexogene mixture with negative oxygen balance (Ivanova et al., 2011). A water-cooled combustion chamber with 3.0 m3 volume was applied. A mixture of diamond blends containing 85% NCDs was obtained. The NCDs were purified by oxidative removal of the non-diamond carbon using a mixture of K2Cr2O7 and H2SO4 according to (Tsoncheva et al., 2006). NCDs were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDS). The XRD spectra were obtained with a Bruker D2Phaser diffractometer with CuKα radiation in 2 theta range between 30 and 110° with a step of 0.02° and a measuring time of 10 s per point. The size and morphology of NCDs were studied by TEM. The experiments were performed with a Philips EM420 transmission electron microscope with accelerating voltage of 120 kV. The sample was suspended by ultrasonic agitation in ethanol at room temperature and an aliquot of the solution was dropped on a holey carbon film supported on a copper grid. The impurities present in the samples were analyzed with an EDAX 9100/70 attached to the microscope.

#### **4.3 Preparation of polymer hydrogels and its lovastatin and NCD loaded forms**

The polymer hydrogels were obtained by dissolving of the PLGA-PEG-PLGA polymers in deionized water at suitable temperatures - below and above the so-called "gelation temperature". Equal amounts of freeze-dried PLGA-PEG-PLGA polymers were dissolved in different amounts of sterile water to prepare samples with desired concentrations (10, 15, 20, and 25% (w/w)). The total solubility of the copolymers was achieved by continuous shaking of

heated to 80 ºC to precipitate, and remove the water-soluble impurities and unreacted

PLGA1000-PEG1000-PLGA1000 8.352 1.665 5.000 97 PLGA900-PEG1000-PLGA900 7.500 1.499 5.000 93 PLGA800-PEG1000-PLGA800 6.665 1.330 5.000 98 PLGA700-PEG1000-PLGA700 5.832 1.165 5.000 87 Table 1. The starting monomer and oligomer amounts for the synthesized block copolymers

The polymers were characterized by 1H NMR (250 MHz) spectroscopy recorded with a Bruker Avance DRX 250 apparatus, Fourier transforms infrared (FTIR) spectroscopy, and Size exclusion chromatography (GPC). GPC was performed on Waters chromatographic system, equipped with a double detection- differential refractometer M410 and M484 UV detector. Data collection and processing were done using Clarity software. The analyses were performed on Ultrastyragel Linear**,** Styragel 100 Å**,** and Styragel 500 Å columns (Waters) calibrated with polyethylene oxide (PEO) standards. Tetrahydrofuran was used as mobile phase with a flow rate of 1.0 ml/min at 45 °C. FTIR spectra (KBr pallets) were recorded on Bruker-Vector 22 FTIR spectrometer at a resolution of 1-2 cm-1 accumulating 50

Shock-wave synthesis was used to produce nanocrystalline diamonds **(**NCDs) by explosive conversion of a trinitrotoluene/hexogene mixture with negative oxygen balance (Ivanova et al., 2011). A water-cooled combustion chamber with 3.0 m3 volume was applied. A mixture of diamond blends containing 85% NCDs was obtained. The NCDs were purified by oxidative removal of the non-diamond carbon using a mixture of K2Cr2O7 and H2SO4 according to (Tsoncheva et al., 2006). NCDs were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy (EDS). The XRD spectra were obtained with a Bruker D2Phaser diffractometer with CuKα radiation in 2 theta range between 30 and 110° with a step of 0.02° and a measuring time of 10 s per point. The size and morphology of NCDs were studied by TEM. The experiments were performed with a Philips EM420 transmission electron microscope with accelerating voltage of 120 kV. The sample was suspended by ultrasonic agitation in ethanol at room temperature and an aliquot of the solution was dropped on a holey carbon film supported on a copper grid. The impurities present in the samples were analyzed with an EDAX

**4.3 Preparation of polymer hydrogels and its lovastatin and NCD loaded forms** 

The polymer hydrogels were obtained by dissolving of the PLGA-PEG-PLGA polymers in deionized water at suitable temperatures - below and above the so-called "gelation temperature". Equal amounts of freeze-dried PLGA-PEG-PLGA polymers were dissolved in different amounts of sterile water to prepare samples with desired concentrations (10, 15, 20, and 25% (w/w)). The total solubility of the copolymers was achieved by continuous shaking of

Sample Lactide, g Glycolide, g PEG, g Yield,%

polyethylene oxide. The purification process was repeated three times.

**4.2 Nanocrystalline diamonds production and characterization**

9100/70 attached to the microscope.

and their yields.

scans.

the samples at a temperature of 4.0 °C for 48 h. The gel-sol transition behavior of the block copolymer solutions was investigated following the procedure described by Lee et al. (Lee et al., 2006) using a WB-4MS water bath-thermostat (Biosan) and also was studied by rheological analysis (Yu et al., 2010). The introduction of NCDs in the polymer hydrogels was made by preparation of sterile aqueous NCD suspensions with different concentrations, used in order to get a final concentration of 0.5 mg/ml in the hydrogels. The sterilization of the NCDs was achieved through stepwise washing with 99% and 45% ethanol and autoclaved deionized water. Lovastatin loaded hydrogels were obtained by dissolution of lovastatin (powder) in the already prepared hydrogels. The final concentration of lovastatin was 825 µM.

#### **4.4** *In vitro* **degradation of the polymeric hydrogels**

The degradation of the polymeric hydrogels (166 µl of 25% (w/w) PLGA1000-PEG1000- PLGA1000 or PLGA900-PEG1000-PLGA900) was determined by their incubation in the presence of 5.0 ml EPCs cell culture media (see below) in 24 well plates, in a humidified 5.0% CO2 incubator at 37 °C for 10, 20, and 30 days. Following the incubation periods, the cell culture media was removed and the remaining hydrogels were washed with distilled H2O several times and dried under vacuum up to a constant weight. The obtained dried polymer residuals were dissolved in CDCl3 and characterized by 1H NMR spectroscopy. The hydrogels degradation was also followed by phase contrast microscopy (Carl Zeiss Teraval 3) equipped with a DCM 300 digital camera (Hangzhou Huaxin IC Technology Inc., China).

#### **4.5 Preparation and surface modification of nanodiamond films for implants functionalization**

The ultrananocrystalline diamond (UNCD) coatings discussed in this chapter were grown by microwave plasma chemical vapour deposition (MWCVD) from gas mixtures containing 17% CH4 in N2. The substrate temperature was kept 600 °C, the working pressure 2.2 kPa, the microwave power 800 W, the deposition time 360 min. Detailed description of the MWCVD set-up is given in (Popov et al., 2006b). Monocrystalline silicon wafers were used as substrates which were ultrasonically pretreated in a suspension of diamond powders with different fractions in n-pentane prior the deposition in order to enhance the diamond nucleation on the surface. Such coatings were prepared also on a number of other materials of biomedical interest, for example, Ti-alloy used for implants (Kulisch & Popov, 2006).

In order to tailor the surface properties of the UNCD films with respect to their wettability, surface conductivity, etc., they were subjected to a number of surface modification processes as follows: i) H2 microwave plasma at 400 °C in the deposition set-up (in the following: *HP*); ii) O2 microwave plasma at room temperature (*OP*); iii) CHF3 plasma in a 13.56 MHz parallel plate reactor also at room temperature (*FP*); iv) NH3/N2 plasma in a 13.56 MHz plasma enhanced chemical vapour deposition set-up at room temperature (*NP*); v) UV/O3 photochemical treatment at room temperature (*UV*) and vi) chemical treatment with aqua regia (HCl/HNO3 with a ratio of 3:1) at room temperature (*AR*). Further experimental details can be found in several references (Koch et al., 2011; Kulisch et al., 2010; Popov et al., 2008b).

#### **4.6 Isolation and characterization of endothelial progenitor cells (EPCs)**

Endothelial progenitor cells were isolated and cultured with minor modifications of the protocols described in earlier works (Fuchs et al., 2006a; Fuchs et al., 2006b). In brief, mononuclear cells were harvested from human peripheral blood buffy coats using Ficoll

Osteoporosis: A Look at the Future 675

developmental aspects, such as growth, differentiation, migration and maturation should be taken into account, as these may dictate substrate properties, like degradation and porosity, or require additional instrumentation of bioactive factors (Bouten et al., 2011). Such scaffolds with a possible application for *in bone injection* have to be designed and studied very carefully, because the progenitor cells derived from bone marrow may also be homed on to

The composition of PLGA-PEG-PLGA block copolymers and its average molecular weight (Mn) were determined by 1H NMR spectroscopy (Fig. 2). The broad chemical shift signal at 3.65 ppm marked as **b** is a characteristic of the methylene protons of the ethylene oxide repeating units. The signals observed at 1.58 ppm (signal **a**) CH3-CH-, 5.2 ppm (signal **d**) CH3-CH-, and 4.8 ppm (signal **c**) -O-CH2-C(O)- are assigned to protons of methyl, methine, and methylene groups in lactide and glycolide units, respectively. The integrations (*Ia, Ib, Ic, Id*) of the signals observed at 1.58, 3.65, 4.8 and 5.2 ppm assigned above were used for calculation of average molecular weight of all block copolymers following the equations:

 Mn (NMR) = [(Ia/3)MLac + (Ic/2)MGly + MEO] × 4IbDPPEG (1) or

 Mn (NMR) = [(IdMLac + (Ic/2)MGly + MEO] × 4IbDPPEG (2) The DPPEG is the degree of polymerization of the PEG block and MLac, MGly and MEO are the molecular weights, of the lactic, glycolic, and ethylene oxide segments. The calculated values of the average molecular weight of the block copolymers are represented in Table 2.

> *Mn, (NMR)*

*Mn, (GPC)* *Mw/Mn , (GPC)* 

*(Theor.)*

Table 2. Characteristics of the PLGA-PEG-PLGA triblock copolymers. Data are

representative from twelve independent syntheses and are expressed as average ± S.D.

The results showed deviations between the theoretically expected and the experimentally calculated molecular masses of all polymers. These deviations could be attributed to the presence of some moisture in the organic substances – monomers and PEG oligomer. The NMR data and those obtained from the GPC analysis reveal similar values for the molecular weight of all polymers. The received dispersity (*Mw/Mn)* indicates the narrow molecular weight distribution(see Table 2), which is typical for polymers prepared by a "living",

The composition of the polymers was also proved by FTIR spectroscopy. The spectra of all polymers show similar bands and signals. Only slight differences in the intensity of the bands attributed to the ester and ether type bonds at 1760 and 1163 cm-1 were observed and referred to the differences between the numbers of monomer units included in the polymer

*PLGA1000-PEG1000-PLGA1000 3000 3150±122 3300±169 1.25 PLGA900-PEG1000-PLGA900 2800 2948±151 3140±115 1.17 PLGA800-PEG1000-PLGA800 2600 2873±177 2700 1.11 PLGA700-PEG1000-PLGA700 2400 2664±113 2621 1.21* 

the bioactive substrate.

controlled polymerization.

chains (data not shown).

**5.1 Characterization of three-dimensional (3D) scaffolds** 

*Sample Mn,*

(Sigma-Aldrich) gradient centrifugation and cultured in endothelial cell growth medium-2 (EGM-2 kit; CC-3162, Lonza, Belgium), 5.0% fetal calf serum (FCS; Lonza, Belgium), and 1.0% penicillin/streptomycin, on collagen-coated (BD Europe, Germany) well plates, where 5×106 cells per well were seeded. The good colonies of EPCs appeared after 3 to 4 weeks in culture. The EPCs' phenotype characterization was done following the protocol of (Fuchs et al., 2006a).

#### **4.7 Transformation of EPCs to osteoblast**

Two days after colony formation, the EPCs were trypsinized to single cells, passed through a 70 µm filter, and plated on 1.0 mm cover slips coated with hydrogels at 25 cells/cm2 in osteoprogenitor medium (IMEM, Invitrogen) supplemented with 10% fetal bovine serum (Lonza, Belgium), 0.1 mM 2-mercaptoethanol, 2.0 mM Glutamax I, 2.0 mM BMP-2, and 0.2 mM ascorbic acid. The cultures were fed with osteoprogenitor medium every 2-3 days and allowed to differentiate up to 21 days to form mature bone nodules on different substrates. The transformation of endothelial phenotype of EPCs into long-term osteoblast culture growing on hydrogels or UNCD coatings was assessed as described previously (Popov et al., 2008a; Trajkovski et al., 2009) and followed by phase contrast and fluorescent microscopy, real time PCR (qPCR) and Western blot **(**Trajkovski et al., 2009**).** RNA and proteins were isolated with Trisol (Invitrogen, USA) following manufacturer's instruction on Day 1, 7, 14 and 21 after EPCs seeding on hydrogels and nanocomposites. Prior to amplification, the RNA purity and integrity were checked on Nanodrop-1000 (Termo-Scientific) and agarose gel electrophoresis. SYBR Green qPCR analysis was performed on RotorGene-6000 (Corbet) with the primers sequences and conditions as described by Woll et al (Woll et al., 2006; Woll & Bronson, 2006).

#### **4.8** *In vivo* **experiments**

This study was carried out in accordance with the guidelines of the Medical University Pleven Ethics Committee (N 20/21.12.2010). A total of 24 adult female Wistar rats (220– 250 g, 3 animals per group) were used. Following i. p. injection of 45 mg/kg b.wt. ketamine a sterile 25% (w/w) PLGA1000-PEG1000-PLGA1000 polymers with or without lovastatin (825 µM) or nanodiamonds (500 µg/ml) were implanted s. c. The animals were sacrificed on day 1 and 30 days later, and the explants were taken for further analysis.

#### **4.9 Statistical analysis**

The data were evaluated by analysis of variance (ANOVA) followed by Tukey's post-hoc test. Differences in the results at the level of p<0.05 were considered statistically significant. The statistical analysis was carried out using the PASW 18.0 statistical software package (IBM) for Windows.

### **5. Results**

The structure-function properties of the native bone tissue pose strong design criteria to the substrates required to engineer their living counterparts or to be used in endogenous repair. When designing these substrates one should consider the macroscopic (organ level, geometry), the microscopic (cell and tissue level, scaffold architecture), and molecular properties relevant for tissue morphogenesis and function (e.g. hematopoiesis). In addition,

(Sigma-Aldrich) gradient centrifugation and cultured in endothelial cell growth medium-2 (EGM-2 kit; CC-3162, Lonza, Belgium), 5.0% fetal calf serum (FCS; Lonza, Belgium), and 1.0% penicillin/streptomycin, on collagen-coated (BD Europe, Germany) well plates, where 5×106 cells per well were seeded. The good colonies of EPCs appeared after 3 to 4 weeks in culture. The EPCs' phenotype characterization was done following the protocol of (Fuchs et

Two days after colony formation, the EPCs were trypsinized to single cells, passed through a 70 µm filter, and plated on 1.0 mm cover slips coated with hydrogels at 25 cells/cm2 in osteoprogenitor medium (IMEM, Invitrogen) supplemented with 10% fetal bovine serum (Lonza, Belgium), 0.1 mM 2-mercaptoethanol, 2.0 mM Glutamax I, 2.0 mM BMP-2, and 0.2 mM ascorbic acid. The cultures were fed with osteoprogenitor medium every 2-3 days and allowed to differentiate up to 21 days to form mature bone nodules on different substrates. The transformation of endothelial phenotype of EPCs into long-term osteoblast culture growing on hydrogels or UNCD coatings was assessed as described previously (Popov et al., 2008a; Trajkovski et al., 2009) and followed by phase contrast and fluorescent microscopy, real time PCR (qPCR) and Western blot **(**Trajkovski et al., 2009**).** RNA and proteins were isolated with Trisol (Invitrogen, USA) following manufacturer's instruction on Day 1, 7, 14 and 21 after EPCs seeding on hydrogels and nanocomposites. Prior to amplification, the RNA purity and integrity were checked on Nanodrop-1000 (Termo-Scientific) and agarose gel electrophoresis. SYBR Green qPCR analysis was performed on RotorGene-6000 (Corbet) with the primers sequences and conditions as described by Woll et

This study was carried out in accordance with the guidelines of the Medical University Pleven Ethics Committee (N 20/21.12.2010). A total of 24 adult female Wistar rats (220– 250 g, 3 animals per group) were used. Following i. p. injection of 45 mg/kg b.wt. ketamine a sterile 25% (w/w) PLGA1000-PEG1000-PLGA1000 polymers with or without lovastatin (825 µM) or nanodiamonds (500 µg/ml) were implanted s. c. The animals were sacrificed on day 1

The data were evaluated by analysis of variance (ANOVA) followed by Tukey's post-hoc test. Differences in the results at the level of p<0.05 were considered statistically significant. The statistical analysis was carried out using the PASW 18.0 statistical software package

The structure-function properties of the native bone tissue pose strong design criteria to the substrates required to engineer their living counterparts or to be used in endogenous repair. When designing these substrates one should consider the macroscopic (organ level, geometry), the microscopic (cell and tissue level, scaffold architecture), and molecular properties relevant for tissue morphogenesis and function (e.g. hematopoiesis). In addition,

and 30 days later, and the explants were taken for further analysis.

al., 2006a).

**4.7 Transformation of EPCs to osteoblast** 

al (Woll et al., 2006; Woll & Bronson, 2006).

**4.8** *In vivo* **experiments** 

**4.9 Statistical analysis** 

(IBM) for Windows.

**5. Results** 

developmental aspects, such as growth, differentiation, migration and maturation should be taken into account, as these may dictate substrate properties, like degradation and porosity, or require additional instrumentation of bioactive factors (Bouten et al., 2011). Such scaffolds with a possible application for *in bone injection* have to be designed and studied very carefully, because the progenitor cells derived from bone marrow may also be homed on to the bioactive substrate.

#### **5.1 Characterization of three-dimensional (3D) scaffolds**

The composition of PLGA-PEG-PLGA block copolymers and its average molecular weight (Mn) were determined by 1H NMR spectroscopy (Fig. 2). The broad chemical shift signal at 3.65 ppm marked as **b** is a characteristic of the methylene protons of the ethylene oxide repeating units. The signals observed at 1.58 ppm (signal **a**) CH3-CH-, 5.2 ppm (signal **d**) CH3-CH-, and 4.8 ppm (signal **c**) -O-CH2-C(O)- are assigned to protons of methyl, methine, and methylene groups in lactide and glycolide units, respectively. The integrations (*Ia, Ib, Ic, Id*) of the signals observed at 1.58, 3.65, 4.8 and 5.2 ppm assigned above were used for calculation of average molecular weight of all block copolymers following the equations:

$$\mathbf{M}\_{\text{n(NMR)}} = \left[ (\mathbf{I}\_{\text{a}} / 3)\mathbf{M}\_{\text{Lac}} + (\mathbf{I}\_{\text{c}} / 2)\mathbf{M}\_{\text{Gly}} + \mathbf{M}\_{\text{EO}} \right] \times 4\mathbf{I}\_{\text{b}}\mathbf{D}\mathbf{P}\_{\text{PRG}}\tag{1}$$

$$\mathbf{M}\_{\text{n(NMR)}} = \left[ (\mathbf{I}\_{\text{d}} \mathbf{M}\_{\text{l.ac}} + (\mathbf{I}\_{\text{c}} / \mathbf{2}) \mathbf{M}\_{\text{Cly}} + \mathbf{M}\_{\text{HD}} \right] \times \mathbf{4} \mathbf{I}\_{\text{b}} \text{DP}\_{\text{FEG}} \tag{2}$$


or


Table 2. Characteristics of the PLGA-PEG-PLGA triblock copolymers. Data are representative from twelve independent syntheses and are expressed as average ± S.D.

The results showed deviations between the theoretically expected and the experimentally calculated molecular masses of all polymers. These deviations could be attributed to the presence of some moisture in the organic substances – monomers and PEG oligomer. The NMR data and those obtained from the GPC analysis reveal similar values for the molecular weight of all polymers. The received dispersity (*Mw/Mn)* indicates the narrow molecular weight distribution(see Table 2), which is typical for polymers prepared by a "living", controlled polymerization.

The composition of the polymers was also proved by FTIR spectroscopy. The spectra of all polymers show similar bands and signals. Only slight differences in the intensity of the bands attributed to the ester and ether type bonds at 1760 and 1163 cm-1 were observed and referred to the differences between the numbers of monomer units included in the polymer chains (data not shown).

Osteoporosis: A Look at the Future 677

Injectable biodegradable copolymer hydrogels, which exhibit a sol–gel phase transition in response to external stimuli, such as temperature changes, have found several biomedical and pharmaceutical applications, such as drug delivery, cell growth, and tissue engineering (Gao et al., 2010; Kan et al., 2005; Kim et al., 2001; Nguyen & Lee, 2010; Qiao et al., 2006; Zentner et al., 2001). Such polymers are also biocompatible and biodegradable, and they represent an ideal system for treatment, being able to overcome the problem of carrier removal after injection (implantation). Hydrogels sensitive to temperature are useful for both *in vitro* and *in vivo* applications. These applications will strongly depend on hydrogels composition and it is important to administer the sol dispersion upon quick transformation to gel under physiological conditions. The composition, morphology and crystallinity of the block copolymers strongly influence the mechanical properties and rate of degradation under specific conditions. To study the *in vitro* behavior of the PLGA1000-PEG1000-PLGA1000 and PLGA900-PEG1000-PLGA900 copolymers we used light microscopy and 1H NMR

Fig. 3. Sol-gel transitions of PLGA1000-PEG1000-PLGA1000 block copolymer (A) and phase

released oligomers and monomers lead to the weight loss of the hydrogels.

Polymer degradation is the key process of erosion and plays a crucial role for all polymers studied. The processes involved in the erosion of a degradable polymer are very complicated. They can be summarized as follows: (1) water enters the polymer bulk; (2) water entrance may be accompanied by swelling; (3) chemical polymer degradation appears, leading to the creation of oligomers and monomers.; (4) the degradation changes the microstructure of the hydrogels through pore formation; (5) oligomers and monomers are released; (6) dissolution and diffusion of oligomers and monomers are undergoing; (7) the pH inside the pores can be changed, depending of polymer composition; (8) finally, the

diagrams of PLGA-PEG-PLGA triblock copolymers (B).

**5.3** *In vitro* **degradation of the polymeric hydrogels** 

spectroscopy.

Fig. 2. Chemical characterization of the polymers. A: 1H NMR spectra of PLGA1000-PEG1000- PLGA1000 (1) and PLGA900-PEG1000-PLGA900 (2) block copolymers; B: Molecular weight distribution overlay patterns of indicated polymers received by gel-permeation chromatography.

The copolymers with an analogous structure but a different ratio between the hydrophilic (PEG) and hydrophobic (PLGA) blocks have been an object of considerable scientific interest and value. The influence of the hydrophilic/hydrophobic ratio between PEG and PLGA blocks, and of the block length on the sol-gel–sol transition properties of PLGA-PEG-PLGA triblock copolymers has been reported (Lee et al., 2001). The authors have also found that the effects of different intramolecular/micellar behavior are accompanied by drastic changes in the area of gel zone.

#### **5.2 Temperature–induced sol-gel phase transition of PLGA-PEG-PLGA block copolymers**

In our work we chose to investigate PLGA-PEG-PLGA copolymers with much lower hydrophobic PLGA content (PEG:PLGA = 1:2 ; 1:1.8). The advantages of such hydrogels and especially their ability to be loaded with bioactive substances, drugs, cells, nanoparticles and etc., prior administration are of particular interests. The phase behavior (reversible sol-gelsol transitions) of the obtained block copolymers in aqueous media at different temperatures and concentrations are illustrated in Fig. 3. As shown, only PLGA1000-PEG1000-PLGA1000 and PLGA900-PEG1000-PLGA900 copolymers possessed the typical gel-sol phase transition behavior upon heating. They have the ability to form temperature dependent micellar aggregates and after further temperature increase form gels due to micelles aggregation and/or packing. In contrast, the other two copolymers showed a vastly different performance. As the longer PLGA chains are present, stronger hydrophobic interactions are detected, leading to an increase in the aggregation and packing between the polymer micelles resulting in the formation of a denser gel state. The gel zone in the phase diagram of PLGA800-PEG1000-PLGA800 and PLGA700-PEG1000-PLGA700 copolymers was not found. Nevertheless, we observed significant alterations of their viscous properties – changes from amber viscous to white viscous state. With additional rise of the temperature, direct transition from white viscous to suspension state was also observed. The slight increase in the temperature enhances the thermal motion of the hydrophobic chains in the PLGA800- PEG1000-PLGA800 and PLGA700-PEG1000-PLGA700 copolymers, which may lead to the disturbance of the micellar structures and polymer precipitation.

Fig. 2. Chemical characterization of the polymers. A: 1H NMR spectra of PLGA1000-PEG1000- PLGA1000 (1) and PLGA900-PEG1000-PLGA900 (2) block copolymers; B: Molecular weight distribution overlay patterns of indicated polymers received by gel-permeation

The copolymers with an analogous structure but a different ratio between the hydrophilic (PEG) and hydrophobic (PLGA) blocks have been an object of considerable scientific interest and value. The influence of the hydrophilic/hydrophobic ratio between PEG and PLGA blocks, and of the block length on the sol-gel–sol transition properties of PLGA-PEG-PLGA triblock copolymers has been reported (Lee et al., 2001). The authors have also found that the effects of different intramolecular/micellar behavior are accompanied by drastic

In our work we chose to investigate PLGA-PEG-PLGA copolymers with much lower hydrophobic PLGA content (PEG:PLGA = 1:2 ; 1:1.8). The advantages of such hydrogels and especially their ability to be loaded with bioactive substances, drugs, cells, nanoparticles and etc., prior administration are of particular interests. The phase behavior (reversible sol-gelsol transitions) of the obtained block copolymers in aqueous media at different temperatures and concentrations are illustrated in Fig. 3. As shown, only PLGA1000-PEG1000-PLGA1000 and PLGA900-PEG1000-PLGA900 copolymers possessed the typical gel-sol phase transition behavior upon heating. They have the ability to form temperature dependent micellar aggregates and after further temperature increase form gels due to micelles aggregation and/or packing. In contrast, the other two copolymers showed a vastly different performance. As the longer PLGA chains are present, stronger hydrophobic interactions are detected, leading to an increase in the aggregation and packing between the polymer micelles resulting in the formation of a denser gel state. The gel zone in the phase diagram of PLGA800-PEG1000-PLGA800 and PLGA700-PEG1000-PLGA700 copolymers was not found. Nevertheless, we observed significant alterations of their viscous properties – changes from amber viscous to white viscous state. With additional rise of the temperature, direct transition from white viscous to suspension state was also observed. The slight increase in the temperature enhances the thermal motion of the hydrophobic chains in the PLGA800- PEG1000-PLGA800 and PLGA700-PEG1000-PLGA700 copolymers, which may lead to the

**5.2 Temperature–induced sol-gel phase transition of PLGA-PEG-PLGA block** 

disturbance of the micellar structures and polymer precipitation.

chromatography.

**copolymers** 

changes in the area of gel zone.

Injectable biodegradable copolymer hydrogels, which exhibit a sol–gel phase transition in response to external stimuli, such as temperature changes, have found several biomedical and pharmaceutical applications, such as drug delivery, cell growth, and tissue engineering (Gao et al., 2010; Kan et al., 2005; Kim et al., 2001; Nguyen & Lee, 2010; Qiao et al., 2006; Zentner et al., 2001). Such polymers are also biocompatible and biodegradable, and they represent an ideal system for treatment, being able to overcome the problem of carrier removal after injection (implantation). Hydrogels sensitive to temperature are useful for both *in vitro* and *in vivo* applications. These applications will strongly depend on hydrogels composition and it is important to administer the sol dispersion upon quick transformation to gel under physiological conditions. The composition, morphology and crystallinity of the block copolymers strongly influence the mechanical properties and rate of degradation under specific conditions. To study the *in vitro* behavior of the PLGA1000-PEG1000-PLGA1000 and PLGA900-PEG1000-PLGA900 copolymers we used light microscopy and 1H NMR spectroscopy.

Fig. 3. Sol-gel transitions of PLGA1000-PEG1000-PLGA1000 block copolymer (A) and phase diagrams of PLGA-PEG-PLGA triblock copolymers (B).

#### **5.3** *In vitro* **degradation of the polymeric hydrogels**

Polymer degradation is the key process of erosion and plays a crucial role for all polymers studied. The processes involved in the erosion of a degradable polymer are very complicated. They can be summarized as follows: (1) water enters the polymer bulk; (2) water entrance may be accompanied by swelling; (3) chemical polymer degradation appears, leading to the creation of oligomers and monomers.; (4) the degradation changes the microstructure of the hydrogels through pore formation; (5) oligomers and monomers are released; (6) dissolution and diffusion of oligomers and monomers are undergoing; (7) the pH inside the pores can be changed, depending of polymer composition; (8) finally, the released oligomers and monomers lead to the weight loss of the hydrogels.

Osteoporosis: A Look at the Future 679

Fig. 5. The relative change in the composition of the block copolymers with the time. Alteration in the molecular weight of PLGA are calculated at a constant PEG content and

lactic ester bonds by lactic and glycolic acids will be slow down.

Fig. 6. Schematic representation of hydrogel formation and erosion/degradation.

Sol-gel transition characteristics, including transition temperature and gel window width are the critical parameters which should be taken into consideration in designing 3D

The mechanism of erosion/degradation of the polymeric hydrogels can be presented by the general scheme shown in Fig. 6. It can be summarized as follow: (1) the amphiphilic PLGA-PEG-PLGA three-block copolymers form stable micelle solutions; (2) under the influence of thermal fluctuations on the polymeric chains, the micelles are packed into a crystalline-like structure – hydrogel; (3) the H2O immerges into hydrogels, and the quick swelling happens; (4) when the swelling equilibrium is reached, the chains start to break as a function of time; (5) the presence of water provokes bulk degradation of the hydrogels via a random hydrolytic scission of the ester linkages in the vicinity of PEG; (6) sustained release of watersoluble PEG and PEG's end-capped with short PLGA tails; (7) the hydrophobicity of the remaining hydrogel should increase; (8) as the experiments were conducted in cell culture media with high buffer capacity, the further hydrolysis of glycolic–glycolic and glycolic–

molecular weight.

Fig. 4 reflects the hydrogels morphology after 10 and 30 days in contact with 5.0 ml EPC culture media at 37 °C. The 20 and 25% (w/w) PLGA1000-PEG1000-PLGA1000 and PLGA900- PEG1000-PLGA900 copolymer samples were about 1.0 mm thick and 1.0 cm in diameter.

Fig. 4. Optical microscopic images of the polymeric hydrogels during their destruction in cell culture environment. Magnification: x100.

Changes in the weight-average molecular weight (MW) of PEG and PLGA components in the scaffolds as a function of degradation time are shown in Fig. 5. It can be seen that the MW of hydrogels decreased with incubation time for all of the scaffolds. There were no significant differences in the degradation rate between 20 and 25% hydrogels. The decrease in molecular weight can be attributed to hydrolysis and macromolecular scission of PLGA (Shih et al., 1996). Monomer release profiles for PLGA have a short induction period. The release rate was higher at early times and declines in a concave manner.

The acidic degradation products of PLGA did not lead to severe decrease in the pH values. It drops from 7.4 (Day 1) to 6.4 (Day 30). To evaluate the degradation of the scaffolds, the medium pH for the specimens was compared to media that was held under the same conditions but did not contain any samples (blank). The EPC's media with a lower pH than the blank one would indicate the release of acidic products from the scaffolds and can lead to faster degradation.

By 24 h the difference between the blank media pH and the pH of the media in contact with hydrogels was negligible and remained so through 1 week. On day 10, the pH of the media incubated with hydrogels began to decrease. This decrease in pH persisted through week 2 and 3, with a maximum deviation from blank pH of - 0.94 ± 0.06 at week 4.

It was noticed that the weight loss was about 39% after 30 days degradation in the cell culture media, however, it was 30% just after 10 days. We used the formulas (1) and (2) mentioned above to calculate the changes in the hydrogels' molecular weight - molar content of the different co-compounds. It should also be kept in mind, that the obtained values are relative and don't represent actual PLGA and PEG content in the hydrogels or overall molecular weight of the polymeric chains, but can be used for the construction of a simple qualitative model describing their destruction. Looking at Fig. 5 we can see significant changes or decreasing in the content of PEG part in hydrogels i. e. decreasing the molecular weight of the corresponding copolymers with the time. Similar indicated changes in the hydrophobicity of the polymeric materials of similar type have also been reported by (Youxin et al., 1994; Zweers et al., 2004).

Fig. 4 reflects the hydrogels morphology after 10 and 30 days in contact with 5.0 ml EPC culture media at 37 °C. The 20 and 25% (w/w) PLGA1000-PEG1000-PLGA1000 and PLGA900- PEG1000-PLGA900 copolymer samples were about 1.0 mm thick and 1.0 cm in diameter.

Fig. 4. Optical microscopic images of the polymeric hydrogels during their destruction in

release rate was higher at early times and declines in a concave manner.

and 3, with a maximum deviation from blank pH of - 0.94 ± 0.06 at week 4.

Changes in the weight-average molecular weight (MW) of PEG and PLGA components in the scaffolds as a function of degradation time are shown in Fig. 5. It can be seen that the MW of hydrogels decreased with incubation time for all of the scaffolds. There were no significant differences in the degradation rate between 20 and 25% hydrogels. The decrease in molecular weight can be attributed to hydrolysis and macromolecular scission of PLGA (Shih et al., 1996). Monomer release profiles for PLGA have a short induction period. The

The acidic degradation products of PLGA did not lead to severe decrease in the pH values. It drops from 7.4 (Day 1) to 6.4 (Day 30). To evaluate the degradation of the scaffolds, the medium pH for the specimens was compared to media that was held under the same conditions but did not contain any samples (blank). The EPC's media with a lower pH than the blank one would indicate the release of acidic products from the scaffolds and can lead

By 24 h the difference between the blank media pH and the pH of the media in contact with hydrogels was negligible and remained so through 1 week. On day 10, the pH of the media incubated with hydrogels began to decrease. This decrease in pH persisted through week 2

It was noticed that the weight loss was about 39% after 30 days degradation in the cell culture media, however, it was 30% just after 10 days. We used the formulas (1) and (2) mentioned above to calculate the changes in the hydrogels' molecular weight - molar content of the different co-compounds. It should also be kept in mind, that the obtained values are relative and don't represent actual PLGA and PEG content in the hydrogels or overall molecular weight of the polymeric chains, but can be used for the construction of a simple qualitative model describing their destruction. Looking at Fig. 5 we can see significant changes or decreasing in the content of PEG part in hydrogels i. e. decreasing the molecular weight of the corresponding copolymers with the time. Similar indicated changes in the hydrophobicity of the polymeric materials of similar type have also been reported by

cell culture environment. Magnification: x100.

to faster degradation.

(Youxin et al., 1994; Zweers et al., 2004).

Fig. 5. The relative change in the composition of the block copolymers with the time. Alteration in the molecular weight of PLGA are calculated at a constant PEG content and molecular weight.

The mechanism of erosion/degradation of the polymeric hydrogels can be presented by the general scheme shown in Fig. 6. It can be summarized as follow: (1) the amphiphilic PLGA-PEG-PLGA three-block copolymers form stable micelle solutions; (2) under the influence of thermal fluctuations on the polymeric chains, the micelles are packed into a crystalline-like structure – hydrogel; (3) the H2O immerges into hydrogels, and the quick swelling happens; (4) when the swelling equilibrium is reached, the chains start to break as a function of time; (5) the presence of water provokes bulk degradation of the hydrogels via a random hydrolytic scission of the ester linkages in the vicinity of PEG; (6) sustained release of watersoluble PEG and PEG's end-capped with short PLGA tails; (7) the hydrophobicity of the remaining hydrogel should increase; (8) as the experiments were conducted in cell culture media with high buffer capacity, the further hydrolysis of glycolic–glycolic and glycolic– lactic ester bonds by lactic and glycolic acids will be slow down.

Fig. 6. Schematic representation of hydrogel formation and erosion/degradation.

Sol-gel transition characteristics, including transition temperature and gel window width are the critical parameters which should be taken into consideration in designing 3D

Osteoporosis: A Look at the Future 681

Next we have examined the release of fibronectin (FN) during the EPCs differentiation to osteoblast. Fluorescent photomicrographs of FN stained cells are shown on Fig. 8. As it can be seen the cell response is heterogeneous and FN distributes in a diffuse, punctate pattern of staining throughout the whole surface. The obtained results can be summarized as follows: (1) FN release was detected on day 7 and this was connected with its adsorption to the surfaces; (2) During the process of EPCs differentiation, the release of FN was favored by the hydrogels + NCDs (day 12, 15, and 21). These observations confirmed the hypothesis suggesting that FN is adsorbed preferentially on hydrophobic surfaces (Nordahl et al., 1995), such as the surface of the hydrogels + NCD undergoing erosion. It has also been suggested that FN plays a unique role during the differentiation of osteoblast cultures connected with the formation of mineralized nodules *in vitro* (Robey, 1996). We have

Fig. 8. Composite induced changes in distribution of fibronectin. Hydrogels were based on 25% (w/w) PLGA1000-PEG1000-PLGA1000 (A); PLGA1000-PEG1000-PLGA1000 + lovastatin (B); PLGA1000-PEG1000-PLGA1000 +nanocrystalline diamonds (C); PLGA1000-PEG1000-PLGA1000 + lovastatin + nanocrystalline diamonds (D). The tracings immediately under each panel are scans of the pixelated fluorescence intensity of fibronectin integrated vertically and scanned

All data obtained by us indicated that not only the PLGA-PEG-PLGA scaffolds are biocompatible with the EPCs as reported (Trajkovski et al., 2009; Ivanova et al., 2011), but composites with NCDs actually promote their growth and transformation. The processes of EPCs differentiation can influence the expression of several important genes. We further

**5.5 qPCR analysis of mRNA derived from endothelial progenitors cells undergoing** 

The expression of four osteogenic markers *Twist1, Runx2, Osterix,* and *Bglap1*, and the endothelial one - platelet endothelial cell adhesion molecule (*PECAM1)*, was analyzed at

reported results confirming this observation (Ivanova et al., 2011).

horizontally across the magnified field.

**transformation to osteoblasts** 

followed these changes by quantitative real time PCR analyses.

scaffold biomaterials. For *in vitro* and *in vivo* application is also necessary to know their behavior in surrounding environments. To follow the biochemical and molecular biological response of the composites prepared by us we investigated the possibility these polymers to be prepared as a cell loaded form.

#### **5.4 Scaffold colonization and seeding efficiency**

The EPCs' attachment and differentiation were performed on sample scaffolds prepared with or without NCDs and lovastatin to evaluate the cell biocompatibility. The experiments suggested that on day 1 of culturing low number of cells was attached to all scaffolds studied. The loading efficiency reached 73.1 ± 5.3% after 3 days on PLGA-PEG-PLGA + NCDs and composites with NCDs and lovastatin (89.6 ± 5.7%) and only 11.4 ± 1.5% for the PLGA-PEG-PLGA + lovastatin. A qualitative analysis of cell adhesion on the scaffolds was carried out by phase contrast microscopy up to 21 days. This study (Fig. 7) demonstrated that the cells were distributed unequally throughout the surface structure with different morphology at the beginning. The results presented in Fig. 7 also confirmed the infiltration and migration of cells deep into the 3D porous network on scaffolds containing NCDs or NCDs + lovastatin only. Cells with distinct rounded nuclei were observed throughout the scaffold, further suggesting normal cell growth on these composites. The results about EPCs adhesion on PLGA-PEG-PLGA scaffolds can found in (Trajkovski et al., 2009; Ivanova et al., 2011).

Fig. 7. Endothelial progenitor cells growing on different type composites based on 25% (w/w) PLGA1000-PEG1000-PLGA1000 hydrogel, containing lovastatin and/or nanocrystalline diamonds (NCDs). Bars 50 µm.

scaffold biomaterials. For *in vitro* and *in vivo* application is also necessary to know their behavior in surrounding environments. To follow the biochemical and molecular biological response of the composites prepared by us we investigated the possibility these polymers to

The EPCs' attachment and differentiation were performed on sample scaffolds prepared with or without NCDs and lovastatin to evaluate the cell biocompatibility. The experiments suggested that on day 1 of culturing low number of cells was attached to all scaffolds studied. The loading efficiency reached 73.1 ± 5.3% after 3 days on PLGA-PEG-PLGA + NCDs and composites with NCDs and lovastatin (89.6 ± 5.7%) and only 11.4 ± 1.5% for the PLGA-PEG-PLGA + lovastatin. A qualitative analysis of cell adhesion on the scaffolds was carried out by phase contrast microscopy up to 21 days. This study (Fig. 7) demonstrated that the cells were distributed unequally throughout the surface structure with different morphology at the beginning. The results presented in Fig. 7 also confirmed the infiltration and migration of cells deep into the 3D porous network on scaffolds containing NCDs or NCDs + lovastatin only. Cells with distinct rounded nuclei were observed throughout the scaffold, further suggesting normal cell growth on these composites. The results about EPCs adhesion on PLGA-PEG-

be prepared as a cell loaded form.

diamonds (NCDs). Bars 50 µm.

**5.4 Scaffold colonization and seeding efficiency** 

PLGA scaffolds can found in (Trajkovski et al., 2009; Ivanova et al., 2011).

Fig. 7. Endothelial progenitor cells growing on different type composites based on 25% (w/w) PLGA1000-PEG1000-PLGA1000 hydrogel, containing lovastatin and/or nanocrystalline Next we have examined the release of fibronectin (FN) during the EPCs differentiation to osteoblast. Fluorescent photomicrographs of FN stained cells are shown on Fig. 8. As it can be seen the cell response is heterogeneous and FN distributes in a diffuse, punctate pattern of staining throughout the whole surface. The obtained results can be summarized as follows: (1) FN release was detected on day 7 and this was connected with its adsorption to the surfaces; (2) During the process of EPCs differentiation, the release of FN was favored by the hydrogels + NCDs (day 12, 15, and 21). These observations confirmed the hypothesis suggesting that FN is adsorbed preferentially on hydrophobic surfaces (Nordahl et al., 1995), such as the surface of the hydrogels + NCD undergoing erosion. It has also been suggested that FN plays a unique role during the differentiation of osteoblast cultures connected with the formation of mineralized nodules *in vitro* (Robey, 1996). We have reported results confirming this observation (Ivanova et al., 2011).

Fig. 8. Composite induced changes in distribution of fibronectin. Hydrogels were based on 25% (w/w) PLGA1000-PEG1000-PLGA1000 (A); PLGA1000-PEG1000-PLGA1000 + lovastatin (B); PLGA1000-PEG1000-PLGA1000 +nanocrystalline diamonds (C); PLGA1000-PEG1000-PLGA1000 + lovastatin + nanocrystalline diamonds (D). The tracings immediately under each panel are scans of the pixelated fluorescence intensity of fibronectin integrated vertically and scanned horizontally across the magnified field.

All data obtained by us indicated that not only the PLGA-PEG-PLGA scaffolds are biocompatible with the EPCs as reported (Trajkovski et al., 2009; Ivanova et al., 2011), but composites with NCDs actually promote their growth and transformation. The processes of EPCs differentiation can influence the expression of several important genes. We further followed these changes by quantitative real time PCR analyses.

#### **5.5 qPCR analysis of mRNA derived from endothelial progenitors cells undergoing transformation to osteoblasts**

The expression of four osteogenic markers *Twist1, Runx2, Osterix,* and *Bglap1*, and the endothelial one - platelet endothelial cell adhesion molecule (*PECAM1)*, was analyzed at

Osteoporosis: A Look at the Future 683

The tissue engineering is based on the method of cell seeding on scaffolds that play a role as a matrix to guide cell growth and to assist the formation of functional new tissues. Scaffolds grant this process by promoting an appropriate surface and sufficient spaces to favor the cell attachment, migration, proliferation and special differentiation in three dimensional ways. The design of the scaffold is the most important because it influences the above mentioned processes. There are many worldwide accepted criteria for ideal scaffolds with application in tissue engineering and the critical ones include the main material, mechanical properties, 3D architecture, surface morphology and chemistry, as well as the scaffold environment before and after the degradation process (Wei G. & Ma P.X., 2007). Other important properties are: (1) to be biocompatible – non-immunogenic and (2) non-toxic for the living cells and tissue. To test the biocompatibility of the scaffolds prepared by us we have

The 3D-scaffolds loaded with lovastatin and/or nanodiamods and injected in rats provide an evidence for biocompatibility and degradability. To examine time-dependent *in vivo* mass loss of polymer hydrogels, we prepared sterile 25% (w/w) PLGA1000-PEG1000-PLGA1000 ± NCDs ± lovastatin and injected them into the rats, subcutaneously. Injectable hydrogels for biomedical applications should be degraded or eliminated from the body after accomplishment of their role. Fig. 10 shows *in situ* gel formation of a polymer solution and separated gels explanted at 2 time points. All polymer solutions transformed to hydrogels,

conducted the *in vivo* experiments.

**5.6** *In vivo* **mass decrease of polymer hydrogels** 

lovastatin + nanocrystalline diamonds (D).

and the hydrogels showed time-dependent mass decrease behaviors.

Fig. 10. *In vivo* time-dependent mass decrease of polymer hydrogels. Photographs of separated polymer explants and *in situ* gelation of the polymer solutions from s.c. injected rats. 25% (w/w) PLGA1000-PEG1000-PLGA1000 (A); PLGA1000-PEG1000-PLGA1000 + lovastatin (B); PLGA1000-PEG1000-PLGA1000 +nanocrystalline diamonds (C); PLGA1000-PEG1000-PLGA1000 +

The hydrogels containing PLGA1000-PEG1000-PLGA1000 alone showed the fastest mass decrease, but they still remained detectable within 4 weeks. The mass of the polymer hydrogels was decreased in the order of PLGA1000-PEG1000-PLGA1000 + NCDs ≅ PLGA1000-PEG1000-PLGA1000 + lovastatin + NCDs > PLGA1000-PEG1000-PLGA1000 + lovastatin > PLGA1000-PEG1000-PLGA1000. After 4 weeks, the remained masses of hydrogels were 59%, 58%, 48 and 23%, respectively. We also noticed that around 3 of the PLGA1000-PEG1000-PLGA1000 + lovastatin loaded hydrogels less capillaries were present, which can be attributed to the negative effect of lovastatin on endothelial cell proliferation and angiogenesis. It may also be connected with the difference of

various time points throughout the transformation of the EPCs culture to osteoblasts. The results shown on Fig. 9 revealed a gene expression pattern characteristic of the osteoblast differentiation. The expression of *Twist1 and Osterix* was highly upregulated by day 14 and persisted to day 21 without significant differences between scaffolds studied. *Runx2* and *Bgalp1* expression was continuously increasing during the differentiation and it was found to be significantly higher (Day 7: p=0.034; Day14: p=0.011; Day 21: p=0.07) in EPCs growing on composite scaffolds containing NCDs in comparison with hydrogels ± lovastatin. The PECAM-1 was expressed at the beginning of the differentiation process, but decreased on days 7 and 21. Incorporation of the lovastatin in hydrogels increased the expression of PECAM-1 at day 7 about five times.

In comparison to the hydrogels ± lovastatin, the scaffolds ± NCDs leaded to upregulation of the osteoblasts associated markers at two time points of investigation (Day 7 and 21). In accordance with these observations the total protein amount of Runx2, Osteocalcin and Collagen-α1 was also high (Trajkovski et al., 2009). *Runx2 mRNA* and protein amount were found to increase continuously. Their expression is also critical for mature osteoblast function. RUNX2 binding sites were identified in the osteoblast specific genes encoding osteocalcin, bone sialoprotein, and osteopontin, as well as type I collagen (Ducy et al., 1997). The detected earlier expression of *Runx2 mRNA* might positively influence the latter expression of osteocalcin and collagen-α1 observed by us. The registered increase in *PECAM1 mRNA* demonstrated at Day 1 and 7 also showed the ability of EPCs to switch the differentiation process under specific conditions and confirmed that the lovastatin can stimulate the PECAM-1 expression in endothelial cells. The gene expression data provide strong evidence for osteogenic transformation of the EPCs to osteoblasts on all scaffolds tested.

Fig. 9. Quantitative PCR analysis of endothelial progenitor cells (EPCs) undergoing differentiation to osteoblasts. EPCs growing on 25% (w/w) PLGA1000-PEG1000-PLGA1000 (A); PLGA1000-PEG1000-PLGA1000 + lovastatin (B); PLGA1000-PEG1000-PLGA1000 +nanocrystalline diamonds (C); PLGA1000-PEG1000-PLGA1000 + lovastatin + nanocrystalline diamonds (D). Each assay was run in triplicate at three different template concentrations. Relative mRNA expression is normalized to ribosomal protein L7 (Rpl7), displayed relative to Day 1, and presented as a common log plot.

various time points throughout the transformation of the EPCs culture to osteoblasts. The results shown on Fig. 9 revealed a gene expression pattern characteristic of the osteoblast differentiation. The expression of *Twist1 and Osterix* was highly upregulated by day 14 and persisted to day 21 without significant differences between scaffolds studied. *Runx2* and *Bgalp1* expression was continuously increasing during the differentiation and it was found to be significantly higher (Day 7: p=0.034; Day14: p=0.011; Day 21: p=0.07) in EPCs growing on composite scaffolds containing NCDs in comparison with hydrogels ± lovastatin. The PECAM-1 was expressed at the beginning of the differentiation process, but decreased on days 7 and 21. Incorporation of the lovastatin in hydrogels increased the expression of

In comparison to the hydrogels ± lovastatin, the scaffolds ± NCDs leaded to upregulation of the osteoblasts associated markers at two time points of investigation (Day 7 and 21). In accordance with these observations the total protein amount of Runx2, Osteocalcin and Collagen-α1 was also high (Trajkovski et al., 2009). *Runx2 mRNA* and protein amount were found to increase continuously. Their expression is also critical for mature osteoblast function. RUNX2 binding sites were identified in the osteoblast specific genes encoding osteocalcin, bone sialoprotein, and osteopontin, as well as type I collagen (Ducy et al., 1997). The detected earlier expression of *Runx2 mRNA* might positively influence the latter expression of osteocalcin and collagen-α1 observed by us. The registered increase in *PECAM1 mRNA* demonstrated at Day 1 and 7 also showed the ability of EPCs to switch the differentiation process under specific conditions and confirmed that the lovastatin can stimulate the PECAM-1 expression in endothelial cells. The gene expression data provide strong evidence for

osteogenic transformation of the EPCs to osteoblasts on all scaffolds tested.

Fig. 9. Quantitative PCR analysis of endothelial progenitor cells (EPCs) undergoing differentiation to osteoblasts. EPCs growing on 25% (w/w) PLGA1000-PEG1000-PLGA1000 (A); PLGA1000-PEG1000-PLGA1000 + lovastatin (B); PLGA1000-PEG1000-PLGA1000 +nanocrystalline diamonds (C); PLGA1000-PEG1000-PLGA1000 + lovastatin + nanocrystalline diamonds (D). Each assay was run in triplicate at three different template concentrations. Relative mRNA expression is normalized to ribosomal protein L7 (Rpl7), displayed relative to Day 1, and

PECAM-1 at day 7 about five times.

presented as a common log plot.

The tissue engineering is based on the method of cell seeding on scaffolds that play a role as a matrix to guide cell growth and to assist the formation of functional new tissues. Scaffolds grant this process by promoting an appropriate surface and sufficient spaces to favor the cell attachment, migration, proliferation and special differentiation in three dimensional ways. The design of the scaffold is the most important because it influences the above mentioned processes. There are many worldwide accepted criteria for ideal scaffolds with application in tissue engineering and the critical ones include the main material, mechanical properties, 3D architecture, surface morphology and chemistry, as well as the scaffold environment before and after the degradation process (Wei G. & Ma P.X., 2007). Other important properties are: (1) to be biocompatible – non-immunogenic and (2) non-toxic for the living cells and tissue. To test the biocompatibility of the scaffolds prepared by us we have conducted the *in vivo* experiments.

### **5.6** *In vivo* **mass decrease of polymer hydrogels**

The 3D-scaffolds loaded with lovastatin and/or nanodiamods and injected in rats provide an evidence for biocompatibility and degradability. To examine time-dependent *in vivo* mass loss of polymer hydrogels, we prepared sterile 25% (w/w) PLGA1000-PEG1000-PLGA1000 ± NCDs ± lovastatin and injected them into the rats, subcutaneously. Injectable hydrogels for biomedical applications should be degraded or eliminated from the body after accomplishment of their role. Fig. 10 shows *in situ* gel formation of a polymer solution and separated gels explanted at 2 time points. All polymer solutions transformed to hydrogels, and the hydrogels showed time-dependent mass decrease behaviors.

Fig. 10. *In vivo* time-dependent mass decrease of polymer hydrogels. Photographs of separated polymer explants and *in situ* gelation of the polymer solutions from s.c. injected rats. 25% (w/w) PLGA1000-PEG1000-PLGA1000 (A); PLGA1000-PEG1000-PLGA1000 + lovastatin (B); PLGA1000-PEG1000-PLGA1000 +nanocrystalline diamonds (C); PLGA1000-PEG1000-PLGA1000 + lovastatin + nanocrystalline diamonds (D).

The hydrogels containing PLGA1000-PEG1000-PLGA1000 alone showed the fastest mass decrease, but they still remained detectable within 4 weeks. The mass of the polymer hydrogels was decreased in the order of PLGA1000-PEG1000-PLGA1000 + NCDs ≅ PLGA1000-PEG1000-PLGA1000 + lovastatin + NCDs > PLGA1000-PEG1000-PLGA1000 + lovastatin > PLGA1000-PEG1000-PLGA1000. After 4 weeks, the remained masses of hydrogels were 59%, 58%, 48 and 23%, respectively. We also noticed that around 3 of the PLGA1000-PEG1000-PLGA1000 + lovastatin loaded hydrogels less capillaries were present, which can be attributed to the negative effect of lovastatin on endothelial cell proliferation and angiogenesis. It may also be connected with the difference of

Osteoporosis: A Look at the Future 685

Atomic force microscopy (AFM) studies showed for all investigated samples the characteristic topography of UNCD films, composed of structures with diameters of several hundred nanometers, which themselves possess a substructure (Fig. 12A). The root mean square surface roughness values lie in the narrow range of 10 – 14 nm (Kulisch et al., 2011). The nanostructured surface can expect to enhance the attachment of cells on it. The surface composition of the as-grown UNCD films (*AG* in the following) was investigated by XPS. The results showed that the as-grown surfaces are very clean, with oxygen and nitrogen concentrations of about 2.0 and 1.0 at%, respectively. Nuclear reaction analysis (NRA) revealed the depth profiles of hydrogen concentration in the UNCD films and indicated

Fig. 12. AFM image of the surface of UNCD film revealing the typical topography of the coatings (A) and Hydrogen depth distribution determined by nuclear reaction analysis and scanning electron microscopy cross section micrograph of UNCD film (B) (Kulisch et al.,

The surface composition of UNCD films after different modifications, described above, has been also investigated by XPS (Popov et al., 2008b) and the results are summarized in Fig. 13A. As already mentioned, the surface of *AG* is very clean, the same holds for *AR*. The sample *NP* exhibits an increase of the surface nitrogen concentration from 1.0 to 7.4 at% after ammonia treatment (Koch et al., 2011). It was found that the oxygen content of the O2 plasma treated surface is about 12 at% (*OP*) and about 8.5 at% for the UV/O3 treated sample (*UV*), in contrast to 2.0 at% for *AG*, indicating an oxidation of the surface by both processes. The plasma treatment with CHF3 leads to a surface fluorine concentration of almost the same value (ca. 12 at%) showing that a fluorination process has taken place on the surface. Having in mind that the hydrogen surface concentration of the as-grown samples is about 14 at% (Kulisch et al., 2008), the XPS results indicate a change of the surface termination by the plasma processes. Closer analyses of the XPS peaks revealed that in the case of the oxygen plasma treatment the terminating hydrogen atoms are replaced by O and OH groups rather than by carboxylic acid groups (Popov et al., 2008b). CHF3 plasma treatment has led to a substitution of the C-H bonds

by C-F rather than to the deposition of a CxHyFz polymer (Popov et al., 2008b).

The wettability of the differently treated UNCD surfaces against purified water were examined by contact angles measurements. The results presented in Figs. 13B and 14 show

surface H concentration of 12-14 at% (Fig. 12B)(Kulisch et al., 2008).

2008; Popov et al., 2003; Popov et al., 2011).

environment, especially water content. There was no adverse effect during the *in vivo* tests. The observed results suggest that polymeric solutions ± NCDs and ± lovastatin can be used as injectable and biodegradable hydrogels and the degradation rate of the composites can be controlled by adjusting substituent compositions.

Since the scaffolds with NCDs showed very good biocompatibility the next step was to test the opportunity for preparation of new, bio-enhanced UNCD containing films with potential application for implant preparation.

### **5.7 ND film modifications and the role of implant functionalization**

The UNCD films deposited under the conditions described above have been comprehensively characterized with respect to their crystallinity, composition, topography and bonding structure (Popov et al., 2003; Popov et al., 2004; Popov C. & Kulisch W., 2003). The X-ray diffraction and selected area electron diffraction (Fig. 11A) revealed patterns characteristic for diamond phase. Furthermore, the size of the diamond crystallites was determined from the XRD peaks to be on the order of 3-5 nm. These nanocrystallites are embedded in an amorphous carbon matrix with a grain boundary width of 1.0-1.5 nm, as shown by TEM (Popov et al., 2004). The ratio of the volume fractions of the two phasescrystalline and amorphous - estimated from the density of the coatings and from the total crystallinity is close to unity (Popov et al., 2003). Investigations of the UNCD films with Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), electron energy loss spectroscopy (EELS, Fig. 11B) and Auger electron spectroscopy (AES) showed the presence of sp2-bonded carbon atoms (up to 15 at%) localized in the amorphous matrix (Popov et al., 2003; Popov et al., 2004). Although no H2 was added to the precursor gas mixture, the UNCD films contain about 9-10 at% H in the bulk, as revealed by elastic recoil detection (ERD) analysis, originating from the CH4 molecules. Fourier transform infrared spectroscopy showed that hydrogen is bonded predominantly in sp3-CHx groups (Popov & Kulisch, 2003).

Fig. 11. Physico-chemical characterization of the UNCD films. (A) Selected area electron diffraction of UNCD film; (B) Electron energy loss spectra of different carbon materials (Popov et al., 2004; Popov et al., 2011).

environment, especially water content. There was no adverse effect during the *in vivo* tests. The observed results suggest that polymeric solutions ± NCDs and ± lovastatin can be used as injectable and biodegradable hydrogels and the degradation rate of the composites can be

Since the scaffolds with NCDs showed very good biocompatibility the next step was to test the opportunity for preparation of new, bio-enhanced UNCD containing films with

The UNCD films deposited under the conditions described above have been comprehensively characterized with respect to their crystallinity, composition, topography and bonding structure (Popov et al., 2003; Popov et al., 2004; Popov C. & Kulisch W., 2003). The X-ray diffraction and selected area electron diffraction (Fig. 11A) revealed patterns characteristic for diamond phase. Furthermore, the size of the diamond crystallites was determined from the XRD peaks to be on the order of 3-5 nm. These nanocrystallites are embedded in an amorphous carbon matrix with a grain boundary width of 1.0-1.5 nm, as shown by TEM (Popov et al., 2004). The ratio of the volume fractions of the two phasescrystalline and amorphous - estimated from the density of the coatings and from the total crystallinity is close to unity (Popov et al., 2003). Investigations of the UNCD films with Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), electron energy loss spectroscopy (EELS, Fig. 11B) and Auger electron spectroscopy (AES) showed the presence of sp2-bonded carbon atoms (up to 15 at%) localized in the amorphous matrix (Popov et al., 2003; Popov et al., 2004). Although no H2 was added to the precursor gas mixture, the UNCD films contain about 9-10 at% H in the bulk, as revealed by elastic recoil detection (ERD) analysis, originating from the CH4 molecules. Fourier transform infrared spectroscopy showed that hydrogen is

**5.7 ND film modifications and the role of implant functionalization** 

bonded predominantly in sp3-CHx groups (Popov & Kulisch, 2003).

(Popov et al., 2004; Popov et al., 2011).

Fig. 11. Physico-chemical characterization of the UNCD films. (A) Selected area electron diffraction of UNCD film; (B) Electron energy loss spectra of different carbon materials

controlled by adjusting substituent compositions.

potential application for implant preparation.

Atomic force microscopy (AFM) studies showed for all investigated samples the characteristic topography of UNCD films, composed of structures with diameters of several hundred nanometers, which themselves possess a substructure (Fig. 12A). The root mean square surface roughness values lie in the narrow range of 10 – 14 nm (Kulisch et al., 2011). The nanostructured surface can expect to enhance the attachment of cells on it. The surface composition of the as-grown UNCD films (*AG* in the following) was investigated by XPS. The results showed that the as-grown surfaces are very clean, with oxygen and nitrogen concentrations of about 2.0 and 1.0 at%, respectively. Nuclear reaction analysis (NRA) revealed the depth profiles of hydrogen concentration in the UNCD films and indicated surface H concentration of 12-14 at% (Fig. 12B)(Kulisch et al., 2008).

Fig. 12. AFM image of the surface of UNCD film revealing the typical topography of the coatings (A) and Hydrogen depth distribution determined by nuclear reaction analysis and scanning electron microscopy cross section micrograph of UNCD film (B) (Kulisch et al., 2008; Popov et al., 2003; Popov et al., 2011).

The surface composition of UNCD films after different modifications, described above, has been also investigated by XPS (Popov et al., 2008b) and the results are summarized in Fig. 13A. As already mentioned, the surface of *AG* is very clean, the same holds for *AR*. The sample *NP* exhibits an increase of the surface nitrogen concentration from 1.0 to 7.4 at% after ammonia treatment (Koch et al., 2011). It was found that the oxygen content of the O2 plasma treated surface is about 12 at% (*OP*) and about 8.5 at% for the UV/O3 treated sample (*UV*), in contrast to 2.0 at% for *AG*, indicating an oxidation of the surface by both processes. The plasma treatment with CHF3 leads to a surface fluorine concentration of almost the same value (ca. 12 at%) showing that a fluorination process has taken place on the surface. Having in mind that the hydrogen surface concentration of the as-grown samples is about 14 at% (Kulisch et al., 2008), the XPS results indicate a change of the surface termination by the plasma processes. Closer analyses of the XPS peaks revealed that in the case of the oxygen plasma treatment the terminating hydrogen atoms are replaced by O and OH groups rather than by carboxylic acid groups (Popov et al., 2008b). CHF3 plasma treatment has led to a substitution of the C-H bonds by C-F rather than to the deposition of a CxHyFz polymer (Popov et al., 2008b).

The wettability of the differently treated UNCD surfaces against purified water were examined by contact angles measurements. The results presented in Figs. 13B and 14 show

Osteoporosis: A Look at the Future 687

subjected to BSA exposure in the second series of experiments. Immediately after this exposure the surfaces were investigated by time of flight secondary ion mass spectroscopy (ToF-SIMS) and XPS (Kulisch et al., 2007). According to the ToF-SIMS analysis, all surfaces were covered by a BSA layer approaching a monolayer but it turned out to be impossible to quantify the adhesion and to evaluate differences between the various surfaces. More insight into this question was brought by XPS measurements. The surface composition of all samples investigated was far from that of a thick spin-coated BSA layer on a silicon substrate, taken as a reference, indicating that the BSA coverage on the UNCD surfaces is limited. As the composition of the starting surfaces is different as discussed above, it seems to be more useful to look at the compositional changes inferred by the BSA exposure which are shown in Fig. 15A. From this figure it is evident that the changes of the surface composition of the as-grown *AG* surface are only marginal. They are more pronounced for the H2 and O2 plasma treated samples (*HP* and *OP*). The largest changes, and thus the highest adhesion of BSA, were observed for the chemically aqua regia treated surface *AR*. A possible reason is the partial loss of the hydrogen termination caused by the treatment as discussed above. Summarizing, it can be stated that the unspecific adhesion of biomolecules such as the highly fouling bovine serum

In the third series of experiments, the protein adsorption on UNCD surfaces with different terminations was studied by inverted enzyme-linked immunosorbent assay (ELISA) with albumin and fibrinogen (Popov et al., 2009). The ratio of albumin to fibrinogen adsorption was calculated from the individual levels of both proteins adsorbed on the surfaces (Fig. 15B). The oxygen terminated layers exhibit a higher albumin to fibrinogen ratio as compared with the fluorine and hydrogen terminated films. It has been pointed out that the variation of the albumin and fibrinogen adsorption ratio is strongly related to the associated surface energies since fibrinogen, being itself hydrophobic, preferentially adsorbs on hydrophobic surfaces, but albumin (with a hydrophilic nature) on hydrophilic surfaces during competitive binding*.* The O-terminated UNCD layers have a hydrophilic surface (33° contact angle against water), leading to a higher albumin/fibrinogen ratio. The F-terminated films show the lowest protein ratio, which is related to the hydrophobic nature of these surfaces (contact angle of water 91°). Therefore the albumin adsorption is much greater on the oxygen terminated surfaces and vice versa – the fibrinogen is preferentially adsorbed on

Fig. 15. Relative changes of the surface composition caused by the exposure of the samples to bovine serum albumin (A) and Contact angles and BSA/fibrinogen adsorption ratios on

The biocompatibility of UNCD films was studied by direct contact tests with osteoblast-like cells, fibroblasts and endothelia cells (Popov et al., 2006a; Popov et al., 2007; Popov et al.,

albumin on UNCD surfaces can be influenced by the surface termination.

the hydrophobic fluorine and hydrogen terminated surfaces.

differently terminated UNCD surfaces (B) (Popov et al., 2011).

that all applied treatments have resulted in modification of the original surface. Three types of surfaces can be differentiated: hydrophobic (*AG*, *HP*, *FP*), hydrophilic (*OP*, *NP*, *UV*) and surface *AR* in between the two groups. The contact angles qC of the three highly hydrophobic samples are in the order *AG* < *HP* < *FP* varying between 85 and 100°, while all hydrophilic samples possess qC < 10°. The contact angle of *AR* is 67° showing also a hydrophobic character.

Fig. 13. Surface composition of ultananocrystalline diamond/amorphous carbon composite films after different treatments (B – carbon; C- oxygen; D- nitrogen; E- fluorine) (A) and contact angles of same surfaces after different treatments (B) (Popov et al., 2011).

Fig. 14. Contact angles and water droplet profiles on ultananocrystalline diamond/amorphous carbon composite films: as-grown (left), after F-containing plasma modification (middle) and after UV/O3 treatment (right) (Popov et al., 2011).

The interaction of a surface with cells is usually dominated (at least in the initial stage) by adhesion of proteins onto the biomaterial surface, which occurs rather rapidly. For this reason it is important to study the interactions of proteins with UNCD surfaces and to tailor them, if necessary, by suitable surface modifications. Up to now, three series of experiments have been carried out to investigate the UNCD/protein interactions.

In the first series, scanning force spectroscopy measurements have been performed with asgrown UNCD films and glass (as a reference) onto which proteins have been deposited. A silicon cantilever functionalized with bovine serum albumin (BSA) has been used for this purpose. 120 single measurements at different positions have been performed for each of the two samples. On the UNCD sample none of 120 measurements indicated any interaction; all force curves had the structureless shape. In contrast, for the glass sample in 38% of all force/distance measurements an interaction between the BSA-functionalized cantilever and the surface was observed, which gave rise to force curves. Thus it can be concluded that the UNCD surfaces are not prone to unspecific interactions with proteins (Popov et al., 2007).

On the other hand, even on such inactive surfaces there will be adhesion of highly fouling proteins such as BSA. In order to investigate whether the BSA adhesion on UNCD films is influenced by the surface termination, several of the above discussed surfaces have been

that all applied treatments have resulted in modification of the original surface. Three types of surfaces can be differentiated: hydrophobic (*AG*, *HP*, *FP*), hydrophilic (*OP*, *NP*, *UV*) and surface *AR* in between the two groups. The contact angles qC of the three highly hydrophobic samples are in the order *AG* < *HP* < *FP* varying between 85 and 100°, while all hydrophilic samples possess qC < 10°. The contact angle of *AR* is 67° showing also a hydrophobic character.

Fig. 13. Surface composition of ultananocrystalline diamond/amorphous carbon composite films after different treatments (B – carbon; C- oxygen; D- nitrogen; E- fluorine) (A) and contact angles of same surfaces after different treatments (B) (Popov et al., 2011).

diamond/amorphous carbon composite films: as-grown (left), after F-containing plasma

The interaction of a surface with cells is usually dominated (at least in the initial stage) by adhesion of proteins onto the biomaterial surface, which occurs rather rapidly. For this reason it is important to study the interactions of proteins with UNCD surfaces and to tailor them, if necessary, by suitable surface modifications. Up to now, three series of experiments

In the first series, scanning force spectroscopy measurements have been performed with asgrown UNCD films and glass (as a reference) onto which proteins have been deposited. A silicon cantilever functionalized with bovine serum albumin (BSA) has been used for this purpose. 120 single measurements at different positions have been performed for each of the two samples. On the UNCD sample none of 120 measurements indicated any interaction; all force curves had the structureless shape. In contrast, for the glass sample in 38% of all force/distance measurements an interaction between the BSA-functionalized cantilever and the surface was observed, which gave rise to force curves. Thus it can be concluded that the UNCD surfaces are not prone to unspecific interactions with proteins (Popov et al., 2007). On the other hand, even on such inactive surfaces there will be adhesion of highly fouling proteins such as BSA. In order to investigate whether the BSA adhesion on UNCD films is influenced by the surface termination, several of the above discussed surfaces have been

Fig. 14. Contact angles and water droplet profiles on ultananocrystalline

have been carried out to investigate the UNCD/protein interactions.

modification (middle) and after UV/O3 treatment (right) (Popov et al., 2011).

subjected to BSA exposure in the second series of experiments. Immediately after this exposure the surfaces were investigated by time of flight secondary ion mass spectroscopy (ToF-SIMS) and XPS (Kulisch et al., 2007). According to the ToF-SIMS analysis, all surfaces were covered by a BSA layer approaching a monolayer but it turned out to be impossible to quantify the adhesion and to evaluate differences between the various surfaces. More insight into this question was brought by XPS measurements. The surface composition of all samples investigated was far from that of a thick spin-coated BSA layer on a silicon substrate, taken as a reference, indicating that the BSA coverage on the UNCD surfaces is limited. As the composition of the starting surfaces is different as discussed above, it seems to be more useful to look at the compositional changes inferred by the BSA exposure which are shown in Fig. 15A. From this figure it is evident that the changes of the surface composition of the as-grown *AG* surface are only marginal. They are more pronounced for the H2 and O2 plasma treated samples (*HP* and *OP*). The largest changes, and thus the highest adhesion of BSA, were observed for the chemically aqua regia treated surface *AR*. A possible reason is the partial loss of the hydrogen termination caused by the treatment as discussed above. Summarizing, it can be stated that the unspecific adhesion of biomolecules such as the highly fouling bovine serum albumin on UNCD surfaces can be influenced by the surface termination.

In the third series of experiments, the protein adsorption on UNCD surfaces with different terminations was studied by inverted enzyme-linked immunosorbent assay (ELISA) with albumin and fibrinogen (Popov et al., 2009). The ratio of albumin to fibrinogen adsorption was calculated from the individual levels of both proteins adsorbed on the surfaces (Fig. 15B). The oxygen terminated layers exhibit a higher albumin to fibrinogen ratio as compared with the fluorine and hydrogen terminated films. It has been pointed out that the variation of the albumin and fibrinogen adsorption ratio is strongly related to the associated surface energies since fibrinogen, being itself hydrophobic, preferentially adsorbs on hydrophobic surfaces, but albumin (with a hydrophilic nature) on hydrophilic surfaces during competitive binding*.* The O-terminated UNCD layers have a hydrophilic surface (33° contact angle against water), leading to a higher albumin/fibrinogen ratio. The F-terminated films show the lowest protein ratio, which is related to the hydrophobic nature of these surfaces (contact angle of water 91°). Therefore the albumin adsorption is much greater on the oxygen terminated surfaces and vice versa – the fibrinogen is preferentially adsorbed on the hydrophobic fluorine and hydrogen terminated surfaces.

Fig. 15. Relative changes of the surface composition caused by the exposure of the samples to bovine serum albumin (A) and Contact angles and BSA/fibrinogen adsorption ratios on differently terminated UNCD surfaces (B) (Popov et al., 2011).

The biocompatibility of UNCD films was studied by direct contact tests with osteoblast-like cells, fibroblasts and endothelia cells (Popov et al., 2006a; Popov et al., 2007; Popov et al.,

Osteoporosis: A Look at the Future 689

European Social Fund. The whole responsibility for the data contents lies with the Beneficiary and under no circumstances should this collection be regarded as representing the

Aitman, T. J.; Dong, R.; Vyse, T. J.; Norsworthy, P. J.; Johnson, M. D.; Smith, J.; Mangion, J.;

Annefeld, M.; Caviezel, R.; Schacht, E. & Schicketanz, K. H. (1986). The influence of ossein-

Bloemers, F. W.; Blokhuis, T. J.; Patka, P.; Bakker, F. C.; Wippermann, B. W. & Haarman, H.

Bouten, C. V.; Dankers, P. Y.; Driessen-Mol, A.; Pedron, S.; Brizard, A. M. & Baaijens, F. P.

Cappuzzo, F.; Hirsch, F. R.; Rossi, E.; Bartolini, S.; Ceresoli, G. L.; Bemis, L.; Haney, J.; Witta, S.;

de Melo, O. N.; Fukushima, F. B.; de Matos, G. A.; Bueno, D. F.; de Oliveira, T. S. &

de Vries, B. B.; Pfundt, R.; Leisink, M.; Koolen, D. A.; Vissers, L. E.; Janssen, I. M.;

Deng, F. Y.; Zhao, L. J.; Pei, Y. F.; Sha, B. Y.; Liu, X. G.; Yan, H.; Wang, L.; Yang, T. L.; Recker,

*Osteoporosis International*, Vol.21, No.4, pp. 579-587, ISSN/ISBN 0937-941X Ducy, P.; Zhang, R.; Geoffroy, V.; Ridall, A. L. & Karsenty, G. (1997). Osf2/Cbfa1: A

Feuk, L.; Carson, A. R. & Scherer, S. W. (2006). Structural variation in the human genome.

retardation. *Am.J.Hum.Genet.*, Vol.77, No.4, pp. 606-616

non-small-cell lung cancer. *J.Natl.Cancer Inst.*, Vol.97, No.9, pp. 643-655 Conrad, D. F.; Andrews, T. D.; Carter, N. P.; Hurles, M. E. & Pritchard, J. K. (2006). A high-

*Biomaterials*, Vol.66B, No.2, pp. 526-531, ISSN/ISBN 0021-9304

Roberton-Lowe, C.; Marshall, A. J.; Petretto, E.; Hodges, M. D.; Bhangal, G.; Patel, S. G.; Sheehan-Rooney, K.; Duda, M.; Cook, P. R.; Evans, D. J.; Domin, J.; Flint, J.; Boyle, J. J.; Pusey, C. D. & Cook,H.T. (2006). Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans. *Nature*, Vol.439, No.7078,

hydroxyapatite compound ('Ossopan') on the healing of a bone defect. *Curr. Med.* 

J. T. M. (2003). Autologous bone versus calcium-phosphate ceramics in treatment of experimental bone defects. *Journal of Biomedical Materials Research Part B-Applied* 

(2011). Substrates for cardiovascular tissue engineering. *Adv.Drug Deliv.Rev.*,

Danenberg, K.; Domenichini, I.; Ludovini, V.; Magrini, E.; Gregorc, V.; Doglioni, C.; Sidoni, A.; Tonato, M.; Franklin, W. A.; Crino, L.; Bunn, P. A., Jr. & Varella-Garcia,M. (2005). Epidermal growth factor receptor gene and protein and gefitinib sensitivity in

resolution survey of deletion polymorphism in the human genome. *Nat.Genet.*,

Serakides, R. (2006). Idiopathic hypertrophic osteopathy in a cat. *J.Feline.Med.Surg.*,

Reijmersdal, S.; Nillesen, W. M.; Huys, E. H.; Leeuw, N.; Smeets, D.; Sistermans, E. A.; Feuth,T.; van Ravenswaaij-Arts, C. M.; van Kessel,A. G.; Schoenmakers,E. F.; Brunner, H. G. & Veltman, J. A. (2005). Diagnostic genome profiling in mental

R. R.; Papasian, C. J. & Deng, H. W. (2010). Genome-wide copy number variation association study suggested VPS13B gene for osteoporosis in Caucasians.

transcriptional activator of osteoblast differentiation. *Cell*, Vol.89, No.5, pp. 747-754,

official position of the European Union and the Contract Body.

*Res. Opin.*, Vol.10, No.4, pp. 241-250

Vol.63, No.4-5, pp. 221-241

Vol.38, No.1, pp. 75-81

Vol.8, No.5, pp. 345-348

ISSN/ISBN 0092-8674

*Nat.Rev.Genet.*, Vol.7, No.2, pp. 85-97

**9. References** 

pp. 851-855

2008a). All cells showed good adhesion and spreading on the UNCD surfaces following the incubation. After several days of cultivation they formed confluent monolayers; comparisons with cells from control samples showed that the UNCD films are not cytotoxic and do not affect the cell viability and proliferation.The coatings are also bioinert as revealed by simulated body fluid (SBF) tests. The exposure to SFB with a composition close to that of blood plasma for 10 days did not result in the formation of hydroxyapatite as shown by analyses of the SBF composition and of the film surface (Popov et al., 2006a).

### **6. A look at the future**

Extraordinary progress has been made in the last decade towards the design of implants and scaffolds with a suitable multi-scale hierarchical structure. The limitations of the design of current bone implants arise mainly from the lack of firm quantitative mechanical data of bones in different stages of osteoporosis. Whilst it is known that osteoporotic bone is generally not cancellous in nature and has thin walls, the essential design paradigm of implants does not reflect this application.

A simple system for assisted bone repair proposed is the *in bone injection* of "intelligent" polymers combined with progenitor cells. It utilizes autologous stem cells transplantation in combination with supportive bioresorbable matrices and bioactive molecules for enhancing growth and repair. Ideally, endothelial progenitor cells obtained from peripheral blood of the same patient may be cultured *in vitro* in the presence of different stimuli and/or nanoparticles to undergo osteoblasts differentiation, prior to autologous transplantation. This injectable therapy could also be used for: (1) modifying the bone interior morphology, porosity and interconnectivity, which are extremely important for cell adhesion, proliferation and differentiation; (2) prophylactic treatment for high risk patients to prevent fractures, especially the hip and vertebrae; (3) treatment to stabilize loose prostheses for patients who would soon require revision surgery; (4) providing exceptional repair of the osteoporotic bone by releasing pharmaceuticals to the specific sites with the purpose of accelerating healing, promoting angiogenesis, reducing the risk of infection, etc.

The pursuit of effective treatments for osteoporotic disease is an extremely challenging scientific frontier requiring the integration of multiple engineering, biological, chemical, surgical, and pathophysiology related disciplines. It is also necessary to have a better understanding of molecular and cellular mechanisms specific to osteoporosis. Studying genomics, proteomics and diseases biology in parallel is likely to yield transformative insights in this regard. Our results together with the continuously incoming new data could have direct implications in the use of biomaterials in tissue engineering and in combination with the rapid manufacturing techniques will offer great opportunities to generate different scaffolds for bone engineering in near future.

### **7. Acknowledgment**

We are grateful to the National Science Fund of Bulgaria (Grant TKX-1704) for their financial support.

#### **8. Conflict of interest**

This book chapter has been published with the financial support of the "Human Resources Development" Operational Programme, co-financed by the European Union through the European Social Fund. The whole responsibility for the data contents lies with the Beneficiary and under no circumstances should this collection be regarded as representing the official position of the European Union and the Contract Body.

### **9. References**

688 Osteoporosis

2008a). All cells showed good adhesion and spreading on the UNCD surfaces following the incubation. After several days of cultivation they formed confluent monolayers; comparisons with cells from control samples showed that the UNCD films are not cytotoxic and do not affect the cell viability and proliferation.The coatings are also bioinert as revealed by simulated body fluid (SBF) tests. The exposure to SFB with a composition close to that of blood plasma for 10 days did not result in the formation of hydroxyapatite as shown by analyses of the SBF composition and of the film surface (Popov et al., 2006a).

Extraordinary progress has been made in the last decade towards the design of implants and scaffolds with a suitable multi-scale hierarchical structure. The limitations of the design of current bone implants arise mainly from the lack of firm quantitative mechanical data of bones in different stages of osteoporosis. Whilst it is known that osteoporotic bone is generally not cancellous in nature and has thin walls, the essential design paradigm of

A simple system for assisted bone repair proposed is the *in bone injection* of "intelligent" polymers combined with progenitor cells. It utilizes autologous stem cells transplantation in combination with supportive bioresorbable matrices and bioactive molecules for enhancing growth and repair. Ideally, endothelial progenitor cells obtained from peripheral blood of the same patient may be cultured *in vitro* in the presence of different stimuli and/or nanoparticles to undergo osteoblasts differentiation, prior to autologous transplantation. This injectable therapy could also be used for: (1) modifying the bone interior morphology, porosity and interconnectivity, which are extremely important for cell adhesion, proliferation and differentiation; (2) prophylactic treatment for high risk patients to prevent fractures, especially the hip and vertebrae; (3) treatment to stabilize loose prostheses for patients who would soon require revision surgery; (4) providing exceptional repair of the osteoporotic bone by releasing pharmaceuticals to the specific sites with the purpose of

accelerating healing, promoting angiogenesis, reducing the risk of infection, etc.

The pursuit of effective treatments for osteoporotic disease is an extremely challenging scientific frontier requiring the integration of multiple engineering, biological, chemical, surgical, and pathophysiology related disciplines. It is also necessary to have a better understanding of molecular and cellular mechanisms specific to osteoporosis. Studying genomics, proteomics and diseases biology in parallel is likely to yield transformative insights in this regard. Our results together with the continuously incoming new data could have direct implications in the use of biomaterials in tissue engineering and in combination with the rapid manufacturing techniques will offer great opportunities to generate different

We are grateful to the National Science Fund of Bulgaria (Grant TKX-1704) for their financial

This book chapter has been published with the financial support of the "Human Resources Development" Operational Programme, co-financed by the European Union through the

**6. A look at the future** 

implants does not reflect this application.

scaffolds for bone engineering in near future.

**7. Acknowledgment** 

**8. Conflict of interest** 

support.


Osteoporosis: A Look at the Future 691

Kim, Y. J.; Choi, S.; Koh, J. J.; Lee, M.; Ko, K. S., & Kim, S. W. (2001). Controlled release of insulin

Koch, H.; Kulisch, W.; Popov, C.; Merz, R.; Merz, B. & Reithmaier, J. P. (2011). Plasma

Kulisch, W. & Popov, C. (2006). On the growth mechanisms of nanocrystalline diamond films. *Physica Status Solidi (A) Applications and Materials*, Vol.203, pp.203-219 Kulisch, W.; Popov, C.; Bliznakov, S.; Ceccone, G.; Gilliland, D.; Sirghi, L. & Rossi, F. (2007).

Kulisch, W.; Popov, C.; Gilliland, D.; Ceccone, G.; Rossi, F. & Reithmaier, J. P. (2010).

*Surface and Interface Analysis*, Vol.42, No.6-7, pp. 1152-1155, ISSN 01422421 Kulisch, W.; Popov, C.; Sasaki, T.; Sirghi, L.; Rauscher, H.; Rossi, F. & Reithmaier, J. P.

Kulisch, W.; Sasaki, T.; Rossi, F.; Popov, C.; Sippel, C. & Grambole, D. (2008). Hydrogen

*Status Solidi - Rapid Research Letters*, Vol.2, No.2, pp. 77-79, ISSN 18626254 Lee, D. S.; Shim, M. S.; Kim, S. W.; Lee, H.; Park, I. & Chang,T.Y. (2001). Novel

Lee, S. U. J.; Han, B. O. R.; Park, S. Y.; Han, D. K. & Kim, S. C. (2006). Sol-gel transition

Marini, F. & Brandi, M. L. (2010). Genetic determinants of osteoporosis: common bases to

Moroni, A.; Faldini, C.; Marchetti, S.; Manca, M.; Consoli, V. & Giannini, S. (2001).

Moroni, A.; Faldini, C.; Pegreffi, F.; Hoang-Kim, A.; Vannini, F. & Giannini, S. (2005b).

Nguyen, M. K. & Lee, D. S. (2010). Injectable biodegradable hydrogels. *Macromol. Biosci.*,

Niemeyer, P.; Schonberger, T. S.; Hahn, J.; Kasten, P.; Fellenberg, J.; Suedkamp, N.;

254-258, ISSN/ISBN 09259635

No.8, pp. 587-592, ISSN 1022-1336

Vol.44, No.2, pp. 888-899, ISSN 0887624X

*Joint Surg.Am.*, Vol.87 Suppl 2, pp. 42-51

Vol.87, No.4, pp. 753-759

Vol.10, No.6, pp. 563-579

doi:10.4061/2010/394579, ISSN/ISBN 2090-0392

00406090

ISSN 18626300

from injectable biodegradable triblock copolymer. *Pharm.Res.*, Vol.18, No.4, pp. 548-550

amination of ultrananocrystalline diamond/amorphous carbon composite films for the attachment of biomolecules. *Diamond and Related Materials*, Vol.20, No.2, pp.

Surface and bioproperties of nanocrystalline diamond/amorphous carbon nanocomposite films. *Thin Solid Films*, Vol.515, No.23, pp. 8407-8411, ISSN/ISBN

Investigation of the UV/O3 treatment of ultrananocrystalline diamond films.

(2011). On the development of the morphology of ultrananocrystalline diamond films. *Physica Status Solidi (A) Applications and Materials*, Vol.208, No.1, pp. 70-80,

incorporation in ultrananocrystalline diamond/amorphous carbon films. *Physica* 

thermoreversible gelation of biodegradable PLGA-block-PEO-block-PLGA triblock copolymers in aqueous solution. *Macromolecular Rapid Communications*, Vol.22,

behavior of biodegradable three-arm and four-arm star-shaped PLGA-PEG block copolymer aqueous solution. *Journal of Polymer Science, Part A: Polymer Chemistry*,

cardiovascular diseases? *International Journal of Hypertension*, Vol.2010, 16 pages-

Improvement of the bone-pin interface strength in osteoporotic bone with use of hydroxyapatite-coated tapered external-fixation pins. A prospective, randomized clinical study of wrist fractures. *J.Bone Joint Surg.Am.*, Vol.83-A, No.5, pp. 717-721 Moroni, A.; Faldini, C.; Pegreffi, F.; Hoang-Kim, A. & Giannini, S. (2005a). Osteoporotic

pertrochanteric fractures can be successfully treated with external fixation. *J.Bone* 

Dynamic hip screw compared with external fixation for treatment of osteoporotic pertrochanteric fractures. A prospective, randomized study. *J.Bone Joint Surg.Am.*,

Mehlhorn, A. T.; Milz, S. & Pearce, S. (2010). Xenogenic transplantation of human


Freeman, J. L.; Perry, G. H.; Feuk, L.; Redon, R.; McCarroll, S. A.; Altshuler, D. M.;

Fuchs, S.; Hermanns, M. I. & Kirkpatrick, C. J. (2006a). Retention of a differentiated

Fuchs, S.; Motta, A.; Migliaresi, C. & Kirkpatrick, C. J. (2006b). Outgrowth endothelial cells

Gao, Y.; Sun, Y.; Ren, F., & Gao,S. (2010). PLGA-PEG-PLGA hydrogel for ocular drug delivery of dexamethasone acetate. *Drug Dev.Ind.Pharm.*, Vol.36, No.10, pp. 1131-1138 Garrett, I. R.; Gutierrez, G. E.; Rossini, G.; Nyman, J.; McCluskey, B.; Flores, A. & Mundy, G.

Gonzalez, E.; Kulkarni, H.; Bolivar, H.; Mangano, A.; Sanchez, R.; Catano, G.; Nibbs, R. J.;

Grabo, T. & Longyhore, D. (2008). Pharmacological Management, In: *Osteoporosis: Clinical* 

Hench, L. L. (1982). Glass Surfaces - 1982. *Journal de Physique*, Vol.43, No.NC-9, pp. 625-636,

Hench, L. L. (2004). Glasses to turn on genes. *Glass Science and Technology*, Vol.77, pp. 95-103,

Hopper,J.L. (2000). How to determine if, and by how much, genetic variation influences

Iafrate, A. J.; Feuk, L.; Rivera, M. N.; Listewnik, M. L.; Donahoe, P. K.; Qi, Y.; Scherer, S. W.

Ivanova, L.; Popov, C.; Kolev, I.; Shivachev, B.; Karadjov, J.; Tarassov, M.; Kulisch, W.;

Kan, P.; Lin, X. Z.; Hsieh, M. F., & Chang, K. Y. (2005). Thermogelling emulsions for vascular

*Genome Res.*, Vol.16, No.8, pp. 949-961

*Biomaterials*, Vol.27, No.31, pp. 5399-5408

Vol.307, No.5714, pp. 1434-1440

978-0-8261-0276-8, New York, USA

pp. 85-101, ISSN/ISBN 1354-523X

*Nat.Genet.*, Vol.36, No.9, pp. 949-951

Vol.75, No.1, pp. 185-192

*Materials*, Vol.20, No.2, pp. 165-169, ISSN 0925-9635

ISSN/ISBN 0302-0738

ISSN/ISBN 0946-7475

No.1, pp. 79-92

0266

Aburatani, H.; Jones, K. W.; Tyler-Smith, C.; Hurles, M. E.; Carter, N. P.; Scherer, S. W. & Lee, C. (2006). Copy number variation: new insights in genome diversity.

endothelial phenotype by outgrowth endothelial cells isolated from human peripheral blood and expanded in long-term cultures. *Cell Tissue Res.*, Vol.326,

isolated and expanded from human peripheral blood progenitor cells as a potential source of autologous cells for endothelialization of silk fibroin biomaterials.

R. (2007). Locally delivered lovastatin nanoparticles enhance fracture healing in rats. *Journal of Orthopaedic Research*, Vol.25, No.10, pp. 1351-1357, ISSN/ISBN 0736-

Freedman, B. I.; Quinones, M. P.; Bamshad, M.J.; Murthy, K. K.; Rovin, B.H.; Bradley, W.; Clark, R.A.; Anderson, S. A.; O'connell, R. J.; Agan, B. K.; Ahuja, S. S.; Bologna, R.; Sen, L.; Dolan, M. J. & Ahuja, S. K. (2005). The influence of CCL3L1 gene-containing segmental duplications on HIV-1/AIDS susceptibility. *Science*,

*guidelines for prevention, diagnosis and management*, S. Gueldner, T. Grabo, E. Newman, D. Cooper, (Eds.), 47-82, Springer Publishing Company LLC, ISBN 13:

osteoporosis, In: *The genetics of osteoporosis and metabolic bone disease*, Michael J.Econs, (Ed.), 29-44, Humana Press Inc., Totowa, ISBN 0-89603-702-9, New Jersey, USA Horiuchi, N. & Maeda, T. (2006). Statins and bone metabolism. *Oral Diseases*, Vol.12, No.2,

& Lee, C. (2004). Detection of large-scale variation in the human genome.

Reithmaier, J. P. & Apostolova, M. D. (2011). Nanocrystalline diamond containing hydrogels and coatings for acceleration of osteogenesis. *Diamond and Related* 

embolization and sustained release of drugs. *J Biomed.Mater.Res.B Appl.Biomater.*,


Osteoporosis: A Look at the Future 693

Popov C. & Kulisch W. (2011). Nanocrystalline diamond films for biosensor applications, In:

Pramatarova, L.; Pecheva, E.; Stavrev, S.; Spasov, T.; Montgomery, P.; Toth, A.; Dimitrova,

Ralston, S. H. & de Crombrugghe B. (2006). Genetic regulation of bone mass and susceptibility to osteoporosis. *Genes Dev.*, Vol.20, No.18, pp. 2492-2506 Redon, R.; Ishikawa, S.; Fitch, K. R.; Feuk, L.; Perry, G. H.; Andrews, T. D.; Fiegler, H.; Shapero,

number in the human genome. *Nature*, Vol.444, No.7118, pp. 444-454

Repping, S.; van Daalen, S. K.; Brown, L. G.; Korver, C. M.; Lange, J.; Marszalek, J. D.;

Richards, J. B.; Kavvoura, F. K.; Rivadeneira, F.; Styrkarsdottir, U.; Estrada, K.; Halldorsson,

Robey P. (1996). Bone matrix proteoglycans and glycoproteins, In: *Principles of Bone Biology,*

Rozen, N.; Bick, T.; Bajayo, A.; Shamian, B.; Schrift-Tzadok, M.;Gabet,Y.; Yayon, A.; Bab, I.;

Schoumans, J.; Ruivenkamp, C.; Holmberg, E.; Kyllerman, M.; Anderlid, B. M. &

Sebat, J.; Lakshmi, B.; Malhotra, D.; Troge, J.; Lese-Martin, C.; Walsh, T.; Yamrom, B.; Yoon, S.;

hydrogels in vitro. *Pharmazie*, Vol.61, No.3, pp. 199-202

chromosomes. *Nat.Genet.*, Vol.38, No.4, pp. 463-467

(array-CGH). *J.Med.Genet.*, Vol.42, No.9, pp. 699-705

Netherlands

7, San Diego, USA

Vol.45, No.5, pp. 918-924

*Nanotechnological basis for advanced sensors*, J.P. Reithmaier, P. Paunovic, W. Kulisch, C. Popov, P. Petkov (Eds.), 447-469, Springer, ISBN 978-94-007-0905-8, Dordrecht,

M., & Apostolova, M. (2007). Artificial bones through nanodiamonds. *Journal of Optoelectronics and Advanced Materials*, Vol.9, No.1, pp. 236-239, ISSN 1454-4164 Qiao, M.; Chen, D.; Ma, X., & Hu, H. (2006). Sustained release of bee venom peptide from

biodegradable thermosensitive PLGA-PEG-PLGA triblock copolymer-based

M. H.; Carson, A. R.; Chen, W.; Cho, E. K.; Dallaire, S.; Freeman, J. L.; Gonzalez, J. R.; Gratacos, M.; Huang, J.; Kalaitzopoulos, D.;Komura,D.; MacDonald, J. R.; Marshall, C. R.; Mei, R.; Montgomery, L.; Nishimura, K.; Okamura, K.; Shen, F.; Somerville, M. J.; Tchinda, J.; Valsesia, A.; Woodwark, C.; Yang, F.; Zhang, J.; Zerjal, T.; Zhang, J.; Armengol, L.; Conrad, D. F.; Estivill, X.; Tyler-Smith, C.; Carter, N. P.; Aburatani, H.; Lee, C.; Jones, K. W.; Scherer, S. W. & Hurles, M. E. (2006). Global variation in copy

Pyntikova, T.; van, der, V; Skaletsky, H.; Page, D. C. & Rozen, S. (2006). High mutation rates have driven extensive structural polymorphism among human Y

B.V.; Hsu, Y. H.; Zillikens, M. C.; Wilson, S. G.; Mullin, B. H.; Amin, N.; Aulchenko, Y. S.; Cupples, L.A.; Deloukas, P.; Demissie, S.; Hofman, A.; Kong, A.; Karasik, D.; van Meurs, J. B.; Oostra, B. A.; Pols, H. A.; Sigurdsson, G.; Thorsteinsdottir, U.; Soranzo, N.; Williams, F. M.; Zhou, Y.; Ralston, S. H.; Thorleifsson, G.; van Duijn, C. M.; Kiel, D. P.; Stefansson, K.; Uitterlinden, A. G.; Ioannidis, J. P. & Spector, T. D. (2009). Collaborative meta-analysis: associations of 150 candidate genes with osteoporosis and osteoporotic fracture. *Ann.Intern.Med.*, Vol.151, No.8, pp. 528-537

J. Bilezidian, L. Raisz, G. Rodan, (Eds.), 155-166, Academic Press, ISBN 0-12-098650-

Soudry, M. & Lewinson, D. (2009). Transplanted blood-derived endothelial progenitor cells (EPC) enhance bridging of sheep tibia critical size defects. *Bone*,

Nordenskjold, M. (2005). Detection of chromosomal imbalances in children with idiopathic mental retardation by array based comparative genomic hybridisation

Krasnitz, A.; Kendall, J.; Leotta, A.; Pai, D.; Zhang, R.; Lee, Y. H.; Hicks, J.; Spence, S. J.; Lee, A. T.; Puura, K.; Lehtimaki, T.; Ledbetter, D.; Gregersen, P. K.; Bregman, J.; Sutcliffe, J. S.; Jobanputra, V.; Chung, W.; Warburton, D.; King, M. C.; Skuse, D.;

mesenchymal stem cells in a critical size defect of the sheep tibia for bone regeneration. *Tissue Eng Part A*, Vol.16, No.1, pp. 33-43


Nikolovski, J. & Mooney, D. J. (2000). Smooth muscle cell adhesion to tissue engineering

Nordahl, J.; Mengarelliwidholm, S.; Hultenby, K. & Reinholt, F. P. (1995). Ultrastructural

Publishing Company LLC, ISBN 13: 978-0-8261-0276-8, New York, USA Popov C. & Kulisch W. (2003). Investigation of nanocrystalline diamond films prepared by

Popov, C.; Bliznakov, S.; Boycheva, S.; Milinovik, N.; Apostolova, M. D.; Anspach, N.;

Popov, C., Kulisch, W., Boycheva, S. & Jelinek, M. (2003). Nanocrystalline diamond/

*'03*, pp. 148-153, ISBN 80-214-2525-X, Brno, Czech Republic, October, 2003 Popov, C.; Kulisch, W.; Gibson, P. N.; Ceccone, G. & Jelinek, M. (2004). Growth and

Popov, C.; Kulisch, W.; Jelinek, M.; Bock, A. & Strnad, J. (2006a). Nanocrystalline

Popov, C.; Novotny, M.; Jelinek, M.; Boycheva, S.; Vorlicek, V.; Trchova, M. & Kulisch, W.

Popov, C.; Kulisch, W.; Boycheva, S.; Yamamoto, K.; Ceccone, G. & Koga, Y. (2004).

regeneration. *Tissue Eng Part A*, Vol.16, No.1, pp. 33-43

scaffolds. *Biomaterials*, Vol.21, No.20, pp. 2025-2032

October, 2003

09259635

ISSN/ISBN 09259635

pp. 735-739, ISSN/ISBN 09259635

Vol.18, No.5-8, pp. 895-898, ISSN 09259635

mesenchymal stem cells in a critical size defect of the sheep tibia for bone

Immunolocalization of Fibronectin in Epiphyseal and Metaphyseal Bone of Young-Rats. *Calcified Tissue International*, Vol.57, No.6, pp. 442-449, ISSN/ISBN 0171-967X Penrod J.; Smith A., Terwilliger S., & Gueldner S. (2008). Demographic perspective: The

magnitude of concern, In: *Osteoporosis: Clinical guidelines for prevention, diagnosis and management*, S. Gueldner, T. Grabo, E. Newman, D. Cooper, (Eds.), 9-18, Springer

microwave plasma chemical vapor deposition, *Proceedings of the CVD XVI and EUROCVD-14 Conference*, pp. 1079-1085, ISBN 978-1-56677-378-2, Paris, France,

Hammann, C.; Nellen, W.; Reithmaier, J. P. & Kulisch, W. (2008a). Nanocrystalline diamond/amorphous carbon composite coatings for biomedical applications. *Diamond and Related Materials*, Vol.17, No.4-5, pp. 882-887, ISSN/ISBN 09259635 Popov, C.; Kulisch, W.; Bliznakov, S.; Ceccone, G.; Gilliland, D.; Sirghi, L. & Rossi, F. (2008b).

Surface modification of nanocrystalline diamond/amorphous carbon composite films. *Diamond and Related Materials*, Vol.17, No.7-10, pp. 1229-1234, ISSN/ISBN

amorphous carbon composite films prepared by MWCVD, *Proceedings of the International Conference on Nanosciences, Nanotechnologies and Nanomaterials NANO* 

characterization of nanocrystalline diamond/amorphous carbon composite films prepared by MWCVD. *Diamond and Related Materials*, Vol.13, No.4-8, pp. 1371-1376,

diamond/amorphous carbon composite films for applications in tribology, optics and biomedicine. *Thin Solid Films*, Vol.494, No.1-2, pp. 92-97, ISSN/ISBN 00406090 Popov, C.; Kulisch, W.; Reithmaier, J. P.; Dostalova, T.; Jelinek, M.; Anspach, N. &

Hammann, C. (2007). Bioproperties of nanocrystalline diamond/amorphous carbon composite films. *Diamond and Related Materials*, Vol.16, No.4-7 SPEC. ISS.,

(2006b). Chemical bonding study of nanocrystalline diamond films prepared by plasma techniques. *Thin Solid Films*, Vol.506-507, pp. 297-302, ISSN 00406090 Popov, C.; Vasilchina, H.; Kulisch, W.; Danneil, F.; Stüber, M.; Ulrich, S.; Welle, A. &

Reithmaier, J. P. (2009). Wettability and protein adsorption on ultrananocrystalline diamond/amorphous carbon composite films. *Diamond and Related Materials*,

Structural investigation of nanocrystalline diamond/amorphous carbon composite films. *Diamond and Related Materials*, Vol.13, No.11-12, pp. 2071-2075, ISSN 09259635


**34** 

*Germany* 

Christoph Räth et al.\*

**Simulating Bone Atrophy and Its Effects on the** 

According to the world health organization (WHO) osteoporosis is considered to belong to the ten most important diseases worldwide. It is defined as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture (NIH, 2000). Osteoporosis is a metabolic bone disorder in which bones become brittle and prone to fracture. The underlying reason for the occurrence of osteoporosis is that the two processes being responsible for bone remodelling turn out of balance. Bone tissue is continuously resorbed by special bone cells, the so-called osteoclasts. On the other hand, the formation of bone also takes place by the action of other bone cells, namely the osteoblasts. These cells form new bone. The bone formation is, however, not uniform. It is rather controlled by an external mechanical stimulus such that more bone material is produced at those sites where the local stress is larger. This leads to an adaptation of the inner bone structure (trabecular bone) to externally acting forces on the bone (Mullender & Huiskes, 1995). Thereby, a minimal-weight structure, that is adapted to its applied stresses, is formed as it was already correctly conjectured by Julius Wolff as early as in 1892 (Wolff, 1892). In a healthy bone there exists an equilibrium between bone formation and bone resorption, whereas in an osteoporotic bone more bone resorption than formation takes place, which leads to a rarefied network of the trabecular bone. Besides this disease induced effect the rarefication of the inner bone structure can also have other causes like living under zero gravity

conditions, immobility or age-related atrophy as the most common example.

\* Irina Sidorenko1, Roberto Monetti1, Jan Bauer2, Thomas Baum2, Maiko Matsuura3,

*<sup>2</sup>Institut für Roentgendiagnostik, Technische Universität München, Munich, Germany 3Institute of Anatomy, Ludwig Maximilians Universität München, Munich, Germany 4Institute for Lightweight Design and Structural Biomechanics,* 

*1Max-Planck-Institut für extraterrestrische Physik, Garching, Germany* 

*Vienna Universit of Technolgy (TU Wien), Wien, Austria* 

Advances in modern imaging modalities like high resolution magnetic resonance (HRMR) or micro computed tomography (μCT) imaging have led to great improvements of the image quality especially in terms of spatial resolution which now allows for a proper threedimensional visualisation of the trabecular network. Having these highly resolved data at

*5Institute of Anatomy and Musculoskeletal Research, Paracelsus Private Medical University, Salzburg, Austria*

**1. Introduction** 

Philippe Zysset4 and Felix Eckstein5

**Structure and Stability of the Trabecular Bone** 

*Max-Planck-Institut für extraterrestrische Physik, Garching,* 

Geschwind, D. H.; Gilliam, T. C.; Ye, K. & Wigler, M. (2007). Strong association of de novo copy number mutations with autism. *Science*, Vol.316, No.5823, pp. 445-449


## **Simulating Bone Atrophy and Its Effects on the Structure and Stability of the Trabecular Bone**

Christoph Räth et al.\*

*Max-Planck-Institut für extraterrestrische Physik, Garching, Germany* 

### **1. Introduction**

694 Osteoporosis

polymorphism in the human genome. *Science*, Vol.305, No.5683, pp. 525-528 Sharp, A. J.; Locke, D. P.; McGrath, S. D.; Cheng, Z.; Bailey, J. A.; Vallente, R. U.; Pertz, L. M.;

Shih, C.; Waldron, N. & Zentner, G. M. (1996). Quantitative analysis of ester linkages in

Trajkovski, B.; Karadjov, J., Shivachev, B., Dimitrova, A., Stavrev, S. & Apostolova, M. D. (2009).

Tuzun, E.; Sharp, A. J.; Bailey, J. A.; Kaul, R.; Morrison, V. A.; Pertz, L. M.; Haugen, E.; Hayden,

Wei, G. & Ma, P. X. (2007). Polymeric biomaterials, In: *Tissue engineering using ceramics and* 

Woll, N. L. & Bronson, S. K. (2006). Analysis of embryonic stem cell-derived osteogenic

Woll, N. L.; Heaney, J. D. & Bronson, S. K. (2006). Osteogenic nodule formation from single embryonic stem cell-derived progenitors. *Stem Cells Dev.*, Vol.15, No.6, pp. 865-879 Youxin, L.; Volland, C. & Kissel, T. (1994). In-vitro degradation and bovine serum albumin

copolymers in water. *Biomacromolecules.*, Vol.11, No.8, pp. 2169-2178 Zentner, G. M.; Rathi, R.; Shih, C.; McRea, J. C.; Seo, M. H.; Oh, H.; Rhee, B. G.; Mestecky, J.;

poly(ethylene oxide). *J Control Release*, Vol.100, No.3, pp. 347-356

variation of the human genome. *Nat.Genet.*, Vol.37, No.7, pp. 727-732

(Eds.), 525-532, Springer, ISBN 978-1-4020-9915-1, Dordrecht, Netherlands Tsoncheva, T.; Ivanova, L.; Paneva, D.; Dimitrov, M.; Mitov, I.; Stavrev, S. & Minchev, C.

human genome. *Am.J.Hum.Genet.*, Vol.77, No.1, pp. 78-88

Vol.38, No.1, pp. 69-73, ISSN 0168-3659

ISBN 978-1-84569-176-9, England, Cambridge

cultures. *Methods Mol.Biol.*, Vol.330, pp. 149-159

No.2, pp. 492-500

Vol.72, No.1-3, pp. 203-215

Geschwind, D. H.; Gilliam, T. C.; Ye, K. & Wigler, M. (2007). Strong association of de novo copy number mutations with autism. *Science*, Vol.316, No.5823, pp. 445-449 Sebat, J.; Lakshmi, B.; Troge, J.; Alexander, J.; Young, J.; Lundin, P.; Maner, S.; Massa, H.; Walker,

M.; Chi, M.; Navin, N.; Lucito, R.; Healy, J.; Hicks, J.; Ye, K.; Reiner, A.; Gilliam, T. C.; Trask, B.; Patterson, N.; Zetterberg, A. & Wigler,M. (2004). Large-scale copy number

Clark, R. A.; Schwartz, S.; Segraves, R.; Oseroff, V. V.; Albertson, D. G.; Pinkel,D. & Eichler, E. E. (2005). Segmental duplications and copy-number variation in the

poly(DL-lactide) and poly(DL-lactide-co-glycolide). *Journal of Controlled Release*,

Novel nanostructured materials accelerating osteogenesis, In: *Nanostructured Materials for Advanced Technological Applications,* J. P. Reithmaier, P. Petkov, W. Kulisch, C. Popov,

(2006). Iron-oxide-modified nanosized diamond: preparation, characterization, and catalytic properties in methanol decomposition. *J.Colloid Interface Sci.*, Vol.302,

H.; Albertson, D.; Pinkel, D.; Olson, M. V. & Eichler, E. E. (2005). Fine-scale structural

*polymers*, A. Boccaccini, J. Gough (Eds.), 32-50, Woodhead Publishing Limited,

release of the ABA triblock copolymers consisting of poly(L(+)lactic acid), or poly(L(+)lactic acid-co-glycolic acid) A-blocks attached to central polyoxyethylene Bblocks. *Journal of Controlled Release*, Vol.32, No.2, pp. 121-128, ISSN/ISBN 01683659 Yu, L.; Zhang, Z.; Zhang, H. & Ding, J. (2010). Biodegradability and biocompatibility of

thermoreversible hydrogels formed from mixing a sol and a precipitate of block

Moldoveanu, Z.; Morgan, M., & Weitman,S. (2001). Biodegradable block copolymers for delivery of proteins and water-insoluble drugs. *J Control Release*,

Y. J. & Deng, H. W. (2010). Replication study of candidate genes/loci associated with osteoporosis based on genome-wide screening. *Osteoporos.Int.,* Vol.21, pp.785-795 Zweers, M. L.; Engbers, G. H.; Grijpma, D. W. & Feijen, J. (2004). In vitro degradation of

nanoparticles prepared from polymers based on DL-lactide, glycolide and

Zhang, Y. P.; Deng, F. Y.; Chen, Y.; Pei, Y. F.; Fang, Y.; Guo Y. F.; Guo X.; Liu X. G.; Zhou, Q.; Liu

According to the world health organization (WHO) osteoporosis is considered to belong to the ten most important diseases worldwide. It is defined as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture (NIH, 2000). Osteoporosis is a metabolic bone disorder in which bones become brittle and prone to fracture. The underlying reason for the occurrence of osteoporosis is that the two processes being responsible for bone remodelling turn out of balance. Bone tissue is continuously resorbed by special bone cells, the so-called osteoclasts. On the other hand, the formation of bone also takes place by the action of other bone cells, namely the osteoblasts. These cells form new bone. The bone formation is, however, not uniform. It is rather controlled by an external mechanical stimulus such that more bone material is produced at those sites where the local stress is larger. This leads to an adaptation of the inner bone structure (trabecular bone) to externally acting forces on the bone (Mullender & Huiskes, 1995). Thereby, a minimal-weight structure, that is adapted to its applied stresses, is formed as it was already correctly conjectured by Julius Wolff as early as in 1892 (Wolff, 1892). In a healthy bone there exists an equilibrium between bone formation and bone resorption, whereas in an osteoporotic bone more bone resorption than formation takes place, which leads to a rarefied network of the trabecular bone. Besides this disease induced effect the rarefication of the inner bone structure can also have other causes like living under zero gravity conditions, immobility or age-related atrophy as the most common example.

Advances in modern imaging modalities like high resolution magnetic resonance (HRMR) or micro computed tomography (μCT) imaging have led to great improvements of the image quality especially in terms of spatial resolution which now allows for a proper threedimensional visualisation of the trabecular network. Having these highly resolved data at

<sup>\*</sup> Irina Sidorenko1, Roberto Monetti1, Jan Bauer2, Thomas Baum2, Maiko Matsuura3, Philippe Zysset4 and Felix Eckstein5

*<sup>1</sup>Max-Planck-Institut für extraterrestrische Physik, Garching, Germany* 

*<sup>2</sup>Institut für Roentgendiagnostik, Technische Universität München, Munich, Germany 3Institute of Anatomy, Ludwig Maximilians Universität München, Munich, Germany 4Institute for Lightweight Design and Structural Biomechanics,* 

*Vienna Universit of Technolgy (TU Wien), Wien, Austria* 

*<sup>5</sup>Institute of Anatomy and Musculoskeletal Research, Paracelsus Private Medical University, Salzburg, Austria*

Simulating Bone Atrophy and Its Effects on the Structure and Stability of the Trabecular Bone 697

harvested from 73 thoracic and 78 lumbar human vertebrae. The resulting μCT grey-value images with isotropic spatial resolution of 26 μm were segmented using a low-pass filter by convolving the image with a Gaussian kernel with standard deviation 0.8 and support of 1 to remove noise and a fixed global threshold equal to 22% of the maximal grey value to extract the mineralised bone phase (Hildebrand et al 1999). After μCT scanning, the bone samples were cut to the length of 12 mm and tested by applying uniaxial mechanical compressive load using a servo-hydraulic machine (MTS 858 mini Bionix II, MTS Eden Prairie, USA) with a load cell of 1.5 kN. Maximum compressive strength (MCS) was determined in biomechanical experiment as the first local maximum of the forcedisplacement curve and used in correlation analysis as a golden standard for characterisation of bone strength (Eckstein et al., 2007). Main structural characteristics of the bone specimens are given in Table 1. The binarized 3D μCT images were used as a starting structure for the numerical simulations of the bone resorption process. Table 1 summarizes

*Mean value* 62.2 0.13 1.10 0.15 0.87 105 *Standard deviation* 4.7 0.03 0.15 0.02 0.12 30 Table 1. Some characteristics of the data set: Mean and standard deviation of the age, the histomorphometric parameters bone volume *BV/TV*, trabecular number *Tb.N.,* trabecular thickness *Tb.Th*., trabecular spacing *Tb.Sp*., and the mechanical parameter maximum

Minkowski Functionals (MF) (Michielsen & de Raedt 2001) provide a global morphological and topological description of structural properties of multidimensional data. In this

(,,)

1 2 1 1 12 ( ) *I MF dS R R*

> 1 2 1

∂

=

= +

*I MF dS R R* ∂

*iiii*

method binarized images are considered as a union of 3D convex bodies (voxels)

1

<sup>=</sup> 4

*I pxyz* = <sup>=</sup>

According to integral geometry an *n*-dimensional body can be completely characterized by *n+1* functionals, which evaluate both size and shape of the object. In a three-dimensional space the four functionals are represented by the volume (*MF1*), surface area (*MF2*), integral mean curvature (*MF3*) and integral Gaussian curvature (*MF4*). Minkowski Functionals are derived from the theory of convex sets and expressed as volume integral for *MF1* and surface integrals over boundary *S* of the excursion set *I* with the principal radii of curvature

*i*

*MF dV* <sup>=</sup> 3

*Nbone*

*age* **BV/TV Tb.N. Tb.Th. Tb.Sp. MCS [N]** 

some main characteristics of the data set.

compressive strength MCS.

**2.2 Structure measures 2.2.1 Minkowski functionals** 

*R1* and *R2* for other functionals*.*

1 *I*

2

*I MF dS* ∂

hand it now becomes possible to perform a differential analysis of both the structural and the mechanical properties of the trabecular bone.

We determine the *local* structure of the trabecular network by calculating isotropic (α) and anisotropic scaling indices (αx,αy,αz) (Räth et al., 2008). These measures have been proven to be able to discriminate rod- from sheet-like structures and to quantify the alignment of structures with respect to a preferential direction as e.g. given by the direction of the external force.

Another class of texture measures is given by Minkowski Functionals (Michielsen & De Raedt 2001), which - as the scaling indices – also incorporate correlation functions of higher orders and supply global morphological information about structures under study.

The calculation of the local mechanical properties, i.e. the load distribution inside bodies with high porosity and complex architecture is enabled by the use of the finite element method (FEM), which has become a standard tool in bone research.

Bone modelling and remodelling processes can be simulated by describing the action of the osteoblasts and osteoclasts by means of rate equations. The equations are nonlinear partial differential equations, which can thus only be solved numerically (Huiskes et al, 2000). As it is mostly the case in nonlinear systems the solutions depend very sensitively on the choice of parameters of the model, with which e.g. the onset of bone formation is controlled.

In this chapter we propose methods to simulate the effects of bone modelling on the structure and stability of the trabecular bone. We gradually deteriorate the trabecular structure by using some concept of cellular automata (Wolfram 1983, Wolfram 1984) to solve the rate equations numerically. Specifically, both the three space coordinates as well as the time is discretised. The bone resorption, which is described as a continuous process by the partial differential equations, is thus transformed to a consecutive removal of bone surface voxel, which is discrete in space and time. Having simulated bone modelling we then assess the effects of changes in the bone structure on both the structural and mechanical properties of the specimen.

We do not consider the details of the physiological mechanisms of the bone adaptation to mechanical loading and do not describe metabolic activity of the biological cells. Rather, we assume independent action of osteoclasts (bone resorbing cells) and osteoblats (bone forming cells), which is typical for bone modelling process leading to global morphological and topological changes. Among the large variety of bone modelling scenarios we concentrate on bone atrophy due to erosion of the bone surface by osteoblasts resorption activity and decrease of osteoblast formation activity, which is typical for the process of normal aging (often called primary osteoporosis).

This type of bone modelling can be simulated by random resorption of bone voxels at the interface between trabecular bone and bone marrow. We develop three numerical models for simulation of bone loss, which correspond to the different assumptions about age-related atrophy in male and female bones: thinning of the trabecular bone structure with and without preserving of topological connectivity and with preferential loss of aligned rod-like trabecular elements, which were previously identified by a scaling index analysis.

### **2. Material and methods**

#### **2.1 The data set**

For our study we chose 17 specimens of young (50 < age < 70) patients with high maximum compressive strength (MCS > 70 Newton (N)) out of a data set (Räth et al., 2008) of 151 cylindrical specimens with a diameter of 8 mm and a length of 14 mm, which were harvested from 73 thoracic and 78 lumbar human vertebrae. The resulting μCT grey-value images with isotropic spatial resolution of 26 μm were segmented using a low-pass filter by convolving the image with a Gaussian kernel with standard deviation 0.8 and support of 1 to remove noise and a fixed global threshold equal to 22% of the maximal grey value to extract the mineralised bone phase (Hildebrand et al 1999). After μCT scanning, the bone samples were cut to the length of 12 mm and tested by applying uniaxial mechanical compressive load using a servo-hydraulic machine (MTS 858 mini Bionix II, MTS Eden Prairie, USA) with a load cell of 1.5 kN. Maximum compressive strength (MCS) was determined in biomechanical experiment as the first local maximum of the forcedisplacement curve and used in correlation analysis as a golden standard for characterisation of bone strength (Eckstein et al., 2007). Main structural characteristics of the bone specimens are given in Table 1. The binarized 3D μCT images were used as a starting structure for the numerical simulations of the bone resorption process. Table 1 summarizes some main characteristics of the data set.


Table 1. Some characteristics of the data set: Mean and standard deviation of the age, the histomorphometric parameters bone volume *BV/TV*, trabecular number *Tb.N.,* trabecular thickness *Tb.Th*., trabecular spacing *Tb.Sp*., and the mechanical parameter maximum compressive strength MCS.

#### **2.2 Structure measures 2.2.1 Minkowski functionals**

696 Osteoporosis

hand it now becomes possible to perform a differential analysis of both the structural and

We determine the *local* structure of the trabecular network by calculating isotropic (α) and anisotropic scaling indices (αx,αy,αz) (Räth et al., 2008). These measures have been proven to be able to discriminate rod- from sheet-like structures and to quantify the alignment of structures with respect to a preferential direction as e.g. given by the direction of the external force. Another class of texture measures is given by Minkowski Functionals (Michielsen & De Raedt 2001), which - as the scaling indices – also incorporate correlation functions of higher

The calculation of the local mechanical properties, i.e. the load distribution inside bodies with high porosity and complex architecture is enabled by the use of the finite element

Bone modelling and remodelling processes can be simulated by describing the action of the osteoblasts and osteoclasts by means of rate equations. The equations are nonlinear partial differential equations, which can thus only be solved numerically (Huiskes et al, 2000). As it is mostly the case in nonlinear systems the solutions depend very sensitively on the choice of parameters of the model, with which e.g. the onset of bone formation is controlled. In this chapter we propose methods to simulate the effects of bone modelling on the structure and stability of the trabecular bone. We gradually deteriorate the trabecular structure by using some concept of cellular automata (Wolfram 1983, Wolfram 1984) to solve the rate equations numerically. Specifically, both the three space coordinates as well as the time is discretised. The bone resorption, which is described as a continuous process by the partial differential equations, is thus transformed to a consecutive removal of bone surface voxel, which is discrete in space and time. Having simulated bone modelling we then assess the effects of changes in the bone structure on both the structural and mechanical properties

We do not consider the details of the physiological mechanisms of the bone adaptation to mechanical loading and do not describe metabolic activity of the biological cells. Rather, we assume independent action of osteoclasts (bone resorbing cells) and osteoblats (bone forming cells), which is typical for bone modelling process leading to global morphological and topological changes. Among the large variety of bone modelling scenarios we concentrate on bone atrophy due to erosion of the bone surface by osteoblasts resorption activity and decrease of osteoblast formation activity, which is typical for the process of

This type of bone modelling can be simulated by random resorption of bone voxels at the interface between trabecular bone and bone marrow. We develop three numerical models for simulation of bone loss, which correspond to the different assumptions about age-related atrophy in male and female bones: thinning of the trabecular bone structure with and without preserving of topological connectivity and with preferential loss of aligned rod-like

For our study we chose 17 specimens of young (50 < age < 70) patients with high maximum compressive strength (MCS > 70 Newton (N)) out of a data set (Räth et al., 2008) of 151 cylindrical specimens with a diameter of 8 mm and a length of 14 mm, which were

trabecular elements, which were previously identified by a scaling index analysis.

orders and supply global morphological information about structures under study.

method (FEM), which has become a standard tool in bone research.

the mechanical properties of the trabecular bone.

normal aging (often called primary osteoporosis).

**2. Material and methods** 

**2.1 The data set** 

of the specimen.

Minkowski Functionals (MF) (Michielsen & de Raedt 2001) provide a global morphological and topological description of structural properties of multidimensional data. In this method binarized images are considered as a union of 3D convex bodies (voxels)

$$I = \bigcup\_{i=1}^{N\_{\text{low}}} \vec{p}\_i(\mathbf{x}\_{i'} y\_{i'} z\_i)$$

According to integral geometry an *n*-dimensional body can be completely characterized by *n+1* functionals, which evaluate both size and shape of the object. In a three-dimensional space the four functionals are represented by the volume (*MF1*), surface area (*MF2*), integral mean curvature (*MF3*) and integral Gaussian curvature (*MF4*). Minkowski Functionals are derived from the theory of convex sets and expressed as volume integral for *MF1* and surface integrals over boundary *S* of the excursion set *I* with the principal radii of curvature *R1* and *R2* for other functionals*.*

$$\begin{aligned} MF\_1 &= \int\_I dV & MF\_3 &= 1/2 \int\_\partial \left( \frac{1}{R\_1} + \frac{1}{R\_2} \right) dS \\\\ MF\_2 &= \int\_\partial dS & MF\_4 &= \int\_\partial \frac{1}{R\_1 R\_2} dS \end{aligned}$$

Simulating Bone Atrophy and Its Effects on the Structure and Stability of the Trabecular Bone 699

we found that *r =* 8 (in voxel units) is a well-suited choice. By means of the scaling indices α one can then discriminate between voxels belonging to rod-like (α ≅ 1) and sheet-like (α ≅ 2) structural elements of the trabecular network, which is one of most important

It is straightforward to implement anisotropies in the calculation of the scaling indices by

In this study we calculate anisotropic scaling indices αz with respect to the direction of the external force acting in the bone, for which we chose the ratio of 5:1 for the eigenvalues, i.e. λz= 5λx= 5λy, for setting the degree of anisotropy along the z-direction. This setting was found to be well suited especially for the detection of aligned cylindrical structures (i.e. the

For assessing biomechanical strength of trabecular network we apply Finite Element Method (FEM) with linear elastic assumptions (Rietbergen et al., 1995; Rietbergen et al., 1999; Sidorenko et al. 2009). For the bone mineral material we assume isotropic properties

2 *ij ij ij kk*

equations), which states that stress is proportional to the strain up to the elastic limit

με λδ

{ } , , 2 (1 )(1 2 ) 1 *Y Y <sup>Y</sup>* ν

We use Dirichlet boundary conditions with prescribed on the top surface constant strain

*1%* to simulate uniaxial loading in the natural direction (in this work denoted as z axis) applied in biomechanical experiment. We generate a finite element mesh by direct converting image voxels that belong to the bone tissue into equally sized and oriented eight node brick elements. An exact number of nodes depend on the structure characteristics and varies for our data set from 0.7⋅106 for weak bones, dominated by rod-like trabecular elements, up to 2⋅106 for strong bones with a lot of plate–like formations. From discrete nodal displacements obtained from FEM strain and stress components can be recovered at any point of the structure. For correlation analysis with respect to the MCS we use apparent

> *t t F dA r zz* = σ.

ν

*ij i j x i j y i j z i j d pp xx yy zz*

=− = − + − + −

, , ( )( )() *xyz*

λλλ

( )1/2 2 22 22 2

ν

μ

3

1

= = + .

 ε

*k*

ν

σ *tzz*:

= = +− +

components by generalized Hooke's law (constitutive

 ν

which is recalculated from the normal stress

 *= 0.3* and describe relationship

λ and μ

> ε*0 =*

discriminating feature to discern healthy from osteoporotic bone structure.

λλλ

trabeculae) with typical mean breadth and length.

and strain

with Young's modulus *Y = 10 GPa* and Poisson's ratio

ν

total reaction force *Fr* at the top face *At*

component in direction of applied mechanical load

ε

σ

Material properties are included in the model through Lame parameters

 λ

**2.3 Finite element models** 

σ

between stress

introducing an ellipsoidal distance measure with the eigenvalues λx, λy and λz:

The first two functionals *MF1* and *MF2* describe morphology of the structure and coincide with morphometrical parameters bone volume and surface fractions (*MF1 = BV/TV* and *MF2 = BS/TV*). The fourth integral, also known as Euler characteristic χ, characterises the topological connectivity of structures and can be expressed in terms of Betti numbers β*0* (number of connected components), β*<sup>1</sup>* (number of tunnels), β*<sup>2</sup>* (number of cavities):

$$\mathcal{X} = \beta\_0 - \beta\_1 + \beta\_2 \dots$$

In the case of binary images we have exactly four global characteristics, which can be used as texture measures for assessment of bone strength (Monetti et al., 2011).

#### **2.2.2 Isotropic and anisotropic scaling indices**

We calculate isotropic and anisotropic scaling indices (SIM) (Räth & Morfill 1997; Monetti et al. 2004; Müller et al. 2006; Räth et al. 2008) as measures to characterize the complex trabecular network and its degree of alignment relative to a preferential direction.

Generally, scaling indices represent one way to estimate the local scaling properties of a ndimensional point set. Considering binarised three-dimensional μCT-images, a suitable representation of the image information as a set of three-dimensional points is given by

$$
\vec{p}\_i = (\mathbf{x}\_{i\prime} y\_{i\prime} z\_i)\_{\prime\prime}
$$

i = 1, . . . , Nbone, where Nbone denotes the number of (white) bone voxels and *xi, yi, zi* the voxel position. The three-dimensional image can now be regarded as a set of *N* points

$$P = \{\vec{p}\_i\}\,\not\,\not\,\vec{\iota} = \mathbf{1}\,,\dots\,\mathbf{N}\_{bone}\,\dots$$

For each (bone) voxel the logarithmic gradients

$$\alpha\_i = \frac{\partial \log \rho(\vec{p}\_{i\prime}r)}{\partial \log r}\_{\prime},$$

which are called scaling indices, of the cumulative weighted point distribution

$$\rho(\vec{p}\_{i\prime},r) = \sum\_{j=1}^{N\_{\text{low}}} e^{-\left(\frac{d\_{ij}}{r}\right)\_{\text{rev}}}$$

are calculated with

$$\mathcal{A}\_{ij} = \left\| \vec{p}\_i - \vec{p}\_j \right\|\_2 = \left( (\boldsymbol{x}\_i - \boldsymbol{x}\_j)^2 + (\boldsymbol{y}\_i - \boldsymbol{y}\_j)^2 + (\boldsymbol{z}\_i - \boldsymbol{z}\_j)^2 \right)^{1/2}$$

for the isotropic case.

The calculation of the scaling indices depends on the parameters *n* and *r*. The exponent *n* controls the shape of the weighting function. With increasing *n* the weighting function becomes more and more step-like. In all our studies we found that *n* has only little influence on the results. For the following calculations we fixed *n* to *n*=2, which emphasises the connection to Gaussian smoothing or kernel functions. The scale parameter *r* controls to which scales of the image structure the scaling indices are sensitive. From previous studies we found that *r =* 8 (in voxel units) is a well-suited choice. By means of the scaling indices α one can then discriminate between voxels belonging to rod-like (α ≅ 1) and sheet-like (α ≅ 2) structural elements of the trabecular network, which is one of most important discriminating feature to discern healthy from osteoporotic bone structure.

It is straightforward to implement anisotropies in the calculation of the scaling indices by introducing an ellipsoidal distance measure with the eigenvalues λx, λy and λz:

$$\mathcal{A}\_{ij} = \left\| \vec{p}\_i - \vec{p}\_j \right\|\_{\mathcal{A}\_x, \mathcal{A}\_y, \mathcal{A}\_z} = \left( \lambda\_x^2 (\mathbf{x}\_i - \mathbf{x}\_j)^2 + \lambda\_y^2 (y\_i - y\_j)^2 + \lambda\_z^2 (z\_i - z\_j)^2 \right)^{1/2}$$

In this study we calculate anisotropic scaling indices αz with respect to the direction of the external force acting in the bone, for which we chose the ratio of 5:1 for the eigenvalues, i.e. λz= 5λx= 5λy, for setting the degree of anisotropy along the z-direction. This setting was found to be well suited especially for the detection of aligned cylindrical structures (i.e. the trabeculae) with typical mean breadth and length.

#### **2.3 Finite element models**

698 Osteoporosis

The first two functionals *MF1* and *MF2* describe morphology of the structure and coincide with morphometrical parameters bone volume and surface fractions (*MF1 = BV/TV* and *MF2*

topological connectivity of structures and can be expressed in terms of Betti numbers

*<sup>1</sup>* (number of tunnels),

 β β=−+ <sup>012</sup> .

In the case of binary images we have exactly four global characteristics, which can be used

We calculate isotropic and anisotropic scaling indices (SIM) (Räth & Morfill 1997; Monetti et al. 2004; Müller et al. 2006; Räth et al. 2008) as measures to characterize the complex

Generally, scaling indices represent one way to estimate the local scaling properties of a ndimensional point set. Considering binarised three-dimensional μCT-images, a suitable representation of the image information as a set of three-dimensional points is given by

(,,) *i i ii <sup>p</sup>* <sup>=</sup> *xyz* ,

i = 1, . . . , Nbone, where Nbone denotes the number of (white) bone voxels and *xi, yi, zi* the voxel position. The three-dimensional image can now be regarded as a set of *N* points

> { }, 1,..., *P pi N* = = *<sup>i</sup> bone* .

> > *i*

which are called scaling indices, of the cumulative weighted point distribution

( ,)

*pr e*

<sup>2</sup> ( )( )( ) *ij i j i j i j i j d pp xx yy zz* =− = − +− +−

The calculation of the scaling indices depends on the parameters *n* and *r*. The exponent *n* controls the shape of the weighting function. With increasing *n* the weighting function becomes more and more step-like. In all our studies we found that *n* has only little influence on the results. For the following calculations we fixed *n* to *n*=2, which emphasises the connection to Gaussian smoothing or kernel functions. The scale parameter *r* controls to which scales of the image structure the scaling indices are sensitive. From previous studies

*i*

ρ

<sup>∂</sup> <sup>=</sup> <sup>∂</sup>

α

log ( ,) log *i*

1

*j*

= <sup>=</sup>

*bone*

*p r r* ρ

,

*ij*

( )1/2 2 22

 − 

*d <sup>N</sup> <sup>r</sup> <sup>n</sup>*

β

χ

*<sup>2</sup>* (number of cavities):

, characterises the

β*0*

*= BS/TV*). The fourth integral, also known as Euler characteristic

β

as texture measures for assessment of bone strength (Monetti et al., 2011).

χβ

trabecular network and its degree of alignment relative to a preferential direction.

(number of connected components),

**2.2.2 Isotropic and anisotropic scaling indices** 

For each (bone) voxel the logarithmic gradients

are calculated with

for the isotropic case.

For assessing biomechanical strength of trabecular network we apply Finite Element Method (FEM) with linear elastic assumptions (Rietbergen et al., 1995; Rietbergen et al., 1999; Sidorenko et al. 2009). For the bone mineral material we assume isotropic properties with Young's modulus *Y = 10 GPa* and Poisson's ratio ν *= 0.3* and describe relationship between stress σ and strain ε components by generalized Hooke's law (constitutive equations), which states that stress is proportional to the strain up to the elastic limit

$$
\sigma\_{ij} = 2\mu \varepsilon\_{ij} + \lambda \delta\_{ij} \sum\_{k=1}^{3} \varepsilon\_{kk} \ .
$$

Material properties are included in the model through Lame parameters λ and μ

$$\{\mathcal{Y}, \nu\} \Rightarrow \left| \mathcal{X} = \frac{\mathcal{Y}\nu}{(1+\nu)(1-2\nu)} , \quad \mathcal{2}\mu = \frac{\mathcal{Y}}{1+\nu} \right|.$$

We use Dirichlet boundary conditions with prescribed on the top surface constant strain ε*0 = 1%* to simulate uniaxial loading in the natural direction (in this work denoted as z axis) applied in biomechanical experiment. We generate a finite element mesh by direct converting image voxels that belong to the bone tissue into equally sized and oriented eight node brick elements. An exact number of nodes depend on the structure characteristics and varies for our data set from 0.7⋅106 for weak bones, dominated by rod-like trabecular elements, up to 2⋅106 for strong bones with a lot of plate–like formations. From discrete nodal displacements obtained from FEM strain and stress components can be recovered at any point of the structure. For correlation analysis with respect to the MCS we use apparent total reaction force *Fr* at the top face *At* which is recalculated from the normal stress component in direction of applied mechanical load σ *tzz*:

$$F\_r = \int \sigma\_{xx}^t dA^t \, .$$

Simulating Bone Atrophy and Its Effects on the Structure and Stability of the Trabecular Bone 701

Original χ=const

χ≠const χ≠const, αz<2

Fig. 1. Typical change of the trabecular bone structure under numerically simulated bone resorption (upper left: original bone, upper right: connectivity preserving model, lower left: model without preserving connectivity, lower right: model with preferential resorption of

rod-like trabecular elements).

#### **2.4 Bone resorption models**

We develop three numerical models for bone resorption, which correspond to the different assumptions about bone atrophy in male and female bones. Several investigations (Aaron et al., 1987; Khosla et al., 2006) demonstrated sex difference in trabecular bone aging. Although the decrease with age in trabecular bone volume is common for both sexes, male cancellous bone exposes uniform thinning, whereas female trabecular network suffers from loss of connectivity and entire rod-like structural elements (Barger-Lux et al., 2002).

In all three models we simulate the random resorption of bone material by removal surface voxels according to the given value of relative bone loss volume Δ*BV/TV*, but with different topological features. In order to study the role of topological connectivity for bone strength in the first resorption model (Model I), a bone voxel is removed only if does not change topology of the system, i.e. no new cavities or tunnels are created. In terms of global topological characteristics it means that the fourth Minkowski Functional, which coincides with Euler characteristic χis conserved,

#### MF4 = χ = const.

We compare this model with two other models, which both do not preserve the connectivity (χ ≠ *const*). In Model II there isn't any limitations or conditions on the removal of bone surface voxels. In Model III, however, we simulate the preferential destruction of rod-like trabecular elements by only removing surface voxels with local topological anisotropic scaling index α*z < 2*. Typical changes in trabecular bone structure are demonstrated in Fig. 1. Three regions in circles show the differences in structure due to different resorption models: preserving of connectivity and destroying of rod-like elements at the same place in the trabecular network.

#### **3. Results**

As a main characteristic of the statistical analysis we use Pearson's correlation coefficient *rMCS* (Tables 2 - 5) with respect to the Maximum Compressive Strength (MCS) as it was measured in biomechanical uniaxial compressive experiments. In the Figs. 2 - 4 we show the changes of the different texture measures (black curves and left axis) calculated by MF, SIM and FEM as a function of bone loss. The red curves and right axis in the Figs. 2 - 4 and the values in the corresponding Tables 2 - 4 denote Pearson's correlation coefficient *rMCS* as function of bone loss for the three resorption models.

The diagrams for the first Minkowski Functional *MF1 = BV/TV* (Fig. 2, first row) confirm the linear decrease in bone mineral volume due to resorption and thus represent a validation of our implementation of the bone loss models.

Plots for *MF4 =* χ (Fig. 2, last row) proof that in first resorption model (left column) the connectivity is preserved (χ *= const)* and in the two other models the porosity of the structure increases (Δχ *< 0*) during the resorption process. In both models the increase of porosity occurs due to increase of number of tunnels (β*<sup>1</sup>*). In the third model with preferential resorption of rod-like trabecular elements (with χ ≠ *const* and α*z < 2*) the growth of number of tunnels (β*1*) is compensated by an increase of number of separate parts (β*<sup>0</sup>*) and slows down negative increase of χ. For both resorption models without conservation of connectivity for structure with large relative bone loss (Δ*BV/TV > 30%*) the correlation coefficient of *MF4 =* χ with MCS becomes considerably higher than that for original structure (Tables 2).

We develop three numerical models for bone resorption, which correspond to the different assumptions about bone atrophy in male and female bones. Several investigations (Aaron et al., 1987; Khosla et al., 2006) demonstrated sex difference in trabecular bone aging. Although the decrease with age in trabecular bone volume is common for both sexes, male cancellous bone exposes uniform thinning, whereas female trabecular network suffers from loss of

In all three models we simulate the random resorption of bone material by removal surface

topological features. In order to study the role of topological connectivity for bone strength in the first resorption model (Model I), a bone voxel is removed only if does not change topology of the system, i.e. no new cavities or tunnels are created. In terms of global topological characteristics it means that the fourth Minkowski Functional, which coincides

MF4 = χ = const. We compare this model with two other models, which both do not preserve the connectivity (

 *const*). In Model II there isn't any limitations or conditions on the removal of bone surface voxels. In Model III, however, we simulate the preferential destruction of rod-like trabecular elements by only removing surface voxels with local topological anisotropic scaling index

*2*. Typical changes in trabecular bone structure are demonstrated in Fig. 1. Three regions in circles show the differences in structure due to different resorption models: preserving of connectivity and destroying of rod-like elements at the same place in the trabecular network.

As a main characteristic of the statistical analysis we use Pearson's correlation coefficient *rMCS* (Tables 2 - 5) with respect to the Maximum Compressive Strength (MCS) as it was measured in biomechanical uniaxial compressive experiments. In the Figs. 2 - 4 we show the changes of the different texture measures (black curves and left axis) calculated by MF, SIM and FEM as a function of bone loss. The red curves and right axis in the Figs. 2 - 4 and the values in the corresponding Tables 2 - 4 denote Pearson's correlation coefficient *rMCS* as

The diagrams for the first Minkowski Functional *MF1 = BV/TV* (Fig. 2, first row) confirm the linear decrease in bone mineral volume due to resorption and thus represent a validation of

(Fig. 2, last row) proof that in first resorption model (left column) the

*1*) is compensated by an increase of number of separate parts (

 *< 0*) during the resorption process. In both models the increase of

 *= const)* and in the two other models the porosity of the

β

. For both resorption models without conservation of

 *const* and

χ ≠

Δ

with MCS becomes considerably higher than that for original structure

*<sup>1</sup>*). In the third model with

*BV/TV > 30%*) the correlation

*z < 2*) the growth

β*<sup>0</sup>*) and

α

Δ

*BV/TV*, but with different

χ

α*z <* 

connectivity and entire rod-like structural elements (Barger-Lux et al., 2002).

voxels according to the given value of relative bone loss volume

is conserved,

χ

function of bone loss for the three resorption models.

χ

porosity occurs due to increase of number of tunnels (

preferential resorption of rod-like trabecular elements (with

connectivity for structure with large relative bone loss (

χ

our implementation of the bone loss models.

Δχ

β

χ

χ

slows down negative increase of

connectivity is preserved (

**2.4 Bone resorption models** 

with Euler characteristic

≠

**3. Results** 

Plots for *MF4 =* 

structure increases (

of number of tunnels (

coefficient of *MF4 =* 

(Tables 2).

Fig. 1. Typical change of the trabecular bone structure under numerically simulated bone resorption (upper left: original bone, upper right: connectivity preserving model, lower left: model without preserving connectivity, lower right: model with preferential resorption of rod-like trabecular elements).

Simulating Bone Atrophy and Its Effects on the Structure and Stability of the Trabecular Bone 703

Model I

*0%* 0.87 0.50 -0.20 -0.12 *10%* 0.87 0.59 -0.65 -0.12 *20%* 0.87 0.54 0.09 -0.12 *30%* 0.87 **0.70** -0.60 -0.12 *35%* 0.87 **0.71** -0.53 -0.12 *40%* 0.87 0.67 -0.31 -0.12 *45%* 0.87 0.62 0.0 -0.12 *50%* 0.87 0.66 -0.22 -0.12

Model II

*0%* 0.87 0.50 -0.20 -0.12 *10%* 0.87 0.59 -0.64 0.01 *20%* 0.87 0.54 0.05 0.09 *30%* 0.87 **0.70** -0.43 **-0.75**  *35%* 0.87 **0.71** -0.20 **-0.92**  *40%* 0.87 0.68 0.18 **-0.91**  *45%* 0.87 0.63 **0.49 -0.73**  *50%* 0.87 0.61 **0.50 -0.45** 

Model III

*0%* 0.87 0.50 -0.20 -0.12 *10%* 0.87 0.66 -0.57 -0.05 *20%* 0.87 0.64 -0.16 -0.01 *30%* 0.87 0.66 -0.25 **-0.38**  *35%* 0.87 **0.70** -0.08 **-0.83**  *40%* 0.87 **0.74** 0.03 **-0.89**  *45%* 0.87 **0.71 0.40 -0.85**  *50%* 0.87 0.66 **0.59 -0.68** 

Table 2. Pearson's correlation coefficient of the four MF with respect to MCS for the three models of bone loss and eight rareficiation steps ranging from 0% to 50% removal of the

initial bone volume.

**MF1 MF2 MF3 MF4**

**MF1 MF2 MF3 MF4**

**MF1 MF2 MF3 MF4**

Fig. 2. Minkowski Functionals for 17 specimens (black curves and left axis) and correlation coefficient with MCS (red curve and right axis) for three models of bone loss (from left to right).

**Minkowski Functionals** 

χ ≠ *const,* α*z<2* 

χ ≠ *const* 

Fig. 2. Minkowski Functionals for 17 specimens (black curves and left axis) and correlation coefficient with MCS (red curve and right axis) for three models of bone loss (from left to

χ *= const* 

right).


Model I

#### Model II


#### Model III


Table 2. Pearson's correlation coefficient of the four MF with respect to MCS for the three models of bone loss and eight rareficiation steps ranging from 0% to 50% removal of the initial bone volume.

Simulating Bone Atrophy and Its Effects on the Structure and Stability of the Trabecular Bone 705

*SIM* α*z*

> χ≠*const,* α*z<2*

> > α

α

*z)* for 17 bone

*<sup>z</sup>* spectrum (black curves and

χ≠*const* 

Fig. 3b. Probability distribution function of anisotropic scaling index *P(*

(30%: second row, 50% third row). Last row: mean value of

three models of bone loss (from left to right).

specimens with original structure (first row) and with different value of bone resorption

left axis) and correlation coefficient of mean value with MCS (red curve and right axis) for

χ*=const* 

For isotropic SIM (Fig. 3a) there is almost no difference in the *P(*α*)* spectrum for the different resorption models observed, which leads to only a small decrease in the correlation coefficient (last row in Fig. 3a and Table 3a). For anisotropic SIM (Fig. 3b) there is an obvious difference in *P(*α*z)* already at Δ*BV/TV = 10%* and a more considerable decrease of *rMCS* (last row on Fig.3b and Table 3b) in the case of preferential resorption of rod-like trabecular elements (third model with χ ≠ *const* and α*z < 2*).

Fig. 3a. Probability distribution function of isotropic scaling index *P(*α*)* for 17 bone specimens with original structure (first row) and with different value of bone resorption (30%: second row, 50% third row). Last row: mean value of α spectrum (black curves and left axis) and correlation coefficient of mean value with MCS (red curve and right axis) for three models of bone loss (from left to right).

resorption models observed, which leads to only a small decrease in the correlation coefficient (last row in Fig. 3a and Table 3a). For anisotropic SIM (Fig. 3b) there is an obvious

row on Fig.3b and Table 3b) in the case of preferential resorption of rod-like trabecular

**SIM** α

α*z < 2*).

χ≠*const*  α

χ≠*const,* α*z<2* 

α

α

*)* for 17 bone

spectrum (black curves and

*BV/TV = 10%* and a more considerable decrease of *rMCS* (last

*)* spectrum for the different

For isotropic SIM (Fig. 3a) there is almost no difference in the *P(*

Δ

 *const* and

Fig. 3a. Probability distribution function of isotropic scaling index *P(*

(30%: second row, 50% third row). Last row: mean value of

three models of bone loss (from left to right).

specimens with original structure (first row) and with different value of bone resorption

left axis) and correlation coefficient of mean value with MCS (red curve and right axis) for

χ ≠

difference in *P(*

α

elements (third model with

χ*=const* 

*z)* already at

Fig. 3b. Probability distribution function of anisotropic scaling index *P(*α*z)* for 17 bone specimens with original structure (first row) and with different value of bone resorption (30%: second row, 50% third row). Last row: mean value of α*<sup>z</sup>* spectrum (black curves and left axis) and correlation coefficient of mean value with MCS (red curve and right axis) for three models of bone loss (from left to right).

Simulating Bone Atrophy and Its Effects on the Structure and Stability of the Trabecular Bone 707

0% 0.91 0.91 0.91 10% 0.91 0.91 0.89 20% 0.90 0.90 0.88 30% 0.89 0.88 0.86 40% 0.87 0.86 0.83 50% 0.85 0.82 0.77 Table 4. Correlation coefficient of apparent top reaction force *Fr* calculated by FEM with

Fig. 5. Apparent total reaction force *Fr* for 17 bone specimens and for the three resorption

χ*=const* 

*FEM* 0.91 0.87 0.86 0.83 *MF1* 0.87 0.87 0.87 0.87 *MF2* 0.50 0.67 0.68 0.74 *MF3* -0.20 -0.31 0.18 0.03 *MF4* -0.12 -0.12 -0.91 -0.89

*=const*, dashed line:

*)* 0.69 0.60 0.60 0.58

*z)* 0.70 0.66 0.66 0.56

Table 5. Pearson's correlation coefficient with respect to the MCS for bone structure with

χ≠

χ≠*const* 

*const*, dotted line:

χ≠*const,* α*z<2* 

χ≠*const,* α*z<2*.

χ

*original* 

*BV/TV=40%* for original structure and three resorption models

MCS for three models of bone loss.

models with

Δ

Δ

*Mean P(*

*Mean P(*

α

α

*BV/TV=50%* (solid line:

χ=const χ≠const χ≠const, αz<2



Table 3. Correlation coefficient of mean value of α spectrum *(a)* and α*<sup>z</sup>* spectrum *(b)* with MCS for three models of bone loss.

The mechanical strength of the resorbed trabecular structure as determined with FEM (Fig. 4) depends almost linearly on the relative bone loss Δ*BV/TV* and shows small decrease in the correlation with MCS (Table 4). At high bone loss (Δ*BV/TV = 50%*) the mechanical strength of all bone specimens was found to be larger in the case with conservation of connectivity (solid line in Fig. 5). In Table 5 we summarise effect of large bone loss (Δ*BV/TV = 40%*) on different numerical texture measures. FEM and SIM demonstrate small drop in correlation with MCS of initial structure. These methods can be proposed for prediction of osteoporosis: relative strength and local topology do not change considerably under the process of random surface resorpton. In a contrast, after large bone resorption global *MF2* and especially *MF4* improve their correlation with MCS of the original structure. This effect can be used for the diagnostic of the current state of the bone structure.

Fig. 4. Value of the apparent top reaction force *Fr* for 17 bone specimens calculated by FEM (black curves and left axis) and correlation coefficient of *Fr* with MCS (red curve and right axix) for three different resorption models (from left to right).

*a)* 

*b)* 

The mechanical strength of the resorbed trabecular structure as determined with FEM (Fig.

of all bone specimens was found to be larger in the case with conservation of connectivity

different numerical texture measures. FEM and SIM demonstrate small drop in correlation with MCS of initial structure. These methods can be proposed for prediction of osteoporosis: relative strength and local topology do not change considerably under the process of random surface resorpton. In a contrast, after large bone resorption global *MF2* and especially *MF4* improve their correlation with MCS of the original structure. This effect can

> χ≠*const*

Fig. 4. Value of the apparent top reaction force *Fr* for 17 bone specimens calculated by FEM (black curves and left axis) and correlation coefficient of *Fr* with MCS (red curve and right

(solid line in Fig. 5). In Table 5 we summarise effect of large bone loss (

be used for the diagnostic of the current state of the bone structure.

axix) for three different resorption models (from left to right).

α

Δ

Δ

spectrum *(a)* and

α

*BV/TV* and shows small decrease in the

*BV/TV = 50%*) the mechanical strength

Δ

χ≠*const,* α*z<2* 

*<sup>z</sup>* spectrum *(b)* with

*BV/TV = 40%*) on

Table 3. Correlation coefficient of mean value of

4) depends almost linearly on the relative bone loss

correlation with MCS (Table 4). At high bone loss (

MCS for three models of bone loss.

χ*=const* 

0% 0.70 0.70 0.70 10% 0.69 0.69 0.61 20% 0.68 0.68 0.57 30% 0.68 0.68 0.56 40% 0.66 0.66 0.56 50% 0.66 0.64 0.52

χ=const χ≠const χ≠const, αz<2

0% 0.69 0.69 10% 0.67 0.67 0.64 20% 0.64 0.64 0.61 30% 0.63 0.63 0.60 40% 0.60 0.60 0.58 50% 0.57 0.56 0.55

χ=const χ≠const χ≠const, αz<2


Table 4. Correlation coefficient of apparent top reaction force *Fr* calculated by FEM with MCS for three models of bone loss.

Fig. 5. Apparent total reaction force *Fr* for 17 bone specimens and for the three resorption models with Δ*BV/TV=50%* (solid line: χ*=const*, dashed line: χ≠*const*, dotted line: χ≠*const,* α*z<2*.


Table 5. Pearson's correlation coefficient with respect to the MCS for bone structure with Δ*BV/TV=40%* for original structure and three resorption models

Simulating Bone Atrophy and Its Effects on the Structure and Stability of the Trabecular Bone 709

Barger-Lux, M.J. & Recker, R.R. (2002), Bone microstructure in osteoporosis:transilial biopsy

Eckstein, F.; Matsuura M.; Kuhn, V.; Priemel, M.; Müller R.; Link, TM. & Lochmüller, EM.

Hildebrand, T.;Laib, A.; Müller, R.; Dequeker, J. & Rüegsegger, P. (1999). Direct three-

Huiskes, R.; Ruimerman, R.; et al. (2000). Effects of mechanical forces on maintenance,

Khosla, S.; Riggs, B.L.; Atkinson, E.J.; Oberg, A.L.; McDaniel, L.J.; Holets, M.; Peterson, J.M.

Michielsen, K.; De Raedt, H. (2001). Integral-geometry morphological image analysis.

Monetti, R.A.; Böhm, H.; Müller, D.; Rummeny, E.; Link, T. & Räth, C. (2004). Assessing the

Monetti, R.A.; Bauer J.; Sidorenko I.; Müller D.; Rummeny E.; Matsuura M.; Eckstein F.;

Müller, D.; Link, T.M.; Monetti, R.; Bauer, J.; Böhm, H.; Seifert-Klauss, V.; Rummeny, E.J.;

Mullender, M. G.; Huiskes, R. (1995). A proposal for the regulatory mechanism of Wolff 's

Räth, C. & Morfill, G. (1997). Texture detection and texture duscrimination with anisotropic

Räth, C.; Monetti, R.; Bauer, J.; Sidorenko, I.; Müller, D.; Matsuura, M.; Lochmüller, E.-M.;

Rietbergen, B.; Weinans, H.; Huiskes, R. & Odgaard, A. (1995). A new method to determine

Rietbergen, B.; Weinans, H. & Huiskes, R. (1996). Computational strategies for iterative

Zysset, P. & Eckstein, F. (2008). Strength through structure: visualization and local assessment of the trabecular bone structure. *New Journal of Physics,* Vol. 10, pp.

trabecular bone elastic properties and loading using micromechanical finite-

solutions of large FEM applications employing voxel data, Int. J. Num. Meth. Eng.

*Proceedings of Medical Imaging Conference of SPIE,* Vol. 7965 79650K

NIH: National Institutes of Health (2000). NIH Consens. Statement 17, pp. 1 – 45

scaling indices. *J. Opt. Soc. Am. A,* Vol. 14, No. 12, pp. 3208-3215

element models. *J. Bioemchanics,* Vol. 28, No. 1, pp. 69-81

images in vivo. *Osteoporos. Int.,* Vol. 17, pp. 1783-1493

adaptation of form in trabecular bone. *Nature*, Vol. 405, pp. 704-706

Site-Dependent. *J. Bone Miner. Res.* Vol. 22, No 6, pp. 817–824

297-306

pp. 1167-1174

*Res.*, Vol. 21, No. 1, pp.124-131

*Physics Reports,* Vol.347, pp.461-538

*Conference of SPIE,* Vol. 5370, pp. 215-224

law. J. Orthop. Res., 13, pp. 503–512 .

125010-125027

39, pp. 2743-2767

and histomorphometry. *Topics in Magnetic Resonance Imaging,* Vol. 13, No. 5, pp.

(2007). Sex Differences of Human Trabecular Bone Microstructure in Aging Are

dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur iliac crest, and calcaneus. *J Bone Miner Res,* Vol. 14, No. 7,

& Melton, L.J. (2006), Effects of sex and age on bone microstructure at the ultradistal radius: a population-based noninvasive in vivo assessment. *J Bone Miner* 

biomechanical strength of trabecular bone in vitro using 3D anisotropic non- linear texture measures: The Scaling Vector Method. *Proceedings of Medical Imaging* 

Lochmueller E.-M.; Zysset P. & Räth C. (2011). Structure based classification of μ-CT images of human trabecular bone using local Minkowski Functionals.

Morfill, G.E. & Räth, C. (2006). The 3D-based scaling index algorithm: a new structure measure to analyze trabecular bone architecture in hifh-resolution MR

### **4. Summary and conclusions**

We proposed a method based on the ideas of cellular automata to simulate bone atrophy and applied it to a sample of 17 bone probes visualised with high resolution μCT imaging. Although our study is so far restricted to the simulation of bone loss and did not include any processes of bone formation, we could already gain some new and very interesting insights about the important factors determining the strength of bones.

As expected we found that the initial structure determines the relative strength of the bone under random surface bone losses. Patients with stronger bones in young age have better prognosis for age-related bone atrophy. We found that the connectivity plays the most important role in determining the strength of the bone structure: among three resorption models the highest apparent reaction force was calculated for the resorption model which preserved the connectivity (χ *= const*). FEM, isotropic SIM, the first and second MF yielded stable values of the correlation coefficient *rMCS* under the random bone loss process for all numerical resorption models and can be recommended for prediction of bone strength in bone atrophy process.

The mean value of the anisotropic scaling indices α*<sup>z</sup>* demonstrated sensibility for preferential rod-like trabecular loss as simulated by third numerical resorption model (with χ ≠ *const* and α*z < 2*). For this model the scaling index approach shows a decrease of correlation coefficient *rMCS* already at 10% loss of mineral bone fracture.

For the two resorption models without conservation of the connectivity (χ ≠ *const*) the bone surface resorption significantly improves the correlation of the fourth MF with MCS (from *rMCS = -0.12* for original structure up to the *rMCS = -0.92* for bone loss ratio Δ*BV/TV = 35%*). Such an effect suggests that the random surface resorption destroys thin and unimportant connections of the trabeculae and only the strong and thick trabecular elements are taken into account for correlation with MCS. The removal of bone voxels can thus be interpreted as a distillation of the essential skeleton of the trabecular structure, which is a much more sensitive tracer of the mechanical stability of the bone. In fact we found that fourth Minkowski Functional calculated for structure prepared by random surface resorption yields higher correlations with MCS than FEM-based measures, which are so far considered to yield the highest correlations with the mechanical properties of bone probes. Therefore the rarefication procedure as outlined in this study in combination with Minkowski Functionals may lead to a novel technique for the diagnosis of the trabecular bone quality and strength in the prediction of osteoporosis.

#### **5. Acknowledgments**

This study was in part supported by the Deutsche Forschungsgemeinschaft (DFG) under the grants MU 2288/2-2 and BA 4085/1-2.

#### **6. References**

Aaron, J.E.; Makins, N.B. & Sagreiya, K. (1987). The Microanatomy of Trabecular Bone Loss in Normal Aging Men and Women. *Clinical Orthopaedics and Related Research*, No. 215, pp. 260-271

We proposed a method based on the ideas of cellular automata to simulate bone atrophy and applied it to a sample of 17 bone probes visualised with high resolution μCT imaging. Although our study is so far restricted to the simulation of bone loss and did not include any processes of bone formation, we could already gain some new and very interesting

As expected we found that the initial structure determines the relative strength of the bone under random surface bone losses. Patients with stronger bones in young age have better prognosis for age-related bone atrophy. We found that the connectivity plays the most important role in determining the strength of the bone structure: among three resorption models the highest apparent reaction force was calculated for the resorption model which

stable values of the correlation coefficient *rMCS* under the random bone loss process for all numerical resorption models and can be recommended for prediction of bone strength in

preferential rod-like trabecular loss as simulated by third numerical resorption model (with

surface resorption significantly improves the correlation of the fourth MF with MCS (from

Such an effect suggests that the random surface resorption destroys thin and unimportant connections of the trabeculae and only the strong and thick trabecular elements are taken into account for correlation with MCS. The removal of bone voxels can thus be interpreted as a distillation of the essential skeleton of the trabecular structure, which is a much more sensitive tracer of the mechanical stability of the bone. In fact we found that fourth Minkowski Functional calculated for structure prepared by random surface resorption yields higher correlations with MCS than FEM-based measures, which are so far considered to yield the highest correlations with the mechanical properties of bone probes. Therefore the rarefication procedure as outlined in this study in combination with Minkowski Functionals may lead to a novel technique for the diagnosis of the trabecular bone quality

This study was in part supported by the Deutsche Forschungsgemeinschaft (DFG) under the

Aaron, J.E.; Makins, N.B. & Sagreiya, K. (1987). The Microanatomy of Trabecular Bone Loss

in Normal Aging Men and Women. *Clinical Orthopaedics and Related Research*, No.

 *= const*). FEM, isotropic SIM, the first and second MF yielded

α

*z < 2*). For this model the scaling index approach shows a decrease of

*<sup>z</sup>* demonstrated sensibility for

χ ≠

Δ

 *const*) the bone

*BV/TV = 35%*).

insights about the important factors determining the strength of bones.

correlation coefficient *rMCS* already at 10% loss of mineral bone fracture. For the two resorption models without conservation of the connectivity (

*rMCS = -0.12* for original structure up to the *rMCS = -0.92* for bone loss ratio

χ

The mean value of the anisotropic scaling indices

and strength in the prediction of osteoporosis.

grants MU 2288/2-2 and BA 4085/1-2.

215, pp. 260-271

**5. Acknowledgments** 

**6. References** 

**4. Summary and conclusions** 

preserved the connectivity (

α

bone atrophy process.

 *const* and

χ ≠


**35** 

*Italy* 

**Role of Phytoestrogen Ferutinin in** 

*Department of Biomedical Sciences, Section of Human Morphology,* 

*University of Modena and Reggio Emilia* 

**Preventing/Recovering Bone Loss: Results** 

Carla Palumbo, Francesco Cavani, Laura Bertoni and Marzia Ferretti

**from Experimental Ovariectomized Rat Models** 

Osteoporosis is a chronic bone disease, caused by an imbalance between bone resorption and bone formation (Riggs & Melton, 1986), in which the skeleton becomes fragile and leads to an increased risk of fractures. In osteoporosis the bone mineral density is rapidly reduced, the bone microarchitecture is disrupted and the amount/variety of non-collagenous bone proteins is altered (Chestnut, 1995; Paschalis et al., 1997). In menopausal women, the rapid decrease of estrogens is the predominant cause of the imbalance between bone formation and bone resorption that results, in turn, in severe bone loss (Riggs & Melton, 1986). Hormone Replacement Therapy (HRT), based on estrogen administration, is a method to recover both bone loss and incidence of skeletal fractures in postmenopausal women (Turner et al., 1994); however, as it is well known, it increases, as negative side effects, the occurrence of cardiovascular diseases and endometrial/breast/ovarian malignant cancers (Beral, 2003; Genant et al., 1998; Lacey et al., 2002; Termine & Wong, 1998). In addition to HRT, other compounds such as bisphosphonates, calcitonin, calcium products, RANK Ligand, stronzium ranelate, PTH 1-34, thiazide diuretics and ipriflavone (Bruhn, 2010; El-Desoky et al., 2009; Lacroix, 2000; Rybchyn et al., 2011; Schoofs, 2003; Wasnich, 1983; Zhang et al., 2010) are currently used as pharmacological approaches to osteoporosis, but they are often associated with negative side effects. Therefore, the need to find safer and more effective bone protective agents is still prominent. A great number of preparations from medicinal plants was shown to reduce bone loss induced by ovariectomy in rats (Occhiuto et al., 2007) and to increase bone density in postmenopausal women (Clifton-Bligh, 2001). Among natural products increasingly used as an alternative therapy, the phytoestrogenic isoflavones have been shown to increase bone density in postmenopausal women following high dietary intake (Mei et al., 2001). Also in animal studies, the administration of isoflavones or their derivatives prevented bone loss in ovariectomized rats. They are structurally similar to estradiol and their estrogenic-like activity may also depend on their affinity for some estrogen receptors (ERs). Phytoestrogens appear to bind preferentially to the ERβ and have been classified as Selective Estrogen Receptor Modulators (SERMs) (An et al., al., 2001; Brzezinski & Debi, 1999; Messina et al., 2006). ERβ may play a protective role in breast breast cancer development by reducing mammary cell growth, as well as inhibiting the

**1. Introduction** 


Wolfram S. (1984). Cellular automata as models of complexity, Nat. 311, pp. 419-424

## **Role of Phytoestrogen Ferutinin in Preventing/Recovering Bone Loss: Results from Experimental Ovariectomized Rat Models**

Carla Palumbo, Francesco Cavani, Laura Bertoni and Marzia Ferretti *Department of Biomedical Sciences, Section of Human Morphology, University of Modena and Reggio Emilia Italy* 

### **1. Introduction**

710 Osteoporosis

Sidorenko, I.; Bauer, J.; Monetti, R.; Müller, D.; Rummeny, E.; Eckstein, F.; Matsuura, M.;

Wolff, J. (1892). Das Gesetz der Transformation der Knochen, Hirschwald Verlag, Berlin. Wolfram, S. (1983). Statistical Mechanics of Cellular Automata, Rev. Mod. Phys., 55, pp. 603-

Wolfram S. (1984). Cellular automata as models of complexity, Nat. 311, pp. 419-424

the SPIE, 7262, pp. 72620M

644

Lochmüller, E.-M.; Zysset, P.; Räth, C. (2009). Role of trabecular microfractures in failure of human vertebrae estimated by the finite element method, Proceedings of

> Osteoporosis is a chronic bone disease, caused by an imbalance between bone resorption and bone formation (Riggs & Melton, 1986), in which the skeleton becomes fragile and leads to an increased risk of fractures. In osteoporosis the bone mineral density is rapidly reduced, the bone microarchitecture is disrupted and the amount/variety of non-collagenous bone proteins is altered (Chestnut, 1995; Paschalis et al., 1997). In menopausal women, the rapid decrease of estrogens is the predominant cause of the imbalance between bone formation and bone resorption that results, in turn, in severe bone loss (Riggs & Melton, 1986). Hormone Replacement Therapy (HRT), based on estrogen administration, is a method to recover both bone loss and incidence of skeletal fractures in postmenopausal women (Turner et al., 1994); however, as it is well known, it increases, as negative side effects, the occurrence of cardiovascular diseases and endometrial/breast/ovarian malignant cancers (Beral, 2003; Genant et al., 1998; Lacey et al., 2002; Termine & Wong, 1998). In addition to HRT, other compounds such as bisphosphonates, calcitonin, calcium products, RANK Ligand, stronzium ranelate, PTH 1-34, thiazide diuretics and ipriflavone (Bruhn, 2010; El-Desoky et al., 2009; Lacroix, 2000; Rybchyn et al., 2011; Schoofs, 2003; Wasnich, 1983; Zhang et al., 2010) are currently used as pharmacological approaches to osteoporosis, but they are often associated with negative side effects. Therefore, the need to find safer and more effective bone protective agents is still prominent. A great number of preparations from medicinal plants was shown to reduce bone loss induced by ovariectomy in rats (Occhiuto et al., 2007) and to increase bone density in postmenopausal women (Clifton-Bligh, 2001). Among natural products increasingly used as an alternative therapy, the phytoestrogenic isoflavones have been shown to increase bone density in postmenopausal women following high dietary intake (Mei et al., 2001). Also in animal studies, the administration of isoflavones or their derivatives prevented bone loss in ovariectomized rats. They are structurally similar to estradiol and their estrogenic-like activity may also depend on their affinity for some estrogen receptors (ERs). Phytoestrogens appear to bind preferentially to the ERβ and have been classified as Selective Estrogen Receptor Modulators (SERMs) (An et al., al., 2001; Brzezinski & Debi, 1999; Messina et al., 2006). ERβ may play a protective role in breast breast cancer development by reducing mammary cell growth, as well as inhibiting the

Role of Phytoestrogen Ferutinin in Preventing/Recovering

in the problem of ferutinin side effects.

Fig. 1. *Ferula Hermonis*.

the recent past on the topic.

**2.1 Experimental animals and treatments** 

**2. Methods** 

Bone Loss: Results from Experimental Ovariectomized Rat Models 713

2001). Clinical reports about phytoestrogen effect on endometrial cancer are limited to casecontrolled observational studies (Johnson et al., 2001). Hence the interest of the authors also

The authors report the following methods from some animal experiments they performed in

For animal experiments female Sprague-Dawley rats, aged 7 weeks and weighing 170-190 g at the beginning of the experiments, were used, according to the general age-models

stimulatory effects of ERα (An et al., 2001; Strom et al., 2004). Considering the properties of such such natural compounds, phytoestrogens could be employed as Complementary/Alternative Medicine (CAM) instead of HRT, in order to recover menopausal symptoms (Lee et al., 2000; Morris et al., 2000; Morris et al., 2006). Such evidence that SERMs mime estrogens as osteoprotective substances (Albertazzi, 2002; Wang et al., 2006) without displaying the negative side effects on the etiogenesis of some types of cancer (Duffy et al., 2007; Eason et al., 2005; Gallo et al., 2006; Garcia-Perez et al., 2006; Lian et al., 2001; Limer & Speirs, 2004; Murray et al., 2003; Wu et al., 2002) suggests interesting perspectives in planning alternative treatment strategies.

A great number of preparations from medicinal plants, including red clover, hops and black cohosh, have been tested to investigate their influence on ovariectomy-induced bone loss. Red clover (*Trifolium pratense L.*) was shown to reduce bone loss induced by ovariectomy in rats (Occhiuto et al., 2007) and to increase bone density in postmenopausal women (Clifton-Bligh, 2001). The prenylated flavanone contained in hops (*Humulus lupulus L.*), 8 prenylnaringenin (8-PN), and genistein (found in a number of plants including lupin, fava beans, soybeans, kudzu, and psoralea) seem to protect from ovariectomy-induced bone loss in rats, while exhibiting minimal trophic effects on uterus endometrium (Garcìa-Pérez et al., 2006; Hümpel et al., 2005); in particular isoflavone genistein, by enhancing uterine endometrial glandular apoptosis *in vivo* and *in vitro*, may confer protection against uterine carcinoma (Eason et al., 2005). Moreover, a reduced bone resorption was demonstrated also after black cohosh (*Cimicifuga racemosa L.*) therapy and it was ascribed to the significant binding of its components to estrogen receptors (Wuttke et al., 2003). Despite the huge amount of data published *in vitro* and *in vivo* on another phytoestrogen, Ferutinin, extracted from *Ferula hermonis root* (Fig. 1) (Abourashed et al., 2001), whose effect was investigated on calcium-related cellular processes, few observations are reported in literature concerning ferutinin role on the skeleton, particularly on bone metabolism in both the preventing and curative treatment of osteoporosis. Ferutinin shows high affinity for both subtypes of estrogen receptors (ERs ). Even if ferutinin can bind to both ERs, it acts as an agonist for ERα and as agonist/antagonist for ERβ (Ikeda et al., 2002). Thus, this compound may be useful as a selective estrogen receptor modulator (SERM) (Appendino et al., 2002).

Recently, the interest of the authors was to investigate the effects of ferutinin administration on bone metabolism in prevention and in recovery of severe estrogen deficiencyosteoporosis and to compare them with those of estradiol benzoate treatment, in order to propose an alternative solution to the hormone replacement therapy (HRT) commonly used in osteoporotic women. The animal model used, i.e. ovariectomized rat, appears to be an appropriate model for collecting information which could be applied to human postmenopausal osteoporosis, because of the many similarities of the pathophysiological mechanisms (Comelekoglu et al., 2006; Kalu, 1991; Wronski & Yen, 1991).

Further crucial problem correlated to the use of the phytoestrogen ferutinin is to evaluate its side effects, specifically on the organs which are reputed to be the target of estrogen effects, like uterus, vagina, mammary glands. It is well-known that estrogens stimulate endometrial proliferation and their administration in HRT was associated to an increased risk of cancer. Some phytoestrogens are claimed to have beneficial effects with a minor incidence of undesired side effects in comparison with estrogen therapy. Proliferative activity in estrogen-responsive cells can be either enhanced or suppressed by phytoestrogens depending on their concentration and relative potency (Whitten & Patisaul, 2001). Clinical reports about phytoestrogen effect on endometrial cancer are limited to casecontrolled observational studies (Johnson et al., 2001). Hence the interest of the authors also in the problem of ferutinin side effects.

Fig. 1. *Ferula Hermonis*.

### **2. Methods**

712 Osteoporosis

stimulatory effects of ERα (An et al., 2001; Strom et al., 2004). Considering the properties of such such natural compounds, phytoestrogens could be employed as Complementary/Alternative Medicine (CAM) instead of HRT, in order to recover menopausal symptoms (Lee et al., 2000; Morris et al., 2000; Morris et al., 2006). Such evidence that SERMs mime estrogens as osteoprotective substances (Albertazzi, 2002; Wang et al., 2006) without displaying the negative side effects on the etiogenesis of some types of cancer (Duffy et al., 2007; Eason et al., 2005; Gallo et al., 2006; Garcia-Perez et al., 2006; Lian et al., 2001; Limer & Speirs, 2004; Murray et al., 2003; Wu et al., 2002) suggests interesting perspectives in planning alternative

A great number of preparations from medicinal plants, including red clover, hops and black cohosh, have been tested to investigate their influence on ovariectomy-induced bone loss. Red clover (*Trifolium pratense L.*) was shown to reduce bone loss induced by ovariectomy in rats (Occhiuto et al., 2007) and to increase bone density in postmenopausal women (Clifton-Bligh, 2001). The prenylated flavanone contained in hops (*Humulus lupulus L.*), 8 prenylnaringenin (8-PN), and genistein (found in a number of plants including lupin, fava beans, soybeans, kudzu, and psoralea) seem to protect from ovariectomy-induced bone loss in rats, while exhibiting minimal trophic effects on uterus endometrium (Garcìa-Pérez et al., 2006; Hümpel et al., 2005); in particular isoflavone genistein, by enhancing uterine endometrial glandular apoptosis *in vivo* and *in vitro*, may confer protection against uterine carcinoma (Eason et al., 2005). Moreover, a reduced bone resorption was demonstrated also after black cohosh (*Cimicifuga racemosa L.*) therapy and it was ascribed to the significant binding of its components to estrogen receptors (Wuttke et al., 2003). Despite the huge amount of data published *in vitro* and *in vivo* on another phytoestrogen, Ferutinin, extracted from *Ferula hermonis root* (Fig. 1) (Abourashed et al., 2001), whose effect was investigated on calcium-related cellular processes, few observations are reported in literature concerning ferutinin role on the skeleton, particularly on bone metabolism in both the preventing and curative treatment of osteoporosis. Ferutinin shows high affinity for both subtypes of estrogen receptors (ERs ). Even if ferutinin can bind to both ERs, it acts as an agonist for ERα and as agonist/antagonist for ERβ (Ikeda et al., 2002). Thus, this compound may be useful as

a selective estrogen receptor modulator (SERM) (Appendino et al., 2002).

mechanisms (Comelekoglu et al., 2006; Kalu, 1991; Wronski & Yen, 1991).

Recently, the interest of the authors was to investigate the effects of ferutinin administration on bone metabolism in prevention and in recovery of severe estrogen deficiencyosteoporosis and to compare them with those of estradiol benzoate treatment, in order to propose an alternative solution to the hormone replacement therapy (HRT) commonly used in osteoporotic women. The animal model used, i.e. ovariectomized rat, appears to be an appropriate model for collecting information which could be applied to human postmenopausal osteoporosis, because of the many similarities of the pathophysiological

Further crucial problem correlated to the use of the phytoestrogen ferutinin is to evaluate its side effects, specifically on the organs which are reputed to be the target of estrogen effects, like uterus, vagina, mammary glands. It is well-known that estrogens stimulate endometrial proliferation and their administration in HRT was associated to an increased risk of cancer. Some phytoestrogens are claimed to have beneficial effects with a minor incidence of undesired side effects in comparison with estrogen therapy. Proliferative activity in estrogen-responsive cells can be either enhanced or suppressed by phytoestrogens depending on their concentration and relative potency (Whitten & Patisaul,

treatment strategies.

The authors report the following methods from some animal experiments they performed in the recent past on the topic.

### **2.1 Experimental animals and treatments**

For animal experiments female Sprague-Dawley rats, aged 7 weeks and weighing 170-190 g at the beginning of the experiments, were used, according to the general age-models

Role of Phytoestrogen Ferutinin in Preventing/Recovering

treatments for 30 days and the remaining ones for 60 days:

**2.1.2 Recovering study protocol** 

sacrificed.

Bone Loss: Results from Experimental Ovariectomized Rat Models 715

The rats was randomized into four groups (Ferretti el al., 2010). One group of rats were sham operated, while the rats of the other three groups were ovariectomized. Ovariectomy and sham-operation were performed as above described in the protocol for preventing study. Two months after ovariectomy, namely when osteoporosis was obtained by the consequent estrogen deficiency, half of the rats of each group underwent the following

Group 4 (EB-OVX): Ovariectomized treated with estradiol benzoate 1.5 μg/rat twice a week.

The body weight of each animal was recorded at 4 different times: before ovariectomy (i.e., at the start of the experiment), two months after ovariectomy (namely, at the beginning of treatment), and after 30 and 60 days of treatment. At the end of the treatments, all rats were

Soon after the sacrifice, the 4th, 5th lumbar vertebrae and the right femurs were removed, processed and embedded in methyl methacrylate according to standard protocol for light microscopy. Serial sections of 200μm thickness were taken from both lumbar vertebrae and femurs by means of a diamond-saw microtome cutting system. In particular, the 4th lumbar vertebrae were cut according to sagittal planes, whereas the 5th lumbar vertebrae were transversely cut; concerning the femurs, the distal epiphyseal level was sagittally sectioned,

Histomorphometric analysis was performed on Fast-Green or Alizarin-Red stained sections using a light microscope equipped with an image analysis system. In histomorphometric evaluations of vertebral bodies, only trabecular bone was taken into account: it was manually selected, outlining the internal surface of the cortical bone (Fig. 3A,B). In femoral sagittal sections, a constant area (3.5mm2) of trabecular bone was defined by drawing a circular line adjacent to the cartilagineous plate (Fig. 3C). In transversal mid-diaphyseal

the ratio between the *trabecular bone area* (BV) and the *total area* (TV)*,* i.e. the *trabecular* 

the difference between the total cross section area and the medullary canal area , i.e. the

Only in preventing study protocol, in order to obtain a more precise evaluation of the collected data (i.e. to eliminate the effects of body weight on bone histomorphometric parameters), both the ratio BV/TV and the value Ct-B-Ar were "normalized" (i.e. corrected) with respect to body weight (dividing the calculated parameters by the body weight) on the basis that ovariectomy implies a considerable weight increase, while the chronic treatment with both ferutinin and estradiol benzoate (starting the day after ovariectomy) avoids such increment. On the contrary, in recovering study protocol the same treatment was performed 2 months after ovariectomy and after such period, the body weights of all OVX animals (C-OVX, F-OVX, EB-OVX) were all similar; for this reason, histomorphometric parameters (BV

whereas the shaft was transversely sectioned at the mid-diaphyseal level (Fig. 3).

*bone volume* (BV/TV) expressed in percent values, in trabecular bone;

Group 1 (SHAM): Sham-operated controls receiving vehicle (5% Tween 80 in water) Group 2 (C-OVX): Ovariectomized controls receiving vehicle (5% Tween 80 in water)

Group 3 (F-OVX): Ovariectomized treated with ferutinin 2 mg/kg/day

Ferutinin and Estradiol benzoate were used as above described.

femoral sections the cortical bone area was measured (Fig. 3D).

TV and Ct-B-Ar) were not normalized with respect to body weight.

The following parameters were calculated:

*cortical bone area* (Ct-B-Ar), in cortical bone.

**2.2 Histology and histomorphometrical evaluation** 

reported in literature (Fanti et al.,1998; Kalu, 1991). They were housed two per cage and maintained in standard conditions with a 12:12 light/dark cycle, at the temperature of 22 ± 1°C and 55-60% relative humidity. Commercial rat pellets free of estrogenic substances and drinking water were available ad libitum. After a 7-day adaptation period, the animals were randomly divided in different groups according to two protocols (for prevention and recovery of bone loss, respectively). Animal care, maintenance and surgery were conducted in accordance with the Italian law (D.L. n. 116/1992) and European legislation (EEC n. 86/609). The experimental designs and procedures received the approval of the Bioethical Committee of the Italian National Institute of Health.

#### **2.1.1 Preventing study protocol**

The animals were randomly divided in four groups (Palumbo et al., 2009). Rats of group 1 were sham-operated, while rats of other groups were bilaterally ovariectomized (OVX) under ketamine hydrochloride plus xylazine hydrochloride anaesthesia and the ovaries were bilaterally removed; sham-operation was performed in the same way as ovariectomy, but only exposing the ovaries. Starting on the day after the ovariectomy, half of the female rats were submitted to the following treatments for 30 days and the remaining half for 60 days:

Group 1 (SHAM): Sham-operated controls receiving vehicle (5% Tween 80 in water) Group 2 (C-OVX): Ovariectomized controls receiving vehicle (5% Tween 80 in water)

Group 3 (F-OVX): Ovariectomized treated with ferutinin 2 mg/kg/day

Group 4 (EB-OVX): Ovariectomized treated with estradiol benzoate 1.5 μg/rat twice a week.

Ferutinin, whose formula is showed in Figure 2, was solubilized in Tween 80 (5%) and deionized water and administered in the volume of 5 ml/kg by oral gavage (*per os*). The dosage was chosen taking into account previous studies on rat sexual behavior (Zanoli et al., 2005; Zavatti et al., 2006). Control animals (groups 1 and 2) received the same volume of vehicle solution. Estradiol benzoate, used as a reference compound, was dissolved in peanut oil and subcutaneously injected in the volume of 0.3 ml/rat.

Fig. 2. Chemical structure of ferutinin.

The body weights of all animals were recorded before ovariectomy and after 30 and 60 days of treatment. Half of each rat group was sacrificed after 30 days of treatment and the remaining animals at the end of the experiment.

### **2.1.2 Recovering study protocol**

714 Osteoporosis

reported in literature (Fanti et al.,1998; Kalu, 1991). They were housed two per cage and maintained in standard conditions with a 12:12 light/dark cycle, at the temperature of 22 ± 1°C and 55-60% relative humidity. Commercial rat pellets free of estrogenic substances and drinking water were available ad libitum. After a 7-day adaptation period, the animals were randomly divided in different groups according to two protocols (for prevention and recovery of bone loss, respectively). Animal care, maintenance and surgery were conducted in accordance with the Italian law (D.L. n. 116/1992) and European legislation (EEC n. 86/609). The experimental designs and procedures received the approval of the Bioethical

The animals were randomly divided in four groups (Palumbo et al., 2009). Rats of group 1 were sham-operated, while rats of other groups were bilaterally ovariectomized (OVX) under ketamine hydrochloride plus xylazine hydrochloride anaesthesia and the ovaries were bilaterally removed; sham-operation was performed in the same way as ovariectomy, but only exposing the ovaries. Starting on the day after the ovariectomy, half of the female rats were

Group 4 (EB-OVX): Ovariectomized treated with estradiol benzoate 1.5 μg/rat twice a week. Ferutinin, whose formula is showed in Figure 2, was solubilized in Tween 80 (5%) and deionized water and administered in the volume of 5 ml/kg by oral gavage (*per os*). The dosage was chosen taking into account previous studies on rat sexual behavior (Zanoli et al., 2005; Zavatti et al., 2006). Control animals (groups 1 and 2) received the same volume of vehicle solution. Estradiol benzoate, used as a reference compound, was dissolved in peanut

submitted to the following treatments for 30 days and the remaining half for 60 days: Group 1 (SHAM): Sham-operated controls receiving vehicle (5% Tween 80 in water) Group 2 (C-OVX): Ovariectomized controls receiving vehicle (5% Tween 80 in water)

Group 3 (F-OVX): Ovariectomized treated with ferutinin 2 mg/kg/day

oil and subcutaneously injected in the volume of 0.3 ml/rat.

H

HO

Fig. 2. Chemical structure of ferutinin.

remaining animals at the end of the experiment.

O

O

The body weights of all animals were recorded before ovariectomy and after 30 and 60 days of treatment. Half of each rat group was sacrificed after 30 days of treatment and the

OH

Committee of the Italian National Institute of Health.

**2.1.1 Preventing study protocol** 

The rats was randomized into four groups (Ferretti el al., 2010). One group of rats were sham operated, while the rats of the other three groups were ovariectomized. Ovariectomy and sham-operation were performed as above described in the protocol for preventing study. Two months after ovariectomy, namely when osteoporosis was obtained by the consequent estrogen deficiency, half of the rats of each group underwent the following treatments for 30 days and the remaining ones for 60 days:

Group 1 (SHAM): Sham-operated controls receiving vehicle (5% Tween 80 in water)

Group 2 (C-OVX): Ovariectomized controls receiving vehicle (5% Tween 80 in water)

Group 3 (F-OVX): Ovariectomized treated with ferutinin 2 mg/kg/day

Group 4 (EB-OVX): Ovariectomized treated with estradiol benzoate 1.5 μg/rat twice a week. Ferutinin and Estradiol benzoate were used as above described.

The body weight of each animal was recorded at 4 different times: before ovariectomy (i.e., at the start of the experiment), two months after ovariectomy (namely, at the beginning of treatment), and after 30 and 60 days of treatment. At the end of the treatments, all rats were sacrificed.

### **2.2 Histology and histomorphometrical evaluation**

Soon after the sacrifice, the 4th, 5th lumbar vertebrae and the right femurs were removed, processed and embedded in methyl methacrylate according to standard protocol for light microscopy. Serial sections of 200μm thickness were taken from both lumbar vertebrae and femurs by means of a diamond-saw microtome cutting system. In particular, the 4th lumbar vertebrae were cut according to sagittal planes, whereas the 5th lumbar vertebrae were transversely cut; concerning the femurs, the distal epiphyseal level was sagittally sectioned, whereas the shaft was transversely sectioned at the mid-diaphyseal level (Fig. 3).

Histomorphometric analysis was performed on Fast-Green or Alizarin-Red stained sections using a light microscope equipped with an image analysis system. In histomorphometric evaluations of vertebral bodies, only trabecular bone was taken into account: it was manually selected, outlining the internal surface of the cortical bone (Fig. 3A,B). In femoral sagittal sections, a constant area (3.5mm2) of trabecular bone was defined by drawing a circular line adjacent to the cartilagineous plate (Fig. 3C). In transversal mid-diaphyseal femoral sections the cortical bone area was measured (Fig. 3D).

The following parameters were calculated:


Only in preventing study protocol, in order to obtain a more precise evaluation of the collected data (i.e. to eliminate the effects of body weight on bone histomorphometric parameters), both the ratio BV/TV and the value Ct-B-Ar were "normalized" (i.e. corrected) with respect to body weight (dividing the calculated parameters by the body weight) on the basis that ovariectomy implies a considerable weight increase, while the chronic treatment with both ferutinin and estradiol benzoate (starting the day after ovariectomy) avoids such increment. On the contrary, in recovering study protocol the same treatment was performed 2 months after ovariectomy and after such period, the body weights of all OVX animals (C-OVX, F-OVX, EB-OVX) were all similar; for this reason, histomorphometric parameters (BV TV and Ct-B-Ar) were not normalized with respect to body weight.

Role of Phytoestrogen Ferutinin in Preventing/Recovering

**2.3 Biochemical assays** 

**2.4 Statistical analysis** 

between groups.

**3.1 Body weights** 

**3. Results** 

were similar.

Bone Loss: Results from Experimental Ovariectomized Rat Models 717

Blood samples from experimental rats were collected in tubes and the serum was immediately separated by centrifugation, aliquoted into small volumes and stored at –20°C for analysis. The serum levels of magnesium, calcium, inorganic phosphorus and alkaline phosphatase (ALP)

One-way analysis of variance (ANOVA) with Newman-Keuls test for post-hoc comparisons between individual treatment groups and controls was performed. Student's *t*-test was used where appropriate. Values of *P*<0.05 indicate significant differences

Both in preventive and recovering studies, initial body weights of the four animal groups

In preventing study (Table 1), as expected, the body weight of C-OVX rats, sacrificed at 30 and 60 days after ovariectomy, was significantly higher than that of SHAM animals. The chronic administration of ferutinin as well as estradiol benzoate significantly counterbalanced body weight increase. It must be stressed that estradiol benzoate (EB) treatment was able to equal the body mass gain of sham-operated control rats, while

Treatment group Initial BW BW at 30th day BW at 60th day

Table 1. Effect of ferutinin and estradiol benzoate on body weight of ovariectomized rats. Values represent mean±SEM. Anova followed by Newman-Keuls post test: aP<0.001 vs. SHAM, bP<0.001 vs. C-OVX, cP<0.001 vs. EB-OVX. SHAM: sham-operated controls receiving vehicle; C-OVX: ovariectomized controls receiving vehicle; F-OVX:

ovariectomized treated with ferutinin; EB-OVX: ovariectomized treated with estradiol

significantly in comparison to C-OVX and it is similar to that of SHAM one.

In recovering study (Fig. 4) two months after ovariectomy (namely, at the beginning of treatment) the body weight of ovariectomized rats (C-OVX, F-OVX and EB-OVX) was significantly higher, as expected, with respect to SHAM, but after both 30 and 60 days of chronic administration of ferutinin as well as of estradiol benzoate the body weight reduces

SHAM 198.9±2.4 249.7±4.1 246.0±5.4 C-OVX 205.1±2.1 308.4±5.6a 335.2±9.8a F-OVX 196.8±1.7 194.4±4.0b,c 246.0±5.4 EB-OVX 204.7±3.1 229.8±1.9b 246.8±3.4b

ferutinin caused a more marked decrease in body weight in comparison to EB.

**TABLE 1** - Body weight in Preventing study

benzoate; BW: body weight.

activity were determined by colorimetry using commercially available test kits.

Fig. 3. Histological sections taken from SHAM group in which the histomorphometrical analyses were performed. (A) sagittal section of 4th lumbar vertebra; (B) transversal section of 5th lumbar vertebra; (C) sagittal section of the distal epiphyseal level of femur; (D) transversal section at the mid-diaphyseal level of femur. The dotted lines indicate the areas in which evaluations were recorded. (Figure by Palumbo et al., 2009).

### **2.3 Biochemical assays**

716 Osteoporosis

Fig. 3. Histological sections taken from SHAM group in which the histomorphometrical analyses were performed. (A) sagittal section of 4th lumbar vertebra; (B) transversal section of 5th lumbar vertebra; (C) sagittal section of the distal epiphyseal level of femur; (D) transversal section at the mid-diaphyseal level of femur. The dotted lines indicate the areas

in which evaluations were recorded. (Figure by Palumbo et al., 2009).

Blood samples from experimental rats were collected in tubes and the serum was immediately separated by centrifugation, aliquoted into small volumes and stored at –20°C for analysis. The serum levels of magnesium, calcium, inorganic phosphorus and alkaline phosphatase (ALP) activity were determined by colorimetry using commercially available test kits.

### **2.4 Statistical analysis**

One-way analysis of variance (ANOVA) with Newman-Keuls test for post-hoc comparisons between individual treatment groups and controls was performed. Student's *t*-test was used where appropriate. Values of *P*<0.05 indicate significant differences between groups.

## **3. Results**

### **3.1 Body weights**

Both in preventive and recovering studies, initial body weights of the four animal groups were similar.

In preventing study (Table 1), as expected, the body weight of C-OVX rats, sacrificed at 30 and 60 days after ovariectomy, was significantly higher than that of SHAM animals. The chronic administration of ferutinin as well as estradiol benzoate significantly counterbalanced body weight increase. It must be stressed that estradiol benzoate (EB) treatment was able to equal the body mass gain of sham-operated control rats, while ferutinin caused a more marked decrease in body weight in comparison to EB.


Table 1. Effect of ferutinin and estradiol benzoate on body weight of ovariectomized rats. Values represent mean±SEM. Anova followed by Newman-Keuls post test: aP<0.001 vs. SHAM, bP<0.001 vs. C-OVX, cP<0.001 vs. EB-OVX. SHAM: sham-operated controls receiving vehicle; C-OVX: ovariectomized controls receiving vehicle; F-OVX: ovariectomized treated with ferutinin; EB-OVX: ovariectomized treated with estradiol benzoate; BW: body weight.

In recovering study (Fig. 4) two months after ovariectomy (namely, at the beginning of treatment) the body weight of ovariectomized rats (C-OVX, F-OVX and EB-OVX) was significantly higher, as expected, with respect to SHAM, but after both 30 and 60 days of chronic administration of ferutinin as well as of estradiol benzoate the body weight reduces significantly in comparison to C-OVX and it is similar to that of SHAM one.

Role of Phytoestrogen Ferutinin in Preventing/Recovering

Bone Loss: Results from Experimental Ovariectomized Rat Models 719

Fig. 5. Preventing study. LM micrographs showing the bone histology from the four experimental animal groups: (A) sagittal sections of 4th lumbar vertebra; (B) transversal sections of 5th lumbar vertebra; (C) sagittal sections of the distal epiphyseal level of femur; (D) transversal sections at the mid-diaphyseal level of femur. (Figure by Palumbo et al., 2009).

Fig. 4. Recovering study. Histograms showing the mean values of body weights-BW (g) recorded from all animal groups at 4 different times: (1) at the start of the experiment (before ovariectomy), (2) two months after ovariectomy, (3) after 30 days of treatment and (4) after 60 days of treatment. Values are expressed as mean SEM. \*\*\**P*<0.001 vs. C-OVX; *P*<0.001 vs. SHAM (Anova followed by Newman-Keuls test). SHAM: sham-operated controls receiving vehicle; C-OVX: ovariectomized controls receiving vehicle; F-OVX: ovariectomized treated with ferutinin; EB-OVX: ovariectomized treated with estradiol benzoate; BW: body weight.

#### **3.2 Histology and histomorphometric analysis 3.2.1 Bone mass in preventing study protocol**

Histological observations of bone sections of vertebrae and femurs from treated and control animal groups underlined, as it is expected, that bone mass is clearly lower in C-OVX rats, with respect to SHAM and treated (F-OVX and EB-OVX) animals (Fig. 5).

The histomorphometric results obtained after 30 and 60 days of treatment showed that ovariectomy induced reduction in bone mass of lumbar vertebrae and femur, which is not observed in the animals treated with ferutinin or estradiol benzoate (Figs. 6 and 7); in particular, comparing the two groups of ovariectomized animals treated with ferutinin (F-OVX) and estradiol benzoate (EB-OVX), the mean values are always higher in F-OVX with respect to EB-OVX, sometimes displaying statistical significance.

Fig. 4. Recovering study. Histograms showing the mean values of body weights-BW (g) recorded from all animal groups at 4 different times: (1) at the start of the experiment (before ovariectomy), (2) two months after ovariectomy, (3) after 30 days of treatment and (4) after 60 days of treatment. Values are expressed as mean SEM. \*\*\**P*<0.001 vs. C-OVX; *P*<0.001 vs. SHAM (Anova followed by Newman-Keuls test). SHAM: sham-operated controls

ovariectomized treated with ferutinin; EB-OVX: ovariectomized treated with estradiol

Histological observations of bone sections of vertebrae and femurs from treated and control animal groups underlined, as it is expected, that bone mass is clearly lower in C-OVX rats,

The histomorphometric results obtained after 30 and 60 days of treatment showed that ovariectomy induced reduction in bone mass of lumbar vertebrae and femur, which is not observed in the animals treated with ferutinin or estradiol benzoate (Figs. 6 and 7); in particular, comparing the two groups of ovariectomized animals treated with ferutinin (F-OVX) and estradiol benzoate (EB-OVX), the mean values are always higher in F-OVX with

receiving vehicle; C-OVX: ovariectomized controls receiving vehicle; F-OVX:

with respect to SHAM and treated (F-OVX and EB-OVX) animals (Fig. 5).

respect to EB-OVX, sometimes displaying statistical significance.

benzoate; BW: body weight.

**3.2 Histology and histomorphometric analysis 3.2.1 Bone mass in preventing study protocol** 

Fig. 5. Preventing study. LM micrographs showing the bone histology from the four experimental animal groups: (A) sagittal sections of 4th lumbar vertebra; (B) transversal sections of 5th lumbar vertebra; (C) sagittal sections of the distal epiphyseal level of femur; (D) transversal sections at the mid-diaphyseal level of femur. (Figure by Palumbo et al., 2009).

Role of Phytoestrogen Ferutinin in Preventing/Recovering

Bone Loss: Results from Experimental Ovariectomized Rat Models 721

Fig. 7. Preventing study. Mean values of histomorphometric normalized parameters,

OVX ovariectomized treated with estradiol benzoate.

expressed as BV/TV (%/g) and Ct-B-Ar (mm2/g), in both trabecular and cortical bone of the four animal groups, after 60 days from ovariectomy. (A) Sagittal section of 4th lumbar vertebra; (B) transversal section of 5th lumbar vertebra; (C) sagittal section of the distal epiphyseal level of femur; (D) transversal section at the mid-diaphyseal level of femur. Values are expressed as mean ± SEM. \*\*\*P<0.001 vs. C-OVX; +P<0.05 vs. EB-OVX (ANOVA followed by Newman–Keuls test). SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin, EB-

Fig. 6. Preventing study. Mean values of histomorphometric normalized parameters, expressed as BV/TV (%/g) and CT-B-Ar (mm2/g), in both trabecular and cortical bone of the four animal groups, after 30 days from ovariectomy. (A) sagittal sections of 4th lumbar vertebra; (B) transversal sections of 5th lumbar vertebra; (C) sagittal sections of the distal epiphyseal level of femur; (D) transversal sections at the mid-diaphyseal level of femur. Values are expressed as mean SEM. \*\*\**P*<0.001 vs. C-OVX; +++*P*<0.01 vs. EB-OVX; #*P*<0.05, ##*P*<0.01, ###*P*<0.001 vs. SHAM (ANOVA followed by Newman–Keuls test). SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin; EB-OVX ovariectomized treated with estradiol benzoate.

Fig. 6. Preventing study. Mean values of histomorphometric normalized parameters, expressed as BV/TV (%/g) and CT-B-Ar (mm2/g), in both trabecular and cortical bone of the four animal groups, after 30 days from ovariectomy. (A) sagittal sections of 4th lumbar vertebra; (B) transversal sections of 5th lumbar vertebra; (C) sagittal sections of the distal epiphyseal level of femur; (D) transversal sections at the mid-diaphyseal level of femur. Values are expressed as mean SEM. \*\*\**P*<0.001 vs. C-OVX; +++*P*<0.01 vs. EB-OVX; #*P*<0.05, ##*P*<0.01, ###*P*<0.001 vs. SHAM (ANOVA followed by Newman–Keuls test). SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with

ferutinin; EB-OVX ovariectomized treated with estradiol benzoate.

Fig. 7. Preventing study. Mean values of histomorphometric normalized parameters, expressed as BV/TV (%/g) and Ct-B-Ar (mm2/g), in both trabecular and cortical bone of the four animal groups, after 60 days from ovariectomy. (A) Sagittal section of 4th lumbar vertebra; (B) transversal section of 5th lumbar vertebra; (C) sagittal section of the distal epiphyseal level of femur; (D) transversal section at the mid-diaphyseal level of femur. Values are expressed as mean ± SEM. \*\*\*P<0.001 vs. C-OVX; +P<0.05 vs. EB-OVX (ANOVA followed by Newman–Keuls test). SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin, EB-OVX ovariectomized treated with estradiol benzoate.

Role of Phytoestrogen Ferutinin in Preventing/Recovering

respect to animal groups treated with estradiol benzoate.

Bone Loss: Results from Experimental Ovariectomized Rat Models 723

estrogen benzoate in increasing uterine weight not only in the preventing study but also in the recovering one. In particular, the morphological and morphometrical preliminary data suggest that ferutinin would act on the uterus in a manner similar to that of estradiol benzoate, stimulating endometrial hypertrophy. Moreover, the treatment with ferutinin is of particular interest because the apoptotic index in both preventing and recovering studies seems to be almost always higher in both luminal and glandular endometrial epithelia with

Fig. 9. Recovering study. Mean values of histomorphometric parameters, expressed as BVTV (%) and Ct-B-Ar (mm2), in both trabecular and cortical bone of the all animal groups after 30 days of treatment. (A) Sagittal section of the 4th lumbar vertebra; (B) transversal section of the 5th lumbar vertebra; (C) sagittal section of the distal epiphysis of femur; (D) transversal section at the mid-diaphyseal level of femur. Values are expressed as mean ± SEM. \**P*< 0.05 vs. C-OVX; #*P*< 0.05, ###*P*< 0.001 vs. SHAM (ANOVA followed by Newman– Keuls test). SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin; EB-OVX

ovariectomized treated with estradiol benzoate.

#### **3.2.2 Bone mass in recovering study protocol**

As regards both periods of time (30 and 60 days), the histological sections of vertebrae and femurs from SHAM and treated animal groups showed higher amount of trabecular bone (Figure 8A-B-C) with respect to C-OVX group, while the amount of cortical bone did not show differences among all groups (Fig. 8D). The histomorphometric analyses clearly showed the different results in trabecular and cortical bone: the amount of trabecular bone (Figures 9A-B-C and 10A-B-C) in F-OVX and EB-OVX animals are always higher with respect to C-OVX ones, although they do not reach the values of SHAM animals; as far as cortical bone (Figures 9D and 10D) is concerned, no statistically significant differences were found in bone area among all groups after both 30 and 60 days of treatments.

Fig. 8. Recovering study. LM micrographs showing the bone histology from the four experimental animal groups after 30 days of treatment. (A) Sagittal sections of the 4th lumbar vertebra; (B) transversal sections of the 5th lumbar vertebra; (C) sagittal sections of the distal epiphysis of femur; (D) transversal sections at the mid-diaphyseal level of femur. (Figure by Ferretti et al., 2010).

#### **3.2.3 Uterine tissues**

Preliminary data, not yet published by the authors, concern also the side effects of the chronic treatment with ferutinin on the uterus of ovariectomized rats, particularly regarding weight, size, morphology and structure. The target was to compare ferutinin side effects with those elicited by estradiol benzoate treatment, both in preventing and recovering protocols. Although data are incomplete, ferutinin would seem to exert the same effect of

As regards both periods of time (30 and 60 days), the histological sections of vertebrae and femurs from SHAM and treated animal groups showed higher amount of trabecular bone (Figure 8A-B-C) with respect to C-OVX group, while the amount of cortical bone did not show differences among all groups (Fig. 8D). The histomorphometric analyses clearly showed the different results in trabecular and cortical bone: the amount of trabecular bone (Figures 9A-B-C and 10A-B-C) in F-OVX and EB-OVX animals are always higher with respect to C-OVX ones, although they do not reach the values of SHAM animals; as far as cortical bone (Figures 9D and 10D) is concerned, no statistically significant differences were

found in bone area among all groups after both 30 and 60 days of treatments.

Fig. 8. Recovering study. LM micrographs showing the bone histology from the four

Ferretti et al., 2010).

**3.2.3 Uterine tissues** 

experimental animal groups after 30 days of treatment. (A) Sagittal sections of the 4th lumbar vertebra; (B) transversal sections of the 5th lumbar vertebra; (C) sagittal sections of the distal epiphysis of femur; (D) transversal sections at the mid-diaphyseal level of femur. (Figure by

Preliminary data, not yet published by the authors, concern also the side effects of the chronic treatment with ferutinin on the uterus of ovariectomized rats, particularly regarding weight, size, morphology and structure. The target was to compare ferutinin side effects with those elicited by estradiol benzoate treatment, both in preventing and recovering protocols. Although data are incomplete, ferutinin would seem to exert the same effect of

**3.2.2 Bone mass in recovering study protocol** 

estrogen benzoate in increasing uterine weight not only in the preventing study but also in the recovering one. In particular, the morphological and morphometrical preliminary data suggest that ferutinin would act on the uterus in a manner similar to that of estradiol benzoate, stimulating endometrial hypertrophy. Moreover, the treatment with ferutinin is of particular interest because the apoptotic index in both preventing and recovering studies seems to be almost always higher in both luminal and glandular endometrial epithelia with respect to animal groups treated with estradiol benzoate.

Fig. 9. Recovering study. Mean values of histomorphometric parameters, expressed as BVTV (%) and Ct-B-Ar (mm2), in both trabecular and cortical bone of the all animal groups after 30 days of treatment. (A) Sagittal section of the 4th lumbar vertebra; (B) transversal section of the 5th lumbar vertebra; (C) sagittal section of the distal epiphysis of femur; (D) transversal section at the mid-diaphyseal level of femur. Values are expressed as mean ± SEM. \**P*< 0.05 vs. C-OVX; #*P*< 0.05, ###*P*< 0.001 vs. SHAM (ANOVA followed by Newman– Keuls test). SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin; EB-OVX ovariectomized treated with estradiol benzoate.

Role of Phytoestrogen Ferutinin in Preventing/Recovering

TABLE 2 Treatment

**30 daytreatment** 

**60 daytreatment**

vs. EB-OVX;

**30 daytreatment** 

**60 daytreatment**

**4. Discussion** 

TABLE 3 Treatment

Bone Loss: Results from Experimental Ovariectomized Rat Models 725

SHAM 2.82±0.02 9.8±0.1 9.06±0.22 110.3±12.0 C-OVX 2.94±0.09 10.2±0.05# 9.85±0.35 106.2±4.9 F-OVX 2.74±0.04 9.6±0.1\*\* ++ 7.59±0.21\*\*\* ## ++ 148.4±17.5 EB-OVX 2.88±0.05 10.1±0.07 9.07±0.26 111.0±8.4

SHAM 2.54±0.01 9.7±0.1 7.81±0.17 90.8±5.1 C-OVX 2.52±0.05 9.7±0.2 7.76±0.34 102.6±4.2 F-OVX 2.55±0.05 9.9±0.1 7.05±0.31 118.0±7.1++ EB-OVX 2.53±0.05 9.7±0.2 7.03±0.33 79.7±7.3

*P*<0.05, *P*<0.01 vs. SHAM. SHAM sham-operated controls receiving vehicle;

SHAM 2.43±0.06 10.6±0.01 6.29±0.43 103±13.65 C-OVX 2.41±0.07 10.18±0.18 7.18±0.3 81.6±3.98 F-OVX 2.43±0.04 10.56±0.1 6.09±0.22 144.6±15.4\*\* # ++ EB-OVX 2.48±0.05 10.52±0.1 6.57±0.25 75±12.5

SHAM 2.57±0.06 10.25±0.16 7.52±0.46 89.5±9.24 C-OVX 2.86±0.4 10.28±0.02 6.2±0.22# 90.2±8.61 F-OVX 2.55±0.07 10.58±0.12 6.37±0.27# 109.2±7.19+ EB-OVX 2.47±0.04 10.58±0.14 6.02±0.17# 72.8±5.91

(mg/dl) ALP (UI/l)

(mg/dl) ALP (UI/l)

group Mg (mg/dl) Ca (mg/dl) P inorganic

Table 2. Preventing study. Effect of ferutinin/estradiol benzoate on serum biochemical values of ovariectomized rats treated for 30 and 60 days. All values are expressed as mean ± SEM. Anova followed by Newman-Keuls post test: \*\**P*<0.01, \*\*\**P*<0.001 vs. C-OVX; ++*P*<0.01

C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with

group Mg (mg/dl) Ca (mg/dl) P inorganic

Table 3. Recovering study. Effect of ferutinin/estradiol benzoate on serum biochemical values of ovariectomized rats (30 and 60 days of treatment). All values are expressed as mean ± SEM. Anova followed by Newman-Keuls post test: \*\**P*<0.01 vs. C-OVX; +*P*<0.05, ++*P*<0.01 vs. EB-OVX; #*P*<0.05 vs. SHAM. SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with

The results so far obtained have clearly suggested that ferutinin displays positive effects on bone mass both in preventing and in curative treatment of estrogen deficiency osteoporosis; more precisely, the observations have indicated that ferutinin seems to exert similar effects to estradiol benzoate in curative treatment (Ferretti et al., 2010), and even it seems to be

ferutinin; EB-OVX ovariectomized treated with estradiol benzoate.

ferutinin; EB-OVX ovariectomized treated with estradiol benzoate.

Fig. 10. Recovering study. Mean values of histomorphometric parameters, expressed as BVTV (%) and Ct-B-Ar (mm2), in both trabecular and cortical bone of the all animal groups after 60 days of treatment. (A) Sagittal section of the 4th lumbar vertebra; (B) transversal section of the 5th lumbar vertebra; (C) sagittal section of the distal epiphysis of femur; (D) transversal section at the mid-diaphyseal level of femur. Values are expressed as mean ± SEM. \**P*< 0.05, \*\**P*< 0.01 vs. C-OVX; #*P*< 0.05, ##*P*< 0.01, ###*P*< 0.001 vs. SHAM (ANOVA followed by Newman–Keuls test). SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin; EB-OVX ovariectomized treated with estradiol benzoate.

#### **3.3 Serum biochemical analysis**

In preventing study protocol, serum level variations of magnesium, calcium and inorganic phosphorous among the groups were more evident after 30 days of treatment rather than after 60 days (Table 2), while in the recovering study protocol, no significant differences in serum levels were recorded (Table 3). As far as serum alkaline phosphatase is concerned, its levels in F-OVX animal groups were always higher with respect to all other groups both in preventive and recovering study protocols after 30 as well as 60 days of treatment.

Fig. 10. Recovering study. Mean values of histomorphometric parameters, expressed as BVTV (%) and Ct-B-Ar (mm2), in both trabecular and cortical bone of the all animal groups after 60 days of treatment. (A) Sagittal section of the 4th lumbar vertebra; (B) transversal section of the 5th lumbar vertebra; (C) sagittal section of the distal epiphysis of femur; (D) transversal section at the mid-diaphyseal level of femur. Values are expressed as mean ± SEM. \**P*< 0.05, \*\**P*< 0.01 vs. C-OVX; #*P*< 0.05, ##*P*< 0.01, ###*P*< 0.001 vs. SHAM (ANOVA followed by Newman–Keuls test). SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin;

In preventing study protocol, serum level variations of magnesium, calcium and inorganic phosphorous among the groups were more evident after 30 days of treatment rather than after 60 days (Table 2), while in the recovering study protocol, no significant differences in serum levels were recorded (Table 3). As far as serum alkaline phosphatase is concerned, its levels in F-OVX animal groups were always higher with respect to all other groups both in

preventive and recovering study protocols after 30 as well as 60 days of treatment.

EB-OVX ovariectomized treated with estradiol benzoate.

**3.3 Serum biochemical analysis** 


Table 2. Preventing study. Effect of ferutinin/estradiol benzoate on serum biochemical values of ovariectomized rats treated for 30 and 60 days. All values are expressed as mean ± SEM. Anova followed by Newman-Keuls post test: \*\**P*<0.01, \*\*\**P*<0.001 vs. C-OVX; ++*P*<0.01 vs. EB-OVX; *P*<0.05, *P*<0.01 vs. SHAM. SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin; EB-OVX ovariectomized treated with estradiol benzoate.


Table 3. Recovering study. Effect of ferutinin/estradiol benzoate on serum biochemical values of ovariectomized rats (30 and 60 days of treatment). All values are expressed as mean ± SEM. Anova followed by Newman-Keuls post test: \*\**P*<0.01 vs. C-OVX; +*P*<0.05, ++*P*<0.01 vs. EB-OVX; #*P*<0.05 vs. SHAM. SHAM sham-operated controls receiving vehicle; C-OVX ovariectomized controls receiving vehicle; F-OVX ovariectomized treated with ferutinin; EB-OVX ovariectomized treated with estradiol benzoate.

### **4. Discussion**

The results so far obtained have clearly suggested that ferutinin displays positive effects on bone mass both in preventing and in curative treatment of estrogen deficiency osteoporosis; more precisely, the observations have indicated that ferutinin seems to exert similar effects to estradiol benzoate in curative treatment (Ferretti et al., 2010), and even it seems to be

Role of Phytoestrogen Ferutinin in Preventing/Recovering

carcinoma.

**5. Conclusion** 

2002).

Bone Loss: Results from Experimental Ovariectomized Rat Models 727

Evans et al., 1990; Nian et al., 2006) suggest that the process of osteogenesis should be triggered in F-OVX group, because ALP value in F-OVX is higher with respect to the other groups. A positive effect on osteoblast activity *in vitro* by other phytoestrogens, like

As far as ferutinin side effects is concerned on the organs commonly targeted by estrogens, the apparent above cited antiapoptotic effect on endometrial epithelia is in line with observations previously recorded for genistein by other authors that administered such phytoestrogen to ovariectomized mice (Eason et al., 2005; Garcìa-Pérez et al., 2006). Since an increased risk of endometrial cancer due to excessive hypertrophy is one of the recognized prejudicial effects of estrogens, the phytoestrogen ferutinin, althought induces thickening of endometrium as well as estrogens, seems to increase the percentage of apoptotic epithelial cells, particularly the glandular ones. This effect might exert a protective role against uterine

On the light of the observations above reported on the effect of ferutinin in preventing/recovering severe osteoporosis secondary to ovariectomy in rats, the authors suggest to enumerate ferutinin among the osteoprotective substances. This fact acquires a more relevant importance in the light of recent tenable evidences, as above cited, reported from some authors concerning the absence of negative side effects by some phytoestrogens (particularly genistein, 8-prenylnaringenin, reveratrol and red clover extract) on the tropism of various organs commonly targeted by estrogens (Burdette et al., 2002; Duffy et al., 2007; Eason et al., 2005; Gallo et al., 2006; Garcia-Perez et al., 2006; Hümpel et al., 2005; Lian et al., 2001; Limer & Speirs, 2004; Murray et al., 2003; Whitsett & Lamartiniere, 2006; Wu et al.,

**C. Palumbo, F. Cavani, L. Bertoni, M. Ferretti-2011** 

genistein, has already been published (Liao et al., 2007; Pan et al., 2005).

*Ferula hermonis*

more effective, compared with estradiol benzoate, in preventing bone loss due to estrogen deficiency (Palumbo et al., 2009). It is to be underlined that, comparing the results from the two protocols, in curative study the values of bone mass of treated animals never reach those of SHAM group; this is due to the fact that the treatment started after the occurrence of a severe osteoporosis (as a consequence of estrogen deficiency secondary to two months of ovariectomy).

It is important to emphasise that one of the most important effects of skeletal diseases, like osteoporosis, is the progressive trabecular bone resorption that, in turn, implies enhanced bone fragility and, consequently, an increased frequency of fractures. According to the literature (Kalu, 1991; Wronski et al., 1987) ovariectomy in rats induces different effects on trabecular bone of the axial with respect to the appendicular skeleton, with more marked bone resorption taking place in the latter; for this reason, both vertebrae and femurs were investigated. Moreover, ovariectomy differently affects trabecular and cortical bone, since in OVX animals the bone mass loss observed in trabecular bone was not equally observed in cortical bone; in fact, the values of femur cortical bone areas are similar in OVX and SHAM groups. These data are in line with older ones showing an earlier started decrease in bone mass, more extensive in the spongiosa than in the compacta of rats fed a low-calcium diet (Lozupone & Favia, 1988). This fact is a consequence of the different pattern of distribution of mechanical stresses acting on the two different bony architectures and it is probably related to the different metabolism of the various skeletal regions that, in turn, affect the bone turnover rate of the different skeletal regions, viz. metaphysis compared with diaphyses (Canè et al., 1982). Other authors have also shown that cortical bone is not very sensitive to bone loss due to ovariectomy standing the increased endosteal osteoblasts (Jee et al., 1990; Liu & Kalu, 1990; Turner et al., 1987). All these considerations make the "Bone Organ" a sophisticated system in which metabolic and mechanical demands are actually sensed and integrated in answering both systemic and loading needs.

As previously mentioned the authors wanted to evaluate whether the chronic administration of ferutinin, starting from the day after ovariectomy, is able to prevent estrogen deficiency effects similarly to HRT. The results obtained clearly showed that the phytoestrogen ferutinin displays positive effects in preventing osteoporosis due to estrogen deficiency; more precisely the observations suggest that ferutinin seems to be more effective in preventing bone loss compared with estradiol benzoate. Another positive aspect of ferutinin treatment is to prevent weight gain that typically occurs after ovariectomy. As above mentioned, ferutinin has been shown to interact with estrogen receptors (Appendino et al., 2002; Ikeda et al., 2002). While the majority of phytoestrogens have a higher relative binding affinity for ERβ than ERα, ferutinin displays a higher affinity for ERα (IC50=33.1 nM) than for ERβ (IC50=180.5 nM) (Ikeda et al., 2002). The different roles of specific estrogen receptors ERα and ERβ on body weight regulation were recently investigated by Wegorzewska and co-workers (2008), using the ovariectomized rat model. OVX rats showed a significant increase in body weight, which was reversed by the daily treatment (for 21 days) with estradiol or PPT (propylpyrazoletriol, a selective ERα agonist), but not by the daily treatment with DPN (diarylpropionitrile, a ERβ agonist); these results confirm the major role of ERα in regulating body weight, as it was previously suggested by other authors (Kraichely et al., 2000; Stauffer et al., 2000) by using ER-specific knockout mice.

Regarding the bone turnover-related serum levels, the recorded values of alkaline phosphatase (the most widely recognized biochemical marker for osteoblastic activity -

Evans et al., 1990; Nian et al., 2006) suggest that the process of osteogenesis should be triggered in F-OVX group, because ALP value in F-OVX is higher with respect to the other groups. A positive effect on osteoblast activity *in vitro* by other phytoestrogens, like genistein, has already been published (Liao et al., 2007; Pan et al., 2005).

As far as ferutinin side effects is concerned on the organs commonly targeted by estrogens, the apparent above cited antiapoptotic effect on endometrial epithelia is in line with observations previously recorded for genistein by other authors that administered such phytoestrogen to ovariectomized mice (Eason et al., 2005; Garcìa-Pérez et al., 2006). Since an increased risk of endometrial cancer due to excessive hypertrophy is one of the recognized prejudicial effects of estrogens, the phytoestrogen ferutinin, althought induces thickening of endometrium as well as estrogens, seems to increase the percentage of apoptotic epithelial cells, particularly the glandular ones. This effect might exert a protective role against uterine carcinoma.

### **5. Conclusion**

726 Osteoporosis

more effective, compared with estradiol benzoate, in preventing bone loss due to estrogen deficiency (Palumbo et al., 2009). It is to be underlined that, comparing the results from the two protocols, in curative study the values of bone mass of treated animals never reach those of SHAM group; this is due to the fact that the treatment started after the occurrence of a severe osteoporosis (as a consequence of estrogen deficiency secondary to two months

It is important to emphasise that one of the most important effects of skeletal diseases, like osteoporosis, is the progressive trabecular bone resorption that, in turn, implies enhanced bone fragility and, consequently, an increased frequency of fractures. According to the literature (Kalu, 1991; Wronski et al., 1987) ovariectomy in rats induces different effects on trabecular bone of the axial with respect to the appendicular skeleton, with more marked bone resorption taking place in the latter; for this reason, both vertebrae and femurs were investigated. Moreover, ovariectomy differently affects trabecular and cortical bone, since in OVX animals the bone mass loss observed in trabecular bone was not equally observed in cortical bone; in fact, the values of femur cortical bone areas are similar in OVX and SHAM groups. These data are in line with older ones showing an earlier started decrease in bone mass, more extensive in the spongiosa than in the compacta of rats fed a low-calcium diet (Lozupone & Favia, 1988). This fact is a consequence of the different pattern of distribution of mechanical stresses acting on the two different bony architectures and it is probably related to the different metabolism of the various skeletal regions that, in turn, affect the bone turnover rate of the different skeletal regions, viz. metaphysis compared with diaphyses (Canè et al., 1982). Other authors have also shown that cortical bone is not very sensitive to bone loss due to ovariectomy standing the increased endosteal osteoblasts (Jee et al., 1990; Liu & Kalu, 1990; Turner et al., 1987). All these considerations make the "Bone Organ" a sophisticated system in which metabolic and mechanical demands are actually

As previously mentioned the authors wanted to evaluate whether the chronic administration of ferutinin, starting from the day after ovariectomy, is able to prevent estrogen deficiency effects similarly to HRT. The results obtained clearly showed that the phytoestrogen ferutinin displays positive effects in preventing osteoporosis due to estrogen deficiency; more precisely the observations suggest that ferutinin seems to be more effective in preventing bone loss compared with estradiol benzoate. Another positive aspect of ferutinin treatment is to prevent weight gain that typically occurs after ovariectomy. As above mentioned, ferutinin has been shown to interact with estrogen receptors (Appendino et al., 2002; Ikeda et al., 2002). While the majority of phytoestrogens have a higher relative binding affinity for ERβ than ERα, ferutinin displays a higher affinity for ERα (IC50=33.1 nM) than for ERβ (IC50=180.5 nM) (Ikeda et al., 2002). The different roles of specific estrogen receptors ERα and ERβ on body weight regulation were recently investigated by Wegorzewska and co-workers (2008), using the ovariectomized rat model. OVX rats showed a significant increase in body weight, which was reversed by the daily treatment (for 21 days) with estradiol or PPT (propylpyrazoletriol, a selective ERα agonist), but not by the daily treatment with DPN (diarylpropionitrile, a ERβ agonist); these results confirm the major role of ERα in regulating body weight, as it was previously suggested by other authors (Kraichely et al.,

Regarding the bone turnover-related serum levels, the recorded values of alkaline phosphatase (the most widely recognized biochemical marker for osteoblastic activity -

sensed and integrated in answering both systemic and loading needs.

2000; Stauffer et al., 2000) by using ER-specific knockout mice.

of ovariectomy).

On the light of the observations above reported on the effect of ferutinin in preventing/recovering severe osteoporosis secondary to ovariectomy in rats, the authors suggest to enumerate ferutinin among the osteoprotective substances. This fact acquires a more relevant importance in the light of recent tenable evidences, as above cited, reported from some authors concerning the absence of negative side effects by some phytoestrogens (particularly genistein, 8-prenylnaringenin, reveratrol and red clover extract) on the tropism of various organs commonly targeted by estrogens (Burdette et al., 2002; Duffy et al., 2007; Eason et al., 2005; Gallo et al., 2006; Garcia-Perez et al., 2006; Hümpel et al., 2005; Lian et al., 2001; Limer & Speirs, 2004; Murray et al., 2003; Whitsett & Lamartiniere, 2006; Wu et al., 2002).

Role of Phytoestrogen Ferutinin in Preventing/Recovering

model, *Clin Reumathol* 26(3): 380-384.

cancer, *CA Cancer J Clin* 57: 260-277.

Bone Loss: Results from Experimental Ovariectomized Rat Models 729

Comelekoglu, U., Bagis, S., Yalin, S., Ogenler, O., Yildiz, A., Sahin, N.O., Oguz, I. &

Duffy, C., Perez, K. & Partridge, A. (2007). Implications of phytoestrogen intake for breast

Eason, R.R., Till, S.R., Velarde, M.C., Geng, Y., Chatman, L. Jr., Gu, L., Badger, T.M.,

El-Desoky, H.S., Beltagi, A.M., Ghoneim, M.M. (2009). Determination of the anti-

voltammetric and chromatographic methods. *J AOAC Int* 92(3): 806-812. Evans, D.B., Bunning, R.A.D. & Russell, R.G.G. (1990) The effect of recombinant human

derived from human bone. *Biochem and Biophis Res Comm* 166: 208-216 Fanti, P., Monier-Faugere, M.C., Geng, Z., Schmidt, J., Morris, P.E., Cohen, D. & Malluche,

Ferretti, M., Bertoni, L., Cavani, F., Zavatti, M., Resca, E., Carnevale, G., Benelli, A., Zanoli,

influence the rodent postmenopausal mammary gland, *Menopause* 13: 72-79. Garcia-Perez, M.A., Noguera, R., del Val, R., Noguera, I., Hermenegildo, C. & Cano, A.

Genant, H.K., Baylink, D.J. & Gallagher, J.C. (1998). Estrogens in the prevention of osteoporosis in post menopausal women, *Am J Obstet Gynecol* 161: 1842-1846. Hümpel, M., Isaksson, P., Schaefer, O., Kaufmann, U., Ciana, P., Maggi, A. & Schleuning,

Ikeda, K., Arao, Y., Otsuka, H., Nomoto, S., Horiguchi, H., Kato, S. & Kayama, F. (2002).

Jee, W.S., Mori, S., Lee, X.J. & Chan, S. (1990). Prostaglandin E2 enhances cortical bone mass

Johnson, E.B., Muto M.G., Yanushpolsky, E.H. & Mutter G.L. Phytoestrogen supplementation and endometrial cancer. *Obstet Gynecol* 2001; 98: 947-50. Kalu, D.N. (1991). The ovariectomized rat model of postmenopausal bone loss, *Bone Miner*

Kraichely, D.M., Sun, J., Katzenellenbogen, J.A. & Katzenellenbogen, B.S. (2000)

ovariectomized rats. II: role in recovering osteoporosis, *J Anat* 217: 48-56. Gallo, D., Zannoni, G.F., Martinelli, E., Ferlini, C., Fabrizi, M., Riva, A., Morazzoni, P.,

ovariectomized rats*. Osteoporos Int* 8(3): 274-281.

ovariectomized mice, *Fertility and Sterility* 86: 1003-1005.

Erβ, *Biochem Biophys Res Commun* 291: 354-360.

*Biol* 97: 299-305.

rats. *Bone* 11: 253-266.

15: 175-191.

Hatungil, R. (2006). Biomechanical evaluation in osteoporosis: ovariectomized rat

Simmen, F.A. & Simmen R.C. (2005). Uterine phenotype of young adult rats exposed to dietary soy or genistein during development, *J Nutr Biochem* 16: 625-632.

osteoporosis drug ipriflavone in pharmaceutical formulation by stripping

interleukine-1ß on cellular proliferation and the production of prostaglandin E2, plasminogen activator, osteocalcin and alkaline phosphatase by osteoblast-like cells

H.H. (1998) The phytoestrogen genistein reduces bone loss in short-term

P. & Palumbo, C. (2010). Influence of ferutinin on bone metabolism in

Bombardelli, E. & Scambia, G. (2006). Estradiol and phytoestrogens differently

(2006). Comparative effects of estradiol, raloxifene, and genistein on the uterus of

W.D. (2005). Tissue specificity of 8-prenylnaringenin: Protection from ovariectomy induced bone loss with minimal trophic effects on the uterus, *J Steroid Biochem Mol* 

Terpenoids found in the Umbelliferae family act as agonists/antagonists for ERα and ERβ: differential transcription activity between ferutinin-liganded ERα and

and activates intracortical bone remodelling in intact and ovariectomized female

Conformational changes and coactivator recruitment by novel ligands for estrogen

In conclusion, the results here reported not only provide evidence that ferutinin can significantly prevent/recover ovariectomy-induced bone loss in rats, but also that it could protect against the onset of uterus cancer. Although the putative undesired estrogenic-like side effects on uterus of such phytoestrogen have not yet been fully investigated, ferutinin could be an interesting safer alternative new candidate for HRT in treatment of postmenopausal symptoms, since it seems to protect from bone loss induced by ovariectomy (Ferretti et al., 2010; Palumbo et al., 2009) and in part to mime the ovarian endocrine function during menopause. The authors are aware that additional studies are required to characterize the mechanism by which ferutinin acts both in improving/resolving severe degrees of bone mass loss and in protecting from uterine cancer onset.

### **6. Acknowledgments**

The researches on ferutinin effects in preventing/recovering estrogen deficiencyosteoporosis were supported by 2009-2010 "Fondazione di Vignola" funds and by "Banca Popolare dell'Emilia Romagna".

### **7. References**


In conclusion, the results here reported not only provide evidence that ferutinin can significantly prevent/recover ovariectomy-induced bone loss in rats, but also that it could protect against the onset of uterus cancer. Although the putative undesired estrogenic-like side effects on uterus of such phytoestrogen have not yet been fully investigated, ferutinin could be an interesting safer alternative new candidate for HRT in treatment of postmenopausal symptoms, since it seems to protect from bone loss induced by ovariectomy (Ferretti et al., 2010; Palumbo et al., 2009) and in part to mime the ovarian endocrine function during menopause. The authors are aware that additional studies are required to characterize the mechanism by which ferutinin acts both in improving/resolving severe

The researches on ferutinin effects in preventing/recovering estrogen deficiencyosteoporosis were supported by 2009-2010 "Fondazione di Vignola" funds and by "Banca

Abourashed, E.A., Galal, A.M., El-Feraly, F.S. & Khan, I.A. (2001). Separation and

Albertazzi, P. (2002). Purified phytoestrogens in postmenopausal bone health: is there a role

An, J., Tzagarakis-Foster, C., Scharschmidt, T.C., Lomri, N. & Leitman, D.C. (2001). Estrogen

Appendino, G., Spagliardi, P., Cravotto, G., Pocock, V. & Milligan, S. (2002). Daucane

Beral, V. & Million women study collaborators. (2003). Breast cancer and hormone replacement therapy in the Million Women Study, *Lancet* 362: 419-427 Bruhn, C. (2010) Denosumab. The first inhibitor of RANK-ligand for treatment of

Brzezinski, A. & Debi, A. (1999). Phytoestrogens: the "natural" selective receptor

Burdette, J.E., Liu, J., Lantvit, D., Lim, E., Booth, N., Bhat, K.P., Hedayat, S., Van Breemen,

Canè, V., Marotti, G., Volpi, G., Zaffe, D., Palazzini, S., Remaggi, F. & Muglia, MA. (1982)

Chestnut, C.H. 3rd (1995). Drug therapy: calcitonin, bisphosphonates, and anabolic steroids,

Clifton-Bligh, P.B., Baber, R.J. & Fulcher, G.R. (2001). The effect of isoflavones extracted from red clover (Rimostil) on lipid and bone metabolism, *Menopause* 8: 259-265.

R.B., Constantinou, A.I., Pezzuto, J.M., Farnsworth, N.R. & Bolton, J.L. (2002). Trifolium pratense (red clover) exhibits estrogenic effects in vivo in ovariectomized

Size and density of osteocyte lacunae in different regions of long bones. *Calcif* 

*in* Riggs BL, Melton III LJ (eds.), *Osteoporosis: Etiology, Diagnosis, and Management*.

phytoestrogens: a structure-activity study, *J Nat Prod* 65: 612-615.

quantification of the major daucane esters of *Ferula hermonis* by HPLC, *Planta Med*

receptor beta-selective transcriptional activity and recruitment of coregulators by

degrees of bone mass loss and in protecting from uterine cancer onset.

**6. Acknowledgments** 

**7. References** 

Popolare dell'Emilia Romagna".

67: 681-682.

for genistein?, *Climateric* 5: 190-196.

phytoestrogens, *J Biol Chem* 276: 17808-17814.

osteoporosis. *Med Monatsschr Pharm* 33(10): 370-375.

Lippincott-Raven Publishers, Philadelphia, p.391.

Sprague-Dawley rats. *J Nutr* 132: 27-30.

*Tissue Int* 34: 558-563.

modulators?, *Eur J Obstet Gynecol Reprod Biol* 85: 47-51.


Role of Phytoestrogen Ferutinin in Preventing/Recovering

osteoporotic bone, *Calcif Tissue Int* 61: 487-492.

hypothalamic/pituitary axis in rats, *J Nutr* 127: 263-269.

*J Biol Chem* 286(27): 23771-23779.

*Metab* 27: 538-545.

476-82.

115-122.

344-347.

67.e5.

1699-1706.

15: 275-300.

*Chem* 43: 4934–4947.

Bone Loss: Results from Experimental Ovariectomized Rat Models 731

Palumbo, C., Ferretti, M., Bertoni, L., Cavani, F., Resca, E., Casolari, B., Carnevale, G.,

Pan, W., Quarles, L.D., Song, L.H., Yu, Y.H., Jiao, C., Tang, H.B., Jiang, C.H., Deng, H.W., Li,

Paschalis, E.P., Betts, F., Di Carlo, E., Mendelsohn, R. & Boskey, A.L. (1997). FTIR

Santell, R.C., Chang, Y.C., Nair, M.G. & Helferich, W.G. (1997). Dietary genistein exerts

Schoofs, M. W. (2003). Thiazide diuretics and the risk for hip fracture. *Ann Intern Med* 139:

Stauffer, S.R., Coletta, C.J., Tedesco, R., Nishiguchi, G., Carlson, K., Sun, J.,

Strom, A., Hartman, J., Foster, J.S., Kietz, S., Wimalasena, J. & Gustafsson, J.A. (2004).

Termine, J.D. & Wong, M. (1998). Post-menopausal women and osteoporosis: available

Turner, T.T., Vandersteenhoven, J.J. & Bell, N.H. (1987) The effects of ovariectomy and 17β-

Turner, R.T., Riggs, B.L. & Spelsberg, T.C. (1994). Skeletal effects of estrogen, *Endocrine Rev*

Wang, Z.L., Sun, J.Y., Wang, D.N., Xie, Y.H., Wang, S.W. & Zhao, W.M. (2006).

Wegorzewska, I.N., Walters, K., Weiser, M.J., Cruthirds, D.F., Ewell, E., Larco, D.O., Handa,

Whitsett, T.G. Jr. & Lamartiniere, C.A. (2006) Genistein and resveratrol: mammary cancer

breast cancer cell line T47D, *Proc Natl Acad Sci* 101: 1566-1571.

choices for maintenance of skeletal health, *Maturitas* 30: 241-245.

Riggs, B.L. & Melton, L.J. (1986). Involutional osteoporosis, *N Engl J Med* 314: 1676-1686. Rybchyn, M.S., Slater, M., Conigrave, A.D. & Mason, R.S. (2011) An Akt-dependent Increase

Zavatti, M., Montanari, C., Benelli, A. & Zanoli, P. (2009). Influence of bone metabolism in ovariectomized rats. I: role in preventing osteoporosis, *J Bone Miner* 

Y.J., Zhou, H.H. & Xiao, Z.S. (2005) Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture. *J Cell Biochem* 94(2): 307-316.

microspectroscopic analysis of human iliac crest biopsies from untreated

in Canonical Wnt Signaling and a Decrease in Sclerostin Protein Levels Are Involved in Strontium Ranelate-induced Osteogenic Effects in Human Osteoblasts.

estrogenic effects upon the uterus, mammary gland and the

Katzenellenbogen, B.S. & Katzenellenbogen, J.A. (2000) Pyrazole ligands: structureaffinity/activity relationships and estrogen receptor-alpha-selective agonists. *J Med* 

Estrogen receptor beta inhibits 17beta-estradiol-stimulated proliferation of the

estradiol on cortical bone histomorphometry in growing rats. *J BoneMiner Res* 2:

Pharmacological studies of the large-scaled purified genistein from Huaijiao (*Sophora japonica*-Leguminosae) on anti-osteoporosis, *Phytomedicine* 13: 718-723. Wasnich, R. D. (1983). Thiazide effect on the mineral content of bone. *N Engl J Med* 309(6):

R.J. & Wu, T.J. (2008) Ovariectomy weight gain in female rats is reversed by estrogen receptor alpha agonist, propylpyrazoletriol. *Am J Obstet Gynecol* 199: 67.e1-

chemoprevention and mechanisms of action in the rat. *Expert Rev Anticancer Ther* 6:

receptor-alpha and estrogen receptor-beta: correlations with biological character and distinct differences among SRC coactivator family members. *Endocrinology* 141: 3534–3545


Lacey, J.V. Jr., Mink, P.J., Lubin, J.H., Sherman, M.E., Troisi, R., Hartge, P., Schatzkin, A. &

LaCroix, A. Z. (2000). Low-dose hydrochlorothiazide and preservation of bone mineral

Lee, M.M., Lin, S.S., Wrensch, M.R., Adler, S.R. & Eisenberg, D. (2000). Alternative therapies

Lian, Z., Niwa, K., Tagami, K., Hashimoto, M., Gao, J., Yokoyama, Y., Mori, H. & Tamaya, T.

Limer, J.L. & Speirs, V. (2004). Phyto-oestrogens and breast cancer chemoprevention, *Breast* 

Liu, C.C., Kalu, D.N. (1990). Human parathyroid hormone-(1-34) prevents bone loss and

Lozupone, E. & Favia, A. (1988). Distribution of resorption processes in the compacta and spongiosa of bones from lactating rats fed a low-calcium diet. *Bone* 9: 215-224. Mei, J., Yeung, S.S. & Kung, A.W. (2001). High dietary phytoestrogen intake is associated

Messina, M., McCaskill-Stevens, W. & Lampe, J.W. (2006). Addressing the soy and breast

Morris, C., Thorpe, J., Ambrosio, L. & Santin, M. (2006). The soybean isoflavone genistein

Morris, K.T., Johnson, N., Homer, L. & Walts, D. (2000). A comparison of complementary

Murray, M.J., Meyer, W.R., Lessey, B.A., Oi, R.H., DeWire, R.E. & Fritz, M.A. (2003). Soy

hyperplasia in postmenopausal women: a pilot trial, *Menopause* 10: 456-464. Nian, H., Qin, L.P., Zhang, Q.Y., Zheng, H.C., Yu, Y. & Huang, B.K. (2006). Antiosteoporotic

Occhiuto, F., De Pasquale, R., Guglielmo, G., Palumbo, D.R, Zangla, G., Samperi, S., Renzo,

related endometrial carcinogenesis in mice, *Jpn J Cancer Res* 92: 726-734. Liao, Q.C., Xiao, Z.S., Qin, Y.F. & Zhou, H.H. (2007). Genistein stimulates osteoblastic

3534–3545

42-47.

cancer, *JAMA* 288: 334-341.

*Intern Med* 133(7): 516-526.

*Pharmacol Sin* 28(10): 1597-1602

women, *J Clin Endocrinol Metab* 86: 5217-5221.

ovariectomized rats. *J Ethnopharmacol* 108(1): 96-102

*Cancer Res* 6: 119-127.

*Inst* 98: 1275-1284.

136(5): 1166-1170.

sites, *Am J Surg* 179: 407-411.

5: 973-982.

receptor-alpha and estrogen receptor-beta: correlations with biological character and distinct differences among SRC coactivator family members. *Endocrinology* 141:

Schairer, C. (2002). Menopausal hormone replacement therapy and risk of ovarian

density in older adults. A randomized, double-blind, placebo-controlled trial. *Ann* 

used by women with breast cancer in four ethnic populations, *J Natl Cancer Inst* 92:

(2001). Preventive effects of isoflavones, genistein and daidzein, on estradiol-17β-

differentiation via p38 MAPK-Cbfa1 pathway in bone marrow culture. *Acta* 

augments bone formation in sexually mature ovariectomized rats. *J Bone Miner Res* 

with higher bone mineral density in postmenopausal but not premenopausal

cancer relationship: review, commentary, and workshop proceedings, *J Natl Cancer* 

induces differentiation of MG63 human osteosarcoma osteoblasts. *J Nutr. May*

therapy use between breast cancer patients and patients with other primary tumor

protein isolate with isoflavones does not prevent estradiol-induced endometrial

activity of Er-Xian Decoction, a traditional Chinese herbal formula, in

A. & Circosta, C. (2007). Effects of phytoestrogenic isoflavones from red clover (Trifolium pratense L.) on experimental osteoporosis, *Phytother Res* 21: 130-134.


**36** 

*Serbia* 

**The Phytoestrogens, Calcitonin and** 

Branko Filipović and Branka Šošić-Jurjević

**Thyroid Hormones: Effects on Bone Tissue** 

The skeleton is a metabolically active organ that undergoes remodeling throughout life. This involves a complex process by which old bone is continuously replaced by new tissue. Bone remodeling refers to the sequential, coupled actions of osteoclasts and osteoblasts. In conditions of sex hormone deficiency during advancing age, after the menopause or andropause, the rate of remodeling increases and bone formation is reduced relative to resorption. These alterations can cause microarchitectural deterioration of bone tissues, which increases bone loss as a predisposition to the occurrence of osteoporosis (Rehman et al., 2005). However, in contrast to postmenopausal osteoporosis in women, age-related bone

Numerous studies attest to the importance of estrogen in bone remodeling, evident from the finding that hormone replacement therapy (HRT) administered in a dose-dependent manner effectively prevented bone loss in postmenopausal women (Lindsay et al., 1976, 1984). However, in addition to protective effects on bone, HRT is associated with an increased risk for breast, endometrial, ovarian or prostate cancers (Davison & Davis, 2003; Loughlin & Richie, 1997; Nelson et al., 2002). Therefore, it is important to examine alternative approaches for prevention and treatment of osteoporosis without side effects. It is well known that the incidence of osteoporosis-related fractures is significantly lower in Southern and Eastern Asian women than in Western women (Tham et al., 1998). One possible reason for this difference is a high intake of phytoestrogen-rich plants, which Asian people eat more often than Western people (Ho et al., 2003). As a result, over the past decade a number of clinical trials for prevention of bone loss have assessed the effectiveness of plant derived non-steroidal phytoestrogens found in a wide variety of foods, most notably soybean. Isoflavones, which include daidzein and genistein are a class of phytoestrogens that act like estrogens. Since these compounds bind to estrogen receptors (ERs) and have estrogen-like activity (Branca, 2003), they have attracted much attention

because of their potential benefit in the prevention and treatment of osteoporosis.

In addition to the phytoestrogen-mediated protective mechanisms against bone loss, recent evidence suggests that daidzein may also act on rat bone tissue through enhancement of thyroid C cell activity (Filipović et al., 2010). Namely, thyroid C cells produce the hormone, calcitonin (CT), which lowers plasma calcium concentration by suppressing osteoclast activity. Synthesis of CT and its release from C cells were decreased in conditions of gonadal hormone

**1. Introduction** 

loss in men is less well defined.

*University of Belgrade, Institute for Biological Research"Siniša Stanković"* 


## **The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue**

Branko Filipović and Branka Šošić-Jurjević *University of Belgrade, Institute for Biological Research"Siniša Stanković" Serbia* 

### **1. Introduction**

732 Osteoporosis

Whitten, P.L. & Patisaul, H.B. (2001) Cross-species and interassay comparisons of

Wronski, T.J., Schenck, P.A., Cintron, M. & Walsh, C.C. (1987) Effect of body weight on

Wronski, T.J. & Yen, C.F. (1991). The ovariectomized rat as an animal model for

Wu, A.H., Wan, P., Hankin, J., Tseng, C.C., Yu, M.C. & Pike, M.C. (2002). Adolescent and

Wuttke, W., Seidlová-Wuttke, D. & Gorkow, C. (2003). The Cimicifuga preparation BNO

menopause symptoms and bone markers, *Maturitas* 44(suppl.1): S67-S77. Zanoli, P., Rivasi, M., Zavatti, M., Brusiani, F., Vezzalini, F. & Baraldi, M. (2005) Activity of

Zavatti, M., Montanari, C. & Zanoli, P. (2006) Role of ferutinin in the impairment of female

Zhang, X., Li, S.W., Wu, J.F., Dong, C.L., Zheng, C.X., Zhang, Y.P. & Du, J. (2010) Effects of

sexual function by *Ferula hermonis*. *Physiol Behav* 89: 656-661.

adult soy intake and risk of breast cancers in Asian Americans, *Carcinogenesis* 23:

1055 vs conjugated estrogens in a double-blind placebo-controlled study: effects on

single components of *Ferula hermonis* on male rat sexual behavior. *Int J Impot Res* 17:

ipriavone on postmenopausal syndrome and osteoporosis. *Gynecol Endocrinol*

phytoestrogen action. *Environ Health Perspectives* 109: 5-20.

ostopenia in ovariectomized rats. *Calcif Tiss Int* 40:155-159.

postmenopausal bone loss, *Cells Mater* suppl 1: 69-74.

1491-1496.

513-518.

26(2): 76-80.

The skeleton is a metabolically active organ that undergoes remodeling throughout life. This involves a complex process by which old bone is continuously replaced by new tissue. Bone remodeling refers to the sequential, coupled actions of osteoclasts and osteoblasts. In conditions of sex hormone deficiency during advancing age, after the menopause or andropause, the rate of remodeling increases and bone formation is reduced relative to resorption. These alterations can cause microarchitectural deterioration of bone tissues, which increases bone loss as a predisposition to the occurrence of osteoporosis (Rehman et al., 2005). However, in contrast to postmenopausal osteoporosis in women, age-related bone loss in men is less well defined.

Numerous studies attest to the importance of estrogen in bone remodeling, evident from the finding that hormone replacement therapy (HRT) administered in a dose-dependent manner effectively prevented bone loss in postmenopausal women (Lindsay et al., 1976, 1984). However, in addition to protective effects on bone, HRT is associated with an increased risk for breast, endometrial, ovarian or prostate cancers (Davison & Davis, 2003; Loughlin & Richie, 1997; Nelson et al., 2002). Therefore, it is important to examine alternative approaches for prevention and treatment of osteoporosis without side effects. It is well known that the incidence of osteoporosis-related fractures is significantly lower in Southern and Eastern Asian women than in Western women (Tham et al., 1998). One possible reason for this difference is a high intake of phytoestrogen-rich plants, which Asian people eat more often than Western people (Ho et al., 2003). As a result, over the past decade a number of clinical trials for prevention of bone loss have assessed the effectiveness of plant derived non-steroidal phytoestrogens found in a wide variety of foods, most notably soybean. Isoflavones, which include daidzein and genistein are a class of phytoestrogens that act like estrogens. Since these compounds bind to estrogen receptors (ERs) and have estrogen-like activity (Branca, 2003), they have attracted much attention because of their potential benefit in the prevention and treatment of osteoporosis.

In addition to the phytoestrogen-mediated protective mechanisms against bone loss, recent evidence suggests that daidzein may also act on rat bone tissue through enhancement of thyroid C cell activity (Filipović et al., 2010). Namely, thyroid C cells produce the hormone, calcitonin (CT), which lowers plasma calcium concentration by suppressing osteoclast activity. Synthesis of CT and its release from C cells were decreased in conditions of gonadal hormone

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 735

with the surface of calcified bone, osteoclasts create resorptive lacunae. Osteoclast function is

Fig. 1. Bone cells – osteoblasts, osteocytes and osteoclasts; unpublished image of Filipović et al. In homeostatic equilibrium, bone resorption and formation are balanced. It appears that osteoclasts and osteoblasts closely collaborate in the remodeling process in what is called a "Basic Multicellular Unit", or BMU. This indicates that a coupling mechanism must exist between formation and resorption (Frost, 1964), although its nature is not known. Organization of the BMU in cortical and trabecular bone differs. Between 2% and 5% of cortical bone is remodeled each year. The remodeling process in trabecular bone is mainly a surface event. Due to the much larger surface to volume ratio, it is more actively remodeled than cortical bone, with remodeling rates that can be up to 10 times higher (Lee & Einhorn,

The remodeling cycle consists of three consecutive phases: resorption, reversal and formation. Resorption begins with the migration of partially differentiated preosteoclasts, which form multinucleated osteoclasts on the bone surface. During the reversal phase, mononuclear cells prepare the resorption lacunae for bone formation and provide signals for osteoblast differentiation and migration (Eriksen et al., 1990). Bone formation starts with activation of preosteoblasts to differentiate into osteoblasts. They secrete bone-matrix proteins to form the organic matrix, which is later mineralized. During this period, osteoblasts completely replace the resorbed bone by new tissue. After this phase, the surface is covered with flattened lining cells and a prolonged resting period ensues until a new remodeling cycle is initiated. Duration of the resorption phase is about 2 weeks, the reversal phase lasts for up to 4 or 5 weeks, while the formation phase can continue for 4 months. At each remodeling site, bone resorption is coupled with bone formation, locally released growth factors and cytokines acting as mediators of this process (Canalis et al., 1988; Mundy, 1995). The decrease of bone mass, which may be due to different causes, is a

regulated both by locally acting cytokines and by systemic hormones.

2001).

deficiency (Filipović et al., 2003, 2007; Isaia et al., 1989; Lu et al., 2000; Sakai et al., 2000). Due to its osteoprotective properties, CT is widely applied in the therapy of osteoporosis.

It is known that parathyroid hormone (PTH) is a major factor involved in the systemic regulation of bone resorption. Phytoestrogens may affect the parathyroid gland and reduce PTH secretion (Wong et al., 2002), suggesting that one way in which these compounds inhibit bone loss may be through reducing PTH levels.

Thyroid hormones are essential for normal bone maturation *in utero* and during early life. In adults an excess of thyroid hormones in the body affects the remodeling system in cortical and trabecular bone and may contribute to the development of osteoporosis (Kung, 1994). Receptors for these hormones are present in bone cells and they may directly increase bone resorption (Abu et al., 1997; Rizzoli et al., 1986). Additionally, thyroid-stimulating hormone (TSH), which stimulates the release of thyroid hormones, positively influences bone remodeling. Therefore, demonstrating both anabolic and antiresorptive effects, TSH may represent a promising candidate for the treatment of osteoporosis (Sendak et al., 2007).

In this chapter we will describe the known effects of phytoestrogens on bone. In addition to the direct action of these plant compounds, special attention will be paid to their influence on thyroid C and follicular cells, as producers of CT and thyroid hormones, using the latest data in the literature and our own results. These hormones, together with PTH may be involved in the indirect effects of phytoestrogens on bone tissue.

#### **2. Bone cells and bone remodeling**

Bone is a dynamic organ that undergoes remodeling throughout life. This process results from the separate action of bone forming cells called osteoblasts and bone resorbing cells called osteoclasts. Osteoblasts are responsible for the production of bone matrix constituents and are found in clusters on bone surfaces (Fig 1). They originate from multipotent mesenchymal stem cells, which have the capacity to differentiate into osteoblasts or other cells, such as adipocytes, chondrocytes, myoblasts and fibroblasts (Bianco et al., 2001). A mature osteoblast that is trapped in the bone matrix and remains isolated in lacunae becomes an osteocyte. (Fig.1). Bone formation involves production and maturation of the osteoid matrix, followed by mineralization of the matrix. Osteoblasts produce growth factors, such as insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF- β) and bone morphometric protein (BMP) (Canalis et al., 1993, 1993a; Chen et al., 2004; Globus et al., 1989; Rydzel et al., 1994). These factors regulate osteoblast activity in an autocrine and paracrine manner.

Osteoclasts are large multinucleate cells responsible for bone resorption. They are derived from hematopoetic cells of the mononuclear lineage (Teitelbaum, 2000) (Fig.1). Osteoclasts have an abundant Golgi complex, mitochondria and transport vesicles loaded with lysosomal enzymes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K. These enzymes are secreted via the specialized (ruffler border) plasma membrane of osteoclasts into the bone-resorbing compartment (Väänänen et al., 2000). The process of osteoclast attachment to the bone is complex and involves binding of integrins expressed in osteoclasts with specific amino acid sequences within proteins at the surface of the bone matrix and cytoskeleton activation (Davies et al., 1989; Reinholt et al., 1990). Dynamic structures, called podozomes allow movement of osteoclasts across the bone surface. Bone resorption occurs due to acidification and proteolysis of the bone matrix. As a result of this resorptive activity in contact

deficiency (Filipović et al., 2003, 2007; Isaia et al., 1989; Lu et al., 2000; Sakai et al., 2000). Due to

It is known that parathyroid hormone (PTH) is a major factor involved in the systemic regulation of bone resorption. Phytoestrogens may affect the parathyroid gland and reduce PTH secretion (Wong et al., 2002), suggesting that one way in which these compounds

Thyroid hormones are essential for normal bone maturation *in utero* and during early life. In adults an excess of thyroid hormones in the body affects the remodeling system in cortical and trabecular bone and may contribute to the development of osteoporosis (Kung, 1994). Receptors for these hormones are present in bone cells and they may directly increase bone resorption (Abu et al., 1997; Rizzoli et al., 1986). Additionally, thyroid-stimulating hormone (TSH), which stimulates the release of thyroid hormones, positively influences bone remodeling. Therefore, demonstrating both anabolic and antiresorptive effects, TSH may represent a promising candidate for the treatment of osteoporosis (Sendak et al., 2007). In this chapter we will describe the known effects of phytoestrogens on bone. In addition to the direct action of these plant compounds, special attention will be paid to their influence on thyroid C and follicular cells, as producers of CT and thyroid hormones, using the latest data in the literature and our own results. These hormones, together with PTH may be

Bone is a dynamic organ that undergoes remodeling throughout life. This process results from the separate action of bone forming cells called osteoblasts and bone resorbing cells called osteoclasts. Osteoblasts are responsible for the production of bone matrix constituents and are found in clusters on bone surfaces (Fig 1). They originate from multipotent mesenchymal stem cells, which have the capacity to differentiate into osteoblasts or other cells, such as adipocytes, chondrocytes, myoblasts and fibroblasts (Bianco et al., 2001). A mature osteoblast that is trapped in the bone matrix and remains isolated in lacunae becomes an osteocyte. (Fig.1). Bone formation involves production and maturation of the osteoid matrix, followed by mineralization of the matrix. Osteoblasts produce growth factors, such as insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF- β) and bone morphometric protein (BMP) (Canalis et al., 1993, 1993a; Chen et al., 2004; Globus et al., 1989; Rydzel et al., 1994). These factors regulate

Osteoclasts are large multinucleate cells responsible for bone resorption. They are derived from hematopoetic cells of the mononuclear lineage (Teitelbaum, 2000) (Fig.1). Osteoclasts have an abundant Golgi complex, mitochondria and transport vesicles loaded with lysosomal enzymes, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K. These enzymes are secreted via the specialized (ruffler border) plasma membrane of osteoclasts into the bone-resorbing compartment (Väänänen et al., 2000). The process of osteoclast attachment to the bone is complex and involves binding of integrins expressed in osteoclasts with specific amino acid sequences within proteins at the surface of the bone matrix and cytoskeleton activation (Davies et al., 1989; Reinholt et al., 1990). Dynamic structures, called podozomes allow movement of osteoclasts across the bone surface. Bone resorption occurs due to acidification and proteolysis of the bone matrix. As a result of this resorptive activity in contact

its osteoprotective properties, CT is widely applied in the therapy of osteoporosis.

inhibit bone loss may be through reducing PTH levels.

involved in the indirect effects of phytoestrogens on bone tissue.

osteoblast activity in an autocrine and paracrine manner.

**2. Bone cells and bone remodeling** 

with the surface of calcified bone, osteoclasts create resorptive lacunae. Osteoclast function is regulated both by locally acting cytokines and by systemic hormones.

Fig. 1. Bone cells – osteoblasts, osteocytes and osteoclasts; unpublished image of Filipović et al.

In homeostatic equilibrium, bone resorption and formation are balanced. It appears that osteoclasts and osteoblasts closely collaborate in the remodeling process in what is called a "Basic Multicellular Unit", or BMU. This indicates that a coupling mechanism must exist between formation and resorption (Frost, 1964), although its nature is not known. Organization of the BMU in cortical and trabecular bone differs. Between 2% and 5% of cortical bone is remodeled each year. The remodeling process in trabecular bone is mainly a surface event. Due to the much larger surface to volume ratio, it is more actively remodeled than cortical bone, with remodeling rates that can be up to 10 times higher (Lee & Einhorn, 2001).

The remodeling cycle consists of three consecutive phases: resorption, reversal and formation. Resorption begins with the migration of partially differentiated preosteoclasts, which form multinucleated osteoclasts on the bone surface. During the reversal phase, mononuclear cells prepare the resorption lacunae for bone formation and provide signals for osteoblast differentiation and migration (Eriksen et al., 1990). Bone formation starts with activation of preosteoblasts to differentiate into osteoblasts. They secrete bone-matrix proteins to form the organic matrix, which is later mineralized. During this period, osteoblasts completely replace the resorbed bone by new tissue. After this phase, the surface is covered with flattened lining cells and a prolonged resting period ensues until a new remodeling cycle is initiated. Duration of the resorption phase is about 2 weeks, the reversal phase lasts for up to 4 or 5 weeks, while the formation phase can continue for 4 months.

At each remodeling site, bone resorption is coupled with bone formation, locally released growth factors and cytokines acting as mediators of this process (Canalis et al., 1988; Mundy, 1995). The decrease of bone mass, which may be due to different causes, is a

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 737

activity through inhibition of tyrosine kinase (Blair et al., 1996). Genistein also caused a significant increase in tyrosine phosphatase activity, which is a negative regulator of osteoclastogenesis and osteoclast-resorbing activity in mutant mice (Aoki et al., 1999; Gao

While investigations in vitro give clues about the effects of isoflavones on individual bone cells, studies in vivo provide knowledge about their influence in intact systems. Aged gonadectomized female and male rodents are suitable animal models for studying osteoporosis (Comelekoglu et al., 2007; Filipović et al., 2007; Pantelić et al., 2010; Vanderschueren et al., 1992.) Using them it has been demonstrated that isoflavones can prevent bone loss in female rats and mice after ovariectomy (Ovx) (Blum et al., 2003; Erlandsson et al., 2005; Fonseca & Ward, 2004; Ishimi et al., 1999; Lee et al., 2004; Om & Shim, 2007; Ren et al., 2007; Wu et al., 2004). The bone-preventing effects of isoflavones were also confirmed in male orchidectomized (Orx) rats and mice (Filipović et al., 2010; Ishimi et al., 2002; Khalil et al., 2005; Soung et al., 2006; Wu et al., 2003). On the contrary, some studies showed that isoflavones had minimal or no effects on bone loss in animal models (Bahr et al., 2005; Nakai et al., 2005; Picherit et al., 2001). Moreover, in the monkey, a nonhuman primate, dietary isoflavones do not effectively prevent ovariectomy-induced bone loss (Register et al., 2003). However, others suggested that soy phytoestrogens were

&Yamaguchi, 2000) (Fig 3).

protective against loss of bone volume (Ham et al., 2004).

Fig. 3. Influence of isoflavones on bone cells; Filipović et al.

consequence of an imbalance between the amount of mineral and matrix removed and that subsequently incorporated into each resorption cavity (Kanis et al., 1990).

### **3. Phytoestrogens in bone protection**

Phytoestrogens are structurally and functionally similar to estrogens and their estrogenic activity may occur through ERs. There are three main classes of phytoestrogens: isoflavonoids, coumestans and lignans (Fig. 2). Due to their estrogenic and anti-estrogenic activity, they are termed - natural selective ER modulators (SERMs). Therefore, soybean isoflavones have received great attention as alternatives to HRT for the prevention of postmenopausal osteoporosis. Genistein and daidzein, the main isoflavones in soybean, may protect against osteoporosis, because they can affect both types of bone cells.

Fig. 2. Structure of 17β estradiol, isoflavones (genistein and daidzein), coumestan (coumestrol) and lignans (metairesinol); Filipović et al.

Isoflavones can stimulate the proliferation and differentiation of osteoblasts. Thus, the presence of genistein or daidzein led to a significant increase in protein synthesis, alkaline phosphatase activity, and DNA content in cultures of osteoblastic MC3T3-E1 cells (Sugimoto & Yamaguchi, 2000, 2000a; Yamaguchi & Sugimoto, 2000).

In addition to a stimulating effect on bone formation, these plant compounds may also suppress osteoclastic bone resorption in vitro. Thus, genistein was found to induce apoptosis of osteoclasts isolated from rat femoral tissues. Daidzein also decreased the number of these bone resorbing cells in rats (Gao & Yamaguchi, 1999) and their development in cultures of porcine bone marrow (Rassi et al., 2002). Osteoclast activity is regulated by phosphorylation of cell membrane constituents, involving tyrosine kinases. As a naturally tyrosine kinase inhibitor, genistein was found to suppress avian osteoclastic

consequence of an imbalance between the amount of mineral and matrix removed and that

Phytoestrogens are structurally and functionally similar to estrogens and their estrogenic activity may occur through ERs. There are three main classes of phytoestrogens: isoflavonoids, coumestans and lignans (Fig. 2). Due to their estrogenic and anti-estrogenic activity, they are termed - natural selective ER modulators (SERMs). Therefore, soybean isoflavones have received great attention as alternatives to HRT for the prevention of postmenopausal osteoporosis. Genistein and daidzein, the main isoflavones in soybean,

may protect against osteoporosis, because they can affect both types of bone cells.

Fig. 2. Structure of 17β estradiol, isoflavones (genistein and daidzein), coumestan

Isoflavones can stimulate the proliferation and differentiation of osteoblasts. Thus, the presence of genistein or daidzein led to a significant increase in protein synthesis, alkaline phosphatase activity, and DNA content in cultures of osteoblastic MC3T3-E1 cells (Sugimoto

In addition to a stimulating effect on bone formation, these plant compounds may also suppress osteoclastic bone resorption in vitro. Thus, genistein was found to induce apoptosis of osteoclasts isolated from rat femoral tissues. Daidzein also decreased the number of these bone resorbing cells in rats (Gao & Yamaguchi, 1999) and their development in cultures of porcine bone marrow (Rassi et al., 2002). Osteoclast activity is regulated by phosphorylation of cell membrane constituents, involving tyrosine kinases. As a naturally tyrosine kinase inhibitor, genistein was found to suppress avian osteoclastic

(coumestrol) and lignans (metairesinol); Filipović et al.

& Yamaguchi, 2000, 2000a; Yamaguchi & Sugimoto, 2000).

subsequently incorporated into each resorption cavity (Kanis et al., 1990).

**3. Phytoestrogens in bone protection** 

activity through inhibition of tyrosine kinase (Blair et al., 1996). Genistein also caused a significant increase in tyrosine phosphatase activity, which is a negative regulator of osteoclastogenesis and osteoclast-resorbing activity in mutant mice (Aoki et al., 1999; Gao &Yamaguchi, 2000) (Fig 3).

While investigations in vitro give clues about the effects of isoflavones on individual bone cells, studies in vivo provide knowledge about their influence in intact systems. Aged gonadectomized female and male rodents are suitable animal models for studying osteoporosis (Comelekoglu et al., 2007; Filipović et al., 2007; Pantelić et al., 2010; Vanderschueren et al., 1992.) Using them it has been demonstrated that isoflavones can prevent bone loss in female rats and mice after ovariectomy (Ovx) (Blum et al., 2003; Erlandsson et al., 2005; Fonseca & Ward, 2004; Ishimi et al., 1999; Lee et al., 2004; Om & Shim, 2007; Ren et al., 2007; Wu et al., 2004). The bone-preventing effects of isoflavones were also confirmed in male orchidectomized (Orx) rats and mice (Filipović et al., 2010; Ishimi et al., 2002; Khalil et al., 2005; Soung et al., 2006; Wu et al., 2003). On the contrary, some studies showed that isoflavones had minimal or no effects on bone loss in animal models (Bahr et al., 2005; Nakai et al., 2005; Picherit et al., 2001). Moreover, in the monkey, a nonhuman primate, dietary isoflavones do not effectively prevent ovariectomy-induced bone loss (Register et al., 2003). However, others suggested that soy phytoestrogens were protective against loss of bone volume (Ham et al., 2004).

Fig. 3. Influence of isoflavones on bone cells; Filipović et al.

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 739

controlling osteoclastogenesis. It was shown that isoflavones may increase the activity of osteoblasts by stimulating the secretion of OPG and RANK-L (De Wilde et al., 2004;

Yamagishi et al., 2001) (Fig. 4).

Fig. 4. Mechanisms of isoflavone action in bone; Filipović et al.

Proinflammatory cytokines, such as interleukin (IL)-1, IL-6, and tumor necrosis factor α (TNFα), stimulate osteoclastogenesis and bone resorption. These effects can be achieved by both RANK-L dependent and RANK-L-independent mechanisms (Collin-Osbody et al., 2001; Katagiri et al., 2002). Isoflavones have been shown to inhibit IL-6 synthesis by MC3T3-E1/4 osteoblast-like cells in vitro (Chen et al., 2003; Suh et al., 2003) and to reduce serum IL-1β and TNF-α concentrations in Ovx rats (Li, 2003). Also, a soy supplemented diet may inhibit serum concentrations of proinflammatory cytokines in postmenopausal women (Huang et al., 2005). In addition to osteoclastogenesis, isoflavones appear to influence osteoclast activity through inhibition of inward rectifier K+channels in osteoclasts. This leads to membrane depolarization, intracellular influx of Ca2+ and inhibition of bone resorption (Okamoto et al., 2001). One beneficial effect of isoflavones on bone is increased intestinal calcium absorption (Fig. 4). However, it is not known whether the mechanism(s) by which isoflavones influence calcium absorption include interactions with intestinal ER and/or

vitamin D receptor-mediated calcium transport or not (Arjmandi et al., 2002).

During recent years, numerous human studies have evaluated the effect of soy proteincontaining isoflavones or pure isoflavones on bone mass. However, the results of these observational and dietary interventional investigations have been variable and conflicting. In general, isoflavone supplementation studies indicate a beneficial effect on bone mass (Huang et al., 2006; Lydeking-Olsen et al., 2004; Newton et al., 2006), no effect (Anderson et al., 2002; Arjmandi et al., 2005; Brink et al., 2008; Wu et al., 2006) or a possible negative effect in terms of increased circulating concentrations of biochemical markers associated with bone resorption (Geppert et al., 2004; Wanger et al., 2000).

The large heterogeneity of these results may be due to study design, differences regarding hormonal status of the subjects, together with the duration, type and dose of isoflavone supplementation. In addition, bone sparing benefits may depend on the extent of conversion of isoflavones to metabolites. Thus, equol binds with greater affinity to ERs than daidzein from which it is derived (Setchell et al., 2002). Equol production is dependent on the intestinal microflora and there are large interindividual differences in this metabolism. Some people produce more equol than others. Also, production of this metabolite may at least partially explain why the beneficial effects of isoflavones observed in laboratory rodents, which consistently produce high levels of equol, have not been easily recapitulated in humans, where this is not the case. Generally, the relative importance of phytoestrogens in human health must be resolved and longer-term studies are needed to determine their effects on human bone tissue.

### **4. Phytoestrogens – Mechanisms of action in bone**

Although the mechanisms by which soy phytoestrogens may alter bone remodeling are still not completely known, Atmaca et al. (2008) state that they act on both osteoblasts and osteoclasts through genomic and nongenomic pathways.

Due to their low molecular weight these plant compounds can pass through cell membranes and interact with receptors and enzymes (Adlercreutz et al., 1998). Phytoestrogens possess estrogenic activity and act as natural SERMs. This suggests that their effect on bone can be achieved by binding to ERs. Both α and β subtypes of ERs have been identified in bone (Arts et al., 1997; Onoe et al., 1997). The protective effect of phytoestrogens is probably achieved mainly through binding to ER-β, the expression of which is increased during bone mineralization (Arts et al., 1997; Kuiper et al., 1998). In addition to ERs, phytoestrogens can bind to androgenic receptors and act as phytoandrogens (Chen & Chang, 2007).

Both genistein and daidzein stimulate osteoblast proliferation, differentiation and activation by an ER-dependent mechanism (De Wilde et al., 2004; Pan et al., 2005). These isoflavones regulated the synthesis of core binding factor-1 (Cbfa-1) and bone morphogenic protein-2 (BMP-2), which is involved in the differentiation of osteoblasts (De Wilde et al., 2004; Jia et al., 2003; Pan et al., 2005). Genistein and daidzein activate peroxisome proliferator activator receptors (PPARs). The balance between PPAR and ER activation may govern the balance between adipogenesis and osteoblastogenesis (Dang et al., 2003, 2004).

Osteoclasts express the receptor activator of nuclear factor kappa B (RANK) (Hsu et al., 1999), while the receptor activator of nuclear factor kappa B ligand (RANK-L) and osteoprotegerin (OPG) is expressed by osteoblasts (Udagawa et al., 1999). Binding of RANKL to RANK stimulates osteoclastogenesis, whereas binding of RANK-L to OPG prevents RANK-L – RANK binding and indirectly inhibits osteoclastogenesis (Fuller et al., 1998; Theoleyre et al., 2004). The relative levels this triad of proteins are important for

During recent years, numerous human studies have evaluated the effect of soy proteincontaining isoflavones or pure isoflavones on bone mass. However, the results of these observational and dietary interventional investigations have been variable and conflicting. In general, isoflavone supplementation studies indicate a beneficial effect on bone mass (Huang et al., 2006; Lydeking-Olsen et al., 2004; Newton et al., 2006), no effect (Anderson et al., 2002; Arjmandi et al., 2005; Brink et al., 2008; Wu et al., 2006) or a possible negative effect in terms of increased circulating concentrations of biochemical markers associated with

The large heterogeneity of these results may be due to study design, differences regarding hormonal status of the subjects, together with the duration, type and dose of isoflavone supplementation. In addition, bone sparing benefits may depend on the extent of conversion of isoflavones to metabolites. Thus, equol binds with greater affinity to ERs than daidzein from which it is derived (Setchell et al., 2002). Equol production is dependent on the intestinal microflora and there are large interindividual differences in this metabolism. Some people produce more equol than others. Also, production of this metabolite may at least partially explain why the beneficial effects of isoflavones observed in laboratory rodents, which consistently produce high levels of equol, have not been easily recapitulated in humans, where this is not the case. Generally, the relative importance of phytoestrogens in human health must be resolved and longer-term studies are needed to determine their

Although the mechanisms by which soy phytoestrogens may alter bone remodeling are still not completely known, Atmaca et al. (2008) state that they act on both osteoblasts and

Due to their low molecular weight these plant compounds can pass through cell membranes and interact with receptors and enzymes (Adlercreutz et al., 1998). Phytoestrogens possess estrogenic activity and act as natural SERMs. This suggests that their effect on bone can be achieved by binding to ERs. Both α and β subtypes of ERs have been identified in bone (Arts et al., 1997; Onoe et al., 1997). The protective effect of phytoestrogens is probably achieved mainly through binding to ER-β, the expression of which is increased during bone mineralization (Arts et al., 1997; Kuiper et al., 1998). In addition to ERs, phytoestrogens can

Both genistein and daidzein stimulate osteoblast proliferation, differentiation and activation by an ER-dependent mechanism (De Wilde et al., 2004; Pan et al., 2005). These isoflavones regulated the synthesis of core binding factor-1 (Cbfa-1) and bone morphogenic protein-2 (BMP-2), which is involved in the differentiation of osteoblasts (De Wilde et al., 2004; Jia et al., 2003; Pan et al., 2005). Genistein and daidzein activate peroxisome proliferator activator receptors (PPARs). The balance between PPAR and ER activation may govern the balance

Osteoclasts express the receptor activator of nuclear factor kappa B (RANK) (Hsu et al., 1999), while the receptor activator of nuclear factor kappa B ligand (RANK-L) and osteoprotegerin (OPG) is expressed by osteoblasts (Udagawa et al., 1999). Binding of RANKL to RANK stimulates osteoclastogenesis, whereas binding of RANK-L to OPG prevents RANK-L – RANK binding and indirectly inhibits osteoclastogenesis (Fuller et al., 1998; Theoleyre et al., 2004). The relative levels this triad of proteins are important for

bind to androgenic receptors and act as phytoandrogens (Chen & Chang, 2007).

between adipogenesis and osteoblastogenesis (Dang et al., 2003, 2004).

bone resorption (Geppert et al., 2004; Wanger et al., 2000).

**4. Phytoestrogens – Mechanisms of action in bone** 

osteoclasts through genomic and nongenomic pathways.

effects on human bone tissue.

controlling osteoclastogenesis. It was shown that isoflavones may increase the activity of osteoblasts by stimulating the secretion of OPG and RANK-L (De Wilde et al., 2004; Yamagishi et al., 2001) (Fig. 4).

Fig. 4. Mechanisms of isoflavone action in bone; Filipović et al.

Proinflammatory cytokines, such as interleukin (IL)-1, IL-6, and tumor necrosis factor α (TNFα), stimulate osteoclastogenesis and bone resorption. These effects can be achieved by both RANK-L dependent and RANK-L-independent mechanisms (Collin-Osbody et al., 2001; Katagiri et al., 2002). Isoflavones have been shown to inhibit IL-6 synthesis by MC3T3-E1/4 osteoblast-like cells in vitro (Chen et al., 2003; Suh et al., 2003) and to reduce serum IL-1β and TNF-α concentrations in Ovx rats (Li, 2003). Also, a soy supplemented diet may inhibit serum concentrations of proinflammatory cytokines in postmenopausal women (Huang et al., 2005). In addition to osteoclastogenesis, isoflavones appear to influence osteoclast activity through inhibition of inward rectifier K+channels in osteoclasts. This leads to membrane depolarization, intracellular influx of Ca2+ and inhibition of bone resorption (Okamoto et al., 2001). One beneficial effect of isoflavones on bone is increased intestinal calcium

absorption (Fig. 4). However, it is not known whether the mechanism(s) by which isoflavones influence calcium absorption include interactions with intestinal ER and/or vitamin D receptor-mediated calcium transport or not (Arjmandi et al., 2002).

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 741

investigated. Administration of ipriflavone to intact rats had a gender-related effect on serum CT, which increased in females, but no significant change was seen in male rats (Watanabe et al., 1992). With regard to the inhibitory effect of testosterone on the synthesis of some enzymes it is possible that testosterone inhibited ipriflavone-stimulated CT

Fig. 5. Ultrastructure of a thyroid C cell; nucleus (N), mitochondria (M), secretory granules

Recently the first experimental data suggesting that daidzein affects thyroid C cells and stimulates CT secretory activity in Orx middle-aged rats were presented (Filipović et al., 2010). The androgen deficiency after Orx strongly affected thyroid C cell structure and reduced the synthesis and release of CT. Daidzein treatment decreased immunoreactivity for CT, significantly increased C cell volume (Fig. 6) and slightly raised serum CT

Daidzein administration also decreased bone turnover, prevented loss of cancellous bone and the plate-like structure was recovered after trabecular bone destruction caused by Orx (Fig. 7). Based on these results, the authors suggested that, besides direct action on the skeleton, daidzein may affect bone structure indirectly through enhancement of thyroid C

synthesis (Weiner & Dias, 1990).

(Sg); unpublished image of Filipović et al.

cell activity (Filipović et al., 2010).

concentration.

Nongenomic effects do not involve ERs. These effects of phytoestrogens include inhibition of tyrosine kinase which directly modulate osteoclastic acid secretion (Blair et al., 1996; Williams et al., 1998) or topoisomerase I and II, which helps to regulate cell differentiation and the cell replication cycle (Okura et al., 1998; Yamagishi et al., 2001).

### **5. Indirect effects of phytoestrogens on bone – The role of calcitonin, parathyroid and thyroid hormones**

#### **5.1 Effects of phytoestrogens on thyroid C cells and calcitonin production**

Thyroid C cells are dispersed neuroendocrine cells that produce many bioregulatory peptides, among which CT is considered the most important. This calcium regulating hormone lowers plasma calcium concentration by inhibiting osteoclast activity. In addition to sex steroids, a voluminous literature has accumulated for therapeutic use of CT in treating osteoporosis. Thus, C cells may also be very important in the pathogenesis of osteoporosis.

The C cells are mostly located in the middle of the thyroid lobes and appear in clusters or as solitary cells between follicular cells and the capillary wall. They have a round, elliptical or polygonal shape and never face the follicular lumen. The nucleus is located in the center of the cell. The most salient ultrastructural feature of C cells is the numerous round secretory granules that fill extensive areas of the cytoplasm. The Golgi complex and endoplasmic reticulum are well developed. There is a moderate number of mitochondria, which are mostly round to elongate in shape and not uniformly distributed. Lysosomes are large and contain acid phosphatase and other lysosomal enzymes (Fig. 5).

CT suppresses the number and motility of osteoclasts (Gao & Yamaguchi, 1999; Zaidi et al., 1990) and induces a change in their contractile elements (Hunter et al., 1989). Also, CT increases osteoblast proliferation by acting on components of the insulin-like growth factor system (Farley et al., 2000) and enhancing alkaline phosphatase activity, which is associated with increased synthesis and deposition of bone matrix collagen (Farley et al., 1988, 1992; Ito et al., 1987). The action of CT bone formation is at least in part, mediated via CT receptors located on osteoblasts, through the cAMP second messenger system (Farley et al., 1992; Villa et al., 2003).

It was shown that gonadal hormone deficiency affects thyroid C cell activity. Thus, synthesis of CT and its release from rat C cells were decreased after Ovx due to lack of estrogens (Filipović et al., 2002, 2003; Sakai et al. 2000). Also, the decline in testosterone level induced by Orx altered thyroid C cell structure and reduced the synthesis and release of CT (Filipović et al., 2007; Lu et al., 2000). The same effects were noticed after Orx or the natural menopause in women (Isaia et al. 1989). On the other hand, estrogen treatment was found to have a stimulatory effect on CT secretory activity of C cells in Ovx rats (Grauer et al., 1993; Filipović et al., 2003), Orx rats (Filipović et al., 2010a) and women (Isaia et al., 1992). In addition to estrogen, chronic calcium administration after Ovx increased the release of CT from C cells without affecting CT synthesis, suggesting that estrogen plays an important role in CT synthesis (Filipović et al., 2005). On the other hand, CT administration, which may be useful for treatment of osteoporosis, negatively affected rat thyroid C cells by a negative feedback mechanism (Sekulić et al., 2005).

Among the few studies concerning the potential effects of phytoestrogens on CT production, the influence of ipriflavone, a derivative of isoflavone, on CT synthesis and secretion was

Nongenomic effects do not involve ERs. These effects of phytoestrogens include inhibition of tyrosine kinase which directly modulate osteoclastic acid secretion (Blair et al., 1996; Williams et al., 1998) or topoisomerase I and II, which helps to regulate cell differentiation

Thyroid C cells are dispersed neuroendocrine cells that produce many bioregulatory peptides, among which CT is considered the most important. This calcium regulating hormone lowers plasma calcium concentration by inhibiting osteoclast activity. In addition to sex steroids, a voluminous literature has accumulated for therapeutic use of CT in treating osteoporosis.

The C cells are mostly located in the middle of the thyroid lobes and appear in clusters or as solitary cells between follicular cells and the capillary wall. They have a round, elliptical or polygonal shape and never face the follicular lumen. The nucleus is located in the center of the cell. The most salient ultrastructural feature of C cells is the numerous round secretory granules that fill extensive areas of the cytoplasm. The Golgi complex and endoplasmic reticulum are well developed. There is a moderate number of mitochondria, which are mostly round to elongate in shape and not uniformly distributed. Lysosomes are large and

CT suppresses the number and motility of osteoclasts (Gao & Yamaguchi, 1999; Zaidi et al., 1990) and induces a change in their contractile elements (Hunter et al., 1989). Also, CT increases osteoblast proliferation by acting on components of the insulin-like growth factor system (Farley et al., 2000) and enhancing alkaline phosphatase activity, which is associated with increased synthesis and deposition of bone matrix collagen (Farley et al., 1988, 1992; Ito et al., 1987). The action of CT bone formation is at least in part, mediated via CT receptors located on osteoblasts, through the cAMP second messenger system (Farley et al., 1992; Villa

It was shown that gonadal hormone deficiency affects thyroid C cell activity. Thus, synthesis of CT and its release from rat C cells were decreased after Ovx due to lack of estrogens (Filipović et al., 2002, 2003; Sakai et al. 2000). Also, the decline in testosterone level induced by Orx altered thyroid C cell structure and reduced the synthesis and release of CT (Filipović et al., 2007; Lu et al., 2000). The same effects were noticed after Orx or the natural menopause in women (Isaia et al. 1989). On the other hand, estrogen treatment was found to have a stimulatory effect on CT secretory activity of C cells in Ovx rats (Grauer et al., 1993; Filipović et al., 2003), Orx rats (Filipović et al., 2010a) and women (Isaia et al., 1992). In addition to estrogen, chronic calcium administration after Ovx increased the release of CT from C cells without affecting CT synthesis, suggesting that estrogen plays an important role in CT synthesis (Filipović et al., 2005). On the other hand, CT administration, which may be useful for treatment of osteoporosis, negatively affected rat thyroid C cells by a

Among the few studies concerning the potential effects of phytoestrogens on CT production, the influence of ipriflavone, a derivative of isoflavone, on CT synthesis and secretion was

and the cell replication cycle (Okura et al., 1998; Yamagishi et al., 2001).

**parathyroid and thyroid hormones** 

et al., 2003).

**5. Indirect effects of phytoestrogens on bone – The role of calcitonin,** 

**5.1 Effects of phytoestrogens on thyroid C cells and calcitonin production** 

Thus, C cells may also be very important in the pathogenesis of osteoporosis.

contain acid phosphatase and other lysosomal enzymes (Fig. 5).

negative feedback mechanism (Sekulić et al., 2005).

investigated. Administration of ipriflavone to intact rats had a gender-related effect on serum CT, which increased in females, but no significant change was seen in male rats (Watanabe et al., 1992). With regard to the inhibitory effect of testosterone on the synthesis of some enzymes it is possible that testosterone inhibited ipriflavone-stimulated CT synthesis (Weiner & Dias, 1990).

Fig. 5. Ultrastructure of a thyroid C cell; nucleus (N), mitochondria (M), secretory granules (Sg); unpublished image of Filipović et al.

Recently the first experimental data suggesting that daidzein affects thyroid C cells and stimulates CT secretory activity in Orx middle-aged rats were presented (Filipović et al., 2010). The androgen deficiency after Orx strongly affected thyroid C cell structure and reduced the synthesis and release of CT. Daidzein treatment decreased immunoreactivity for CT, significantly increased C cell volume (Fig. 6) and slightly raised serum CT concentration.

Daidzein administration also decreased bone turnover, prevented loss of cancellous bone and the plate-like structure was recovered after trabecular bone destruction caused by Orx (Fig. 7). Based on these results, the authors suggested that, besides direct action on the skeleton, daidzein may affect bone structure indirectly through enhancement of thyroid C cell activity (Filipović et al., 2010).

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 743

Fig. 7. Trabecular microarchitecture of the proximal tibial metaphysis in control (a, b), orchidectomized (c, d) and orchidectomized rats treated with daidzein (e, f); azan staining

Parathyroid glands are constituted of chief, clear and oxyphilous cells. The chief cells synthesize and secrete PTH and are arranged in rather dense cords or nests around abundant capillaries. These cells are oval or polygonal in shape. The nucleus is irregularly shaped, with a few spots of chromatin located in the margin, and the nuclear membrane is infolded. The plasma membrane shows interdigitations. Mitochondria are dispersed throughout the cytoplasm. The cisternae of the rough-surfaced endoplasmic reticulum are arranged in parallel arrays or randomly distributed in the cytoplasm. The Golgi complexes are well developed. Storage granules are filled with finely particulate electron-dense

PTH plays an important role in calcium homeostasis and has a critical role in bone turnover. It antagonizes CT produced by thyroid C cells and acts directly on bone and kidney to increase Ca influx into the blood circulation. This hormone increases the tubular re-

**5.2 Effects of phytoestrogens on parathyroid hormone production** 

method; unpublished image of Filipović et al.

material (Fig. 8).

Fig. 6. Calcitonin producing thyroid C cells in control (a, b), orchidectomized (c, d) and orchidectomized rats treated with daidzein (e, f); immuno-staining for calcitonin; unpublished image of Filipović et al.

Fig. 6. Calcitonin producing thyroid C cells in control (a, b), orchidectomized (c, d) and orchidectomized rats treated with daidzein (e, f); immuno-staining for calcitonin;

unpublished image of Filipović et al.

Fig. 7. Trabecular microarchitecture of the proximal tibial metaphysis in control (a, b), orchidectomized (c, d) and orchidectomized rats treated with daidzein (e, f); azan staining method; unpublished image of Filipović et al.

### **5.2 Effects of phytoestrogens on parathyroid hormone production**

Parathyroid glands are constituted of chief, clear and oxyphilous cells. The chief cells synthesize and secrete PTH and are arranged in rather dense cords or nests around abundant capillaries. These cells are oval or polygonal in shape. The nucleus is irregularly shaped, with a few spots of chromatin located in the margin, and the nuclear membrane is infolded. The plasma membrane shows interdigitations. Mitochondria are dispersed throughout the cytoplasm. The cisternae of the rough-surfaced endoplasmic reticulum are arranged in parallel arrays or randomly distributed in the cytoplasm. The Golgi complexes are well developed. Storage granules are filled with finely particulate electron-dense material (Fig. 8).

PTH plays an important role in calcium homeostasis and has a critical role in bone turnover. It antagonizes CT produced by thyroid C cells and acts directly on bone and kidney to increase Ca influx into the blood circulation. This hormone increases the tubular re-

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 745

Mimicking the effect of estrogen, phytoestrogens can modulate the action of PTH on bone. Thus, one study in vitro showed that pre-treatment of SaOS-2 osteoblastic cells with genistein enhanced PTH-induced ALP activity and attenuated PTH up regulation of RANKL mRNA expression and PTH down regulation of OPG mRNA expression (Chen & Wong, 2006).

**5.3 Effects of phytoestrogens on thyroid glands and thyroid hormones production**  Hypothalamic–pituitary–thyroid axis (HPT) plays a key role in skeletal development, attainment of peak bone mass and regulation of adult bone turnover (Gogakos et al., 2010; Roef еt al., 2011). Additionally, thyroid disorders are associated with alterations in bone

Soy-food, soy-based infant formula, as well as dietary supplements containing purified soybean isoflavones, genistein and daidzein, are increasingly consumed in typical "Western" diet in the recent years. Commonly cited reasons for using soy infant formula are to feed infants who are allergic to dairy products or are intolerant of lactose, galactose, or cow-milk protein (Tuohy, 2003). In elderly, reason is potential health benefit of soybean isoflavones in protection of age-related diseases, including osteoporosis (Setchell, 1998). Structurally, soybean isoflavones genistein and daidzein are polyphenolic compounds, similar to estradiol-17β and bind with a weaker potency to both types of ERs, with higher affinity for ERβ (Kuiper et al., 1998). Despite the numerous beneficial effects of soy isoflavones, epidemiological and experimental data also exist showing an adverse effect on human health, namely on reproductive and thyroid axis. The association between high soy isoflavones intake and goitrogenesis, as well as protective effect of adequate iodine intake, was reported both in humans (Chorazy et al.1995; Van Wyk et al., 1959) and in different

Therefore, besides the direct beneficial effect of soybean phytoestrogens on bone tissue, isoflavones may also act indirectly, through endocrine disruption and interference with HPT axis. Most researchers who examined osteoprotective potential of isoflavones did not include in their research examining of the thyroid status. We will address that aspect in this

Normal thyroid function in childhood is essential for development of endochondral and intramembranous bone, for normal linear growth, as well as for establishing peak bone mass. Hypothyroidism in children causes growth arrest, delayed bone maturation, and epiphyseal dysgenesis, while T4 replacement results in rapid catch-up growth (Basset & Williams, 2003). Еxposure to soybean isoflavones during development may alter thyroid hormone concentrations and disturb feedback regulation of HPT axis, and these effects can

Soy infant formula is fed to infants as a replacement for human milk, or as an alternative to cow milk formula. Genistein is the predominant isoflavone found in soy infant formula (58- 67%), followed by daidzein (29-34%) and glycitein (5-8%) and infants fed soy infant formula have higher daily intakes of genistein and other isoflavones than other populations (Patisaul & Jefferson, 2010). The question of whether or not soy infant formula is safe has been widely debated for more than a decade, and early epidemiological studies demonstrated that infants fed adapted soy formula without iodine supply were hypothyroid (Van Wyk et al., 1959). This effect was eliminated by supplementing commercial soy infant formulas with iodine, or by

animal models (Ikeda et al., 2000; Kimura et al., 1976; McCarrison, 1933).

**5.3.1 Phytoestrogens, thyroid hormones and skeletal development** 

metabolism (Lakatos, 2003).

subchapter.

be more serious than in the adults.

absorption of calcium and induces increased conversion of 25(OH)-D to 1,25(OH)2-D, which enhances intestinal calcium absorption and increases skeletal calcium mobilization.

PTH has a biphasic effect on bone, as it stimulates bone formation when given intermittently, whereas continuous infusion reduces bone mass (Kim et al., 2003). Treatment with PTH significantly increases ALP activity, which suggests that this hormone modulates SaOS-2 osteoblastic cell differentiation and has an anabolic effect on bone. However, increases in RANKL mRNA and decreased OPG mRNA expression in SaOS-2 cells due to PTH indicates induction of bone resorption (Chen & Wong, 2006).

Elevated PTH secretion contributes to the greater bone resorption in osteoporosis which is related to estrogen deficiency. Estrogen therapy prevented the increase in PTH levels associated with the menopause (Khosla et al., 1997). Similarly, phytoestrogens behave as estrogen and may prevent the bone loss caused by estrogen deficiency in female animals and women through reduction of PTH levels. It was shown that phytoestrogens from medical plants can lower serum PTH levels in aged menopausal monkeys (Trisomboon et al. 2004). Also, postmenopausal women with habitually high intakes of dietary isoflavones had significantly lower levels of serum PTH and higher BMD (Mei et al., 2001). These plant compounds bind to ERs in the kidney, gastrointestinal tract and bone and improve calcium absorption resulting in a secondary decrease in the PTH level. Moreover, phytoestrogens may directly reduce PTH secretion from the parathyroid gland (Wong et al., 2002).

Fig. 8. Ultrastructure of parathyroid chief cells; nucleus (N), mitochondria (M), interdigitations of the plasma membrane (I); unpublished image of Pantelić et al.

absorption of calcium and induces increased conversion of 25(OH)-D to 1,25(OH)2-D, which

PTH has a biphasic effect on bone, as it stimulates bone formation when given intermittently, whereas continuous infusion reduces bone mass (Kim et al., 2003). Treatment with PTH significantly increases ALP activity, which suggests that this hormone modulates SaOS-2 osteoblastic cell differentiation and has an anabolic effect on bone. However, increases in RANKL mRNA and decreased OPG mRNA expression in SaOS-2 cells due to

Elevated PTH secretion contributes to the greater bone resorption in osteoporosis which is related to estrogen deficiency. Estrogen therapy prevented the increase in PTH levels associated with the menopause (Khosla et al., 1997). Similarly, phytoestrogens behave as estrogen and may prevent the bone loss caused by estrogen deficiency in female animals and women through reduction of PTH levels. It was shown that phytoestrogens from medical plants can lower serum PTH levels in aged menopausal monkeys (Trisomboon et al. 2004). Also, postmenopausal women with habitually high intakes of dietary isoflavones had significantly lower levels of serum PTH and higher BMD (Mei et al., 2001). These plant compounds bind to ERs in the kidney, gastrointestinal tract and bone and improve calcium absorption resulting in a secondary decrease in the PTH level. Moreover, phytoestrogens

enhances intestinal calcium absorption and increases skeletal calcium mobilization.

may directly reduce PTH secretion from the parathyroid gland (Wong et al., 2002).

Fig. 8. Ultrastructure of parathyroid chief cells; nucleus (N), mitochondria (M), interdigitations of the plasma membrane (I); unpublished image of Pantelić et al.

PTH indicates induction of bone resorption (Chen & Wong, 2006).

Mimicking the effect of estrogen, phytoestrogens can modulate the action of PTH on bone. Thus, one study in vitro showed that pre-treatment of SaOS-2 osteoblastic cells with genistein enhanced PTH-induced ALP activity and attenuated PTH up regulation of RANKL mRNA expression and PTH down regulation of OPG mRNA expression (Chen & Wong, 2006).

### **5.3 Effects of phytoestrogens on thyroid glands and thyroid hormones production**

Hypothalamic–pituitary–thyroid axis (HPT) plays a key role in skeletal development, attainment of peak bone mass and regulation of adult bone turnover (Gogakos et al., 2010; Roef еt al., 2011). Additionally, thyroid disorders are associated with alterations in bone metabolism (Lakatos, 2003).

Soy-food, soy-based infant formula, as well as dietary supplements containing purified soybean isoflavones, genistein and daidzein, are increasingly consumed in typical "Western" diet in the recent years. Commonly cited reasons for using soy infant formula are to feed infants who are allergic to dairy products or are intolerant of lactose, galactose, or cow-milk protein (Tuohy, 2003). In elderly, reason is potential health benefit of soybean isoflavones in protection of age-related diseases, including osteoporosis (Setchell, 1998).

Structurally, soybean isoflavones genistein and daidzein are polyphenolic compounds, similar to estradiol-17β and bind with a weaker potency to both types of ERs, with higher affinity for ERβ (Kuiper et al., 1998). Despite the numerous beneficial effects of soy isoflavones, epidemiological and experimental data also exist showing an adverse effect on human health, namely on reproductive and thyroid axis. The association between high soy isoflavones intake and goitrogenesis, as well as protective effect of adequate iodine intake, was reported both in humans (Chorazy et al.1995; Van Wyk et al., 1959) and in different animal models (Ikeda et al., 2000; Kimura et al., 1976; McCarrison, 1933).

Therefore, besides the direct beneficial effect of soybean phytoestrogens on bone tissue, isoflavones may also act indirectly, through endocrine disruption and interference with HPT axis. Most researchers who examined osteoprotective potential of isoflavones did not include in their research examining of the thyroid status. We will address that aspect in this subchapter.

### **5.3.1 Phytoestrogens, thyroid hormones and skeletal development**

Normal thyroid function in childhood is essential for development of endochondral and intramembranous bone, for normal linear growth, as well as for establishing peak bone mass. Hypothyroidism in children causes growth arrest, delayed bone maturation, and epiphyseal dysgenesis, while T4 replacement results in rapid catch-up growth (Basset & Williams, 2003). Еxposure to soybean isoflavones during development may alter thyroid hormone concentrations and disturb feedback regulation of HPT axis, and these effects can be more serious than in the adults.

Soy infant formula is fed to infants as a replacement for human milk, or as an alternative to cow milk formula. Genistein is the predominant isoflavone found in soy infant formula (58- 67%), followed by daidzein (29-34%) and glycitein (5-8%) and infants fed soy infant formula have higher daily intakes of genistein and other isoflavones than other populations (Patisaul & Jefferson, 2010). The question of whether or not soy infant formula is safe has been widely debated for more than a decade, and early epidemiological studies demonstrated that infants fed adapted soy formula without iodine supply were hypothyroid (Van Wyk et al., 1959). This effect was eliminated by supplementing commercial soy infant formulas with iodine, or by

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 747

Based on analyses of rare monogenic diseases and the results of animal studies, it was proposed that T3 play a key role in bone development, while TSH is not required for normal skeletal development (Bassett et al., 2008). T3 enters the nucleus and binds to its nuclear receptors (TR). There are three functional TRs: TRα1, TRβ1 and TRβ2, encoded by the THRA and THRB genes. These receptors act as hormone inducible transcription factors that regulate expression of T3-responsive target genes (Yen, 2001). Both TRα1 and TRβ1 isoforms are expressed in bone and TRα1 levels are at least 10-fold greater than TRβ1. These findings support the opinion that TRα1 is the principal mediator of T3 action in bone (Bassett &

In vitro experiments demonstrated that effects of T3 in osteoblastic cell lines and primary osteoblast cultures depend on species, cell type, anatomic origin, differentiation phase and duration of the treatment. T3 was reported to increase expression of osteocalcin, osteopontin, type I collagen, alkaline phosphatase, IGF-I and its regulatory binding proteins IGF1BP-2 and -4 (Milne et al., 2001; Pereira et al., 1999; Varga et al., 2004). Therefore, T3 may exert its stimulatory effect on osteoblasts via complex pathways involving many growth factors and

Similar to osteoporosis, thyroid diseases are much more common in elderly women than in men and is associated with significant morbidity if left untreated (Schindler 2003; Suchartwatnachai et al., 2002). Still, this fact does not imply a causal relationship between the two diseases and many patients may independently develop both. Hypothyroidism occurs in 10% of females and 2% of males in patients older than 60 years. The prevalence of hyperthyroidism in the elderly is approximately 2% (Maugeri et al., 1996), though other authors reported that 10 to 15% of elderly patients were hyperthyroid (Kennedy & Caro, 1996). Thyrotoxicosis increase risk in developing secondary osteoporosis (Amashukeli et al.,

Thyroid hormones play a significant role in maintaining adult bone homeostasis. Results of clinical and experimental studies are consistent and demonstrate that hypothyroid state slows down bone turnover and affect overall gain in bone mass and mineralization. By contrast, bone resorption and formation are accelerated in hyperthyroidism, while the remodeling cycle is shortened (Davies et al., 2005). Increased bone turnover and osteoporosis in thyrotoxicosis are attributed to the thyroid hormone excess and are not a consequence of deficient TSH receptor (TSHR) signaling. However, TSH may play a direct role in regulation of bone turnover, since TSH receptor was identified in osteoblasts. The experiment with ovariectomized rats, which were treated with low doses of TSH (insufficient to alter serum T3, T4 or TSH levels), demonstrated that TSH treatment prevented bone loss and increased bone mass (Sampath et al., 2007; Sun et al., 2008). Although the TSHR is expressed in osteoblasts, current data from in vitro studies are contradictory and suggest that TSH may enhance, inhibit or have no effect on osteoblast

Prevention and treatment of osteoporosis involve Ca and vitamin D supplementation, as well as different drug therapy approaches, which include bisphosphonate, salmon CT and estrogen or androgen replacement therapy for menopausal women and andropausal men, respectively. In addition, in recent years, numerous discussions on safety and benefit of synthetic steroids (both estrogens and androgens) favor the trend towards consumption of

**5.3.2 Phytoestrogens, thyroid hormones and osteoporosis prevention** 

Williams, 2009; O'Shea et al., 2003).

cytokines.

2010; Lakatos, 2003).

differentiation and function (Bassett et al., 2008).

switching to cow milk (Chorazy et al., 1995). Today, soy formula is regularly supplied with iodine and a more recent study demonstrated no significant changes in the serum level of bone alkaline phosphatase, osteocalcin, intact PTH, and the urinary levels of the markers of bone metabolism in children (mean age of 37 months) fed with soy formula (Giampietro et al., 2004). However, infants with congenital hypothyroidism fed with iodine supplemented diet still need higher doses of L-thyroxine (Jabbar et al., 1997). This finding is of particular importance, keeping in mind that the consequence of congenital and juvenile acquired hypothyroidism is retardation of skeletal development and that the effects of T4 replacement (achievement of predicted adult height) strongly depend on the duration of untreated hypothyroidism (Rivkees et al., 1988).

Soybean isoflavones may functionally disrupt the thyroid hormone (TH) system by influencing different steps such as synthesis, transport, action and metabolism of TH. Genistein and daidzein inhibit the activity of thyroid peroxidase (TPO), the key enzyme in the synthesis of thyroid hormones, both in vitro and in vivo (Chang & Doerge, 2000; Divi et al., 1997; Doerge & Chang, 2002). Besides the inhibitory effects of isoflavones on TPO, iodine deficiency is important risk factor for thyroid dysfunction and goiter development, both in humans and in rats. An adequate iodine supply is a way to prevent goitrogenic effects of soy bean isoflavones, especially in the high-risk group of patients with congenital hypothyroidism. Besides the serum concentrations of TH, biological activity of T3 on bone tissue is determined by the membrane transporters of TH, local expression and activity of deiodinase enzymes and receptors for TSH and TH. Polymorphisms in above mentioned genes are associated with important chronic skeletal diseases, including osteoporosis and osteoarthritis (Andersen et al., 2002, 2003; Peeters et al., 2006).

Entry of T3 and T4 into target cells is determined by the active uptake of free hormones by specific cell membrane transporters: monocarboxylate transporter-8 (MCT8), MCT10 and organic acid transporter protein-1c1 (OATP1c1) (van der Deure et al., 2010). MCT8 is expressed in growth plate chondrocytes, bone forming osteoblasts and bone resorbing osteoclasts at all stages of cell differentiation, and its expression is regulated by thyroid status (Capelo et al., 2009), although its functional importance is still unclear. It seems that OATP1c1 is not expressed in the mouse skeleton (Capelo et al., 2009), but there are still no data regarding expression of MCT10. Tyrosine kinase inhibitors sunitinib and imatinib inhibit MCT8 – mediated iodothyroinine transport (Schweizer et al., 2010), but there are still no data regarding possible effects of genistein, which is a potent thyrosine kinase inhibitor as well, on cellular transport of TH.

Deiodinase (Dio) enzymes determine the intracellular levels of bioactive T3 and thus cellspecific gene expression. Expression of deiodinases is tissue specific: Dio 1 enzyme is not expressed in bone, while Dio 2 plays an important role in local regulation of thyroid hormone signaling during fetal bone development. In the adult skeleton Dio 2 activity is restricted to osteoblasts (Williams et al., 2008). Dio 2 expression and activity are inhibited by high concentrations of substrate (T4) and thus are maximal in hypothyroidism and suppressed in thyrotoxicosis. Locally regulated activity of Dio 2 in osteoblasts maintains intra-cellular T3 concentrations constant over the euthyroid range and preserves optimal bone mineralization. Inactivating deiodinase type 3 (Dio 3) is expressed in the skeleton, although the highest levels of enzyme activity occur in growth plate chondrocytes prior to weaning (Yen, 2001). Genistein inhibit both Dio 1 and Dio 2 activity in vitro (Mori et al., 1996), but the physiological importance of this mechanism is still unclear.

switching to cow milk (Chorazy et al., 1995). Today, soy formula is regularly supplied with iodine and a more recent study demonstrated no significant changes in the serum level of bone alkaline phosphatase, osteocalcin, intact PTH, and the urinary levels of the markers of bone metabolism in children (mean age of 37 months) fed with soy formula (Giampietro et al., 2004). However, infants with congenital hypothyroidism fed with iodine supplemented diet still need higher doses of L-thyroxine (Jabbar et al., 1997). This finding is of particular importance, keeping in mind that the consequence of congenital and juvenile acquired hypothyroidism is retardation of skeletal development and that the effects of T4 replacement (achievement of predicted adult height) strongly depend on the duration of untreated

Soybean isoflavones may functionally disrupt the thyroid hormone (TH) system by influencing different steps such as synthesis, transport, action and metabolism of TH. Genistein and daidzein inhibit the activity of thyroid peroxidase (TPO), the key enzyme in the synthesis of thyroid hormones, both in vitro and in vivo (Chang & Doerge, 2000; Divi et al., 1997; Doerge & Chang, 2002). Besides the inhibitory effects of isoflavones on TPO, iodine deficiency is important risk factor for thyroid dysfunction and goiter development, both in humans and in rats. An adequate iodine supply is a way to prevent goitrogenic effects of soy bean isoflavones, especially in the high-risk group of patients with congenital hypothyroidism. Besides the serum concentrations of TH, biological activity of T3 on bone tissue is determined by the membrane transporters of TH, local expression and activity of deiodinase enzymes and receptors for TSH and TH. Polymorphisms in above mentioned genes are associated with important chronic skeletal diseases, including osteoporosis and

Entry of T3 and T4 into target cells is determined by the active uptake of free hormones by specific cell membrane transporters: monocarboxylate transporter-8 (MCT8), MCT10 and organic acid transporter protein-1c1 (OATP1c1) (van der Deure et al., 2010). MCT8 is expressed in growth plate chondrocytes, bone forming osteoblasts and bone resorbing osteoclasts at all stages of cell differentiation, and its expression is regulated by thyroid status (Capelo et al., 2009), although its functional importance is still unclear. It seems that OATP1c1 is not expressed in the mouse skeleton (Capelo et al., 2009), but there are still no data regarding expression of MCT10. Tyrosine kinase inhibitors sunitinib and imatinib inhibit MCT8 – mediated iodothyroinine transport (Schweizer et al., 2010), but there are still no data regarding possible effects of genistein, which is a potent thyrosine kinase inhibitor

Deiodinase (Dio) enzymes determine the intracellular levels of bioactive T3 and thus cellspecific gene expression. Expression of deiodinases is tissue specific: Dio 1 enzyme is not expressed in bone, while Dio 2 plays an important role in local regulation of thyroid hormone signaling during fetal bone development. In the adult skeleton Dio 2 activity is restricted to osteoblasts (Williams et al., 2008). Dio 2 expression and activity are inhibited by high concentrations of substrate (T4) and thus are maximal in hypothyroidism and suppressed in thyrotoxicosis. Locally regulated activity of Dio 2 in osteoblasts maintains intra-cellular T3 concentrations constant over the euthyroid range and preserves optimal bone mineralization. Inactivating deiodinase type 3 (Dio 3) is expressed in the skeleton, although the highest levels of enzyme activity occur in growth plate chondrocytes prior to weaning (Yen, 2001). Genistein inhibit both Dio 1 and Dio 2 activity in vitro (Mori et al.,

1996), but the physiological importance of this mechanism is still unclear.

hypothyroidism (Rivkees et al., 1988).

as well, on cellular transport of TH.

osteoarthritis (Andersen et al., 2002, 2003; Peeters et al., 2006).

Based on analyses of rare monogenic diseases and the results of animal studies, it was proposed that T3 play a key role in bone development, while TSH is not required for normal skeletal development (Bassett et al., 2008). T3 enters the nucleus and binds to its nuclear receptors (TR). There are three functional TRs: TRα1, TRβ1 and TRβ2, encoded by the THRA and THRB genes. These receptors act as hormone inducible transcription factors that regulate expression of T3-responsive target genes (Yen, 2001). Both TRα1 and TRβ1 isoforms are expressed in bone and TRα1 levels are at least 10-fold greater than TRβ1. These findings support the opinion that TRα1 is the principal mediator of T3 action in bone (Bassett & Williams, 2009; O'Shea et al., 2003).

In vitro experiments demonstrated that effects of T3 in osteoblastic cell lines and primary osteoblast cultures depend on species, cell type, anatomic origin, differentiation phase and duration of the treatment. T3 was reported to increase expression of osteocalcin, osteopontin, type I collagen, alkaline phosphatase, IGF-I and its regulatory binding proteins IGF1BP-2 and -4 (Milne et al., 2001; Pereira et al., 1999; Varga et al., 2004). Therefore, T3 may exert its stimulatory effect on osteoblasts via complex pathways involving many growth factors and cytokines.

### **5.3.2 Phytoestrogens, thyroid hormones and osteoporosis prevention**

Similar to osteoporosis, thyroid diseases are much more common in elderly women than in men and is associated with significant morbidity if left untreated (Schindler 2003; Suchartwatnachai et al., 2002). Still, this fact does not imply a causal relationship between the two diseases and many patients may independently develop both. Hypothyroidism occurs in 10% of females and 2% of males in patients older than 60 years. The prevalence of hyperthyroidism in the elderly is approximately 2% (Maugeri et al., 1996), though other authors reported that 10 to 15% of elderly patients were hyperthyroid (Kennedy & Caro, 1996). Thyrotoxicosis increase risk in developing secondary osteoporosis (Amashukeli et al., 2010; Lakatos, 2003).

Thyroid hormones play a significant role in maintaining adult bone homeostasis. Results of clinical and experimental studies are consistent and demonstrate that hypothyroid state slows down bone turnover and affect overall gain in bone mass and mineralization. By contrast, bone resorption and formation are accelerated in hyperthyroidism, while the remodeling cycle is shortened (Davies et al., 2005). Increased bone turnover and osteoporosis in thyrotoxicosis are attributed to the thyroid hormone excess and are not a consequence of deficient TSH receptor (TSHR) signaling. However, TSH may play a direct role in regulation of bone turnover, since TSH receptor was identified in osteoblasts. The experiment with ovariectomized rats, which were treated with low doses of TSH (insufficient to alter serum T3, T4 or TSH levels), demonstrated that TSH treatment prevented bone loss and increased bone mass (Sampath et al., 2007; Sun et al., 2008). Although the TSHR is expressed in osteoblasts, current data from in vitro studies are contradictory and suggest that TSH may enhance, inhibit or have no effect on osteoblast differentiation and function (Bassett et al., 2008).

Prevention and treatment of osteoporosis involve Ca and vitamin D supplementation, as well as different drug therapy approaches, which include bisphosphonate, salmon CT and estrogen or androgen replacement therapy for menopausal women and andropausal men, respectively. In addition, in recent years, numerous discussions on safety and benefit of synthetic steroids (both estrogens and androgens) favor the trend towards consumption of

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 749

treatment (unpublished data). In vitro studies with FTRL-5 cells demonstrated that Ca did not affect the morphology of these cells, but when administered together with TSH, it acted directly, by reducing the thyrotropin stimulatory effect (Gaberscek et al., 1998). Isoform VI of adenilyl cyclase, the enzyme crucial for TSH-induced activation of thyroid follicular cells, was found negatively modulated by Ca in human and dog thyroids (Vanvooren et al., 2000). Doses of Ca were chosen to mimic human exposure to high doses of Ca in treatment of osteoporosis. We can speculate that slowing down of thyroid hormone synthesis may be an indirect mechanism, which lead to decreased bone turnover detected after Ca treatment

Sex steroids, estrogen and testosterone, play an important role in bone physiology and pathology. Endogenous estrogens are regularly produced in bone via aromatase enzyme activity, and exert their effects through ER, which are also detected in male bones (Carani et al., 1997; Grumbach & Auchus, 1999; Korach, 1994). Bone cells are sensitive to both estrogens and androgens, and aromatase inhibition causes similar degree of osteoporosis in

There is a close relationship between sex steroids and thyroid function. Epidemiological studies suggest that the use of estrogens may contribute to the pathogenesis of thyroid tumors (Ron et al., 1987). Experimental studies on rodents demonstrated numerous sexrelated differences in thyroid function and, in general, adult male rodents have higher levels of TSH than females associated with lower T4 and higher plasma levels of T3 (Capen, 1997). The results related to treatment effects of sex steroids on different set points of thyroid function are inconsistent and depend on experimental conditions: type of experimental animal, animal's age and applied dose (Chen & Wallfish, 1978; Henderson et al., 1982; Sekulić et al., 2007). Our previous results demonstrated an inhibitory effect of pharmacologic doses of estradiol (previously used in human studies for treatment of osteoporosis) on thyroid follicular cells in ovariectomized young adult and ovarium-intact young and middle-aged rats, (Sekulić et al., 2006; Šošić -Jurjević et al., 2005, 2006a), as well as after treatments of orchidectomized 16-month-old rat males with 10 times lesser dose of estradiol dipropionate (Sekulić et al., 2010). We choose the dose of estradiol in the experiment which was previously reported to prevent bone loss in males (Fitts et al., 2001; Vanderput et al., 2001). Consistent with literature data, we also detected decreased serum osteocalcin levels, accompanied by decreased urinary Ca concentration in Orx rats treated with EDP (unpublished data). Contrary to effects of estradiol, testosterone treatment of castrated middle-aged males moderately increased serum TSH and total T4 levels (Sekulić et al., 2010), but similarly to estradiol treatment, decreased both serum osteocalcin levels and urinary Ca concentration (unpublished data). Therefore, it seems that the direct effect of sex steroids on bone tissue is more relevant for the net result of replacement therapy on bone

protection then the indirect effect, mediated through modulation of thyroid function.

Direct negative effect of isoflavones on thyroid hormone synthesis, by significant blocking of TPO activity (more than 60%), has been well described. Genistein and daidzein were demonstrated to block both TPO-catalyzed reactions: iodination of thyrosine residues of Tg, and T4 formation by coupling reactions, but this effect was eliminated by iodine (Chang & Doerge, 2000; Divi et al., 1997; Doerge & Chang 2002). Despite significant inactivation of this enzyme, serum thyroid hormone levels were unaffected by isoflavone treatments in young adult rats of both sexes. The authors supposed that soy could cause goiter, but only in animals or humans consuming diets marginally adequate in iodine, or who were predisposed to develop goiter. Most other authors, who performed their studies on young adult animals of both sexes, also reported that soy or isoflavones alone, in the absence of

under our experimental conditions.

male animals as orchidectomy (Vanderschueren et al., 1998).

"green" natural "phytosteroids" or "phyto-selective modulator of ERs". That is why nutritional supplements and concentrated extracts containing purified soybean phyto-SERMs genistein and daidzein are increasingly used as alternative therapy for osteoporosis and other age-related diseases in both sexes (Ramos, 2007; Setchell, 1998; Tham et al., 1998). However, all these treatments may affect thyroid function as well.

Not so many researchers have tried to link effects of supplementation or drug treatment on bone metabolism with modulation of thyroid hormone levels. Rodents are considered useful models for thyroid studies, even though significant differences between rodent and human thyroid physiology have been reported (Choksi et al., 2003; Poirier et al., 1999). Rat thyrocytes are characterized by abundant granular endoplasmic reticulum, well developed Golgi, prominent lysosomes, luminal (apical) microvilli, small mitochondria, and round nuclei with homogeneous chromatin (Fig. 9).

Fig. 9. Ultrastructure of thyroid follicular cell; nucleus (N), mitochondria (M), rough endoplasmatic reticulum (RER), lysosomes (Ly), colloidal droplets (Cd), colloid (C); unpublished image of Šošić-Jurjević et al.

In our laboratory we demonstrated that chronic Ca administration to middle-aged female rats significantly decreased the volume density of the thyroid follicular epithelium, epithelium's height and the index of activation rate, which are morphometric parameters of TH synthetic and secretory potential of thyrocytes (Šošić -Jurjević et al., 2002). Consistent with histomorphometric changes, reduced serum levels of total T4 and T3 were detected (Šošić -Jurjević et al., 2006). At the same time, we determined significant decrease of serum osteocalcin and urinary Ca, as biochemical parameters of reduced bone turnover after Ca

"green" natural "phytosteroids" or "phyto-selective modulator of ERs". That is why nutritional supplements and concentrated extracts containing purified soybean phyto-SERMs genistein and daidzein are increasingly used as alternative therapy for osteoporosis and other age-related diseases in both sexes (Ramos, 2007; Setchell, 1998; Tham et al., 1998).

Not so many researchers have tried to link effects of supplementation or drug treatment on bone metabolism with modulation of thyroid hormone levels. Rodents are considered useful models for thyroid studies, even though significant differences between rodent and human thyroid physiology have been reported (Choksi et al., 2003; Poirier et al., 1999). Rat thyrocytes are characterized by abundant granular endoplasmic reticulum, well developed Golgi, prominent lysosomes, luminal (apical) microvilli, small mitochondria, and round

Fig. 9. Ultrastructure of thyroid follicular cell; nucleus (N), mitochondria (M), rough endoplasmatic reticulum (RER), lysosomes (Ly), colloidal droplets (Cd), colloid (C);

In our laboratory we demonstrated that chronic Ca administration to middle-aged female rats significantly decreased the volume density of the thyroid follicular epithelium, epithelium's height and the index of activation rate, which are morphometric parameters of TH synthetic and secretory potential of thyrocytes (Šošić -Jurjević et al., 2002). Consistent with histomorphometric changes, reduced serum levels of total T4 and T3 were detected (Šošić -Jurjević et al., 2006). At the same time, we determined significant decrease of serum osteocalcin and urinary Ca, as biochemical parameters of reduced bone turnover after Ca

However, all these treatments may affect thyroid function as well.

nuclei with homogeneous chromatin (Fig. 9).

unpublished image of Šošić-Jurjević et al.

treatment (unpublished data). In vitro studies with FTRL-5 cells demonstrated that Ca did not affect the morphology of these cells, but when administered together with TSH, it acted directly, by reducing the thyrotropin stimulatory effect (Gaberscek et al., 1998). Isoform VI of adenilyl cyclase, the enzyme crucial for TSH-induced activation of thyroid follicular cells, was found negatively modulated by Ca in human and dog thyroids (Vanvooren et al., 2000). Doses of Ca were chosen to mimic human exposure to high doses of Ca in treatment of osteoporosis. We can speculate that slowing down of thyroid hormone synthesis may be an indirect mechanism, which lead to decreased bone turnover detected after Ca treatment under our experimental conditions.

Sex steroids, estrogen and testosterone, play an important role in bone physiology and pathology. Endogenous estrogens are regularly produced in bone via aromatase enzyme activity, and exert their effects through ER, which are also detected in male bones (Carani et al., 1997; Grumbach & Auchus, 1999; Korach, 1994). Bone cells are sensitive to both estrogens and androgens, and aromatase inhibition causes similar degree of osteoporosis in male animals as orchidectomy (Vanderschueren et al., 1998).

There is a close relationship between sex steroids and thyroid function. Epidemiological studies suggest that the use of estrogens may contribute to the pathogenesis of thyroid tumors (Ron et al., 1987). Experimental studies on rodents demonstrated numerous sexrelated differences in thyroid function and, in general, adult male rodents have higher levels of TSH than females associated with lower T4 and higher plasma levels of T3 (Capen, 1997). The results related to treatment effects of sex steroids on different set points of thyroid function are inconsistent and depend on experimental conditions: type of experimental animal, animal's age and applied dose (Chen & Wallfish, 1978; Henderson et al., 1982; Sekulić et al., 2007). Our previous results demonstrated an inhibitory effect of pharmacologic doses of estradiol (previously used in human studies for treatment of osteoporosis) on thyroid follicular cells in ovariectomized young adult and ovarium-intact young and middle-aged rats, (Sekulić et al., 2006; Šošić -Jurjević et al., 2005, 2006a), as well as after treatments of orchidectomized 16-month-old rat males with 10 times lesser dose of estradiol dipropionate (Sekulić et al., 2010). We choose the dose of estradiol in the experiment which was previously reported to prevent bone loss in males (Fitts et al., 2001; Vanderput et al., 2001). Consistent with literature data, we also detected decreased serum osteocalcin levels, accompanied by decreased urinary Ca concentration in Orx rats treated with EDP (unpublished data). Contrary to effects of estradiol, testosterone treatment of castrated middle-aged males moderately increased serum TSH and total T4 levels (Sekulić et al., 2010), but similarly to estradiol treatment, decreased both serum osteocalcin levels and urinary Ca concentration (unpublished data). Therefore, it seems that the direct effect of sex steroids on bone tissue is more relevant for the net result of replacement therapy on bone protection then the indirect effect, mediated through modulation of thyroid function.

Direct negative effect of isoflavones on thyroid hormone synthesis, by significant blocking of TPO activity (more than 60%), has been well described. Genistein and daidzein were demonstrated to block both TPO-catalyzed reactions: iodination of thyrosine residues of Tg, and T4 formation by coupling reactions, but this effect was eliminated by iodine (Chang & Doerge, 2000; Divi et al., 1997; Doerge & Chang 2002). Despite significant inactivation of this enzyme, serum thyroid hormone levels were unaffected by isoflavone treatments in young adult rats of both sexes. The authors supposed that soy could cause goiter, but only in animals or humans consuming diets marginally adequate in iodine, or who were predisposed to develop goiter. Most other authors, who performed their studies on young adult animals of both sexes, also reported that soy or isoflavones alone, in the absence of

The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 751

increased serum TSH, in orchidectomized (Orx) middle-aged male rats fed a iodinesufficient soy-free diet (Šošić -Jurjević et al., 2010). Our research team obtained that both genistein and daidzein increased bone mass following orchidectomy of middle-aged males (Filipovic et al., 2010 and unpublished data). Therefore, decreased serum level of TH might contribute to the detected increase in trabecular bone mass, and decrease in bone turnover

Phytoestrogens have the potential to maintain bone health. Owing to their properties, these plant-derived non-steroidal compounds have a potential beneficial role in delaying or preventing osteoporosis. Therefore, they have attracted much attention as alternatives to HRT. As SERM, phytoestrogens may generate a bone protective effect via stimulation of osteoblastic bone formation and inhibition of osteoclastic bone resorption. Proposed molecular mechanisms are based on their ER-mediated effects. In addition to direct action, phytoestrogens can affect bone structure indirectly, by stimulating or inhibiting the synthesis of certain hormones, i.e. through increased synthesis of CT from thyroid C cells, as

This work was supported by the Ministry of Education and Science of the Republic of Serbia, Grant No. 173009. The authors express their gratitude to the late Dr Dana Brunner for her guidance and contribution, to Mrs. Anna Nikolić and Mr. Kristijan Jurjević for

Abu, EO., Bord, S., Horner, A., Chatterjee, VK. & Compston, JE. (1997). The expression of thyroid hormone receptors in human bone. *Bone*, Vol. 21, pp. 137-142 Adlercreutz, H. (1998). Evolution, nutrition, intestinal microflora, and prevention of cancer:

Amashukeli, M., Giorgadze, E., Tsagareli, M., Nozadze, N. & Jeiranashvili, N. (2010). The

Andersen, S., Bruun, NH., Pedersen, KM. & Laurberg, P. (2003). Biologic variation is

Andersen, S., Pedersen, KM., Bruun, NH. & Laurberg, P. (2002). Narrow individual

subclinical thyroid disease*. J Clin Endocrinol Metab*, Vol. 87, pp. 1068-1072 Anderson, JJ., Chen, X., Boass, A., Symons, M., Kohlmeier, M., Renner, JB. & Garner, SC.

Aoki, K., Didomenico, E., Sims, NA., Mukhopadhyay, K., Neff, L., Houghton, A., Amling,

impact of thyroid diseases on bone metabolism and fracture risk. *Georgian Med* 

important for interpretation of thyroid function tests. *Thyroid*, Vol. 13, pp. 1069-1078

variations in serum T(4) and T(3) in normal subjects: a clue to the understanding of

(2002). Soy isoflavones: no effects on bone mineral content and bone mineral density in healthy, menstruating young adult women after one year. *J Am Coll Nutr*,

M., Levy, JB., Horne, WC. & Baron, R. (1999). The tyrosine phosphatase SHP-1 is a

a hypothesis. *Proc Soc Exp Biol Med*, Vol. 217, pp. 241–246

in aged male orchidectomized rat model.

well as reduction of PTH and thyroid hormone levels.

assistance with English manuscript preparation.

News, Vol. 184-185, pp. 34-39

Vol. 21, pp. 388–393

**6. Conclusion** 

**7. Acknowledgment** 

**8. References** 

other goitrogenic stimulus, did not affect thyroid weights, histopathology and the serum levels of TSH and thyroid hormones (Chang & Doerge, 2000; Schmutzler et al., 2004). The thyroid function becomes impaired with aging in rodents, and the number of thyroid dysfunction increase in elderly population (Donda & Lemarchand-Béraud, 1989; Reymond et al., 1992). We were the first who demonstrated that therapeutic doses of both genistein and daidzein induce hypertrophy of Tg-immunopositive follicular epithelium and colloid depletion (Fig. 10), and reduce the level of serum thyroid hormones, accompanied by

Fig. 10. Thyroid gland tissue of control (a, b), orchidectomized (c, d) and orchidectomized rats treated with daidzein (e, f); hematoxylin-eosin and immuno-staining for thyroglobulin; unpublished image of Šošić-Jurjević et al.

increased serum TSH, in orchidectomized (Orx) middle-aged male rats fed a iodinesufficient soy-free diet (Šošić -Jurjević et al., 2010). Our research team obtained that both genistein and daidzein increased bone mass following orchidectomy of middle-aged males (Filipovic et al., 2010 and unpublished data). Therefore, decreased serum level of TH might contribute to the detected increase in trabecular bone mass, and decrease in bone turnover in aged male orchidectomized rat model.

### **6. Conclusion**

750 Osteoporosis

other goitrogenic stimulus, did not affect thyroid weights, histopathology and the serum levels of TSH and thyroid hormones (Chang & Doerge, 2000; Schmutzler et al., 2004). The thyroid function becomes impaired with aging in rodents, and the number of thyroid dysfunction increase in elderly population (Donda & Lemarchand-Béraud, 1989; Reymond et al., 1992). We were the first who demonstrated that therapeutic doses of both genistein and daidzein induce hypertrophy of Tg-immunopositive follicular epithelium and colloid depletion (Fig. 10), and reduce the level of serum thyroid hormones, accompanied by

Fig. 10. Thyroid gland tissue of control (a, b), orchidectomized (c, d) and orchidectomized rats treated with daidzein (e, f); hematoxylin-eosin and immuno-staining for thyroglobulin;

unpublished image of Šošić-Jurjević et al.

Phytoestrogens have the potential to maintain bone health. Owing to their properties, these plant-derived non-steroidal compounds have a potential beneficial role in delaying or preventing osteoporosis. Therefore, they have attracted much attention as alternatives to HRT. As SERM, phytoestrogens may generate a bone protective effect via stimulation of osteoblastic bone formation and inhibition of osteoclastic bone resorption. Proposed molecular mechanisms are based on their ER-mediated effects. In addition to direct action, phytoestrogens can affect bone structure indirectly, by stimulating or inhibiting the synthesis of certain hormones, i.e. through increased synthesis of CT from thyroid C cells, as well as reduction of PTH and thyroid hormone levels.

### **7. Acknowledgment**

This work was supported by the Ministry of Education and Science of the Republic of Serbia, Grant No. 173009. The authors express their gratitude to the late Dr Dana Brunner for her guidance and contribution, to Mrs. Anna Nikolić and Mr. Kristijan Jurjević for assistance with English manuscript preparation.

### **8. References**


The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 753

Canalis, E., Pash, J. & Varghese, S. (1993). Skeletal growth factors. *Crit Rev Eukaryot Gene* 

Canalis, E., Pash, J., Gabbitas, B., Rydziel, S. & Varghese, S. (1993a). Growth factors regulate

Capelo, LP., Beber, EH., Fonseca, TL. & Gouveia, CH. (2009). The monocarboxylate

skeletons and in osteoblastic MC3T3-E1 cells. *Thyroid*, Vol. 19, pp. 171-178 Capen, C. (1997). Mehanicistic data and risk assesment of selected toxic end points of the

Carani, C., Qin, K., Simoni, M., Faustini-Fustini, M., Serpente, S., Boyd, J., Korach, KS. &

Chang, HC. & Doerge, DR. (2000). Dietary genistein inactivates rat thyroid peroxidase in

Chen, D., Zhao, M. & Mundy, GR. (2004). Bone morphogenetic proteins. *Growth Factors*, Vol.

Chen, HJ. & Walfish, PG. (1978). Effects of estradiol benzoate on thyroid-pituitary function

Chen, JJ. & Chang, HC. (2007). By modulating androgen receptor coactivators, daidzein may

Chen, WF. & Wong, MS. (2006). Genistein modulates the effects of parathyroid hormone in

Chen, XW., Garner, SC., Quarles, LD. & Anderson, JJB. (2003). Effects of genistein on

Choksi, NY., Jahnke, GD., St Hilaire, C. & Shelby, M. (2003). Role of thyroid hormones in

Chorazy, PA., Himelhoch, S., Hopwood, NJ., Greger, NG. & Postellon, DC. (1995). Persistent

Collin-Osdoby, P., Rothe, L., Anderson, F., Nelson, M., Maloney, W. & Osdoby, P. (2001).

Comelekoglu, U., Bagis, S., Yalin, S., Ogenler, O., Yildiz, A., Sahin, NO., Oguz, I. & Hatungil,

Dang, ZC., Audinot, V., Papapoulos, SE., Boutin, JA. & Lowik, C. (2003). Peroxisome

human osteoclastogenesis. *J Biol Chem*, Vol. 276, pp. 20659– 20672

soy phytoestrogen genistein. *J Biol Chem*, Vol. 278, pp. 962–967

expression cell of bone markers during MC3T3-E1 osteoblastic differentiation. *J* 

human and laboratory animal reproductive health. Birth Defects *Res B Dev Reprod*

hypothyroidism in an infant receiving a soy formula: case report and review of the

Receptor activator of NF-kappa B and osteoprotegerin expression by human microvascular endothelial cells, regulation by inflammatory cytokines, and role in

R. (2007). Biomechanical evaluation in osteoporosis: ovariectomized rat model. *Clin* 

proliferator-activated receptor gamma (PPAR gamma) as a molecular target for the

human osteoblastic SaOS-2 cells. *Br J Nutr*, Vol. 95, pp. 1039-1047

thyroid gland. *Toxicologic Pathology*, Vol. 25, pp. 39-48

in female rats. *Endocrinology*, Vol. 103, pp. 1023-1030

act as a phytoandrogen. *Prostate*, Vol. 67, pp. 457-462

*Nutr Biochem*, Vol. 14, pp. 342–349

literature. *Pediatrics*, Vol. 96, pp. 148-150

*Toxicol*, Vol. 68, pp. 479-491

*Rheumatol*, Vol. 26, pp. 380-384

deficiency. *N Engl J Med*, Vol. 337, pp. 91-95

the synthesis of insulin-like growth factor-I in bone cell cultures. *Endocrinology*, Vol.

transporter 8 and L-type amino acid transporters 1 and 2 are expressed in mouse

Simpson, ER. (1997). Effect of testosterone and estradiol in a man with aromatase

vivo without an apparent hypothyroid effect. *Toxicol Appl Pharmacol*, Vol. 168, pp.

*Expr*, Vol. 3, pp. 155-166

133, pp. 33-38

244-252

22, 233-241

negative regulator of osteoclastogenesis and osteoclast resorbing activity: Increased resorption and osteopenia in mev/mev mutant mice. Bone, Vol. 25, pp. 261-267


Arjmandi, BH., Lucas, EA., Khalil, DA., Devareddy, L., Smith, BJ., McDonald, J., Arquitt,

Arts, J., Kuiper, GG., Janssen, JM., Gustafsson, JA., Lowik, CW., Pols, HA. & van Leeuwen,

Atmaca, A., Kleerekoper, M., Bayraktar, M. & Kucuk, O. (2008). Soy isoflavones in the management of postmenopausal osteoporosis. *Menopause*, Vol. 15, pp. 748–757 Bahr, JM., Nakai, M., Rivera, A., Walsh, J., Evans, GL., Lotinun, S., Turner, RT., Black, M. &

Bassett, JH. & Williams, GR. (2003). The molecular actions of thyroid hormone in

Bassett, JH., Williams, AJ., Murphy, E., Boyde, A., Howell, PG., Swinhoe. R., Archanco, M.,

Bassett, JH. & Williams, GR. (2009). The skeletal phenotypes of TRalpha and TRbeta mutant

Bianco, P., Riminucci, M., Gronthos, S. & Robey, PG. (2001). Bone marrow stromal stem cells: nature, biology, and potential applications. *Stem Cells*, Vol. 19, pp. 180-192 Blair, HC., Jordan, SE., Peterson, TG. & Barnes, S. (1996). Variable effects of tyrosine kinase

Blum, SC., Heaton, SN., Bowman, BM., Hegsted, M. & Miller, SC. (2003). Dietary soy protein

Branca, F. (2003). Dietary phyto-oestrogens and bone health. *Proc Nutr Soc*, Vol. 62, pp. 877–

Brink, E., Coxam, V., Robins, S., Wahala, K., Cassidy, A. & Branca, F. (2008). Long-term

Canalis, E., McCarthy, T. & Centrella, M. (1988). Growth factors and the regulation of bone

*Calcif Tissue Int*, Vol. 70, pp. 483-487

Dawley rats. *Menopause*, Vol. 12, pp. 165-173

mice. *J Mol Endocrinol*, Vol. 42, pp. 269-282

ovariectomized rats. *J Cell Biochem*, Vol. 61, pp. 629-637

ovariectomized rats*. J Nutr*, Vol. 133, pp. 1244-1249

remodeling. *J Clin Invest*, Vol. 81, pp. 277-281

bone.*Trends Endocrinol Metab*, Vol. 14, pp. 356-164

women. *Nutr J,* Vol. 4, pp. 8

pp. 5067- 5070

pp. 501-512

887

761–770

negative regulator of osteoclastogenesis and osteoclast resorbing activity: Increased resorption and osteopenia in mev/mev mutant mice. Bone, Vol. 25, pp. 261-267 Arjmandi, BH., Khalil, DA. & Hollis, BW. (2002). Soy protein: its effects on intestinal calcium

transport, serum vitamin D, and insulin-like growth factor-I in ovariectomized rats.

AB., Payton, ME. & Mason, C. (2005). One year soy protein supplementation has positive effects on bone formation markers but not bone density in postmenopausal

JP. (1997). Differential expression of estrogen receptors alpha and beta mRNA during differentiation of human osteoblast SV-HFO cells. *Endocrinology,* Vol. 138,

Jeffery, EH. (2005). Dietary soy protein and isoflavones: minimal effects on bone and no effect on the reproductive tract of sexually mature ovariectomized Sprague-

Flamant , F., Samarut, J., Costagliola, S., Vassart, G., Weiss, RE., Refetoff, S. & Williams, GR. (2008). A lack of thyroid hormones rather than excess thyrotropin causes abnormal skeletal development in hypothyroidism. *Mol Endocrinol*, Vol. 22,

inhibitors on avian osteoclastic activity and reduction of bone loss in

maintains some indices of bone mineral density and bone formation in

consumption of isoflavone-enriched foods does not affect bone mineral density, bone metabolism, or hormonal status in early postmenopausal women: a randomized, double-blind, placebo controlled study. *Am J Clin Nutr*, Vol. 87, pp.


The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 755

Filipović, B., Šošić -Jurjević, B., Manojlović Stojanoski, M., Nestorović, N., Milošević V. &

Filipović, B., Šošić -Jurjević, B., Ajdžanović, V., Trifunović, S., Manojlović Stojanoski, M.,

Filipović, B., Šošić -Jurjević, B., Ajdzanović, V., Brkić, D., Manojlović-Stojanoski, M.,

Filipović B., Šošić -Jurjević B., Ajdžanović V., Pantelić J., Nestorović N. & Sekulić M. Estardiol

Fitts, JM., Klein, RM. & Powers, CA. (2001). Estrogen and tamoxifen interplay with T(3) in

Fonseca, D. & Ward, WE. (2004). Daidzein together with high calcium preserve bone mass

Frost, HM. (1964). Dynamics of bone remodeling. In: *Frost HM (ed) Bone Biodynamics. Littel,* 

Fuller, K., Wong, B., Fox, S., Choi, Y. & Chambers, TJ. (1998). TRANCE is necessary and

Gaberscek, S., Stiblar-Martincic, D. & Kalisnik, M. (1998). The influence of calcium on thyroid follicular cells FRTL-5 in vitro. *Folia Biol (Praha)*, Vol. 44, pp. 49-52 Gao, YH. & Yamaguchi, M. (1999). Suppressive effect of genistein on rat bone osteoclasts: apoptosis is induced through Ca2+ signaling. *Biol Pharm Bull,* Vol. 22, pp. 805–809 Gao, YH. & Yamaguchi, M. (2000) Suppressive effect of genistein on rat bone osteoclasts:

Geppert, J., Baier, S., Zehn, N., Gouni-Berthold, I., Berthold, HK., Reinsberg, J. & Stehle, P.

Giampietro, PG., Bruno, G., Furcolo, G., Casati, A., Brunetti, E., Spadoni, GL. & Galli, E.

Globus, RK., Plouet, J. & Gospodarowicz, D. (1989). Cultured bovine bone cells synthesize

ovariectomized rats. *Life Sci,* Vol. 77, pp. 121-129

*Histochem Cell Biol*, Vol. 128, pp. 153–159

*Int,* Vol. 21, pp. 1609-1616

*Brown, Boston*, pp 315-333

*Med*, Vol. 188, pp. 997– 1001

activation. *Int J Mol Med*, Vol. 5, pp. 261-267

*Pediatr Endocrinol Metab*, Vol. 17, pp. 191-196

*Endocrinology*, Vol. 124, pp. 1539-1547

pp. 489-497

108

Rouen, France, July 11-15, 2010

*Endocrinology*, Vol. 142, pp. 4223-4235

Sekulić M. (2005). The effect of chronic calcium treatment on thyroid C cells in

Ristić, N., Nestorović, N., Milošević, V. & Sekulić M. (2007) The effect of orchidectomy on thyroid C cells and bone histomorphometry in middle-aged rats.

Milosević, V. & Sekulić M. (2010). Daidzein administration positively affects thyroid C cells and bone structure in orchidectomized middle-aged rats. *Osteoporos* 

effects the function of neuroendocrine C cells in orchidectomized middle-aged rat thyroid gland. (2010a). *The 7th International Congress of Neuroendocrinology*, p. 180,

male rats: pharmacologically distinct classes of estrogen responses affecting growth, bone, and lipid metabolism, and their relation to serum GH and IGF-I.

and biomechanical strength at multiple sites in ovariectomized mice. *Bone*, Vol. 35,

sufficient for osteoblast-mediated activation of bone resorption in osteoclasts. *J Exp* 

involvement of protein kinase inhibition and protein tyrosine phosphatase

(2004). Short-term effects of high soy supplementation on sex hormones, bone markers, and lipid parameters in young female adults. *Eur J Nutr*, Vol. 43, pp. 100–

(2004). Soy protein formulas in children: no hormonal effects in long-term feeding. *J* 

basic fibroblast growth factor and store it in their extracellular matrix.


Dang, ZC. & Lowik, C. (2004). The balance between concurrent activation of ERs and PPARs

Davies, J., Warwick, J., Totty, N., Philp, R., Helfrich, M. & Horton, M. (1989). The osteoclast

DeWilde, A., Lieberherr, M., Colin, C. & Pointillart, A. (2004). A low dose of daidzein acts

Divi, RL., Chang, HC. & Doerge, DR. (1997). Anti-thyroid isoflavones from soybean:

Doerge, DR. & Chang, HC. (2002). Inactivation of thyroid peroxidase by soy isoflavones, in

Donda, A. & Lemarchand-Béraud, T. (1989). Aging alters the activity of 5'-deiodinase in the

Eriksen, EF., Hodgson, SF., Eastell, R., Cedel, SL., O'Fallon, WM. & Riggs, BL. (1990).

Erlandsson, MC., Islander, U., Moverare, S., Ohlsson, C. & Carlsten, H. (2005). Estrogenic

Farley, JR., Tarbaux, NM., Hall, SL., Linkhart, TA. & Baylink, DJ. (1988). The anti-bone-

Farley, JR., Hall, SL., Herring, S. & Tarbaux, NM. (1992). Two biochemical indices of mouse

Farley, J., Dimai, HP., Stilt-Coffing, B., Farley, P., Pham, T. & Mohan, S. (2000). Calcitonin

Filipović, B., Šošić -Jurjević, B., Manojlović-Stojanoski, M., Nestorović, N., Milošević, V. &

Filipović, B., Šošić -Jurjević, B., Nestorović, N., Manojlović Stojanoski, M., Kostić N.,

related to the vitronectin receptor. *J Cell Biol*, Vol. 109, pp. 1817-1826 Davies, TF, Ando, T., Lin, RY., Tomer, Y. & Latif, R. (2005). Thyrotropin receptor-associated diseases: from adenomata to Graves disease. *J Clin Invest*, Vol. 115, pp. 1972-1983 Davison, S. & Davis, SR. (2003). Hormone replacement therapy: current controversies. *Clin* 

19, pp. 853–861

pp. 1087-1096

pp. 1305-1309

Vol. 50, pp. 67–73

*Endocrinology,* Vol. 58, pp. 249–261

*Cell Physiol*, Vol. 200, pp. 253–262

levels. *J Bone Miner Res*, Vol. 5, pp. 311-319

Y*ugoslov Med Biohem*, Vol. 21, pp. 345-350

lymphopoiesis in mice. *APMIS*, Vol. 113, pp. 317-323

bone cell proliferation. *Endocrinology*, Vol. 123, pp. 159–167

human osteoblast-line cells. *Calcif Tissue Int,* Vol. 67, pp. 247–254

with estradiol. *Histochem Cell Biol,* Vol. 120, pp. 409-414

determines daidzein-induced osteogenesis and adipogenesis. *J Bone Miner Res*, Vol.

functional antigen, implicated in the regulation of bone resorption, is biochemically

as an ER beta-selective agonist in trabecular osteoblasts of young female piglets. *J* 

isolation, characterization, and mechanisms of action. *Biochem Pharmacol*, Vol. 54,

vitro and in vivo. *J Chromatogr B Analyt Technol Biomed Life Sc*, Vol. 777, pp. 269-279

adenohypophysis, thyroid gland, and liver of the male rat. *Endocrinology*. Vol. 124,

Cancellous bone remodeling in type I (postmenopausal) osteoporosis: quantitative assessment of rates of formation, resorption, and bone loss at tissue and cellular

agonism and antagonism of the soy isoflavone genistein in uterus, bone and

resorptive agent calcitonin also acts in vitro to directly increase bone formation and

bone formation are increased, in vivo, in response to calcitonin. *Calcif Tissue Int*,

increases the concentration of insulin-like growth factors in serum-free cultures of

Sekulić, M. (2002). The effect of ovariectomy on thyroid C cells of adult rats.

Milošević, V. & Sekulić M. (2003). The thyroid C cells of ovariectomized rats treated


The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 757

Ishimi, Y., Yoshida, M., Wakimoto, S., Wu, J., Chiba, H., Wang, X Takeda, K. & Miyaura C.

Ito, N., Yamazaki, H., Nakazaki, M., Miyahara, T., Kozuka, H. & Sudo, H. (1987). Response

Jia, TL., Wang, HZ., Xie, LP., Wang, XY. & Zhang, RQ. (2003). Daidzein enhances osteoblast

Kanis, JA., Aaron, JE., Evans, D., Thavarajah, M. & Beneton, M. (1990). Bone loss and age-

Katagiri, T. & Takahashi, N. (2002). Regulatory mechanisms of osteoblast and osteoclast

Kennedy, JW. & Caro, JF. (1996). The ABC of managing hyperthyroidism in the older

Khalil, DA., Lucas, EA., Smith, BJ., Soung, DY., Devareddy, L., Juma, S., Akhter, MP.,

Khosla, S., Atkinson, EJ., Melton, LJ III. & Riggs, BL. (1997). Effects of age and estrogen

Kim, CH., Takai, E., Zhou, H., von Stechow, D., Müller, R., Dempster, DW. & Guo, XE.

Kimura, S., Suwa, J., Ito, M. (1976). Sato, H. Development of malignant goiter by defatted

Korach, KS. (1994). Insights from the study of animals lacking functional estrogen receptor.

Kuiper, GG., Lemmen, JG., Carlsson, B., Corton, JC., Safe, SH., van der Saag, PT., van der

Lakatos, P. (2003). Thyroid hormones: beneficial or deleterious for bone? *Calcif Tissue Int*,

Lee, CA. & Einhorn, T. (2001). In: *Osteoporosis,* edited by Marcus, Feldman & Kelsey, pp. 3-20

soybean with iodine-free diet in rats. *Gann*, Vol. 67, pp. 763-765

Recker, R. & Arjmandi, BH. (2005). Soy isoflavones may protect against orchidectomy-induced bone loss in aged male rats. *Calcif Tissue Int.* Vol. 76, pp. 56–

status on serum parathyroid hormone levels and biochemical markers of bone turnover in women: a populationbased study. *J Clin Endocrinol Metab*, Vol. 82, pp.

(2003). Trabecular bone response to mechanical and parathyroid hormone stimulation: the role of mechanical microenvironment. *J Bone Miner Res*, Vol. 18, pp.

Burg, B. & Gustafsson, JA. (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. *Endocrinology,* Vol. 139, pp. 4252–4263 Kuiper, GG., Lemmen, JG., Carlsson, B., Corton, JC., Safe, SH., van der Saag, PT., van der

Burg, B. & Gustafsson, JA. (1998). Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta. *Endocrinology*, Vol. 139, pp. 4252–4263 Kung, AWC. (1994). The effect of thyroid hormone on bone metabolism and osteoporosis. *J* 

prevents bone loss in castrated male mice. *Bone*, Vol. 31, pp. 180-185

density or differentiation stage. *Calcif Tissue Int*, Vol. 40, pp. 200–205 Jabbar, MA., Larrea, J. & Shaw, RA. (1997). Abnormal thyroid function tests in infants with

production. *Biochem Pharmacol*, Vol. 65, pp. 709–715

related fractures. *Exp Gerontol*, Vol. 25, pp. 289-296

differentiation. *Oral Dis*, Vol. 8, pp. 147– 159

patient. *Geriatrics*, Vol. 51, pp. 22-32

*Science*, Vol. 266, pp. 1524-1527

Vol. 73, pp. 205-209

*Hong Kong Med Assoc*, Vol. 46, pp. 247-251

16, pp. 280-282

62

1522–1527

2116-2125

(2002). Genistein, a soybean isoflavone, affects bone marrow lymphopoiesis and

of osteoblastic clonal cell line (MC3T3-E1) to [Asu]eel calcitonin at a specific cell

congenital hypothyroidism: the influence of soy-based formula. *J Am Coll Nutr*, Vol.

growth that may be mediated by increased bone morphogenetic protein (BMP)


Gogakos, AI., Duncan Bassett, JH. & Williams, GR. (2010). Thyroid and bone. *Arch Biochem* 

Grauer, A., Klein, P., Naveh-Many, T., Silver, J., Ziegler, R. & Raue, F. (1993). Diminished

Grumbach, MM. & Auchus, RJ. (1999). Estrogen: consequences and implications of human mutations in synthesis and action. *J Clin Endocrinol Metab*, Vol. 84, pp. 4677-4694 Ham, KD. & Carlson CS. (2004). Effects of estrogen replacement therapy on bone turnover in

Henderson, KM., McNeilly, AS. & Swanston, IA. (1982). Gonadotrophin and steroid

Ho, SC., Woo, J., Lam, S., Chen, YM., Sham, A. & Lau, J. (2003). Soy protein consumption

Hsu, HL., Lacey, DL., Dunstan, CR., Solovyev, I., Colombero, A., Timms, E., Tan, HL.,

Huang, YF., Cao, SM., Nagamani, M., Anderson, KE., Grady, JJ. & Lu, LJW. (2005).

Huang, HY., Yang, HP., Yang, HT., Yang, TC., Shieh, MJ. & Huang, SY. (2006). One-year soy

Hunter, SJ., Schraer, H. & Gay, CV. (1989). Characterization of the cytoskeleton of isolated chick osteoclasts: effect of calcitonin. *J Histochem Cytochem,* Vol. 37, pp. 1529–1537 Ikeda, T., Nishikawa, A., Imazawa, T., Kimura, S. & Hirose, M. (2000). Dramatic synergism

Isaia, GC., Campagnoli, C., Mussetta, M., Massobrio, M., Salamono, G., Gallio, M. &

Isaia, GC., Mussetta, M., Massobrio, M., Sciolla, A., Gallio, M. & Molinatti, GM. (1992). Influence of estrogens on calcitonin secretion. *J Endocrinol Invest*, Vol. 15, pp. 59–62

Ishimi, Y., Miyaura, C., Ohmura, M., Onoe, Y., Sato, T., Uchiyama, Y., Ito, M., Wang, X.,

dosedependent effect. *J Nutr Biochem,* Vol. 17, pp. 509–517

thyroid hyperplasia. *Carcinogenesis*, Vol. 21, pp. 707-713

content in the rat. *Horm Metab Res*, Vol. 25, pp. 389–390

cynomolgus monkeys. *J Bone Miner Res*, Vol. 19, pp. 823-829

calcitonin secretion after ovariectomy without apparent reduction in calcitonin

subchondral bone and epiphyseal metaphyseal cancellous bone of ovariectomized

concentrations in bovine follicular fluid and their relationship to follicle size. *J* 

and bone mass in early postmenopausal Chinese women. *Osteoporos Int,* Vol. 14,

Elliott, G., Kelley, MJ., Sarosi, I., Wang, L., Xia, XZ., Elliott, R., Chiu, L., Black, T., Scully, S., Capparelli, C., Morony, S., Shimamoto, G., Bass, MB. & Boyle, WJ. (1999). Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. *Proc Nat Acad Sci*,

Decreased circulating levels of tumor necrosis factor-alpha in postmenopausal women during consumption of soy-containing isoflavones. *J Clin Endocr Metab*, Vol.

isoflavone supplementation prevents early postmenopausal bone loss but without a

between excess soybean intake and iodine deficiency on the development of rat

Molinatti, GM. (1989). Calcitonin and lumbar bone mineral content during oestrogen-progesterone administration in postmenopausal women. *Maturitas*, Vol.

Suda, T. & Ikegami, S. (1999). Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency. *Endocrinology*, Vol.

Biophys, Vol. 503, pp. 129-136

*Reprod Fertil*, Vol. 65, pp. 467-473

pp. 835–842

Vol. 96, pp. 3540–3545

90, pp. 3956–3962

11, pp. 287–294

140, pp. 1893-1900


The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 759

Okura, A., Arakawa, H., Oka, H., Yoshinari, T. & Monden, Y. (1998). Effect of genistein on

Om, AS. & Shim, JY. (2007). Effect of daidzein, a soy isoflavone, on bone metabolism in Cd-

Onoe, Y., Miyaura, C., Ohta, H., Nozawa, S. & Suda, T. (1997). Expression of estrogen

O'Shea, PJ., Harvey, CB., Suzuki, H., Kaneshige, M., Kaneshige, K., Cheng, SY. & Williams,

with resistance to thyroid hormone. *Mol Endocrinol*, Vol. 17, pp. 1410-1424 Pan, W., Quarles, LD., Song, LH., Yu, YH., Jiao, C., Tang, HB., Jiang, CH., Deng, HW., Li,

Pantelić, J., Filipović, B., Šošić -Jurjević, B., Medigović, I. & Sekulić, M. (2010). Effects of

Patisaul, HB. & Jefferson, W. (2010). The pros and cons of phytoestrogens. *Front* 

Peeters, RP., van der Deure, WM. & Visser, TJ. (2006). Genetic variation in thyroid hormone

Pereira, RC., Jorgetti, V. & Canalis, E. (1999). Triiodothyronine induces collagenase-3 and gelatinase B expression in murine osteoblasts. *Am J Physiol*, Vol. 77, pp. E496-E504 Picherit, C., Bennetau-Pelissero, C., Chanteranne, B., Lebecque, P., Davicco, MJ., Barlet, JP. &

Poirier, LA., Doerge, DR., Gaylor, DW, Miller, MA, Lorentzen, RJ., Casciano, DA., Kadlubar,

Ramos, S. (2007). Effects of dietary flavonoids on apoptotic pathways related to cancer

Rassi, CM., Lieberherr, M., Chaumaz, G., Pointillart, A. & Cournot, G. (2002). Down-

Register, TC., Jayo, MJ. & Anthony, MS. (2003). Soy phytoestrogens do not prevent bone loss in postmenopausal monkeys. *J Clin Endocrinol Metab*, Vol. 88, pp. 4362-4370 Rehman, HU. & Masson, EA. (2005). Neuroendocrinology of female aging. *Gender Medicine*,

Reinholt, FP., Hultenby, K., Oldberg, A. & Heinegård, D. (1990). Osteopontin--a possible anchor of osteoclasts to bone. *Proc Natl Acad Sci U S A*, Vol. 87, pp. 4473-4475

treated ovariectomized rats. *Acta Biochim Pol,* Vol. 54, pp. 641-646

cells. *Biochem Biophys Res Commun*, Vol. 157, pp. 183-189

receptor in rat bone. *Endocrinology,* Vol. 138, pp. 4509-4512

pp. 143-144, Belgrade, Serbia, October 11-12, 2010

deiodinases. *Eur J Endocrinol*, Vol. 155, pp. 655-662

chemoprevention. *J Nutr Biochem*, Vol. 18, pp. 427-442

*Neuroendocrinol*, Vol. 31, pp. 400-419

*Pharmacol*, Vol. 30 pp. 217-222

Vol. 17, pp. 630-638

Vol. 2, pp. 41-56

307–316

pp. 723-728

topoisomerase activity and on the growth of [val 12] Ha-ras transformed NIH 3T3

GR. (2003). A thyrotoxic skeletal phenotype of advanced bone formation in mice

YJ., Zhou, HH. & Xiao, ZS. (2005). Genistein stimulates the osteoblastic differentiation via NO/cGMP in bone marrow culture. *J Cell Biochem*, Vol. 94, pp.

testosterone and estradiol treatment on bone histomorphometry in orchidectomized middle-aged rats. *Proceedings of 4th Serbian Congress for Microscopy,*

pathway genes; polymorphisms in the TSH receptor and the iodothyronine

Coxam, V. (2001). Soybean isoflavones dose-dependently reduce bone turnover but do not reverse established osteopenia in adult ovariectomized rats. *J Nutr*, Vol. 131,

FF. & Schwetz, BA. (1999). An FDA review of sulfamethazine toxicity. *Regul Toxicol* 

regulation of osteoclast differentiation by daidzein via caspase 3. *J Bone Miner Res,*


Lee, YB., Lee, HJ., Kim, KS., Lee, JY., Nam, SY., Cheon, SH. & Sohn HS. (2004). Evaluation of

Li, BB. & Yu, SF. (2003). Genistein prevents bone resorption diseases by inhibiting bone resorption and stimulating bone formation. *Biol Pharm Bull*, Vol. 26, pp. 780–786 Lindsay, R., Hart, DM., Aitken, JM., MacDonald, ED., Anderson, JB. & Clarke, AC. (1976).

Lindsay, R., Hart, DM. & Clark, DM. (1984). The minimum effective dose of estrogen for prevention of postmenopausal bone loss. *Obstet Gynecol*, Vol. 63, pp. 759–763 Loughlin, K. & Richie, J. (1997). Prostate cancer after exogenous testosterone treatment for

Lu, CC., Tsai, SC., Chien, EJ., Tsai, CL. & Wang, PS. (2000). Age-related differences in the

Lydeking-Olsen, E., Beck-Jensen, JE., Setchell, KD. & Holm-Jensen, T. (2004). Soymilk or

Maugeri, D., Salvatore Russo, M., Carnazzo, G., Di Stefano, F., Catanzaro, S., Campagna, S.,

Milne, M., Quail, JM., Rosen, CJ. & Baran, DT. (2001). Insulin-like growth factor binding

Mori, K., Stone, S., Braverman, LE. & Devito, WJ. (1996). Involvement of tyrosine

Mundy, GR. (1995). Local control of bone formation by osteoblasts. *Clin Orthop Relat Res*,

Nakai, M., Cook, L., Pyter, LM., Black, M., Sibona, J., Turner, RT., Jeffery, EH. & Bahr, JM.

Nelson, HD., Humphrey, LL., Nygren, P., Teutsch, SM. & Allan, JD. (2002). Postmenopausal hormone replacement therapy: scientific review. *JAMA*, Vol. 288, pp. 872–881 Newton, KM., LaCroix, AZ., Levy, L., Li, SS., Qu, P., Potter, JD. & Lampe, JW. (2006) Soy

Okamoto, F., Okabe, K. & Kajiya, H. (2001). Genistein, a soybean isoflavone, inhibits inward rectifier K+ channels in rat osteoclasts. *Jap J Physiol*, Vol. 51, pp. 501–509

McCarrison, R. (1993). A Paper on FOOD AND GOITRE. *Br Med J,* Vol. 14, pp. 671-675 Mei, J., Yeung, SS. & Kung, AW. (2001). High dietary phytoestrogen intake is associated

women. *J Clin Endocrinol Metab*, Vol. 86, pp. 5217-5221

rat astrocytes. *Endocrinology*, Vol. 137, pp. 1313-1318

female rats. *Menopause*, Vol. 12, pp. 291-298

*Maturitas,* Vol. 55, pp. 270–277

progesterone for prevention of bone loss: a 2 year randomized, placebo- ontrolled

Romano, G., Franze, C., Motta, M., Panebianco, P. (1996). Altered laboratory thyroid parameters indicating hyperthyroidism in elderly subjects. *Arch Gerontol* 

with higher bone mineral density in postmenopausal but not premenopausal

proteins in femoral and vertebral bone marrow stromal cells: expression and regulation by thyroid hormone and dexamethasone. *J Cell Biochem*, Vol. 81, pp. 229-

phosphorylation in the regulation of 5'-deiodinases in FRTL-5 rat thyroid cells and

(2005). Dietary soy protein and isoflavones have no significant effect on bone and a potentially negative effect on the uterus of sexually mature intact Sprague-Dawley

protein and bone mineral density in older men and women: a randomized trial.

secretion of calcitonin in male rats. *Metabolism*, Vol. 49, pp. 253–258

*Bioscience, Biotechnology and Biochemistry,* Vol. 68, pp. 1040-1045

pp. 1038–1041

impotence. *J Urology,* Vol. 157, pp.1845

trial. *Eur J Nutr*. Vol. 43, pp. 246–257

*Geriatr*, Vol. 22, pp. 145-153

240

Vol. 313, pp. 19-26

the preventive effect of isoflavone extract on bone loss in ovariectomized rats.

Long-term prevention of postmenopausal osteoporosis by oestrogen. *Lancet,* Vol. 1,


The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 761

Sekulić, M., Šošić -Jurjević, B., Filipović, B., Ajdzanović, V., Pantelić, J., Nestorović, N.,

Sendak, RA., Sampath, TK. & McPherson, JM. (2007). Newly reported roles of thyroid-

Setchell, KD. (1998). Phytoestrogens: the biochemistry, physiology, and implications for human health of soy isoflavones. *Am J Clin* Nutr, Vol. 68, pp. 1333S-1346S Setchell, KDR., Brown, NM. & Lydeking-Olsen, E. (2002). The clinical importance of the

Šošić -Jurjević, B., Filipović, B., Nestorović, N., Lovren, M. & Sekulić, M. (2002), Effect of

Šošić -Jurjević, B., Filipović, B., Milosević, V., Nestorović, N., Manojlović-Stojanoski, M.,

Šošić -Jurjević, B., Filipović, B., Milosević, V., Nestorović, N., Negić, N. & Sekulić, M. (2006a).

Šošić -Jurjević, B., Filipović, B., Stojanoski-Manojlović, M. & Sekulić, M. (2006). Calcium

Šošić -Jurjević, B., Filipović, B., Ajdzanović, V., Savin, S., Nestorović, N., Milosević, V. &

Soung, DY., Devareddy, L., Khalil, DA., Hooshmand, S., Patade, A., Lucas, EA. & Arjmandi,

Suchartwatnachai, C., Thepppisai, U. & Jirapinyo, M. (2002). Screening for hypothyroidism

Sugimoto, E. & Yamaguchi, M. (2000). Anabolic effect of genistein in osteoblastic MC3T3-E1

Sugimoto, E. & Yamaguchi, M. (2000a). Stimulatory effect of Daidzein in osteoblastic

Suh, KS., Koh, G., Park, CY., Woo, JT., Kim, SW., Kim, JW., Park, IK. & Kim, YS. (2003).

Soybean isoflavones inhibit tumor necrosis factor-alpha-induced apoptosis and the production of interleukin-6 and prostaglandin E-2 in osteoblastic cells.

at a menopause clinic. *Int J Gynaecol Obstet*, Vol. 77, pp. 39-40

MC3T3-E1 cells. *Biochem Pharmacol,* Vol. 59, pp. 471-475

rats. *14th International Thyroid Congress, Paris* p. 0302

aged rat females. *Jug Med Biochem*, Vol. 21, pp. 261-267

axis in adult rats. *Life Sci*, Vol. 79, pp. 890-897

*Biological Sciences*, Vol. 58, pp. 31-32

cells. *Int J Mol Med,* Vol. 5, pp. 515-520

*Phytochemistry*, Vol. 63, pp. 209–215

*Orthop*, Vol. 31, pp. 753-757

132, pp. 3577-3584

*Res*, Vol. 63, pp. 48-54

235, pp. 590-598

Vol. 78, pp. 385–391

44-49

histochemical, stereological, and ultrastructural study. *Microsc Res Tech*, Vol. 70, pp.

Manojlović–Stojanoski, M. & Milosević V. (2010). Testosterone and estradiol differently affect thyroid structure and function i orchidectomized middle—aged

stimulating hormone and follicle-stimulating hormone in bone remodelling. *Int* 

metabolite equolVa clue to the effectiveness of soy and its isoflavones. *J Nutr*, Vol.

calcium on structural and morphometric features of thyroid gland tissue in middle-

Brkić, B. & Sekulić, M. (2005). Chronic estradiol exposure modulates thyroid structure and decreases T4 and T3 serum levels in middle-aged female rats. *Horm* 

Effects of ovariectomy and chronic estradiol administration on pituitary-thyroid

administration decreases thyroid functioning in middle-aged female rats *Archives of* 

Sekulić, M. (2010). Suppressive effects of genistein and daidzein on pituitarythyroid axis in orchidectomized middle-aged rats. *Exp Biol Med (Maywood)*, Vol.

BH. (2006). Soy affects trabecular microarchitecture and favorably alters select bone-specific gene expressions in a male rat model of osteoporosis. *Calcif Tissue Int*,


Ren, P., Ji, H., Shao, Q., Chen, X., Han, J. & Sun, Y. (2007).Protective effects of sodium

Reymond, F., Dénéréaz, N. & Lemarchand-Béraud, T. (1992). Thyrotropin action is impaired in the thyroid gland of old rats. *Acta Endocrinol (Copenh)*, Vol. 126, pp. 55-63 Rivkees, SA., Bode, HH. & Crawford, JD. (1988). Long-term growth in juvenile acquired

Rizzoli, R., Poser, J. & Bürgi, U. (1986). Nuclear thyroid hormone receptors in cultured bone

Roef, G., Lapauw, B., Goemaere, S., Zmierczak, H., Fiers, T., Kaufman, J.M. & Taes Y. (2011).

Ron, E., Kleinerman, RA., Boice, JDJr., LiVolsi, VA., Flannery, JT. & Fraumeni, JFJr. (1987). A

Rydziel, S., Shaikh, S. & Canalis, E. (1994). Platelet-derived growth factor-AA and -BB

Sakai, K., Yamada, S. & Yamada, K. (2000). Effect of ovariectomy on parafollicular cells in

Sampath, TK., Simic, P., Sendak, R., Draca, N., Bowe, AE., O'Brien, S., Schiavi, SC.,

Schindler, AE. (2003). Thyroid function and postmenopause. *Gynecol* Endocrinol, Vol. 17, pp.

Schmutzler, C., Hamann, I., Hofmann, PJ., Kovacs, G., Stemmler, L., Mentrup, B.,

Schweizer, U., Braun, D., Köhrle, J. & Hershman J. (2010). Tyrosine kinase inhibitors non –

Sekulić, M., Šošić -Jurjević, B., Filipović, B., Milošević, V., Nestorović, N. & Manojlović-

pp. 129-136

pp. 599-602

1027-1034

pp. 1-12

79-85

cells. *Metabolism*, Vol. 35, pp. 71-74

*Endocrinology*, Vol. 134, pp. 2541-2546

*Res*, Vol. 22, pp. 849-859

the rat. *Okajimas Folia Anat Jpn*, Vol. 76, pp. 311–319

heart and kidney. *Toxicology*, Vol. 205, pp. 95-102

Thyroid Congress, Paris, 11-16 September, LB-12

daidzein sulfonate on trabecular bone in ovariectomized rats. *Pharmacology,* Vol. 79,

hypothyroidism: the failure to achieve normal adult stature. *N Engl J Med*, Vol. 318,

Thyroid hormone status within the physiological range affects bone mass and density in healthy men at the age of peak bone mass. *Eur J Endocrinol*, Vol. 164, pp.

population-based case-control study of thyroid cancer. *J Natl Cancer Inst*, Vol. 79,

(PDGF-AA and -BB) enhance the synthesis of PDGF-AA in bone cell cultures.

McPherson, JM. & Vukicevic, S. (2007). Thyroid-stimulating hormone restores bone volume, microarchitecture, and strength in aged ovariectomized rats. *J Bone Miner* 

Schomburg, L., Ambrugger, P., Grüters, A., Seidlova-Wuttke, D., Jarry, H., Wuttke, W. & Köhrle, J. (2004). Endocrine active compounds affect thyrotropin and thyroid hormone levels in serum as well as endpoints of thyroid hormone action in liver,

competitively inhibit MCT8-mediated iodothyronine transport. 14th International

Stojanoski, M. (2005). The effects of synthetic salmon calcitonin on thyroid C and follicular cells in adult female rats. *Folia Histochem Cytobiol*. Vol. 43, pp. 103-108 Sekulić, M., Šošić -Jurjević, B., Filipović, B., Manojlović-Stojanoski, M. & Milosević, V. (2006).

Immunoreactive TSH cells in juvenile and peripubertal rats after estradiol and human chorionic gonadotropin treatment. *Acta Histochem*, Vol. 108, pp. 117-123 Sekulić, M., Šošić -Jurjević, B., Filipović, B., Nestorović, N., Negić, N., Stojanoski, MM. &

Milosević, V. (2007). Effect of estradiol and progesterone on thyroid gland in pigs: a

histochemical, stereological, and ultrastructural study. *Microsc Res Tech*, Vol. 70, pp. 44-49


The Phytoestrogens, Calcitonin and Thyroid Hormones: Effects on Bone Tissue 763

Varga, F., Spitzer, S. & Klaushofer, K. (2004). Triiodothyronine (T3) and 1,25-

3043–3048

Vol. 4, pp. 1791–1798

Vol. 43, pp. 126-134

53, pp. 942-948

dihydroxyvitamin D3 (1,25D3) inversely regulate OPG gene expression in dependence of the osteoblastic phenotype. *Calcif Tissue Int*, Vol. 74, pp. 382-387 Villa, I., Dal,Fiume, C., Maestroni, A., Rubinacci, A., Ravasi, F. & Guidobono, F. (2003).

Human osteoblast-like cell proliferation induced by calcitonin-related peptides involves PKC activity. *Am J Physiol Endocrinol Metab*, Vol. 284, pp. E627–E633 Wangen, KE., Duncan, AM., Merz-Demlow, BE., Xu, X., Marcus, R., Phipps, WR. & Kurzer,

MS. (2000). Effects of soy isoflavones on markers of bone turnover in premenopausal and postmenopausal women. *J Clin Endocr Metab*, Vol. 85, pp.

ornithine decarboxylase messenger RNA levels in rat Sertoli cells. *Mol Endocrinol*,

Williams, GR. (2008). Iodothyronine deiodinase enzyme activities in bone. *Bone,* 

modulators inhibit the effects of insulin-like growth factors in

Combined intervention of exercise and genistein preventedand rogen deficiency-

Ishimi, Y. (2004). Combined intervention of soy isoflavone and moderate exercise prevents body fat elevation and bone loss in ovariectomized mice. *Metabolism*, Vol.

S., Yamada, K. & Ishimi, Y. (2006). Effects of isoflavone and exercise on BMD and fat mass in postmenopausal Japanese women: a 1-year randomized placebo-

for osteoclast differentiation factor and OPG in osteogenic stromal cells by genistein: evidence for the involvement of topoisomerase II in osteoclastogenesis.

protein synthesis in osteoblastic MC3T3-E1 cells: activation of aminoacyl-tRNA

Watanabe, K., Takekoshi, S. & Kakudo, K. (1992). Effects of ipriflavone on calcitonin synthesis in C cells of the rat thyroid. *Calcif Tissue Int*, Vol. 51, pp. S27–S29 Weiner, KX. & Dias, JA. (1990). Protein synthesis is required for testosterone to decrease

Williams, AJ., Robson, H., Kester, MH., van Leeuwen, JP., Shalet, SM., Visser, TJ. &

Williams, JP., Jordan, SE., Barnes, S. & Blair, HC. (1998). Tyrosine kinase inhibitor effects on avian osteoclastic acid transport. *Am J Clin Nutr*, Vol. 68, pp. 1369S-1374S Wong, C., Lai, T., Hilly, JM., Stewart, CE. & Farndon, JR. (2002). Selective estrogen receptor

Wu, J., Wang, XX., Chiba, H., Higuchi, M., Takasaki, M., Ohta, A. & Ishimi, Y. (2003).

Wu, J., Wang, X., Chiba, H., Higuchi, M., Nakatani, T., Ezaki, O., Cui, H., Yamada, K. &

Wu, J., Oka, J., Tabata, I., Higuchi, M., Toda, T., Fuku, N., Ezaki, J., Sugiyama, F., Uchiyama,

Yamagishi, T., Otsuka, E. & Hagiwara, H. (2001). Reciprocal control of expression of mRNAs

Yamaguchi, M. & Sugimoto, E. (2000). Stimulatory effect of genistein and daidzein on

Yen, PM. (2001). Physiological and molecular basis of thyroid hormone action. *Physiol Rev*,

hyperparathyroidism. *Surgery*, Vol. 132, pp. 998-1006

controlled trial. *J Bone Miner Res*, Vol. 21, pp. 780–789

synthetase. *Mol Cell Biochem,* Vol. 214, pp. 97-102

*Endocrinology*, Vol. 142, pp. 3632-3637

Vol. 81, pp. 1097-1142

induced bone loss in mice. *J Appl Physiol*, Vol. 94, pp. 335–342


Sun, L., Vukicevic, S., Baliram, R., Yang, G., Sendak, R., McPherson, J., Zhu, L.L., Iqbal, J.,

Teitelbaum, SL. (2000). Bone resorption by osteoclasts. *Science*, Vol. 289, pp. 1504-1508 Tham, DM., Gardner, CD. & Haskell, WL. (1998). Clinical review 97: potential health

mechanistic evidence. *J Clin Endocrinol Metab*, Vol. 83, pp. 2223–2235 Theoleyre, S., Wittrant, Y., Tat, SK., Fortun, Y., Redini, F. & Heymann, D. (2004). The

pp. 4289-4294

*Dev*, Vol. 50, pp. 639-645

factor. *Bone*, Vol. 25, pp. 517–523

function. *J Cell Sci*, Vol. 113, pp. 377-381

*Biophys Res Commun*, Vol. 285, pp. 70-76

475

pp. 401-405

Latif, R., Natrajan, A., Arabi, A., Yamoah, K., Moonga, BS., Gabet, Y., Davies, TF., Bab, I., Abe, E., Sampath, K. & Zaidi, M. (2008). Intermittent recombinant TSH injections prevent ovariectomy-induced bone loss. *Proc Natl Acad Sci USA*, Vol. 105,

benefits of dietary phytoestrogens: a review of the clinical, epidemiological, and

molecular triad OPG/RANK/RANKL: involvement in the orchestration of pathophysiological bone remodeling. *Cytokine Growth Factor Rev*, Vol. 15, pp. 457–

Long-term treatment effects of Pueraria mirifica phytoestrogens on parathyroid hormone and calcium levels in aged menopausal cynomolgus monkeys. *J Reprod* 

Yasuda, H., Goto, M., Tsuda, E., Higashio, K., Gillespie, MT., Martin, TJ. & Suda, T. (1999). Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colony-stimulating

transporters, including MCT8, MCT10, and OATPs, and the effects of genetic

A., Boonen, S., Bouillon, R. & Vanderschueren, D. (2001). Testosterone prevents orchidectomy-induced bone loss in estrogen receptor-alpha knockout mice. *Biochem* 

(1992). Bone and mineral metabolism in aged male rats: short- and long-term effects

Dumont, JE. (2000). Expression of multiple adenylyl cyclase isoforms in human and

Trisomboon, H., Malaivijitnond, S., Suzuki, J., Hamada, Y., Watanabe, G. & Taya, K. (2004).

Tuohy, PG. (2003). Soy infant formula and phytoestrogens. *J Paediatr Child Health*, Vol. 39,

Udagawa, N., Takahashi, N., Jimi, E., Matsuzaki, K., Tsurukai, T., Itoh, K., Nakagawa, N.,

Väänänen, HK., Zhao, H., Mulari, M. & Halleen JM. (2000). The cell biology of osteoclast

van der Deure, WM., Peeters, RP. & Visser, TJ. (2010). Molecular aspects of thyroid hormone

Van Wyk, JJ., Arnold, MB., Wynn, J. & Pepper, F. (1959). The effects of a soybean product on

Vandenput, L., Ederveen, AG., Erben, RG., Stahr, K., Swinnen, JV., Van Herck, E., Verstuyf,

Vanderschueren, D., Van Herck, E., Suiker, AMH., Visser, WJ., Schot, LPC. & Bouillon, R.

Vanderschueren, D., Boonen, S. & Bouillon, R. (1998). Action of androgens versus estrogens

Vanvooren, V., Allgeier, A., Cosson, E., Van Sande, J., Defer, N., Pirlot, M., Hanoune, J. &

variation in these transporters. *J Mol Endocrinol*, Vol. 44, pp. 1-11

thyroid function in humans. *Pediatrics*, Vol. 24, pp. 752-760

of androgen deficiency. *Endocrinology*, Vol. 130, pp. 2906–2916

in male skeletal homeostasis. *Bone*, Vol. 23, pp. 391-394

dog thyroid. *Mol Cell Endocrinol*, Vol. 170, pp. 185-196


**37** 

*Japan* 

Junji Ohtani, Fujita Tadashi,

R.A. Marquez Hernandez, Toshitsugu Kawata,

Masato Kaku, Masahide Motokawa and Kazuo Tanne

*University Graduate School of Biomedical Sciences, Hiroshima,* 

**Nutrition for Enhancing Bone Volume in Mice** 

*Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima* 

It is well known that postmenopausal osteoporosis is related to severe decreases in the serum estrogen levels. Estrogen deficiency produces an imbalance in the bone remodeling balance which is associated with a reduced bone volume and higher fracture risk. Androgen deficiency also produces osteoporosis by directly affecting bone cells. In addition, androgen was demonstrated to be present during the fetal period, and it is considered to play an essential role in the sexual differentiation of males. Therefore, both sex hormones, androgen and estrogen, are great important for bone homeostasis not only in females but also in males. This is further supported by the fact that both males and females express androgen receptor (AR), estrogen receptor-alpha (ER) and estrogen receptor-beta (ER). Androgen and estrogen react with the AR and ER and ERrespectively. It is thus speculated that

Up to present, various studies have examined the roles of estrogen and its receptors to explore potential methods for preventing or treating postmenopausal osteoporosis (Judd et al., 1983; Kousteni et al., 2002; Martin-Milan et al., 2010; Pietschmann et al., 2008). Recent clinical studies have reported that the prevention of hip bone fractures and vertebral deformities is highly pertinent to improving the quality of life for older people (Chang et al., 2004; O'Neill et al., 2009). To avoid these incidents, we have to try to achieve adequate peak bone mass during adolescent growth, it is also important to understand the bone growth, because this is currently an under-investigated area. In adult animal experiments, orchiectomy and ovariectomy reduced both the bone volume and density. Furthermore, the bone volume loss was induced not only in the long bones but also in the mandibular condyles (Fujita et al., 2001). In addition, we have reported that these phenomena were also shown in immature mice (Fujita et al., 2006). Therefore, the influence of sex hormones on bone remodeling has been demonstrated in the craniofacial region. In orthodontic and orthopedic treatment, it is especially, difficult to predict bone growth including craniofacial and mandibular growth during adolescence. The full nature of bone growth, in association

The ratio of osteoporosis patient in Japan based on the diagnosed by Japanese society for bone and mineral research is reported 24% of over fifty-year-old people and it is indicated the number of patients is higher than those in USA and European countries (Orimo, 2000).

they mutually regulate themselves during bone development and remodeling.

with sex hormones remains to be fully elucidated.

**1. Introduction** 

Zaidi, M., Datta, HK., Moonga, BS. & MacIntyre, I. (1990). Evidence that the action of calcitonin on rat osteoclasts is mediated by two G proteins acting via separate postreceptor pathways. *J Endocrinol,* Vol. 126, pp. 473–481

## **Nutrition for Enhancing Bone Volume in Mice**

Junji Ohtani, Fujita Tadashi,

R.A. Marquez Hernandez, Toshitsugu Kawata, Masato Kaku, Masahide Motokawa and Kazuo Tanne *Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan* 

### **1. Introduction**

764 Osteoporosis

Zaidi, M., Datta, HK., Moonga, BS. & MacIntyre, I. (1990). Evidence that the action of

receptor pathways. *J Endocrinol,* Vol. 126, pp. 473–481

calcitonin on rat osteoclasts is mediated by two G proteins acting via separate post-

It is well known that postmenopausal osteoporosis is related to severe decreases in the serum estrogen levels. Estrogen deficiency produces an imbalance in the bone remodeling balance which is associated with a reduced bone volume and higher fracture risk. Androgen deficiency also produces osteoporosis by directly affecting bone cells. In addition, androgen was demonstrated to be present during the fetal period, and it is considered to play an essential role in the sexual differentiation of males. Therefore, both sex hormones, androgen and estrogen, are great important for bone homeostasis not only in females but also in males. This is further supported by the fact that both males and females express androgen receptor (AR), estrogen receptor-alpha (ER) and estrogen receptor-beta (ER). Androgen and estrogen react with the AR and ER and ERrespectively. It is thus speculated that they mutually regulate themselves during bone development and remodeling.

Up to present, various studies have examined the roles of estrogen and its receptors to explore potential methods for preventing or treating postmenopausal osteoporosis (Judd et al., 1983; Kousteni et al., 2002; Martin-Milan et al., 2010; Pietschmann et al., 2008). Recent clinical studies have reported that the prevention of hip bone fractures and vertebral deformities is highly pertinent to improving the quality of life for older people (Chang et al., 2004; O'Neill et al., 2009). To avoid these incidents, we have to try to achieve adequate peak bone mass during adolescent growth, it is also important to understand the bone growth, because this is currently an under-investigated area. In adult animal experiments, orchiectomy and ovariectomy reduced both the bone volume and density. Furthermore, the bone volume loss was induced not only in the long bones but also in the mandibular condyles (Fujita et al., 2001). In addition, we have reported that these phenomena were also shown in immature mice (Fujita et al., 2006). Therefore, the influence of sex hormones on bone remodeling has been demonstrated in the craniofacial region. In orthodontic and orthopedic treatment, it is especially, difficult to predict bone growth including craniofacial and mandibular growth during adolescence. The full nature of bone growth, in association with sex hormones remains to be fully elucidated.

The ratio of osteoporosis patient in Japan based on the diagnosed by Japanese society for bone and mineral research is reported 24% of over fifty-year-old people and it is indicated the number of patients is higher than those in USA and European countries (Orimo, 2000).

Nutrition for Enhancing Bone Volume in Mice 767

clarified that special protein containing many phosphoric acid called CPP. Generally, calcium is the fifth abundant element by mass in the human body, where it is a common cellular ionic messenger with many functions, and serves also as a structural element in bone. And it has four stable isotopes (40Ca, 42Ca, 43Ca and 44Ca) plus two more isotopes (46Ca and 48Ca). 97% of naturally occurring calcium is in the form of 40Ca which is a one of daughter products of 40K decay, along with 40Ar (Mueller Boehm, 2011; Skulan DePaolo, 1999). Calcium is hard to be absorbed in human body since it combines with negative ion such as a phosphoric acid in a small intestine. However, CPP prevents own insoluble and promote its efficiency of absorption in a small intestine due to a part of serine residue is phosphorylated and the ionic bond of CPP is carried out to calcium with

Moreover, the function in the promotion of absorption efficacy of CPP, it is positively applied in the form of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and is widely well-known as Recaldent. CPP from the major protein of milk have the remarkable ability to stabilize calcium, phosphate and fluoride ions as water soluble amorphous complexes that provide bioavairable calcium, phosphate and fluoride ions to the tooth. Animal and short-term human clinical trials to repair early staged of tooth decay by replacing the calcium and phosphate ions lost due to decay (Reynolds, 2009; Llena et al., 2009). Long-term controlled clinical trials also have demonstrated the efficacy of CPP-ACP is slowing the progression of dental caries and in regressing early stages of tooth decay (Andersson et al., 2007; Morgan et al., 2008). Moreover, it was reported that it is effective rather than the 500ppm sodium fluoride paste which the dentists have used conventionally for the purpose of prevention of tooth decay (Zhang et al., 2011;

On the other hand, it was reported that the calcium in food exists 50~60% in milk, approximately 20% in small fish and approximately 30% in vegetable. But, absorption efficacy of calcium in human body is not much excellent. Therefore, CPP is further developed as "food suitable for the people of calcium shortage" such as suger-free gum, tooth cream, dairy milk and another drink and Japanese tofu. However, with such professional tool, food and drink form, we speculated that it will be probably difficult to

Fifty male and fifty female C57BL/6J mice of twelve-week-old and thirty male and thirty female C57BL/6J mice of five-day-old (Jackson Laboratory, Bar Harbar, ME, USA) were used in this experiment and divided respectively into eight groups with ten in each sex. In the experimental groups, orchiectomy (ORX) and ovariectomy (OVX) were performed for ten mice of twelve-week-old in each gender at the beginning of experiment to create the situation of osteoporosis model. ORX and OVX were performed by means of a stereoscopic microscope (SZX9, Olympus Optical Co., Tokyo, Japan) under general anesthesia with sodium pentobarbital, whereas the control mice underwent sham operation. The detail of time schedules in adult and growing mice during a series of experiment are shown in Table 1 and 2, respectively. These animals were treated under the ethical regulations defined by

take freely and daily for the purpose of increasing the absorption efficacy of calcium.

the Ethics Committee, Hiroshima University Faculty of Dentistry.

the negative ion of phosphate group.

Altenburger et al., 2010).

**3. Animal study** 

**3.1 Materials and methods** 

Therefore, we all recognize osteoporosis is one of serious social problems in Japan. This fact indicates that the influences of sex hormones on bone metabolism are very complicated, and the mechanism is very difficult to be understood.

Peak bone mass, in general, is acquired from childhood to adolescence, but 35% of cortical bone and 50% of trabecular bone are lost gradually thereafter after. The importance of preventive medicine thus has been gradually recognized in the field of orthopaedic surgery with a concept that peak bone mass is to be increased and saved during childhood as much as possible (Lapauw et al., 2009; Kaufman, 2009). Such an idea has been attracting a special attention in the field of clinical medicine for the prevention of osteoporosis. The importance of normal development and growth of bone until adolescence has also been reported (Karlsson et al., 2008; Wang et al., 2008). This fact suggested that if we are able to increase the peak bone mass during adolescence as possible by providing some effective nutrition for increasing bone volume, it may leads to prevent the risk and to delay the appearance of osteoporosis.

According to the prevention and treatment of guideline in osteoporosis, it was recommended to intake daily dose of calcium is higher (Rosen Gallagher, 2011). However, the amount of calcium intake is approximately 500mg per one day in practically, and even added the consumption applied by supplement, could not reach daily objective intake.

A newly developed snack used in our experiments contains appropriate amounts of minerals (calcium and magnesium ) and casein phosphopeptide (CPP) as well as soybean isoflavone which has a sex hormone-like action, which is recognized as a specified food for health, is also included. The objective of this chapter was to introduce the effects of bone remodeling including bone volume, density and strength in osteoporosis and healthy growing mice fed a special composition nutrition.

### **2. Nutrition**

#### **2.1 Function of nutrition**

Recently, it was focused on the prevention of osteoporosis and life style related disease like a diabetic and heart disease. Under such situation, people are well considered the food function. It is well known that the function of foodstuff has mainly for nutrition and taste. Additionally, it has also the important function as a decreasing of risk in some disease. The development of foodstuff which has the specific function for living body adjustment is going forward actively in nowadays, therefore the daily food which we intake were diversify. Now it was reported that there are detected and demonstrated in some nutrition that the function of anti-thrombosis, anti-oxidation, encouragement to assimilate calcium, controlling of blood pressure and improvement of cholesterol readings as a food for special health uses and Japanese government had approved it (Yamada et al., 2008). For example, beta-carotene was known as a great nutrition because of its essential function, foodstuff factor as a provitamin, taste factor by its color in vegetable and fruit, and the role of antioxidation (Kim et al., 2010).

#### **2.2 Casein phosphopeptide**

CPP is also well known as a factor of encouragement to assimilate calcium. Milk and dairy products contains CPP together with functional protein like a milky basic protein and lactoferrin. Casein as a milk protein is a protein containing phosphoric acid, and is decomposed into oligopeptide of various sizes by the digestive enzyme. It became

Therefore, we all recognize osteoporosis is one of serious social problems in Japan. This fact indicates that the influences of sex hormones on bone metabolism are very complicated, and

Peak bone mass, in general, is acquired from childhood to adolescence, but 35% of cortical bone and 50% of trabecular bone are lost gradually thereafter after. The importance of preventive medicine thus has been gradually recognized in the field of orthopaedic surgery with a concept that peak bone mass is to be increased and saved during childhood as much as possible (Lapauw et al., 2009; Kaufman, 2009). Such an idea has been attracting a special attention in the field of clinical medicine for the prevention of osteoporosis. The importance of normal development and growth of bone until adolescence has also been reported (Karlsson et al., 2008; Wang et al., 2008). This fact suggested that if we are able to increase the peak bone mass during adolescence as possible by providing some effective nutrition for increasing bone volume, it may leads to prevent the risk and to delay the appearance of

According to the prevention and treatment of guideline in osteoporosis, it was recommended to intake daily dose of calcium is higher (Rosen Gallagher, 2011). However, the amount of calcium intake is approximately 500mg per one day in practically, and even added the consumption applied by supplement, could not reach daily objective intake. A newly developed snack used in our experiments contains appropriate amounts of minerals (calcium and magnesium ) and casein phosphopeptide (CPP) as well as soybean isoflavone which has a sex hormone-like action, which is recognized as a specified food for health, is also included. The objective of this chapter was to introduce the effects of bone remodeling including bone volume, density and strength in osteoporosis and healthy

Recently, it was focused on the prevention of osteoporosis and life style related disease like a diabetic and heart disease. Under such situation, people are well considered the food function. It is well known that the function of foodstuff has mainly for nutrition and taste. Additionally, it has also the important function as a decreasing of risk in some disease. The development of foodstuff which has the specific function for living body adjustment is going forward actively in nowadays, therefore the daily food which we intake were diversify. Now it was reported that there are detected and demonstrated in some nutrition that the function of anti-thrombosis, anti-oxidation, encouragement to assimilate calcium, controlling of blood pressure and improvement of cholesterol readings as a food for special health uses and Japanese government had approved it (Yamada et al., 2008). For example, beta-carotene was known as a great nutrition because of its essential function, foodstuff factor as a provitamin, taste factor by its color in vegetable and fruit, and the role of anti-

CPP is also well known as a factor of encouragement to assimilate calcium. Milk and dairy products contains CPP together with functional protein like a milky basic protein and lactoferrin. Casein as a milk protein is a protein containing phosphoric acid, and is decomposed into oligopeptide of various sizes by the digestive enzyme. It became

the mechanism is very difficult to be understood.

growing mice fed a special composition nutrition.

osteoporosis.

**2. Nutrition** 

**2.1 Function of nutrition** 

oxidation (Kim et al., 2010).

**2.2 Casein phosphopeptide** 

clarified that special protein containing many phosphoric acid called CPP. Generally, calcium is the fifth abundant element by mass in the human body, where it is a common cellular ionic messenger with many functions, and serves also as a structural element in bone. And it has four stable isotopes (40Ca, 42Ca, 43Ca and 44Ca) plus two more isotopes (46Ca and 48Ca). 97% of naturally occurring calcium is in the form of 40Ca which is a one of daughter products of 40K decay, along with 40Ar (Mueller Boehm, 2011; Skulan DePaolo, 1999). Calcium is hard to be absorbed in human body since it combines with negative ion such as a phosphoric acid in a small intestine. However, CPP prevents own insoluble and promote its efficiency of absorption in a small intestine due to a part of serine residue is phosphorylated and the ionic bond of CPP is carried out to calcium with the negative ion of phosphate group.

Moreover, the function in the promotion of absorption efficacy of CPP, it is positively applied in the form of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), and is widely well-known as Recaldent. CPP from the major protein of milk have the remarkable ability to stabilize calcium, phosphate and fluoride ions as water soluble amorphous complexes that provide bioavairable calcium, phosphate and fluoride ions to the tooth. Animal and short-term human clinical trials to repair early staged of tooth decay by replacing the calcium and phosphate ions lost due to decay (Reynolds, 2009; Llena et al., 2009). Long-term controlled clinical trials also have demonstrated the efficacy of CPP-ACP is slowing the progression of dental caries and in regressing early stages of tooth decay (Andersson et al., 2007; Morgan et al., 2008). Moreover, it was reported that it is effective rather than the 500ppm sodium fluoride paste which the dentists have used conventionally for the purpose of prevention of tooth decay (Zhang et al., 2011; Altenburger et al., 2010).

On the other hand, it was reported that the calcium in food exists 50~60% in milk, approximately 20% in small fish and approximately 30% in vegetable. But, absorption efficacy of calcium in human body is not much excellent. Therefore, CPP is further developed as "food suitable for the people of calcium shortage" such as suger-free gum, tooth cream, dairy milk and another drink and Japanese tofu. However, with such professional tool, food and drink form, we speculated that it will be probably difficult to take freely and daily for the purpose of increasing the absorption efficacy of calcium.

### **3. Animal study**

### **3.1 Materials and methods**

Fifty male and fifty female C57BL/6J mice of twelve-week-old and thirty male and thirty female C57BL/6J mice of five-day-old (Jackson Laboratory, Bar Harbar, ME, USA) were used in this experiment and divided respectively into eight groups with ten in each sex. In the experimental groups, orchiectomy (ORX) and ovariectomy (OVX) were performed for ten mice of twelve-week-old in each gender at the beginning of experiment to create the situation of osteoporosis model. ORX and OVX were performed by means of a stereoscopic microscope (SZX9, Olympus Optical Co., Tokyo, Japan) under general anesthesia with sodium pentobarbital, whereas the control mice underwent sham operation. The detail of time schedules in adult and growing mice during a series of experiment are shown in Table 1 and 2, respectively. These animals were treated under the ethical regulations defined by the Ethics Committee, Hiroshima University Faculty of Dentistry.

Nutrition for Enhancing Bone Volume in Mice 769

Figure 1 shows the photographs of femur section examined by pQCT at the end of experiment. Irrespective of the sex differences, the trabecular bone volume surrounded by the thick cortical bone was maintained at high level in the control groups, in sham operated mice fed NCD in particular (group 1). Meanwhile, trabecular bone volume was decreased moderately in the experimental group 2 with LCD as compared with control mice. Furthermore, the group 4, gonadectomized mice fed LCD exhibited an excessive decrease in bone volume as compared with the groups 1 and 2. On the other hand, the femur section in the group 5, osteoporosis model mice given SCD, presented a prominent recovery of

As a result of pQCT analysis, similar phenomena were observed as mentioned above for the femur structures. The trabecular bone density of gonadectomized mice fed SCD was significantly increased as compared with sham opereated mice given LCD in both genders (Fig. 2). Moreover, the bone density was also significantly increased only in the female mice

For the cortical bone density, different findings from above mentioned results were obtained. Improvement effect was especially revealed in the gonadectomized mice given SCD (group 5) (Fig. 3). Additionally, it was also shown in the sham operated mice fed SCD (group 3) compared with the sham operated mice fed LCD (group 2). These tendencies were

**3.2 In ORX and OVX mice** 

trabecular bone volume.

Fig. 1. Photographs of femoral section in adult mice.

observed in both sexes.

of group 5 as compared with sham operated mice fed NCD.


Table 1. Time schedule during experiments in adult mice

Table 2. Time schedule during experiments in growing mice

We prepared three types of powder diet *e.g.*: normal calcium diet (NCD, Ca : 0.9%, Clea Japan Co., Tokyo, Japan), low calcium diet (LCD, Ca : 0.63%, Clea Japan Co.,) and special diet (SCD, Ca : 0.9%) developed as a new bean snack. NCD with 0.9% calcium was made on basis of objective consumption of calcium intake in humans defined by the Ministry of Health, Labour and Welfare (MHLW) in Japan. LCD including 0.63% calcium was made on the basis of actual calcium consumption in Japanese. The SCD containing LCD and the newly developmental snack, was supplied as a diet including calcium content same as NCD. The newly developmental snack was composed of calcium, magnesium, CPP and black soybean. As the mainly composition, 1.3% of calcium, 0.6% of magnesium, 0.15% of CPP and 12.8% of black soybean were included in this snack. NCD was given up to six weeks after sham operation in the group 1 and 6. Other six groups were given LCD until six weeks after gonadectomy. Six weeks after surgery, group 3, 5 and 8 in each sex were given SCD including the newly developed bean snack.

Eighteen weeks after surgery, all the animals were sacrified under general anesthesia and the femur was fixed with 4% formaldehyde and prepared for histomorphometric analysis. Peripheral Quantitive Computed Tomography (XCT Research SA, Stratec Medizintechnik GmbH, Pforzheim, Germany) was used to quantify bone density and bone mineral content. Femur was measured at a point 1.4mm distal area from chondrocyte growth plate. The cortical bone area was defined as over 690mg/cm3 threshold and the trabecular bone area was defined as under 395mg/cm3 threshold. Moreover, bone strength of femur performed by under three point flexural test was also executed in growing mice.

We performed pairwise comparisons (Fisher) to examine the difference in measured values between the groups with a confidence level greater than 95%. All the data are presented as means±standard deviations.

### **3.2 In ORX and OVX mice**

768 Osteoporosis

We prepared three types of powder diet *e.g.*: normal calcium diet (NCD, Ca : 0.9%, Clea Japan Co., Tokyo, Japan), low calcium diet (LCD, Ca : 0.63%, Clea Japan Co.,) and special diet (SCD, Ca : 0.9%) developed as a new bean snack. NCD with 0.9% calcium was made on basis of objective consumption of calcium intake in humans defined by the Ministry of Health, Labour and Welfare (MHLW) in Japan. LCD including 0.63% calcium was made on the basis of actual calcium consumption in Japanese. The SCD containing LCD and the newly developmental snack, was supplied as a diet including calcium content same as NCD. The newly developmental snack was composed of calcium, magnesium, CPP and black soybean. As the mainly composition, 1.3% of calcium, 0.6% of magnesium, 0.15% of CPP and 12.8% of black soybean were included in this snack. NCD was given up to six weeks after sham operation in the group 1 and 6. Other six groups were given LCD until six weeks after gonadectomy. Six weeks after surgery, group 3, 5 and 8 in each sex were given SCD

Eighteen weeks after surgery, all the animals were sacrified under general anesthesia and the femur was fixed with 4% formaldehyde and prepared for histomorphometric analysis. Peripheral Quantitive Computed Tomography (XCT Research SA, Stratec Medizintechnik GmbH, Pforzheim, Germany) was used to quantify bone density and bone mineral content. Femur was measured at a point 1.4mm distal area from chondrocyte growth plate. The cortical bone area was defined as over 690mg/cm3 threshold and the trabecular bone area was defined as under 395mg/cm3 threshold. Moreover, bone strength of femur performed

We performed pairwise comparisons (Fisher) to examine the difference in measured values between the groups with a confidence level greater than 95%. All the data are presented as

by under three point flexural test was also executed in growing mice.

Table 1. Time schedule during experiments in adult mice

Table 2. Time schedule during experiments in growing mice

including the newly developed bean snack.

means±standard deviations.

Figure 1 shows the photographs of femur section examined by pQCT at the end of experiment. Irrespective of the sex differences, the trabecular bone volume surrounded by the thick cortical bone was maintained at high level in the control groups, in sham operated mice fed NCD in particular (group 1). Meanwhile, trabecular bone volume was decreased moderately in the experimental group 2 with LCD as compared with control mice. Furthermore, the group 4, gonadectomized mice fed LCD exhibited an excessive decrease in bone volume as compared with the groups 1 and 2. On the other hand, the femur section in the group 5, osteoporosis model mice given SCD, presented a prominent recovery of trabecular bone volume.

Fig. 1. Photographs of femoral section in adult mice.

As a result of pQCT analysis, similar phenomena were observed as mentioned above for the femur structures. The trabecular bone density of gonadectomized mice fed SCD was significantly increased as compared with sham opereated mice given LCD in both genders (Fig. 2). Moreover, the bone density was also significantly increased only in the female mice of group 5 as compared with sham operated mice fed NCD.

For the cortical bone density, different findings from above mentioned results were obtained. Improvement effect was especially revealed in the gonadectomized mice given SCD (group 5) (Fig. 3). Additionally, it was also shown in the sham operated mice fed SCD (group 3) compared with the sham operated mice fed LCD (group 2). These tendencies were observed in both sexes.

Nutrition for Enhancing Bone Volume in Mice 771

The results of trabecular bone density were shown in Figure 6. The mice fed NCD revealed around 40 to 50 mg/cm3 in density, but by the cause of changing food to LCD, it lead to decrease in both sexes. Under such situation, in case of group 8, mice fed SCD from after six weeks to the end of experiment, trabecular bone density showed significantly increased

Fig. 4. Total bone density in adult mice

Fig. 5. Bone mineral density in adult mice

**3.3 In young mice** 

Fig. 2. Trabecular bone density in adult mice

Fig. 3. Cortical bone density in adult mice

Subsequently, the total bone density was apparently increased as compared with other sham operated mice. The reason may be due to SCD supplied to the gonadectomized mice, especially in the female mice (Fig. 4).

For bone mineral content, the groups 3 and 5 mice given SCD exhibited a significant increase as compared with the groups 1 and 2, especially in female mice. It is demonstrated from these findings that the newly development snack is very effective for the improvement of reduced bone quality and controlling of osteoporosis (Fig. 5).

Subsequently, the total bone density was apparently increased as compared with other sham operated mice. The reason may be due to SCD supplied to the gonadectomized mice,

For bone mineral content, the groups 3 and 5 mice given SCD exhibited a significant increase as compared with the groups 1 and 2, especially in female mice. It is demonstrated from these findings that the newly development snack is very effective for the improvement

Fig. 2. Trabecular bone density in adult mice

Fig. 3. Cortical bone density in adult mice

of reduced bone quality and controlling of osteoporosis (Fig. 5).

especially in the female mice (Fig. 4).

Fig. 4. Total bone density in adult mice

Fig. 5. Bone mineral density in adult mice

#### **3.3 In young mice**

The results of trabecular bone density were shown in Figure 6. The mice fed NCD revealed around 40 to 50 mg/cm3 in density, but by the cause of changing food to LCD, it lead to decrease in both sexes. Under such situation, in case of group 8, mice fed SCD from after six weeks to the end of experiment, trabecular bone density showed significantly increased

Nutrition for Enhancing Bone Volume in Mice 773

In the results of bone mineral content, Male mice after fed SCD showed no significant differences as compared with the control mice (Fig. 9). On the other hand, female mice of group 7 and 8 showed significantly reduced bone mineral content as compared with mice of

According to the results of total bone density and bone mineral content, the results of three point flexural test also showed same tendency in male mice (Fig. 10). Meanwhile, although the results of group 8 showed significant lower than the one of group 6 in total bone density

and bone mineral content, group 7 and 8 demonstrated same level of group 6.

Fig. 8. Total bone density in growing mice

Fig. 9. Bone mineral content in growing mice

group 6.

compared with mice fed LCD, therefore, there is no significant differences between control mice of group 6 and mice fed food which is changing to SCD after feeding LCD of group 8. In the results of cortical bone density, similar phenomena were observed with trabecular bone density, the cortical bone density of mice fed LCD revealed significantly decreased compared with mice given NCD (Fig. 7). However, by the feed changing to SCD, cortical bone density wasn't getting better as contrasted with the results of trabecular bone density and the results of cortical bone density in adult mice. These tendencies were observed in both sexes. As a result, only in female mice, total bone density also showed the sufficient effect of improvement (Fig. 8). There is no significant difference between group 6 and 8 in male mice.

Fig. 6. Trabecular bone density in growing mice

Fig. 7. Cortical bone density in growing mice

compared with mice fed LCD, therefore, there is no significant differences between control mice of group 6 and mice fed food which is changing to SCD after feeding LCD of group 8. In the results of cortical bone density, similar phenomena were observed with trabecular bone density, the cortical bone density of mice fed LCD revealed significantly decreased compared with mice given NCD (Fig. 7). However, by the feed changing to SCD, cortical bone density wasn't getting better as contrasted with the results of trabecular bone density and the results of cortical bone density in adult mice. These tendencies were observed in both sexes. As a result, only in female mice, total bone density also showed the sufficient effect of improvement (Fig.

8). There is no significant difference between group 6 and 8 in male mice.

Fig. 6. Trabecular bone density in growing mice

Fig. 7. Cortical bone density in growing mice

Fig. 8. Total bone density in growing mice

In the results of bone mineral content, Male mice after fed SCD showed no significant differences as compared with the control mice (Fig. 9). On the other hand, female mice of group 7 and 8 showed significantly reduced bone mineral content as compared with mice of group 6.

According to the results of total bone density and bone mineral content, the results of three point flexural test also showed same tendency in male mice (Fig. 10). Meanwhile, although the results of group 8 showed significant lower than the one of group 6 in total bone density and bone mineral content, group 7 and 8 demonstrated same level of group 6.

Fig. 9. Bone mineral content in growing mice

Nutrition for Enhancing Bone Volume in Mice 775

This snack we had developed was composed of calcium, magnesium, CPP and black soybean. We have examined into body weight and height of all mice during experimental period. However, all mice of supplied food in these measurements were no significant difference compared with the control groups in both sexes (data were not shown). In addition, the amount of all nutrition including supplied food was restrained according the instructions of MHLW in Japan. Therefore, we assumed that supplied special diet food has

It is well known that calcium intake decreases the risk of bone fracture. It was also reported that the calcium supplementation suppress bone formation when magnesium is deficient (Mora Gilsanz, 2003). Based on these reports, we designed to mix calcium and magnesium at an appropriate proportion of 2 to 1. These contents in this snack were compounded to

It was also reported that addition of CPP will have a beneficial effect on the absorption of calcium (Dontas Yiannakopoulos, 2007). In Japan, CPP was accredited as a food for qualified health uses by MHLW. Accordingly, we add a certain amount of CPP to absorb

The black soybean contains isoflavone which is widely accepted to have a weak estrogen activity, and to be able to bind estrogen receptor. Actually, Brandi and Miyauchi et al., reported that isoflavone had either effect for the suppression of bone resorption or enhancement of bone formation, affecting directly both osteoclasts and osteoblasts (Brandi, 2003; Miyauchi et al., 1996). In addition, isoflavone was revealed to affect bone metabolism similarly to the sex hormone-like effect in male (Chavarro et al., 2008). Recently, it was also reported that isoflavones have revealed inhibition of bone loss in castrated male mice and growing male mice respectively (Fujioka et al., 2007; Ishimi et al., 2002; Khalil et al., 2005). Under such background, a new snack was developed by use of black soybean. The amount of isoflavone in this snack was determined with a special reference to the amount of safety

Decrease in the trabecular and cortical bone volumes after gonadectomy has already been demonstrated by our previously reports (Fujita et al., 2001, 2004). We also reported that bone growth was significantly suppressed in the gonadectomized mice immediately after birth (Fujita et al., 2006). Moreover, we clarified that decrease in bone volume was occurred four weeks after gonadectomy. According to these results, we examined bone density of the

The density of trabecular and cortical bones in the gonadectomized mice given LCD was significantly lower than in the sham operated mice given NCD and LCD, respectively. It was revealed that the deficiency of calcium intake caused decrease in bone density, under sex hormone disturbances in particular. Therefore, it is speculated that the bone density is below the optimal level in Japanese and fracture risk may become higher for aged people. These findings also support that the incidence of primary osteoporosis is higher in Japan

The bone metabolism is classified into two types, high turnover type to accelerate both bone formation and resorption and low turnover type caused by degradation of bone formation. In this study, twelve-week-old mice with sham operation fed LCD are regarded as young growing humans with low turnover type, whereas gonadectomy mice to simulate sex hormone disturbance in the experimental groups are assumed to be under the condition of

fulfill ninety percentage of the daily-required nutrition defined by MHLW in Japan.

no occurrence of side effect following snack administration.

mineral content easily.

surplus nutrition per day defined by MHLW.

femur six weeks after ovariectomy and orchiectomy.

than in American and European countries.

Fig. 10. Three point flexural test in growing mice

#### **4. Discussion**

Osteoporosis is generally thought of as a disease that affects females, because the prevalence of osteoporosis and the rate of fracture are much higher in postomenomausal females than in older males. However, the absolute number of male patients affected by osteoporosis and fractures has been reported to be large. Older Asian males with low serum estradiol levels also display elevated bone loss and increased risk of fractures similar to the findings in Caucasians (Woo et al., 2011; Moayyeri et al., 2009). The main concern of treatment in osteoporosis research is to prevent bone volume loss by decreasing the progression of bone resorption. Nevertheless, the current knowledge is not sufficient to identify the precise causes of osteoporosis and all of the subjects at risk. As a result, recent studies have indicated that weight-bearing activity and possibly calcium supplements are beneficial if they are begun during childhood, preferably before puberty. The achievement of optimal peak bone mass is important to prevent the risk of bone fracture due to osteoporosis in the future.

The sex hormones are known to be important for the regulation of reproductive functions. They induce sexual differentiation before birth, and sexual maturation during puberty in both genders. They also exert influences on the nervous and cardiovascular systems and are important in the development of the skeletal structure. There are two types of sex hormones, androgens and estrogens. These hormones are secreted mainly from the testis and ovaries. Testosterone can activate the androgen receptor either directly or indirectly after conversion to DHT by 5-reductase. Moreover, it is well known that testosterone is also converted into 17-estradiol by P450 aromatase, which activates the estrogen receptors (alpha and beta). Based on this facts, Ovariectmized and Orchiectomized animals provide an excellent model to study osteoporosis due to estrogen and androgen deficiency in both genders. Both OVX and ORX mice exhibit marked bone loss with increased bone resorption. It was also reported that a loss in bone mineral density in ORX mice was evident between 1 to 4 months post-surgery, and histomorphometric evaluations revealed that this occurred more rapidly and with greater sensitivity in a rat model.

Osteoporosis is generally thought of as a disease that affects females, because the prevalence of osteoporosis and the rate of fracture are much higher in postomenomausal females than in older males. However, the absolute number of male patients affected by osteoporosis and fractures has been reported to be large. Older Asian males with low serum estradiol levels also display elevated bone loss and increased risk of fractures similar to the findings in Caucasians (Woo et al., 2011; Moayyeri et al., 2009). The main concern of treatment in osteoporosis research is to prevent bone volume loss by decreasing the progression of bone resorption. Nevertheless, the current knowledge is not sufficient to identify the precise causes of osteoporosis and all of the subjects at risk. As a result, recent studies have indicated that weight-bearing activity and possibly calcium supplements are beneficial if they are begun during childhood, preferably before puberty. The achievement of optimal peak bone mass is

The sex hormones are known to be important for the regulation of reproductive functions. They induce sexual differentiation before birth, and sexual maturation during puberty in both genders. They also exert influences on the nervous and cardiovascular systems and are important in the development of the skeletal structure. There are two types of sex hormones, androgens and estrogens. These hormones are secreted mainly from the testis and ovaries. Testosterone can activate the androgen receptor either directly or indirectly after conversion to DHT by 5-reductase. Moreover, it is well known that testosterone is also converted into 17-estradiol by P450 aromatase, which activates the estrogen receptors (alpha and beta). Based on this facts, Ovariectmized and Orchiectomized animals provide an excellent model to study osteoporosis due to estrogen and androgen deficiency in both genders. Both OVX and ORX mice exhibit marked bone loss with increased bone resorption. It was also reported that a loss in bone mineral density in ORX mice was evident between 1 to 4 months post-surgery, and histomorphometric evaluations revealed that this occurred more rapidly

important to prevent the risk of bone fracture due to osteoporosis in the future.

Fig. 10. Three point flexural test in growing mice

and with greater sensitivity in a rat model.

**4. Discussion** 

This snack we had developed was composed of calcium, magnesium, CPP and black soybean. We have examined into body weight and height of all mice during experimental period. However, all mice of supplied food in these measurements were no significant difference compared with the control groups in both sexes (data were not shown). In addition, the amount of all nutrition including supplied food was restrained according the instructions of MHLW in Japan. Therefore, we assumed that supplied special diet food has no occurrence of side effect following snack administration.

It is well known that calcium intake decreases the risk of bone fracture. It was also reported that the calcium supplementation suppress bone formation when magnesium is deficient (Mora Gilsanz, 2003). Based on these reports, we designed to mix calcium and magnesium at an appropriate proportion of 2 to 1. These contents in this snack were compounded to fulfill ninety percentage of the daily-required nutrition defined by MHLW in Japan.

It was also reported that addition of CPP will have a beneficial effect on the absorption of calcium (Dontas Yiannakopoulos, 2007). In Japan, CPP was accredited as a food for qualified health uses by MHLW. Accordingly, we add a certain amount of CPP to absorb mineral content easily.

The black soybean contains isoflavone which is widely accepted to have a weak estrogen activity, and to be able to bind estrogen receptor. Actually, Brandi and Miyauchi et al., reported that isoflavone had either effect for the suppression of bone resorption or enhancement of bone formation, affecting directly both osteoclasts and osteoblasts (Brandi, 2003; Miyauchi et al., 1996). In addition, isoflavone was revealed to affect bone metabolism similarly to the sex hormone-like effect in male (Chavarro et al., 2008). Recently, it was also reported that isoflavones have revealed inhibition of bone loss in castrated male mice and growing male mice respectively (Fujioka et al., 2007; Ishimi et al., 2002; Khalil et al., 2005). Under such background, a new snack was developed by use of black soybean. The amount of isoflavone in this snack was determined with a special reference to the amount of safety surplus nutrition per day defined by MHLW.

Decrease in the trabecular and cortical bone volumes after gonadectomy has already been demonstrated by our previously reports (Fujita et al., 2001, 2004). We also reported that bone growth was significantly suppressed in the gonadectomized mice immediately after birth (Fujita et al., 2006). Moreover, we clarified that decrease in bone volume was occurred four weeks after gonadectomy. According to these results, we examined bone density of the femur six weeks after ovariectomy and orchiectomy.

The density of trabecular and cortical bones in the gonadectomized mice given LCD was significantly lower than in the sham operated mice given NCD and LCD, respectively. It was revealed that the deficiency of calcium intake caused decrease in bone density, under sex hormone disturbances in particular. Therefore, it is speculated that the bone density is below the optimal level in Japanese and fracture risk may become higher for aged people. These findings also support that the incidence of primary osteoporosis is higher in Japan than in American and European countries.

The bone metabolism is classified into two types, high turnover type to accelerate both bone formation and resorption and low turnover type caused by degradation of bone formation. In this study, twelve-week-old mice with sham operation fed LCD are regarded as young growing humans with low turnover type, whereas gonadectomy mice to simulate sex hormone disturbance in the experimental groups are assumed to be under the condition of

Nutrition for Enhancing Bone Volume in Mice 777

Pietschmann, P., Rauner, M., Sipos, W., Kerschan-Schindl, K. P. (2008). Osteoporosis: an

Chang, K. P., Center, J. R., Nguyen, T. V., Eisman, J. A. (2004). Incidence of hip and other

O'Neill, T. W., Felsenberg, D., Varlow, J., Cooper, C., Kanis, J. A., Silman, A. J. (1996). The

Fujita, T., Kawata, T., Tokimasa, C., Tanne, K. (2001). Influence of oestrogen and androgen

Fujita, T., Ohtani, J., Shigekawa, M., Kawata, T., Kaku, M., Kohno, S., Tsutsui, K., Tenjo, K.,

Orimo, H. (2001) New diagnostic criteria of primary osteoporosis. *Clinical Calcium,* Vol. 11,

Lapauw, B., Taes, Y., Bogaert, V., Vanbillemont, G., Goemaere, S., Zmierczak, H., De

Wang, Q., Seeman, E. (2008). Skeletal growth and peak bone mass. *Best Practice and* 

Rosen, C. J., Gallagher, J. C. (2011). The 2011 IOM report on vitamin D and calcium

Schoenau, E., Fricke, O. (2008). Mechanical influences on bone development in children.

Yamada, K., Sato-Mito, N., Nagata, J., Umegaki, K. (2008). Health claim evidence

No. 9, (September 2001), pp. 1133-1139. ISSN 20010917-5857

ISSN 0888-8809

(October 2008), pp. 3-12, ISSN 1423-0003

(December 2009), pp. 1010-1018, ISSN 1523-4681

(January 2004), pp. 950-254, ISSN 1554-0591

2004), pp. 532-536 ISSN 1523-4681

pp. 57-65, ISSN 1879-1506

(October 2008), ISSN 1654-661X

2, (April 2011), pp. 79-84, ISSN 1094-6950

687-700, ISSN 1532-1908

1192S-8S ISSN 1541-6100

1479-683X

cortical bone. *Molecular endocrinology,* Vol. 24, No. 2, (January 2010), pp. 323-334,

age-related and gender-specific disease: a mini-review. *Gerontology*, Vol. 55, No. 1,

osteoporotic fractures in elderly men and women: Dubbo Osteoporosis Epidemiology Study. *Journal of Bone and Mineral Research*, Vol. 19, No. 4, (January

prevalence of vertebral deformity in european men and women: the European Vertebral Osteoporosis Study. *Journal of Bone and Mineral Research*, Vol. 11, No. 7,

on modelling of the mandibular condylar bone in ovariectomized and orchiectomized growing mice. *Archives Oral Biology,* Vol. 46, No. 1, (January 2001),

Motokawa, M., Tohma, Y., Tanne, K. (2006) Effects of sex hormone disturbances on craniofacial growth in newborn mice. *Journal of Dental Research,* Vol. 83, No. 3,

Bacquer, D., Kaufman, J. (2009). Serum estradiol is associated with volumetric bone mineral density and modulates the impact of physical activity on bone size at the age of peak bone mass; a study in healty male siblings. *Journal of Bone and Mineral Research*, Vol. 24, No. 6, (December 2008), pp. 1075-1085, ISSN 1523-4681 Karlsson, M. K., Nordqvist, A., Karlsson, C. (2008). Physical activity increase bone mass

during growth. *Food and Nutrition Research,* Vol. 52 doi: 10.3402/fnr.v52i0.1871.

*Research. Clinical Endocrinology and Metabolism,* Vol. 22, No. 5, (October 2008), pp.

requirements for north America: clinical implications for providers treating patients with low bone mineral density. *Journal of Clinical Densitometry*, Vol. 14, No.

*European Journal of Endoclinology,* Vol. 159, No. 1, (September 2008), pp. 27-31 ISSN

requirements in Japan. *Journal of Nutrition,* Vol. 138, No. 6, (January 2008), pp.

postmenopausal osteoporosis as a high turnover type. Irrespective of the turnover types, however, the bone density in the group 5 given appropriate amount of calcium by supplying SCD exhibited a remarkable increase. It is thus suggested that sufficient calcium quantity through nutrition of newly developed bean snack enhanced bone formation irrespective of age.

It is reported that postmenopausal women with daily calcium intake of less than 400mg experience significant bone loss and that calcium intake of 800mg per day is effective for improving postmenopausal bone loss (Dawson-Hughes et al., 1990; Reid et al., 1993). On the other hand, it is well known that improvement effect against bone volume loss by calcium intake is available only at the initial stage of treatment. In addition, Riggs et al., clarified that the effect on bone loss is weaker than those reported for estrogen and bisphosphonates therapy, indicating that calcium supplements alone can't substitute treatment for osteoporosis (Riggs et al., 1998). In this study, longitudinal effect of newly developmental snack intake was not examined. However, It is hopefully anticipated that new bean snack could contributed to the enrichment of QOL as a nutrition function food, because of it was contain of several ingredients to promote assimilation efficiency of calcium.

### **5. Conclusions**

The new snack we developed included proper amount of calcium, magnesium, CPP and black soybean. When this product was given to the osteoporosis model mice, bone density of the femur was significantly increased. From these results, it is suggested that this product supplement promote bone formation irrespective of gender and age. We demonstrated that newly developmental snack supplements may be a useful preventive measure for the people whose bone mineral density values are less than the ideal condition.

### **6. Acknowledgement**

This investigation was supported by Grants-in Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology in Japan (19890131) and Hiroshima University collaborative research (052010).

#### **7. References**


postmenopausal osteoporosis as a high turnover type. Irrespective of the turnover types, however, the bone density in the group 5 given appropriate amount of calcium by supplying SCD exhibited a remarkable increase. It is thus suggested that sufficient calcium quantity through nutrition of newly developed bean snack enhanced bone formation

It is reported that postmenopausal women with daily calcium intake of less than 400mg experience significant bone loss and that calcium intake of 800mg per day is effective for improving postmenopausal bone loss (Dawson-Hughes et al., 1990; Reid et al., 1993). On the other hand, it is well known that improvement effect against bone volume loss by calcium intake is available only at the initial stage of treatment. In addition, Riggs et al., clarified that the effect on bone loss is weaker than those reported for estrogen and bisphosphonates therapy, indicating that calcium supplements alone can't substitute treatment for osteoporosis (Riggs et al., 1998). In this study, longitudinal effect of newly developmental snack intake was not examined. However, It is hopefully anticipated that new bean snack could contributed to the enrichment of QOL as a nutrition function food, because of it was

The new snack we developed included proper amount of calcium, magnesium, CPP and black soybean. When this product was given to the osteoporosis model mice, bone density of the femur was significantly increased. From these results, it is suggested that this product supplement promote bone formation irrespective of gender and age. We demonstrated that newly developmental snack supplements may be a useful preventive measure for the

This investigation was supported by Grants-in Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology in Japan (19890131) and Hiroshima

Judd, H. L., Meldrum, D. R., Deftos, L. J., Henderson, B. E. (1983). Estrogen replacement

Kousteni, S., Chen, J., Bellido R. T., Han, L., Ali, A. A., O'Brien, C. A., Plotkin, L., Fu, Q.,

Martin-Milan, M., Almeida, M., Ambrogini, E., Han, L., Zhao, H., Weinstein, R. S., Jilka, R.

therapy: indications and complications. *Annual of Internal Medicine,* Vol. 98, No. 2,

Mancino, A. T., Wen, Y., Vertino, A.M., Powers, C.C., Stewart, S.A., Ebert, R., Parfitt, A.M., Weinstein, R.S., Jilka, R.L., Manolagas, S.C. (2002). Reversal of bone loss in mice by nongenotropic signaling of sex steroids. *Science*, Vol. 298, No. 5594,

L., O'Brien, C. A., Manolagas, S. C. (2010). The estrogen receptor-alpha in osteoclasts mediates the protective effects of estrogens on cancellous but not

contain of several ingredients to promote assimilation efficiency of calcium.

people whose bone mineral density values are less than the ideal condition.

(February 1983), pp. 195-205, ISSN 1539-3704

(February 2003), pp. 843-846, ISSN 1095-9203

irrespective of age.

**5. Conclusions** 

**6. Acknowledgement** 

**7. References** 

University collaborative research (052010).

cortical bone. *Molecular endocrinology,* Vol. 24, No. 2, (January 2010), pp. 323-334, ISSN 0888-8809


Nutrition for Enhancing Bone Volume in Mice 779

Dontas, I. A., Yiannakopoulos, C. K. (2007). Risk factors and prevention of osteoporosis-

Brandi, M. L. (2003). Management of post-menopausal osteoporosis: something new on the

Miyauchi, A., Notoya, K., Taketomi, S., Takagi, Y., Fujii, Y., Jinnai, K., Takahashi, K.,

Chavarro, J. E., Toth, T. L., Sadio, S. M., Hauser, R. (2008). Soy food and isoflavone intake

Fujioka, M., Sudo, Y., Okumura, M., Wu, J., Uehara, M., Takeda, K., Hosokawa, Y., Yamada,

Ishimi, Y., Yoshida, M., Wakimoto, S., Wu, J., Chiba, H., Wang, X., Takeda, K., Miyaura, C.

Khalil, D. A., Lucas, E.A., Smith, B. J., Soung, D. Y., Devareddy, L., Juma, S., Akhter, M. P.,

Fujita, T., Kawata, T., Ohtani, J., Kaku, M., Tokimasa, C., Kohno, S., Tsutsui, K., Tenjo, K.,

Fujita, T., Ohtani, J., Shigekawa, M., Kawata, T., Kaku, M., Kohno, S., Motokawa, M.,

Dawson-Hughes, B., Dallal, G. E., Krall, E. A., Sadowski, L., Sahyoun, N., Tannenbaum, S.

*Orthodontics,* Vol. 28, No. 2, (April 2006), pp. 190~193, ISSN 1460-2210 Fujita, T., Ohtani, J., Shigekawa, M., Kawata, T., Kaku, M., Kohno, S., Tsutsui, K., Tenjo, K.,

Vol. 56, No. 8, (August 2008), pp. 1142-1148, ISSN 1532-8600

76, No. 1, (January 2005), pp. 56-62, ISSN 1432-0827

(March 2006), pp. 250-254, ISSN 1544-0591

(September 1990), pp. 878-883, ISSN 1533-4406

2009), pp. 268-272, ISSN 1108-7161

8, (August 1996), pp. 3544-3550, ISSN 1945-7170

170-173, ISSN 1720-8386

185, ISSN 1873-2763

2350

0544

related fractures. *Journal of musculoskelet Neuronal Interact*, Vol. 7, No. 3, (September

horizon?. *Journal of Endocrinology Investigation,* Vol. 26, No. 2, (Feburary 2003), pp.

Chihara, K., Fujita, T. (1996). Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function. *Endocrinology*, Vol. 137, No.

in relation to semen quality parameters among men from an infertility clinic. *Human Reproduction,* Vol. 23, No. 11, (November 2008), pp. 2584-2590, ISSN 1460-

K., Ikegami, S., Ishimi, Y. (2007). Differential effects of isoflavones on bone formation in growing male and female mice. *Metabolism Clinical and Experiment,*

(2002). Genistein, a soybean isoflavone, affects bone marrow lymphopoiesis and prevents bone loss in castrated male mice. *Bone,* Vol. 31, No. 1, (July 2002), pp. 180-

Recker, R., Arjmandi, B. H. (2005). Soy isoflavones may protect against orchidectomy-induced bone loss in aged male rats. *Calcified Tissue International,* Vol.

Motokawa, M., Tanne, K. (2001). Breadth of the mandibular condyle affected by disturbances of the sex hormones in ovariectomized and orchiectomized mice. *Clinical Orthodontic Research,* Vol. 4, No. 3, (August 2001), pp. 291-294, ISSN 1600-

Tohma, Y., Tanne, K. (2006). Influence of sex hormone disturbances on the internal structure of the mandible in newborn mice. *European Journal of* 

Motokawa, M., Tohma, Y., Tanne, K. (2006). Effects of sex hormone disturbances on craniofacial growth in newborn mice. *Journal of Dental Research,* Vol. 83, No. 3,

(1990). A controlled trial of the effect of calcium supplementation on bone density in postmenopausal women. *The New England Journal of Medicine,* Vol. 323, No. 13,


Kim, Y. K., Wassef, L., Chung, S., Jiang, H., Wyss, A., Blaner, W. S., Quadro, L. (2011).

Mueller, L., Boehm, V. (2011). Antioxidant activity of beta-carotene compounds in

Skulan, J. DePaolo, D. J. (1999). Calcium isotope fractionation between soft and

Reynolds, E. C. (2009). Casein phosphopeptide-amorphous calcium phosphate: the scientific

Llena, C., Forner, L., Baca, P. (2009). Anticariogenicity of casein phosphopeptide-

Andersson, A., Sköld-Larsson, K., Hallgren, A., Petersson, L. G., Twetman, S. (2007).

Morgan, M. V., Adams, G. G., Bailey, D. L., Tsao, C. E., Fischman, S. L., Reynolds, E. C.

Zhang, Q., Zou, J., Yang, R., Zhou, X. (2011). Remineralization effects of casein

Altenburger, M. J., Gmeiner, B., Hellwig, E., Wrbas, K. T., Schirrmeister, J. F. (2010). The

Moayyeri, A., Kaptoge, S., Luben, R. N., Wareham, N. J., Bingham, S., Reeve, J., Khaw, K.

*Epidemiology*, Vol. 24, No. 5, (April 2009), pp. 259-266 ISSN 1573-7284 Mora, S., Gilsanz, V. (2003). Establishment of peak bone mass. *Endocrinology and* 

*Dental Practice,* Vol. 10, No. 3, (May 2009), pp. 1-9, ISSN 1602-1622

(November 1993), pp. 13709–13713, ISSN 1091-6490

5, No. 3, (January 2007), pp. 229-233, ISSN 1602-1622

(January 2011), pp. 374-381, ISSN 1365-263X

10.1007/s00198-001-1552-y

1558-4410

1652, ISSN 1530-6860

1420-3049

1544-0737

976X

Beta-carotene and its cleavage enzyme beta-carotene-15, 15'-oxygenase (CMOI) affect retinoid metabolism in developing tissues. *Official Publication of the Federation of American Societies for Experimental Biology*, Vol. 25, No. 5, (May 2011), pp. 1641-

different in vitro assays. *Molecules.* Vol. 25, No. 16, (January 2011), pp. 1055-69 ISSN

mineralized tissues as a monitor of calcium use in vertebrates. *Proceedings of the National Academy of Sciences of the United States of America,* Vol. 96, No. 24,

evidence. *Advances in Dental Research,* Vol. 21, No. 1, (August 2009), pp. 25-29, ISSN

amorphous calcium phosphate: a review of the literature. *Journal of Contemporary* 

Effect of a dental cream containing amorpous complexs on white spot lesion regression assessed by laser fluorescence. *Oral Health and Preventive Dentistry,* Vol.

(2008). The anticariogenic effect of sugar-free gum containing CPP-ACP nanocomplexes on approximal caries determined using digital bitewing radiography. *Caries Research,* Vol. 42, No. 3, (April 2008), pp. 171-184, ISSN 1421-

phosphopeptide-amorphous calcium phosphate crème on artificial early enamel lesions of primary teeth. *International Journal of Pediatric Dentistry,* Vol. 21, No. 5,

evaluation of fluorescence changes after application of casein phosphopeptides (CPP) and amorphous calcium phosphate (ACP) on early carious lesions. *American Journal of Dentistry,* Vol. 23, No. 4, (August 2010), pp. 188-192, ISSN 0894-8275 Woo, J., Kwok, T., Leung, J. C., Ohlsson, C., Vandenput, L., Leung, P. C. (2011). Sex

steroids and bone health in older Chinese men. *Osteoporosis International*, doi:

T. (2009). Estimation of absolute fracture risk among middle-aged and older men and women: the EPIC-Norfolk population cohort study. *European Journal of* 

*Metabolism Clinics of North America,* Vol. 32, No. 1, (March 2003), pp. 39-63, ISSN


**1. Introduction** 

**38** 

*Japan* 

**Osteoporosis and Bone Regeneration** 

Majority of skeletal conditions generate or heal normally due to inherent capacity. However, compromised conditions such as induced by congenital or acquired diseases may sometimes lead to uncompleted development or regenerate. The quality and volume of bone is an important factor to be considered in orthopaedic and dental fields. We occasionally encounter difficult clinical cases because of insufficient bone. Bone is a tissue that is being constantly remodelled, and bone mass at any given time depends on the balance between the rate of osteoblastic bone formation and osteoclastic bone resorption. These cellular

Osteoporosis is a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture (1993). The term osteoporosis was first introduced in France and Germany during the last century, meaning "porous bone" and initially implied a histological diagnosis, but was later refined for bone which was normally mineralized but reduced in quantity. Estrogen deficiency in postmenopausal women elicits bone loss in the vertebrae and long bones resulting in bone fractures, and this condition is called postmenopausal osteoporosis

To the anatomical site features, a bone defect repair rate is mainly dependent on the bone wound size (J.P. Schmitz & J.O. Hollinger, 1986). Theoretically, an experimental osseous injury performed to study repair mechanisms should be wide enough to preclude a spontaneous healing. In order to do this, the non-regeneration threshold of bone tissue was investigated in the studied models, inducing a so-called critical-sized defect (CSD). The CSD may be defined as "the smallest size intraosseous wound in a particular bone and species of animal that will not heal spontaneously during the lifetime of the animal" (J.P. Schmitz & J.O. Hollinger, 1986; J.O. Hollinger & J.C. Kleinschmidt, 1990). The CSD may be therefore considered the prototype of discontinuity defects, as a condition of failed osteogenesis for

Although autologous or allogous bone transplant is partially effective, a simple and effective method for bone augmentation and regeneration is clinically desired in the orthopedic and dental fields. In the 1960s, Urist proposed the existence of bone-inducing molecules, which he termed bone morphogenetic proteins (BMP) (M.R. Urist, 1965). In 1988, the cDNA of BMPs was characterized, and human recombinant BMP2 (rhBMP2) (H.D. Zegzula et al., 1997; D.L. Wheeler et al., 1998; R.D. Welch et al., 1998; J.R. Lieberman et al., 1999; J.L. Dragoo et al., 2003) and BMP7 (X. Chen et al., 2002; F.C. den Boer et al., 2003; M.M. Abu-Serriah et al., 2004) are currently available. However, large amounts of rhBMP2 or 7 are required for

functions are controlled by various systemic and local factors.

overcoming the threshold of physiologic repair processes.

(T.J. Wronski et al., 1985, 1988, 1989a).

Shinji Kuroda, Kanako Noritake and Shohei Kasugai

*Tokyo Medical and Dental University* 


## **Osteoporosis and Bone Regeneration**

Shinji Kuroda, Kanako Noritake and Shohei Kasugai

*Tokyo Medical and Dental University Japan* 

### **1. Introduction**

780 Osteoporosis

Reid, I. R., Ames, R. W., Evans, M. C., Gamble, G. D., Sharpe, S. J. (1993). Effect of calcium

*of Medicine,* Vol. 328, No.7, (February 1993), pp. 460-464, ISSN 1533-4406 Riggs, B. L., O'Fallon W. M., Muhs, J., O'Connor M. K., Kumar, R., Melton L. J. (1998).

*Research*, Vol. 13, No. 2, (February, 1998), pp. 168-174, ISSN 1523-4681

supplementation on bone loss in postmenopausal women. *The New England Journal* 

Long-term effects of calcium supplementation on serum parathyroid hormone level, bone turnover, and bone loss in elderly women. *Journal of Bone and Mineral* 

> Majority of skeletal conditions generate or heal normally due to inherent capacity. However, compromised conditions such as induced by congenital or acquired diseases may sometimes lead to uncompleted development or regenerate. The quality and volume of bone is an important factor to be considered in orthopaedic and dental fields. We occasionally encounter difficult clinical cases because of insufficient bone. Bone is a tissue that is being constantly remodelled, and bone mass at any given time depends on the balance between the rate of osteoblastic bone formation and osteoclastic bone resorption. These cellular functions are controlled by various systemic and local factors.

> Osteoporosis is a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture (1993). The term osteoporosis was first introduced in France and Germany during the last century, meaning "porous bone" and initially implied a histological diagnosis, but was later refined for bone which was normally mineralized but reduced in quantity. Estrogen deficiency in postmenopausal women elicits bone loss in the vertebrae and long bones resulting in bone fractures, and this condition is called postmenopausal osteoporosis (T.J. Wronski et al., 1985, 1988, 1989a).

> To the anatomical site features, a bone defect repair rate is mainly dependent on the bone wound size (J.P. Schmitz & J.O. Hollinger, 1986). Theoretically, an experimental osseous injury performed to study repair mechanisms should be wide enough to preclude a spontaneous healing. In order to do this, the non-regeneration threshold of bone tissue was investigated in the studied models, inducing a so-called critical-sized defect (CSD). The CSD may be defined as "the smallest size intraosseous wound in a particular bone and species of animal that will not heal spontaneously during the lifetime of the animal" (J.P. Schmitz & J.O. Hollinger, 1986; J.O. Hollinger & J.C. Kleinschmidt, 1990). The CSD may be therefore considered the prototype of discontinuity defects, as a condition of failed osteogenesis for overcoming the threshold of physiologic repair processes.

> Although autologous or allogous bone transplant is partially effective, a simple and effective method for bone augmentation and regeneration is clinically desired in the orthopedic and dental fields. In the 1960s, Urist proposed the existence of bone-inducing molecules, which he termed bone morphogenetic proteins (BMP) (M.R. Urist, 1965). In 1988, the cDNA of BMPs was characterized, and human recombinant BMP2 (rhBMP2) (H.D. Zegzula et al., 1997; D.L. Wheeler et al., 1998; R.D. Welch et al., 1998; J.R. Lieberman et al., 1999; J.L. Dragoo et al., 2003) and BMP7 (X. Chen et al., 2002; F.C. den Boer et al., 2003; M.M. Abu-Serriah et al., 2004) are currently available. However, large amounts of rhBMP2 or 7 are required for

Osteoporosis and Bone Regeneration 783

trabecular and cortically rich regions, respectively. Also, the mandibles were scanned from the medial plane to the distal plane of the molar region with pQCT at interval of 0.5 mm as indicated in Fig. 1, following which the trabecular BMD and cortical BMD of the mandibular

In OVX rats, radio opacity of the femur attenuated after OVX was observed by soft X-ray radiography, and both uterus weight (data not shown) and femoral total BMD in DEXA analysis (Fig. 3) decreased by 80.8 and 13.7%, respectively. Although the total bone mineral density and the incisor mineral density of the mandible of the ovariectomized rats were similar to those of the sham-operated rats, the bone mineral density of the condylar region in the ovariectomized rats had markedly decreased by 14%. In pQCT analysis, decrease of 30% at the 17-mm section in trabecular BMD of the femur was prominent in OVX rats whereas ovariectomy did not affect BMD of the cortical bone of the femur. The molar region of the mandible excluding the molar showed decrease of maximal 13% in trabecular bone mineral density at the eighth and its adjoining slices; on the hand, cortical BMD was not affected in any of the slices (data not shown). This study revealed regional differences in

sections, excluding the incisor and molar, were measured using computer software.

bone mineral density decrease in the mandible in ovariectomized rats.

Fig. 1. Areas and positions for BMD measurement (S. Kuroda et al., 2003).

External incisal ridge and mandibular condyle in the areas surrounded with rectangles (4x6 mm) and square (2x2 mm) were scanned by DEXA, respectively. The sections including molars at interval of 0.5 mm were scanned by pQCT. Trabecular and cortically rich regions

Fig. 2. Soft X-ray Radiography of the femurs. Radio opacity decreased after OVX (S. Kuroda

of the femur were scanned at 2 mm and 17 mm from the growth plate, respectively.

**2.1.3 Results** 

et al., 2003).

use in clinical treatment. Although treatment with rhBMPs is effective, the extremely high cost of their clinical application is a barrier to their use. Development of an efficient carrier for BMPs or characterizing inhibitors of BMPs and blocking them may solve this problem in the future. Furthermore, fibroblast growth factor (FGF) family members also participate in regulating osteogenesis during fracture repair. FGF2 has been shown to be widely secreted around the fracture site in the processes of wound healing and bone regeneration; however, FGF4, which plays important roles in bone development during embryogenesis, has not yet been detected in such cases in postnatal ages.

In this chapter, several histological events concerning bone are demonstrated: first, an osteoporotic effect on mandibular bone is introduced; second, calvarial healing in the bone defect was demonstrated by GBR; and subsequently, it is shown that systemic and local deliveries of FGF4 and locally carried BMP2 can contribute to osteognesis in animal experiments.

### **2. Experimental cases and discussion**

#### **2.1 Osteoporosis of mandible**

#### **2.1.1 Background**

Bone is constantly remodelled, and bone mass at any given time is controlled by bone formation and bone resorption. These cellular events and functions are intimately associated with various systemic and local factors. Estrogen deficiency in postmenopausal women elicits bone loss in the vertebrae and long bones resulting in bone fractures, and this condition is called postmenopausal osteoporosis (T.J. Wronski et al., 1985, 1988, 1989a). Compared with the vertebrae and long bones, considerably less information is available on bone loss of the mandibles under estrogen deficient conditions. The bone mineral content of edentulous mandibles decreases with aging especially post menopause, but this is not evident in elderly men (E. Klemetti et al., 1993a, 1993b; C.W. Ulm et al., 1994; H. May et al., 1995). The ovariectomized (OVX) rat provides an experimental model of postmenopausal osteoporosis. It has been introduced that the bone mineral content and mechanical properties of the mandibles of OVX rats are similar to those of sham-operated rats; however, maxillary molar extraction causes bone loss from the mandibles of OVX rats (R.P. Elovic et al., 1994, 1995; E. Klemetti & P. Vainio, 1994). These previous studies indicate that estrogen deficiency somehow affects the mandible in both humans and experimental animals. To investigate precise region of the affected mandible, rats were ovariectomized and then subjected to longitudinal scanning for bone mineral density (BMD) measurement.

#### **2.1.2 Materials and methods**

Adult female Sprague–Dawley rats were bilaterally OVX- or sham-operated under Nembutal anaesthesia. The mandibles and femurs were dissected out after 3 months immersed in 70% ethanol. The uteri were also retrieved and weighed to check the effects of ovariectomy. Soft X-ray images of the mandibles and femurs were taken with a soft X-ray radiographic apparatus (SPOM50; Sofron, Tokyo, Japan). Total femoral BMD was initially measured with a DEXA (DCS-600R; Aloka, Tokyo, Japan), and then calculated. Total mandible BMD was measured using DEXA, after which the BMD of the incisal edge and condylar region of the mandible was measured. The trabecular and cortical BMD of the femurs were measured at cross sections 2 mm and 17 mm from the growth plate, respectively, perpendicular to the long axis with peripheral quantitative computed tomography (pQCT) (XCT 960 A; Stratec, Pforzheim, Germany) (Fig. 1). The positions are trabecular and cortically rich regions, respectively. Also, the mandibles were scanned from the medial plane to the distal plane of the molar region with pQCT at interval of 0.5 mm as indicated in Fig. 1, following which the trabecular BMD and cortical BMD of the mandibular sections, excluding the incisor and molar, were measured using computer software.

### **2.1.3 Results**

782 Osteoporosis

use in clinical treatment. Although treatment with rhBMPs is effective, the extremely high cost of their clinical application is a barrier to their use. Development of an efficient carrier for BMPs or characterizing inhibitors of BMPs and blocking them may solve this problem in the future. Furthermore, fibroblast growth factor (FGF) family members also participate in regulating osteogenesis during fracture repair. FGF2 has been shown to be widely secreted around the fracture site in the processes of wound healing and bone regeneration; however, FGF4, which plays important roles in bone development during embryogenesis, has not yet

In this chapter, several histological events concerning bone are demonstrated: first, an osteoporotic effect on mandibular bone is introduced; second, calvarial healing in the bone defect was demonstrated by GBR; and subsequently, it is shown that systemic and local deliveries of FGF4 and locally carried BMP2 can contribute to osteognesis in animal

Bone is constantly remodelled, and bone mass at any given time is controlled by bone formation and bone resorption. These cellular events and functions are intimately associated with various systemic and local factors. Estrogen deficiency in postmenopausal women elicits bone loss in the vertebrae and long bones resulting in bone fractures, and this condition is called postmenopausal osteoporosis (T.J. Wronski et al., 1985, 1988, 1989a). Compared with the vertebrae and long bones, considerably less information is available on bone loss of the mandibles under estrogen deficient conditions. The bone mineral content of edentulous mandibles decreases with aging especially post menopause, but this is not evident in elderly men (E. Klemetti et al., 1993a, 1993b; C.W. Ulm et al., 1994; H. May et al., 1995). The ovariectomized (OVX) rat provides an experimental model of postmenopausal osteoporosis. It has been introduced that the bone mineral content and mechanical properties of the mandibles of OVX rats are similar to those of sham-operated rats; however, maxillary molar extraction causes bone loss from the mandibles of OVX rats (R.P. Elovic et al., 1994, 1995; E. Klemetti & P. Vainio, 1994). These previous studies indicate that estrogen deficiency somehow affects the mandible in both humans and experimental animals. To investigate precise region of the affected mandible, rats were ovariectomized and then

subjected to longitudinal scanning for bone mineral density (BMD) measurement.

Adult female Sprague–Dawley rats were bilaterally OVX- or sham-operated under Nembutal anaesthesia. The mandibles and femurs were dissected out after 3 months immersed in 70% ethanol. The uteri were also retrieved and weighed to check the effects of ovariectomy. Soft X-ray images of the mandibles and femurs were taken with a soft X-ray radiographic apparatus (SPOM50; Sofron, Tokyo, Japan). Total femoral BMD was initially measured with a DEXA (DCS-600R; Aloka, Tokyo, Japan), and then calculated. Total mandible BMD was measured using DEXA, after which the BMD of the incisal edge and condylar region of the mandible was measured. The trabecular and cortical BMD of the femurs were measured at cross sections 2 mm and 17 mm from the growth plate, respectively, perpendicular to the long axis with peripheral quantitative computed tomography (pQCT) (XCT 960 A; Stratec, Pforzheim, Germany) (Fig. 1). The positions are

been detected in such cases in postnatal ages.

**2. Experimental cases and discussion** 

**2.1 Osteoporosis of mandible** 

**2.1.2 Materials and methods** 

experiments.

**2.1.1 Background** 

In OVX rats, radio opacity of the femur attenuated after OVX was observed by soft X-ray radiography, and both uterus weight (data not shown) and femoral total BMD in DEXA analysis (Fig. 3) decreased by 80.8 and 13.7%, respectively. Although the total bone mineral density and the incisor mineral density of the mandible of the ovariectomized rats were similar to those of the sham-operated rats, the bone mineral density of the condylar region in the ovariectomized rats had markedly decreased by 14%. In pQCT analysis, decrease of 30% at the 17-mm section in trabecular BMD of the femur was prominent in OVX rats whereas ovariectomy did not affect BMD of the cortical bone of the femur. The molar region of the mandible excluding the molar showed decrease of maximal 13% in trabecular bone mineral density at the eighth and its adjoining slices; on the hand, cortical BMD was not affected in any of the slices (data not shown). This study revealed regional differences in bone mineral density decrease in the mandible in ovariectomized rats.

Fig. 1. Areas and positions for BMD measurement (S. Kuroda et al., 2003).

External incisal ridge and mandibular condyle in the areas surrounded with rectangles (4x6 mm) and square (2x2 mm) were scanned by DEXA, respectively. The sections including molars at interval of 0.5 mm were scanned by pQCT. Trabecular and cortically rich regions of the femur were scanned at 2 mm and 17 mm from the growth plate, respectively.

Osteoporosis and Bone Regeneration 785

To investigate the efficacy of the collagen membrane for enhancement of bone regeneration in rat parietal bone defects, two symmetrical full thickness bone defects (5 mm diameter) were created at the calvarial bone of adult male Wistar rats. The defects were covered with a collagen membrane (Koken Tissue Guide, Koken, Japan) for GBR for 1 to 12 weeks. And then the specimens of the bone defect along with surrounding bone and soft tissues were collected and denuded of the skin. The samples were subjected to X-ray imaging using a µCT scanner (InspeXio; Shimadzu Science East Corporation, Tokyo, Japan) with a voxel size of 70 µm/pixel. Tri/3D-Bon software (RATOC System Engineering Co. Ltd, Tokyo, Japan) to make a 3D reconstruction from the resulting set of scans, and were also analysed by

After radiographical analyses, the samples were fixed in 10% neutralized formalin for 1 week, followed by decalcification in 10% EDTA for 4 weeks. After decalcification, an incision was made precisely through the midpoint of the bone defects to ensure that the microtome sections were made in the ROI and dehydrated in ascending grades of ethanol. The samples were consequently embedded in paraffin to allow for the preparation, staining with hematoxylin–eosin, and observation of 5-μm-thick coronal sections under an optical

The swelling and scabbing at the incised area attenuated by 2 weeks. Alteration of the skeletal defects was visualized in the µCT images over the period (Fig. 5). The opacity of newly formed bone was not found to reach the level of the surrounding host bone in the defects of the collagen group, showing incomplete healing at 4 weeks and bone recovery became abundant at 8 weeks after surgery (Fig. 5); and on the other hand, although new bone apposition was observed partially in the control defects, they acquired newly generated bone only along the defect rim with the similar opacity to that of the collagen

Fig. 5. µCT images of the parietal bone covered with the collagen membrane at 4 weeks

Observation of the bone development process evenly and gradually replaced the collagen membrane. As shown in Fig. 6, bone regeneration as well as membrane absorption was evident in the collagen group, but the thickness of the new bone was significantly higher for the control group over the study period. Notably, osteogenesis was observed to occur

**2.2.2 Materials and methods** 

DEXA to measure the bone morphology in the defect area.

covered defect and did not heal completely afterward.

(right) and without membrane at 8 weeks (left) after surgery.

primarily inside the collagen membrane.

microscope (BZ-8000; Keyence, Osaka).

**2.2.3 Results** 

Fig. 3. (A) Total bone mineral density of femur by DEXA. (B) Trabecular and cortical bone mineral densities of femur at the sections shown in Fig. 1. Values are presented as Mean ± SD, n = 6. \*p < 0.05 was regarded as statistically significant. (S. Kuroda et al., 2003)

Fig. 4. (A) The total mandible, the external part of incisor and the condylar region of mandible by DEXA. (B) Trabecular bone mineral densities of mandible at the sections shown in Fig. 1. Values are presented as Mean ± SD, n = 6. \*p < 0.05 was regarded as statistically significant. (S. Kuroda et al., 2003)

#### **2.2 Guided bone regeneration with a collagen membrane 2.2.1 Background**

Guided Bone Regeneration (GBR) technique is clinically used to acquire the sufficient bone volume, which has been developed by Nyman and Dahlin (C. Dahlin et al., 1988, 1989, 1990). The concept of this technique is that the application of a membrane creates a secluded space to facilitate proliferation of angiogenic and osteogenic cells from the basal bone into the defect without interference by fibroblasts. The membranes for GBR are mainly divided into three types: expanded polytetrafluoroethylene (ePTFE), synthetic biodegradable polyeters and collagen. Collagen is a material of resorbable membranes, which has several advantages such as hemostatic function, allowance of an early wound stabilization, chemotactic properties to attract fibroblasts and facilitating nutrient transfer. Therefore, collagen membranes are currently the membrane of choice for most GBR procedures.

### **2.2.2 Materials and methods**

784 Osteoporosis

**mg/cm3**

**1000**

> **0** ~

~ ~ ~~

**BMD**

Fig. 3. (A) Total bone mineral density of femur by DEXA. (B) Trabecular and cortical bone mineral densities of femur at the sections shown in Fig. 1. Values are presented as Mean ± SD, n = 6. \*p < 0.05 was regarded as statistically significant. (S. Kuroda et al., 2003)

**BMD**

Fig. 4. (A) The total mandible, the external part of incisor and the condylar region of

**2.2 Guided bone regeneration with a collagen membrane** 

significant. (S. Kuroda et al., 2003)

**2.2.1 Background** 

mandible by DEXA. (B) Trabecular bone mineral densities of mandible at the sections shown in Fig. 1. Values are presented as Mean ± SD, n = 6. \*p < 0.05 was regarded as statistically

Guided Bone Regeneration (GBR) technique is clinically used to acquire the sufficient bone volume, which has been developed by Nyman and Dahlin (C. Dahlin et al., 1988, 1989, 1990). The concept of this technique is that the application of a membrane creates a secluded space to facilitate proliferation of angiogenic and osteogenic cells from the basal bone into the defect without interference by fibroblasts. The membranes for GBR are mainly divided into three types: expanded polytetrafluoroethylene (ePTFE), synthetic biodegradable polyeters and collagen. Collagen is a material of resorbable membranes, which has several advantages such as hemostatic function, allowance of an early wound stabilization, chemotactic properties to attract fibroblasts and facilitating nutrient transfer. Therefore,

collagen membranes are currently the membrane of choice for most GBR procedures.

**SHAM OVX <sup>0</sup>**

∗ ∗

**2 864 1210 th**

**OVX SHAM**

**Slice**

∗ ∗

**<sup>2000</sup> Trabecular bone**

**Cortical bone**

∗

To investigate the efficacy of the collagen membrane for enhancement of bone regeneration in rat parietal bone defects, two symmetrical full thickness bone defects (5 mm diameter) were created at the calvarial bone of adult male Wistar rats. The defects were covered with a collagen membrane (Koken Tissue Guide, Koken, Japan) for GBR for 1 to 12 weeks. And then the specimens of the bone defect along with surrounding bone and soft tissues were collected and denuded of the skin. The samples were subjected to X-ray imaging using a µCT scanner (InspeXio; Shimadzu Science East Corporation, Tokyo, Japan) with a voxel size of 70 µm/pixel. Tri/3D-Bon software (RATOC System Engineering Co. Ltd, Tokyo, Japan) to make a 3D reconstruction from the resulting set of scans, and were also analysed by DEXA to measure the bone morphology in the defect area.

After radiographical analyses, the samples were fixed in 10% neutralized formalin for 1 week, followed by decalcification in 10% EDTA for 4 weeks. After decalcification, an incision was made precisely through the midpoint of the bone defects to ensure that the microtome sections were made in the ROI and dehydrated in ascending grades of ethanol. The samples were consequently embedded in paraffin to allow for the preparation, staining with hematoxylin–eosin, and observation of 5-μm-thick coronal sections under an optical microscope (BZ-8000; Keyence, Osaka).

### **2.2.3 Results**

The swelling and scabbing at the incised area attenuated by 2 weeks. Alteration of the skeletal defects was visualized in the µCT images over the period (Fig. 5). The opacity of newly formed bone was not found to reach the level of the surrounding host bone in the defects of the collagen group, showing incomplete healing at 4 weeks and bone recovery became abundant at 8 weeks after surgery (Fig. 5); and on the other hand, although new bone apposition was observed partially in the control defects, they acquired newly generated bone only along the defect rim with the similar opacity to that of the collagen covered defect and did not heal completely afterward.

Fig. 5. µCT images of the parietal bone covered with the collagen membrane at 4 weeks (right) and without membrane at 8 weeks (left) after surgery.

Observation of the bone development process evenly and gradually replaced the collagen membrane. As shown in Fig. 6, bone regeneration as well as membrane absorption was evident in the collagen group, but the thickness of the new bone was significantly higher for the control group over the study period. Notably, osteogenesis was observed to occur primarily inside the collagen membrane.

Osteoporosis and Bone Regeneration 787

Several FGF family members exert anabolic effects in bone when either systemically administered or locally applied (P. Aspenberg & L.S. Lohmander, 1989; H. Kawaguchi et al., 1994; T. Nakamura et al., 1995, 1998). FGF4 consists of 206 amino acid residues (M. Taira et al., 1987, T. Yoshida et al., 1987) and it has been reported that the FGF family plays a major role in the stimulation of cellular proliferation (M. Seno et al., 1990). In this study, the effects of rhFGF4s were clarified in mice after its systemic injection and in rat femurs after local

In 1988, the cDNA of BMPs was characterized (J.M. Wozney et al., 1988). Gene therapy using genes of osteogenic proteins, such as BMPs, has been a focus of considerable attention (J. Fang et al., 1996; J. Bonadio et al., 1999, 2000; J.R. Lieberman et al., 1999; R.T. Franceschi et al., 2000; N. Abe et al., 2002; Y. Chen et al., 2002; J.L. Dragoo et al., 2003; C.H. Rundle et al., 2003; H. Tsuda et al., 2003; A.L. Bertone et al., 2004; I. Ono et al., 2004). When osteogenic genes are transferred to local cells, protein secretion begins and the stimulation of osteogenesis by the protein continues for a longer time than that seen in protein therapy. Experimental studies on osteogenic gene transfer have been emerging, and there are several gene transfer techniques used to stimulate bone regeneration: *ex vivo* (J.R. Lieberman et al., 1999; R.T. Franceschi et al., 2000; N. Abe et al., 2002; Y. Chen et al., 2002; J.L. Dragoo et al., 2003; C.H. Rundle et al., 2003; H. Tsuda et al., 2003; A.L. Bertone et al., 2004) and *in vivo* (J. Fang et al., 1996; J. Bonadio et al., 1999; J. Bonadio, 2000; K. Honma et al., 2001; H. Uusitalo et al., 2001; A. Sano et al., 2003; I. Ono et al., 2004) gene transfers and gene transfers with viral vector (J.R. Lieberman et al., 1999; R.T. Franceschi et al., 2000; H. Uusitalo et al., 2001; N. Abe et al., 2002; Y. Chen et al., 2002; J.L. Dragoo et al., 2003; C.H. Rundle et al., 2003; H. Tsuda et al., 2003; A.L. Bertone et al., 2004) or with nonviral vector (J. Bonadio et al., 1999; K. Honma et al., 2001; A. Sano et al., 2003; I. Ono et al., 2004). Here demonstrated is an *in vivo* gene transfer using nonviral vectors. This study was to examine whether our designed matrix, which consists of collagen, CaP, and a plasmid vector encoding for BMP2, enhances

Human FGF4 cDNAs vector were ligated to pET-29(+) vector (pET system, Novagen). After subcloning in JM109 and plasmid purification, the plasmids were transferred into BL21(DE3)pLysS, an E. coli strain used for protein expression. Protein expression was induced with isopropyl-b-Dthiogalactopyranoside (IPTG). The proteins were then purified using the STag Purification Kit (Novagen). The purified proteins were dialyzed against

Forty male ddY mice, 6 weeks old, were divided into eight groups and subcutaneously injected with rhFGF4s at doses of 0.03, 0.1, and 0.3 mg/kg every day for 2 weeks, which stimulated cellular proliferation of NIH3T3 cells, at doses of 0.03, 0.1, and 0.3 mg/kg. These rhFGF4s were dissolved in PBS containing 0.1% bovine serum albumin and injected. The

After the 2 week injection course, the femurs were removed and contact microradiographs (CMRs) were taken of these ground sections using a soft X-ray radiographic apparatus

water using a minidialysis system (Bio-Tech International), and then freeze-dried.

five mice in the control group were injected with vehicle only.

administration.

bone tissue regeneration in a rat bone defect model.

**2.3.2 Materials and methods**  *Systemic administration of rhFGF4* 

(Sofron, SPO-M50).

Fig. 6. Photomicrographs of the calvarial defects at 2 and 4 weeks (Hematoxylin and eosin, original magnification ×4).

The bone regenerate with the membrane was significantly prominent with BMC of newly formed bone in the defect at 8 and 12 weeks after surgery. Furthermore, the BMD of newly formed bone was significant at 12 weeks. These results indicate that covering the bone defects with collagen membranes has an ability to deliver the suitable space for bone regeneration and make the regenerated bone better quality (Fig. 7).

Fig. 7. (A) BMC of the defects by DEXA. (B) BMD of the defects by DEXA. \*Statistically different from the uncovered control, *p* < 0.05.

#### **2.3 Osteoconductive/osteoinductive proteins stimulate bone 2.3.1 Background**

Gene expressions and productions of cytokines and growth factors in local regions that were traumatically or surgically injured are very crucial for tissue regeneration and engineering. However, cascades of the mechanisms and interactions of their roles have not been completely represented. Therefore, progress of such studies may lead to therapeutic aid.

Collagen

Control

original magnification ×4).

2 w 4 w

Fig. 6. Photomicrographs of the calvarial defects at 2 and 4 weeks (Hematoxylin and eosin,

The bone regenerate with the membrane was significantly prominent with BMC of newly formed bone in the defect at 8 and 12 weeks after surgery. Furthermore, the BMD of newly formed bone was significant at 12 weeks. These results indicate that covering the bone defects with collagen membranes has an ability to deliver the suitable space for bone

(A)

(B)

Fig. 7. (A) BMC of the defects by DEXA. (B) BMD of the defects by DEXA. \*Statistically

Gene expressions and productions of cytokines and growth factors in local regions that were traumatically or surgically injured are very crucial for tissue regeneration and engineering. However, cascades of the mechanisms and interactions of their roles have not been completely represented. Therefore, progress of such studies may lead to therapeutic aid.

different from the uncovered control, *p* < 0.05.

**2.3.1 Background** 

**2.3 Osteoconductive/osteoinductive proteins stimulate bone** 

regeneration and make the regenerated bone better quality (Fig. 7).

Several FGF family members exert anabolic effects in bone when either systemically administered or locally applied (P. Aspenberg & L.S. Lohmander, 1989; H. Kawaguchi et al., 1994; T. Nakamura et al., 1995, 1998). FGF4 consists of 206 amino acid residues (M. Taira et al., 1987, T. Yoshida et al., 1987) and it has been reported that the FGF family plays a major role in the stimulation of cellular proliferation (M. Seno et al., 1990). In this study, the effects of rhFGF4s were clarified in mice after its systemic injection and in rat femurs after local administration.

In 1988, the cDNA of BMPs was characterized (J.M. Wozney et al., 1988). Gene therapy using genes of osteogenic proteins, such as BMPs, has been a focus of considerable attention (J. Fang et al., 1996; J. Bonadio et al., 1999, 2000; J.R. Lieberman et al., 1999; R.T. Franceschi et al., 2000; N. Abe et al., 2002; Y. Chen et al., 2002; J.L. Dragoo et al., 2003; C.H. Rundle et al., 2003; H. Tsuda et al., 2003; A.L. Bertone et al., 2004; I. Ono et al., 2004). When osteogenic genes are transferred to local cells, protein secretion begins and the stimulation of osteogenesis by the protein continues for a longer time than that seen in protein therapy. Experimental studies on osteogenic gene transfer have been emerging, and there are several gene transfer techniques used to stimulate bone regeneration: *ex vivo* (J.R. Lieberman et al., 1999; R.T. Franceschi et al., 2000; N. Abe et al., 2002; Y. Chen et al., 2002; J.L. Dragoo et al., 2003; C.H. Rundle et al., 2003; H. Tsuda et al., 2003; A.L. Bertone et al., 2004) and *in vivo* (J. Fang et al., 1996; J. Bonadio et al., 1999; J. Bonadio, 2000; K. Honma et al., 2001; H. Uusitalo et al., 2001; A. Sano et al., 2003; I. Ono et al., 2004) gene transfers and gene transfers with viral vector (J.R. Lieberman et al., 1999; R.T. Franceschi et al., 2000; H. Uusitalo et al., 2001; N. Abe et al., 2002; Y. Chen et al., 2002; J.L. Dragoo et al., 2003; C.H. Rundle et al., 2003; H. Tsuda et al., 2003; A.L. Bertone et al., 2004) or with nonviral vector (J. Bonadio et al., 1999; K. Honma et al., 2001; A. Sano et al., 2003; I. Ono et al., 2004). Here demonstrated is an *in vivo* gene transfer using nonviral vectors. This study was to examine whether our designed matrix, which consists of collagen, CaP, and a plasmid vector encoding for BMP2, enhances bone tissue regeneration in a rat bone defect model.

### **2.3.2 Materials and methods**

### *Systemic administration of rhFGF4*

Human FGF4 cDNAs vector were ligated to pET-29(+) vector (pET system, Novagen). After subcloning in JM109 and plasmid purification, the plasmids were transferred into BL21(DE3)pLysS, an E. coli strain used for protein expression. Protein expression was induced with isopropyl-b-Dthiogalactopyranoside (IPTG). The proteins were then purified using the STag Purification Kit (Novagen). The purified proteins were dialyzed against water using a minidialysis system (Bio-Tech International), and then freeze-dried.

Forty male ddY mice, 6 weeks old, were divided into eight groups and subcutaneously injected with rhFGF4s at doses of 0.03, 0.1, and 0.3 mg/kg every day for 2 weeks, which stimulated cellular proliferation of NIH3T3 cells, at doses of 0.03, 0.1, and 0.3 mg/kg. These rhFGF4s were dissolved in PBS containing 0.1% bovine serum albumin and injected. The five mice in the control group were injected with vehicle only.

After the 2 week injection course, the femurs were removed and contact microradiographs (CMRs) were taken of these ground sections using a soft X-ray radiographic apparatus (Sofron, SPO-M50).

Osteoporosis and Bone Regeneration 789

Soft X-ray images revealed an increase in trabecular bone was evident dose-dependently in

Fig. 8. Contact microradiographs (CMRs) of the longitudinal sections of femurs after

Histomorphometric analysis revealed an increase in BV/TV and Tb.N, which represents an increase in trabecular bone. Furthermore, bone formation parameters (MS/BS and OS/BS) increased in a dosedependent manner, whereas a bone resorption parameter (ES/BS) was

> **BV/TV (%) 18.3 ± 2.5 23.4 ± 1.9 28.9 ± 1.5 31.1 ± 2.3 ES/BS (%) 10.5 ± 1.2 9.0 ± 1.6 9.8 ± 0.9 11.8 ± 1.1 MS/BS (%) 18.5 ± 2.7 20.0 ± 2.7 39.4 ± 2.5 43.1 ± 2.5 OS/BS (%) 17.5 ± 1.0 19.0 ± 2.6 30.4 ± 2.1 42.2 ± 1.9 Tb.Th (µm) 30.9 ± 1.3 28.8 ± 1.5 30.1 ± 2.3 33.7 ± 2.3 Tb.N (µm) 2.7 ± 0.2 3.0 ± 0.1 3.8 ± 0.2 3.8 ± 0.2**

Table 1. The increase of trabecular bone after administration with rhFGF4 was measured and confirmed with histomorphometric parameters. Data are presented as the mean ± SE

There were no visible or weight differences in the rats between the 2 groups at each time point, and neither the shapes nor sizes of the tibiae were affected by the local injection of rhFGF4 (data not shown). However, soft X-ray images demonstrated less radiolucence in the rhFGF4 group (Fig. 9A). DEXA analysis revealed increased BMD of the cancellous bone-rich zone of tibiae after the local injection of 1.0 mg of rhFGF4 and significance between the 2 groups at day 10 (Fig. 9B). Similarly, based on pQCT analysis (Fig. 9C), the trabecular BMD increased significantly in the rhFGF4 group from day 7 to day 10. Further, the higher BMD was maintained by the rhFGF4 injection over time. On the other hand, the cortical BMD

(n=5). a: Significantly different from controls, *p*<0.05. (S. Kuroda et al., 1999)

exhibited no difference either between the groups or over time (data not shown).

**0 (Control) 0.03 0.1 0.3**

**rhFGF4 (mg/kg)**

**2.3.3 Results** 

not affected (Table 1).

*Local administration of rhFGF4* 

*Systemic administration of rhFGF4* 

the CMRs in the rhFGF4-administered group (Fig. 8).

administration with rhFGF4 (S. Kuroda et al., 1999).

For histomorphometric analysis, the sections were further ground down to 30 µm and stained with toluidine blue. Then, histomorphometric measurements were using an image analysis system (IBAS 2000, Carl Zeiss) to measure the histomorphometric parameters on the images of the areas. The measured parameters and the calculated parameters follow the previous report described in JBMR (A.M. Parfitt et al., 1987).

#### *Local administration of rhFGF4*

Thirty-two 10-week-old male Sprague-Dawley rats were divided into two equal groups; one group received local injection of rhFGF4 and the other received local injection of vehicle as control. An injection of 1 µg rhFGF4 (0.1 µg/µl) was given from the left tibial proximal intercondyler notch into the midshaft of the marrow cavity directly with a 21G needle.

The animals were killed under chloroform anesthesia on days 7 and 10. Tibiae were removed and photographed using soft X-rays. The bone mineral densities were measured with dual-energy X-ray absorptimetry. The trabecular and cortical bone marrow densities of the tibiae were measured with pQCT (Fig. 9A). After BMD measurements, the tibiae were fixed in 10% formalin, dehydrated, and embedded in methyl methacrylate resin (OsteoResin, Wako, Osaka, Japan). Then, longitudinal sections of 5-µm thickness were made via a microtome (Microtome 2050 Supercut, Reichert-Jung, Kandel Electronics, Inc., Oreland, PA) and stained by the Villanueva bone staining method. The x20 objectives of both light and fluorescence microscopes (Axiophot, Carl Zeiss, Oberkochen, Germany) were used to take optical images of the sections. Histomorphometric measurements were performed of the tibia using an image analysis system (IBAS 2000, Carl Zeiss).

#### *BMP2 gene transfer at fracture site*

cDNA of hBMP2 was inserted into pEGFP-N1 plasmid vector (Clontech, Mountain View, CA). hBMP2 encoding plasmid (bmp2) was precipitated in CaP solution (CalPhos Mammalian Transfection Kit, Clontech). An equal volume of 2% bovine type I atelocollagen solution (Atelocollagen Implant, Koken, Tokyo, Japan) was then added. Twelve micrograms of this mixture (50 µl) were lyophilized and designated "bmp2-CaP-collagen".

The bone segments across a 5-mm segmental tibial defect of male Wistar rats were fixed with stainless-steel screws. Implants were placed and held in the osteotomy. The specimens were fixed in 10% neutral formalin. Some specimens were embedded in methylmethacrylate resin and longitudinal sections that included the bone defects were then prepared. Undemineralized sections were stained with toluidine. For mechanical tests, the other samples were supported at the proximal and distal points on the jig, and force was applied to the middle of the bone defect perpendicularly at a displacement rate of 1 mm/min using a materials testing machine (Instron 1123, Cauton, MA). Force and displacement data were stored in the computer.

The sites of the bone defects were collected and homogenized immediately for total RNA extraction (Isogen, Nippon Gene, Tokyo, Japan). And then RT-PCR was performed (SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA; PureTaq Ready-To-Go PCR Beads, Amersham Biosciences, Piscataway, NJ). Initially, denaturing was carried out at 95°C for 5 min, followed by optimizing cycles: 95°C for 30 s for denaturing, optimized temperature for 30 s for annealing, and 72°C for 30 s for extension. Each RT-PCR product was electrophoresed in 2% agarose gel in TAE buffer and stained with ethidium bromide, followed by photography under ultraviolet light.

### **2.3.3 Results**

788 Osteoporosis

For histomorphometric analysis, the sections were further ground down to 30 µm and stained with toluidine blue. Then, histomorphometric measurements were using an image analysis system (IBAS 2000, Carl Zeiss) to measure the histomorphometric parameters on the images of the areas. The measured parameters and the calculated parameters follow the

Thirty-two 10-week-old male Sprague-Dawley rats were divided into two equal groups; one group received local injection of rhFGF4 and the other received local injection of vehicle as control. An injection of 1 µg rhFGF4 (0.1 µg/µl) was given from the left tibial proximal intercondyler notch into the midshaft of the marrow cavity directly with a 21G needle. The animals were killed under chloroform anesthesia on days 7 and 10. Tibiae were removed and photographed using soft X-rays. The bone mineral densities were measured with dual-energy X-ray absorptimetry. The trabecular and cortical bone marrow densities of the tibiae were measured with pQCT (Fig. 9A). After BMD measurements, the tibiae were fixed in 10% formalin, dehydrated, and embedded in methyl methacrylate resin (OsteoResin, Wako, Osaka, Japan). Then, longitudinal sections of 5-µm thickness were made via a microtome (Microtome 2050 Supercut, Reichert-Jung, Kandel Electronics, Inc., Oreland, PA) and stained by the Villanueva bone staining method. The x20 objectives of both light and fluorescence microscopes (Axiophot, Carl Zeiss, Oberkochen, Germany) were used to take optical images of the sections. Histomorphometric measurements were performed of

cDNA of hBMP2 was inserted into pEGFP-N1 plasmid vector (Clontech, Mountain View, CA). hBMP2 encoding plasmid (bmp2) was precipitated in CaP solution (CalPhos Mammalian Transfection Kit, Clontech). An equal volume of 2% bovine type I atelocollagen solution (Atelocollagen Implant, Koken, Tokyo, Japan) was then added. Twelve micrograms

The bone segments across a 5-mm segmental tibial defect of male Wistar rats were fixed with stainless-steel screws. Implants were placed and held in the osteotomy. The specimens were fixed in 10% neutral formalin. Some specimens were embedded in methylmethacrylate resin and longitudinal sections that included the bone defects were then prepared. Undemineralized sections were stained with toluidine. For mechanical tests, the other samples were supported at the proximal and distal points on the jig, and force was applied to the middle of the bone defect perpendicularly at a displacement rate of 1 mm/min using a materials testing machine (Instron 1123, Cauton, MA). Force and displacement data were

The sites of the bone defects were collected and homogenized immediately for total RNA extraction (Isogen, Nippon Gene, Tokyo, Japan). And then RT-PCR was performed (SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA; PureTaq Ready-To-Go PCR Beads, Amersham Biosciences, Piscataway, NJ). Initially, denaturing was carried out at 95°C for 5 min, followed by optimizing cycles: 95°C for 30 s for denaturing, optimized temperature for 30 s for annealing, and 72°C for 30 s for extension. Each RT-PCR product was electrophoresed in 2% agarose gel in TAE buffer and stained with ethidium

of this mixture (50 µl) were lyophilized and designated "bmp2-CaP-collagen".

previous report described in JBMR (A.M. Parfitt et al., 1987).

the tibia using an image analysis system (IBAS 2000, Carl Zeiss).

bromide, followed by photography under ultraviolet light.

*Local administration of rhFGF4* 

*BMP2 gene transfer at fracture site* 

stored in the computer.

*Systemic administration of rhFGF4* 

Soft X-ray images revealed an increase in trabecular bone was evident dose-dependently in the CMRs in the rhFGF4-administered group (Fig. 8).

Fig. 8. Contact microradiographs (CMRs) of the longitudinal sections of femurs after administration with rhFGF4 (S. Kuroda et al., 1999).

Histomorphometric analysis revealed an increase in BV/TV and Tb.N, which represents an increase in trabecular bone. Furthermore, bone formation parameters (MS/BS and OS/BS) increased in a dosedependent manner, whereas a bone resorption parameter (ES/BS) was not affected (Table 1).


Table 1. The increase of trabecular bone after administration with rhFGF4 was measured and confirmed with histomorphometric parameters. Data are presented as the mean ± SE (n=5). a: Significantly different from controls, *p*<0.05. (S. Kuroda et al., 1999)

### *Local administration of rhFGF4*

There were no visible or weight differences in the rats between the 2 groups at each time point, and neither the shapes nor sizes of the tibiae were affected by the local injection of rhFGF4 (data not shown). However, soft X-ray images demonstrated less radiolucence in the rhFGF4 group (Fig. 9A). DEXA analysis revealed increased BMD of the cancellous bone-rich zone of tibiae after the local injection of 1.0 mg of rhFGF4 and significance between the 2 groups at day 10 (Fig. 9B). Similarly, based on pQCT analysis (Fig. 9C), the trabecular BMD increased significantly in the rhFGF4 group from day 7 to day 10. Further, the higher BMD was maintained by the rhFGF4 injection over time. On the other hand, the cortical BMD exhibited no difference either between the groups or over time (data not shown).

Osteoporosis and Bone Regeneration 791

Histological examinations indicated that the group treated with bmp2-CaP-collagen showed the most abundant osteogenesis (Fig. 10). The osteotomy site was connected with fibrous tissue only at 2 weeks. It was connected with callus and the gap was filled with newly formed cartilage and bone at 4 weeks after the operation. At 6 weeks, although there were still remnants of cartilage in the center, newly formed bone was remodeled to the cortical or cancellous bone and fused to the stump of the host bone, and the osteotomy sites became unclear. When treated with bmp2-collagen, the bone defect was bridged at 6 weeks but the bridged area was smaller and newly formed bone was less mature than that of the bmp2- CaP-collagen group. In the group treated with only collagen, a residue remained at 6 weeks, and although small callus formation from the host bone was observed, the defects were

**bmp2-CaP-collagen bmp2-collagen collagen**

Fig. 10. Histological images of the defects treated with different implants (M. Endo et al.,

Longitudinal sections were prepared at 6 weeks after the operation. Sections were stained

Mechanical strength to fracture at the osteotomy sites is presented in Fig. 11. The groups treated with bmp2 and collagen could be measured; however, the single implant of collagen did not induce the bone bridge. At 4 weeks, the bone treated with bmp2-CaP-collagen was stronger than that treated with bmp2-collagen. The mechanical strength of bone treated with bmp2-CaP-collagen was closely similar to that of the contralateral tibia at 6 weeks. In this

Fig. 11. Mechanical strength of the tibiae treated with different implants (M. Endo et al.,

group the fracture did not occur at the osteotomy site, but at the host bone.

**\***

*BMP2 gene transfer at fracture site* 

mainly filled with fibrous tissue.

with toluidine blue (original magnification x 40).

**% of strength of intact tibia**

2006).

2006).

Fig. 9. (A) Soft X-ray images of the tibiae. (B) BMD of the tibiae by DEXA. (C) Trabecular BMD of the mid shafts of the tibiae by pQCT. a Significantly increased by time, b Statistically different between the groups, p < 0.05. (S. Kuroda et al., 2007)

Histomorphometric analysis elucidated increases in BV/TV, OS/BS, Ob.S/BS, MS/BS, ES/BS and Oc.S/BS in the rhFGF4 group at day 7 (Table 2), which represents high turnover of bone remodelling and derived increase of trabecular BMD. However, the ratios of parameters to BS were decreased at day 10; in particular, the ratios of OS, Ob.S, ES and Oc.S to BS were significantly decreased from day 7.


Table 2. Histomorphometry was analysed with following parameters. BV: bone volume; TV: tissue volume; BS: bone surface; OS: osteoid surface; MS: mineralized surface (single labeled by calcein); ES: eroded surface; Ob.S: osteoblast surface; Oc.S: osteoclast surface; Tb.Th: trabecular thickness; Tb.N: trabecular number. Data represents the mean of 4 samples from the both groups ± SD. a indicates values that are significantly different from control, p < 0.05. b indicates values that become significantly different in the same group, p < 0.05. (S. Kuroda et al., 2007)

(A) (B)

(C)

**<sup>b</sup> <sup>a</sup>**

**Control rhFGF4 <sup>b</sup>**

> **0 7 10 Day**

Vehicle rhFGF4 Vehicle rhFGF4

Day 7 Day 10

a

a

a

a

a

13.4±1.8 17.5±1.6

12.6±0.7 23.6±7.2

10.4±1.8 16.2±1.1

10.3±1.8 14.1±2.0

11.2±2.4 14.1±3.3

a

b

a

ab

b

b

a

Fig. 9. (A) Soft X-ray images of the tibiae. (B) BMD of the tibiae by DEXA. (C) Trabecular BMD of the mid shafts of the tibiae by pQCT. a Significantly increased by time, b Statistically

Histomorphometric analysis elucidated increases in BV/TV, OS/BS, Ob.S/BS, MS/BS, ES/BS and Oc.S/BS in the rhFGF4 group at day 7 (Table 2), which represents high turnover of bone remodelling and derived increase of trabecular BMD. However, the ratios of parameters to BS were decreased at day 10; in particular, the ratios of OS, Ob.S, ES and Oc.S

BV/TV 15.3±6.7 23.9±3.8 15.0±5.6 30.6±7.8

Tb.Th 22.5±9.6 26.3±9.9 22.9±6.9 28.7±7.8 Tb.N 6.6±0.8 9.6±2.2 5.6±0.6 11.3±2.3

Table 2. Histomorphometry was analysed with following parameters. BV: bone volume; TV: tissue volume; BS: bone surface; OS: osteoid surface; MS: mineralized surface (single labeled by calcein); ES: eroded surface; Ob.S: osteoblast surface; Oc.S: osteoclast surface; Tb.Th: trabecular thickness; Tb.N: trabecular number. Data represents the mean of 4 samples from the both groups ± SD. a indicates values that are significantly different from control, p < 0.05. b indicates values that become significantly different in the same group, p < 0.05. (S. Kuroda et al., 2007)

**Trabecular BMD (mg/cm3)**

different between the groups, p < 0.05. (S. Kuroda et al., 2007)

OS/BS 16.5±3.1 29.3±3.5

MS/BS 18.6±3.0 31.7±2.5

ES/BS 16.6±4.0 23.5±1.4

Ob.S/BS 10.7±3.4 24.4±4.7

Oc.S/BS 10.6±2.9 22.3±2.8

to BS were significantly decreased from day 7.

B

#### *BMP2 gene transfer at fracture site*

Histological examinations indicated that the group treated with bmp2-CaP-collagen showed the most abundant osteogenesis (Fig. 10). The osteotomy site was connected with fibrous tissue only at 2 weeks. It was connected with callus and the gap was filled with newly formed cartilage and bone at 4 weeks after the operation. At 6 weeks, although there were still remnants of cartilage in the center, newly formed bone was remodeled to the cortical or cancellous bone and fused to the stump of the host bone, and the osteotomy sites became unclear. When treated with bmp2-collagen, the bone defect was bridged at 6 weeks but the bridged area was smaller and newly formed bone was less mature than that of the bmp2- CaP-collagen group. In the group treated with only collagen, a residue remained at 6 weeks, and although small callus formation from the host bone was observed, the defects were mainly filled with fibrous tissue.

### **bmp2-CaP-collagen bmp2-collagen collagen**

Fig. 10. Histological images of the defects treated with different implants (M. Endo et al., 2006).

Longitudinal sections were prepared at 6 weeks after the operation. Sections were stained with toluidine blue (original magnification x 40).

Mechanical strength to fracture at the osteotomy sites is presented in Fig. 11. The groups treated with bmp2 and collagen could be measured; however, the single implant of collagen did not induce the bone bridge. At 4 weeks, the bone treated with bmp2-CaP-collagen was stronger than that treated with bmp2-collagen. The mechanical strength of bone treated with bmp2-CaP-collagen was closely similar to that of the contralateral tibia at 6 weeks. In this group the fracture did not occur at the osteotomy site, but at the host bone.

Fig. 11. Mechanical strength of the tibiae treated with different implants (M. Endo et al., 2006).

Osteoporosis and Bone Regeneration 793

susceptibility of the bone of the molar region of the mandible to estrogen deficient condition

In the present study, we found trabecular bone decrease of the molar region of the mandible in OVX-rats. Although the mechanism of the low susceptibility of the molar region of the mandible to estrogen deficient condition is not clear, it is likely that mechanical stress derived from functional occlusion is preventing the bone loss in this pathological condition. Elovic and his collaborators have clearly demonstrated that maxillary molar extracion together with ovariectomy causes more bone loss in the mandible than maxillary molar extraction alone (E. Klemetti et al., 1993a, 1993b, 1994; R.P. Elovic et al., 1994; E. Klemetti & P. Vainio, 1994; R.P. Elovic et al., 1995; L. Jahangiri et al., 1997), which supports this

Cell migration is likely influenced by the size of the inter-fibrous space in the molecular construction of the membranes (J. Behring et al., 2008). As such, cells were able to migrate inside the collagen membrane. The collagen was found to perform well as a new bone space maintainer excluding the adjacent soft tissue. Several cell-culture studies have explored the biocompatibility of GBR membranes by comparing their levels of inflammatory-related gene expression (A. Friedmann et al., 2008) and osteogenic markers (S.B. Idris et al.). However, only a few *in vivo* researches before this study had done so by developing and comparing gene expression profiles rather than performing radiological and histological analysis during osteogenesis in the bone defect (M. Nyan et al.). By allowing for observation of differences in cellular events between the experimental (with membrane) and control (no membrane) conditions, the development and comparison of gene expression profiles will

The most characteristic feature of the systemic effects of FGF4 is likely the stimulation of endosteal but not periosteal bone formation. This is elicited by proliferation of preosteoblastic cells in bone marrow, followed by recruitment of osteoblasts from preosteoblastic cells (T. Nakamura et al., 1995; S. Kuroda et al., 1999). Subsequently, the increase of cancellous bone becomes prominent (H. Mayahara et al., 1993; T. Nakamura et al., 1995). As suggested, FGF family members are of great importance for bone development and morphogenesis (B. Feldman et al., 1995; H. Ohuchi et al., 1995; R.A. Buckland et al., 1998). Further, the expression of FGF family members such as basic FGF is often upregulated during fracture repair and may contribute to the regeneration process (M.E. Bolander, 1992). Some reports have suggested that exogenous FGF family members accelerate bone fracture healing and wound healing when locally applied (H. Kawaguchi et al., 1994; T. Nakamura et al., 1998) and also recover bone mass that has been pathologically damaged because of ovariectomy or diabetes, for instance (H. Kawaguchi et al., 1994; C.R. Dunstan et al., 1999). Therefore, although FGF4 has not been detected in postnatal stages, it is assumed that exogenous FGF4 during tissue regeneration might have important aspects as well as basic FGF or other growth factors (R.K. Globus et al., 1989; M. Noda & J.J. Camilliere, 1989; M.E. Joyce et al., 1990; M.E. Bolander, 1992), and exogenous FGF4 as well as basic FGF may possibly enhance the local regeneration (M.

permit molecular examination of the bone-healing process in the future study.

*Systemic/local administration of rhFGF4* 

Noda & R. Vogel, 1989).

was low compared to the bones of condylar region of the mandible and the femur.

speculation.

**GBR** 

Values are presented as mean ± standard deviation. The statistically significant difference is observed between G1 and G2 at 4 weeks (p<0.05). G1: bmp2-CaP-collagen; G2: bmp2 collagen; G3: collagen.

Both rat and human BMP2 gene expression were detected throughout the experimental period in Fig. 12: human BMP2 gene expression level did not alter so much with time, and, similarly, the expression level of rat BMP2 gene remained up to 8 weeks. The expression level of osteocalcin gene was elevated up to 8 weeks. On the other hand, VEGF genes were evenly expressed during the period. RANK and RANKL gene expression were enhanced initially, and the levels were maintained until 8 weeks.

Fig. 12. Gene expression at the defect site treated with bmp2-CaP-collagen (M. Endo et al., 2006). One to five µg of total RNA of each sample was reverse-transcribed to cDNA in 20 µl, 1 µl of which was used for PCR amplification in 50 µl. And then 8 µl out of 50 µl was run for a gel electrophoresis.

### **3. Discussion and conclusion**

#### **OVX**

In OVX rats, both bone formation and resorption is accelerated; however, the unbalance, more bone resorption than bone formation, causes trabecular bone loss in long bones and vertebrates (Y. Otawara et al., 1983; T.J. Wronski et al., 1989a, 1989b; B.C. Toolan et al., 1992). In the present study, total femoral BMD of OVX rats decreased in DEXA analysis. This BMD decrease in OVX rats was due to the decrease in trabecular bone and cortical bone was not affected, which was revealed in pQCT analysis. These results are the same as previously reported by other investigators (P. Pastoureau et al., 1995; C.M. Bagi et al., 1996; S.A. Breen et al., 1996).

Firstly, BMD measurement of condylar region demonstrated BMD decrease in OVX rats, minus 14% from BMD of shame-operated one. Percent BMD decrease of this region was similar to the decrease of femoral BMD in OVX rats. Secondary, BMD of trabecular bone of molar region of mandible decreased in OVX rats, which was revealed in pQCT analysis. Notably, the extent of this BMD decrease (maximal 13% decrease) was less than the decrease of trabecular bone of the femur in OVX rats (30% decrease at the 17-mm-section). Thus, the susceptibility of the bone of the molar region of the mandible to estrogen deficient condition was low compared to the bones of condylar region of the mandible and the femur.

In the present study, we found trabecular bone decrease of the molar region of the mandible in OVX-rats. Although the mechanism of the low susceptibility of the molar region of the mandible to estrogen deficient condition is not clear, it is likely that mechanical stress derived from functional occlusion is preventing the bone loss in this pathological condition. Elovic and his collaborators have clearly demonstrated that maxillary molar extracion together with ovariectomy causes more bone loss in the mandible than maxillary molar extraction alone (E. Klemetti et al., 1993a, 1993b, 1994; R.P. Elovic et al., 1994; E. Klemetti & P. Vainio, 1994; R.P. Elovic et al., 1995; L. Jahangiri et al., 1997), which supports this speculation.

#### **GBR**

792 Osteoporosis

Values are presented as mean ± standard deviation. The statistically significant difference is observed between G1 and G2 at 4 weeks (p<0.05). G1: bmp2-CaP-collagen; G2: bmp2-

Both rat and human BMP2 gene expression were detected throughout the experimental period in Fig. 12: human BMP2 gene expression level did not alter so much with time, and, similarly, the expression level of rat BMP2 gene remained up to 8 weeks. The expression level of osteocalcin gene was elevated up to 8 weeks. On the other hand, VEGF genes were evenly expressed during the period. RANK and RANKL gene expression were enhanced

**period 2 4 6 8 (week)**

Fig. 12. Gene expression at the defect site treated with bmp2-CaP-collagen (M. Endo et al., 2006). One to five µg of total RNA of each sample was reverse-transcribed to cDNA in 20 µl, 1 µl of which was used for PCR amplification in 50 µl. And then 8 µl out of 50 µl was run for

In OVX rats, both bone formation and resorption is accelerated; however, the unbalance, more bone resorption than bone formation, causes trabecular bone loss in long bones and vertebrates (Y. Otawara et al., 1983; T.J. Wronski et al., 1989a, 1989b; B.C. Toolan et al., 1992). In the present study, total femoral BMD of OVX rats decreased in DEXA analysis. This BMD decrease in OVX rats was due to the decrease in trabecular bone and cortical bone was not affected, which was revealed in pQCT analysis. These results are the same as previously reported by other investigators (P. Pastoureau et al., 1995; C.M. Bagi et al., 1996; S.A. Breen

Firstly, BMD measurement of condylar region demonstrated BMD decrease in OVX rats, minus 14% from BMD of shame-operated one. Percent BMD decrease of this region was similar to the decrease of femoral BMD in OVX rats. Secondary, BMD of trabecular bone of molar region of mandible decreased in OVX rats, which was revealed in pQCT analysis. Notably, the extent of this BMD decrease (maximal 13% decrease) was less than the decrease of trabecular bone of the femur in OVX rats (30% decrease at the 17-mm-section). Thus, the

collagen; G3: collagen.

a gel electrophoresis.

**OVX** 

et al., 1996).

**3. Discussion and conclusion** 

initially, and the levels were maintained until 8 weeks.

**GAPDH BMP2 hBMP2 OC VEGF Col I RANK RANKL**

Cell migration is likely influenced by the size of the inter-fibrous space in the molecular construction of the membranes (J. Behring et al., 2008). As such, cells were able to migrate inside the collagen membrane. The collagen was found to perform well as a new bone space maintainer excluding the adjacent soft tissue. Several cell-culture studies have explored the biocompatibility of GBR membranes by comparing their levels of inflammatory-related gene expression (A. Friedmann et al., 2008) and osteogenic markers (S.B. Idris et al.). However, only a few *in vivo* researches before this study had done so by developing and comparing gene expression profiles rather than performing radiological and histological analysis during osteogenesis in the bone defect (M. Nyan et al.). By allowing for observation of differences in cellular events between the experimental (with membrane) and control (no membrane) conditions, the development and comparison of gene expression profiles will permit molecular examination of the bone-healing process in the future study.

#### *Systemic/local administration of rhFGF4*

The most characteristic feature of the systemic effects of FGF4 is likely the stimulation of endosteal but not periosteal bone formation. This is elicited by proliferation of preosteoblastic cells in bone marrow, followed by recruitment of osteoblasts from preosteoblastic cells (T. Nakamura et al., 1995; S. Kuroda et al., 1999). Subsequently, the increase of cancellous bone becomes prominent (H. Mayahara et al., 1993; T. Nakamura et al., 1995). As suggested, FGF family members are of great importance for bone development and morphogenesis (B. Feldman et al., 1995; H. Ohuchi et al., 1995; R.A. Buckland et al., 1998). Further, the expression of FGF family members such as basic FGF is often upregulated during fracture repair and may contribute to the regeneration process (M.E. Bolander, 1992). Some reports have suggested that exogenous FGF family members accelerate bone fracture healing and wound healing when locally applied (H. Kawaguchi et al., 1994; T. Nakamura et al., 1998) and also recover bone mass that has been pathologically damaged because of ovariectomy or diabetes, for instance (H. Kawaguchi et al., 1994; C.R. Dunstan et al., 1999). Therefore, although FGF4 has not been detected in postnatal stages, it is assumed that exogenous FGF4 during tissue regeneration might have important aspects as well as basic FGF or other growth factors (R.K. Globus et al., 1989; M. Noda & J.J. Camilliere, 1989; M.E. Joyce et al., 1990; M.E. Bolander, 1992), and exogenous FGF4 as well as basic FGF may possibly enhance the local regeneration (M. Noda & R. Vogel, 1989).

Osteoporosis and Bone Regeneration 795

The mechanism of how the GAM system stimulates tissue regeneration is speculated as the following (J. Fang et al., 1996; J. Bonadio et al., 1999, 2000). CaP, in which plasmid vector is incorporated, has been used for *in vitro* gene transfer (C.F. Graham, 1973; A. Loyter et al., 1982a, 1982b; S. Kato et al., 1986; E. Orrantia & P.L. Chang, 1990; M. Werner et al., 1990; A.V. Zelenin et al., 1991; J.K. Burkholder et al., 1993; J.C. Sanford et al., 1993; T.A. Thompson et al., 1993). Since CaP stabilizes nucleic acids (A. Loyter et al., 1982a, 1982b), we speculated that CaP would be also useful for *in vivo* gene transfer (Y.W. Yang & J.C. Yang, 1997; S.Y. Watanabe et al., 1999; P. Batard et al., 2001). In the regeneration process, the cells surrounding GAM migrate into the matrix. Fundamentally, it is likely that the cells migrating into GAM are mainly fibroblastic cells and some of these cells can be regarded as targets for the plasmid. They engulf the plasmid vector, subsequently starting to produce an encoded protein. Thus, GAM acts as a bio-reactor for producing an encoded protein, which was human BMP2 in the present study. After transplanting our modified GAM, human BMP2 gene expression was observed at almost the same level throughout the experimental period. It is clear that human BMP2 produced by transfected cells enhanced bone regeneration in the present study. The duration of the gene expression in this gene transfer system depends on the site of the application and the size of the GAM, which presumably influences the period of the matrix degradation and the duration of gene expression. Although we did not observe cessation of human BMP2 gene expression, the expression of human BMP2 gene declined until time suggesting temporality of the expression of the

The results of the radiographic and histological analyses demonstrated that this critical size bone defect was bridged when it was treated with bmp2 and collagen. Notably, the regeneration of the defects treated with bmp2-CaP-collagen was more prominent than that of the defects treated with bmp2-collagen. These results were also confirmed in the

Ideas to reduce and avoid the emergence of compromised bone status such as osteoporosis, fractures and critical skeletal defects, and to increase bone mineral density and bone volume must be a theme for minimizing the burden of fractures through interventions that help to achieve optimal peak bone mass, reduce excessive skeletal resorption, enhance bone

The authors give many thanks to Professor Keiichi Ohya, D.D.S., Ph.D., Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, and Associate Professor Hisatomo Kondo, D.D.S., Ph.D., Section of Oral Implantology, Department of Prosthetic Dentistry, School of Dentistry, Iwate Medical University, Morioka, Japan, for technical advices and assistance.

Abe, N., Lee, Y.P., Sato, M., Zhang, X., Wu, J., Mitani, K. & Lieberman, J.R. (2002).

Enhancement of bone repair with a helper-dependent adenoviral transfer of bone

transfect gene.

biomechanical test.

**4. Acknowledgment** 

**5. References** 

formation.

The ratio of bone volume to tissue volume indicated that new bone formation seemed to have started before day 7 and this value became significantly higher on day 10 than that in the control group. The changes of these histomorphometric parameters might be attributed to an increase in the endosteum bone callous, which was supported by a prominent increase in the affected trabecular BMD but not the cortical BMD. Thus, rhFGF4 serves as an anabolic molecule in bone.

These studies were designed to analyze changes of bone mineral density or content with DEXA under osteoporosis, external growth factor intake and GBR in a skeletal defect, respectively. DEXA is widely used for obtaining an averaged mineral density of each part of a sample by directional scanning in as narrow a width as approximately 1 mm. These averages can be sammed for the total bone area to observe the total mineral content and density. The densities are expressed in a unit of mg/cm2 because the bone, originally threedimensional, is recognized as a flat picture during the scanning process. While pQCT and micro computed tomography (µCT) are provided for three-dimensional analyses of bone density and/or digital reconstructing of bone for several indices of morphometry, DEXA allows much easier and faster settings of samples and calculating of densities in resions of interest (ROI). Therefore, the key point and the characteristics of DEXA are giving an initial idea to get to know time-dependent changes of a sample or difference among samples. Overall, DEXA, pQCT and µCT can provide convenient and prompt tools, which can perform acculate comparisons.

#### *Gene trasnfer for BMP2*

There are three phases in the bone regeneration process: 1) the early inflammatory phase; 2) the repair phase; and 3) the remodeling phase (V.I. Sikavitsas et al., 2001). In the early inflammatory phase, the hemorrhage and the subsequent hematoma are followed by infiltration of inflammatory cells and fibroblasts to the repair area. These events lead to vascularization and the formation of granulation tissue. The second phase is the repair phase, which is characterized by a callus. This phase begins with vascular ingrowth, osteoid secretion and the presence of collagenous fibers. A temporary callus consisting of cartilage is produced. In the remodeling phase, osteoblasts are active and the cartilage tissue is replaced with immature cancellous bone. Some cancellous bone is then converted to mature dense bone. Although histological examination was performed at the limited time points in the present study, it is likely that these sequential events occurred in the present bone defect. This repair process was clearly modified by the different combinations of implanted materials.

Constant expression of VEGF indicates importance of neovascularization during the healing process. Since RANK, receptor activator for NFκB on the cellular membrane of osteoclast progenitors and osteoclasts, and its ligand RANKL, which is secreted from osteoblasts, are involved in osteoclast maturation and activation, maintained RANK expression suggests onward activation of osteoclasts leading to the bone remodeling including the callus. On the other hand, osteocalcin is a bone-specific matrix protein which is produced by mature osteoblasts. Thus, the increase of osteocalcin gene expression at 6 weeks implicates reaching the maturation of the defect site. Although rat BMP2 gene as well as human BMP2 gene diminished gradually, the gene expression remained detected over the period, suggesting enhanced osteoblast incorporation into the fractured site.

The ratio of bone volume to tissue volume indicated that new bone formation seemed to have started before day 7 and this value became significantly higher on day 10 than that in the control group. The changes of these histomorphometric parameters might be attributed to an increase in the endosteum bone callous, which was supported by a prominent increase in the affected trabecular BMD but not the cortical BMD. Thus, rhFGF4 serves as an anabolic

These studies were designed to analyze changes of bone mineral density or content with DEXA under osteoporosis, external growth factor intake and GBR in a skeletal defect, respectively. DEXA is widely used for obtaining an averaged mineral density of each part of a sample by directional scanning in as narrow a width as approximately 1 mm. These averages can be sammed for the total bone area to observe the total mineral content and density. The densities are expressed in a unit of mg/cm2 because the bone, originally threedimensional, is recognized as a flat picture during the scanning process. While pQCT and micro computed tomography (µCT) are provided for three-dimensional analyses of bone density and/or digital reconstructing of bone for several indices of morphometry, DEXA allows much easier and faster settings of samples and calculating of densities in resions of interest (ROI). Therefore, the key point and the characteristics of DEXA are giving an initial idea to get to know time-dependent changes of a sample or difference among samples. Overall, DEXA, pQCT and µCT can provide convenient and prompt tools, which can

There are three phases in the bone regeneration process: 1) the early inflammatory phase; 2) the repair phase; and 3) the remodeling phase (V.I. Sikavitsas et al., 2001). In the early inflammatory phase, the hemorrhage and the subsequent hematoma are followed by infiltration of inflammatory cells and fibroblasts to the repair area. These events lead to vascularization and the formation of granulation tissue. The second phase is the repair phase, which is characterized by a callus. This phase begins with vascular ingrowth, osteoid secretion and the presence of collagenous fibers. A temporary callus consisting of cartilage is produced. In the remodeling phase, osteoblasts are active and the cartilage tissue is replaced with immature cancellous bone. Some cancellous bone is then converted to mature dense bone. Although histological examination was performed at the limited time points in the present study, it is likely that these sequential events occurred in the present bone defect. This repair process was clearly modified by the different combinations of implanted

Constant expression of VEGF indicates importance of neovascularization during the healing process. Since RANK, receptor activator for NFκB on the cellular membrane of osteoclast progenitors and osteoclasts, and its ligand RANKL, which is secreted from osteoblasts, are involved in osteoclast maturation and activation, maintained RANK expression suggests onward activation of osteoclasts leading to the bone remodeling including the callus. On the other hand, osteocalcin is a bone-specific matrix protein which is produced by mature osteoblasts. Thus, the increase of osteocalcin gene expression at 6 weeks implicates reaching the maturation of the defect site. Although rat BMP2 gene as well as human BMP2 gene diminished gradually, the gene expression remained detected over the period, suggesting

enhanced osteoblast incorporation into the fractured site.

molecule in bone.

perform acculate comparisons.

*Gene trasnfer for BMP2* 

materials.

The mechanism of how the GAM system stimulates tissue regeneration is speculated as the following (J. Fang et al., 1996; J. Bonadio et al., 1999, 2000). CaP, in which plasmid vector is incorporated, has been used for *in vitro* gene transfer (C.F. Graham, 1973; A. Loyter et al., 1982a, 1982b; S. Kato et al., 1986; E. Orrantia & P.L. Chang, 1990; M. Werner et al., 1990; A.V. Zelenin et al., 1991; J.K. Burkholder et al., 1993; J.C. Sanford et al., 1993; T.A. Thompson et al., 1993). Since CaP stabilizes nucleic acids (A. Loyter et al., 1982a, 1982b), we speculated that CaP would be also useful for *in vivo* gene transfer (Y.W. Yang & J.C. Yang, 1997; S.Y. Watanabe et al., 1999; P. Batard et al., 2001). In the regeneration process, the cells surrounding GAM migrate into the matrix. Fundamentally, it is likely that the cells migrating into GAM are mainly fibroblastic cells and some of these cells can be regarded as targets for the plasmid. They engulf the plasmid vector, subsequently starting to produce an encoded protein. Thus, GAM acts as a bio-reactor for producing an encoded protein, which was human BMP2 in the present study. After transplanting our modified GAM, human BMP2 gene expression was observed at almost the same level throughout the experimental period. It is clear that human BMP2 produced by transfected cells enhanced bone regeneration in the present study. The duration of the gene expression in this gene transfer system depends on the site of the application and the size of the GAM, which presumably influences the period of the matrix degradation and the duration of gene expression. Although we did not observe cessation of human BMP2 gene expression, the expression of human BMP2 gene declined until time suggesting temporality of the expression of the transfect gene.

The results of the radiographic and histological analyses demonstrated that this critical size bone defect was bridged when it was treated with bmp2 and collagen. Notably, the regeneration of the defects treated with bmp2-CaP-collagen was more prominent than that of the defects treated with bmp2-collagen. These results were also confirmed in the biomechanical test.

Ideas to reduce and avoid the emergence of compromised bone status such as osteoporosis, fractures and critical skeletal defects, and to increase bone mineral density and bone volume must be a theme for minimizing the burden of fractures through interventions that help to achieve optimal peak bone mass, reduce excessive skeletal resorption, enhance bone formation.

### **4. Acknowledgment**

The authors give many thanks to Professor Keiichi Ohya, D.D.S., Ph.D., Section of Pharmacology, Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, and Associate Professor Hisatomo Kondo, D.D.S., Ph.D., Section of Oral Implantology, Department of Prosthetic Dentistry, School of Dentistry, Iwate Medical University, Morioka, Japan, for technical advices and assistance.

### **5. References**

Abe, N., Lee, Y.P., Sato, M., Zhang, X., Wu, J., Mitani, K. & Lieberman, J.R. (2002). Enhancement of bone repair with a helper-dependent adenoviral transfer of bone

Osteoporosis and Bone Regeneration 797

Chen, Y., Cheung, K.M., Kung, H.F., Leong, J.C., Lu, W.W. & Luk, K.D. (2002). In vivo new

protein-4 gene, Biochem Biophys Res Commun, Vol.298, No.1 pp.121-127 Dahlin, C., Gottlow, J., Linde, A. & Nyman, S. (1990). Healing of maxillary and mandibular

Dahlin, C., Linde, A., Gottlow, J. & Nyman, S. (1988). Healing of bone defects by guided

Dahlin, C., Sennerby, L., Lekholm, U., Linde, A. & Nyman, S. (1989). Generation of new

Dragoo, J.L., Choi, J.Y., Lieberman, J.R., Huang, J., Zuk, P.A., Zhang, J., Hedrick, M.H. &

Dunstan, C.R., Boyce, R., Boyce, B.F., Garrett, I.R., Izbicka, E., Burgess, W.H. & Mundy, G.R.

Elovic, R.P., Hipp, J.A. & Hayes, W.C. (1994). Maxillary molar extraction decreases

Elovic, R.P., Hipp, J.A. & Hayes, W.C. (1995). Maxillary molar extraction causes increased

Endo, M., Kuroda, S., Kondo, H., Maruoka, Y., Ohya, K. & Kasugai, S. (2006). Bone

Fang, J., Zhu, Y.Y., Smiley, E., Bonadio, J., Rouleau, J.P., Goldstein, S.A., McCauley, L.K.,

Feldman, B., Poueymirou, W., Papaioannou, V.E., DeChiara, T.M. & Goldfarb, M. (1995).

Franceschi, R.T., Wang, D., Krebsbach, P.H. & Rutherford, R.B. (2000). Gene therapy for

Friedmann, A., Dehnhardt, J., Kleber, B.M. & Bernimoulin, J.P. (2008). Cytobiocompatibility

Scand J Plast Reconstr Surg Hand Surg, Vol.24, No.1 pp.13-19

marrow, J Orthop Res, Vol.21, No.3 pp.521-528

human fat, J Orthop Res, Vol.21, No.4 pp.622-629

defects in rats, Tissue Eng, Vol.12, No.3 pp.489-497

expressing BMP7, J Cell Biochem, Vol.78, No.3 pp.476-486

Res, Vol.14, No.6 pp.953-959

pp.1735-1741

pp.1087-1093

pp.5753-5758

No.5195 pp.246-249

Res A, Vol.86, No.4 pp.935-941

tissue regeneration, Plast Reconstr Surg, Vol.81, No.5 pp.672-676

study in rabbits, Int J Oral Maxillofac Implants, Vol.4, No.1 pp.19-25 den Boer, F.C., Wippermann, B.W., Blokhuis, T.J., Patka, P., Bakker, F.C. & Haarman, H.J.

bone formation by direct transfer of adenoviral-mediated bone morphogenetic

bone defects using a membrane technique. An experimental study in monkeys,

bone around titanium implants using a membrane technique: an experimental

(2003). Healing of segmental bone defects with granular porous hydroxyapatite augmented with recombinant human osteogenic protein-1 or autologous bone

Benhaim, P. (2003). Bone induction by BMP-2 transduced stem cells derived from

(1999). Systemic administration of acidic fibroblast growth factor (FGF-1) prevents bone loss and increases new bone formation in ovariectomized rats, J Bone Miner

stiffness of the mandible in ovariectomized rats, J Dent Res, Vol.73, No.11

bone loss in the mandible of ovariectomized rats, J Bone Miner Res, Vol.10, No.7

regeneration by modified gene-activated matrix: effectiveness in segmental tibial

Davidson, B.L. & Roessler, B.J. (1996). Stimulation of new bone formation by direct transfer of osteogenic plasmid genes, Proc Natl Acad Sci U S A, Vol.93, No.12

Requirement of FGF-4 for postimplantation mouse development, Science, Vol.267,

bone formation: in vitro and in vivo osteogenic activity of an adenovirus

of collagen and ePTFE membranes on osteoblast-like cells in vitro, J Biomed Mater

morphogenetic protein-2, Biochem Biophys Res Commun, Vol.297, No.3 pp.523- 527


Abu-Serriah, M.M., Odell, E., Lock, C., Gillar, A., Ayoub, A.F. & Fleming, R.H. (2004).

Anonymous (1993). Consensus development conference: diagnosis, prophylaxis, and

Aspenberg, P. & Lohmander, L.S. (1989). Fibroblast growth factor stimulates bone

Bagi, C.M., DeLeon, E., Ammann, P., Rizzoli, R. & Miller, S.C. (1996). Histo-anatomy of the

Batard, P., Jordan, M. & Wurm, F. (2001). Transfer of high copy number plasmid into

Behring, J., Junker, R., Walboomers, X.F., Chessnut, B. & Jansen, J.A. (2008). Toward guided

Bertone, A.L., Pittman, D.D., Bouxsein, M.L., Li, J., Clancy, B. & Seeherman, H.J. (2004).

Bolander, M.E. (1992). Regulation of fracture repair by growth factors, Proc Soc Exp Biol

Bonadio, J. (2000). Tissue engineering via local gene delivery, J Mol Med, Vol.78, No.6

Bonadio, J., Smiley, E., Patil, P. & Goldstein, S. (1999). Localized, direct plasmid gene

Breen, S.A., Millest, A.J., Loveday, B.E., Johnstone, D. & Waterton, J.C. (1996). Regional

Buckland, R.A., Collinson, J.M., Graham, E., Davidson, D.R. & Hill, R.E. (1998). Antagonistic

Burkholder, J.K., Decker, J. & Yang, N.S. (1993). Rapid transgene expression in lymphocyte

Chen, X., Kidder, L.S. & Lew, W.D. (2002). Osteogenic protein-1 induced bone formation

ulnar osteotomy model, J Orthop Res, Vol.22, No.6 pp.1261-1270

protein-7, Br J Oral Maxillofac Surg, Vol.42, No.5 pp.410-418

treatment of osteoporosis, Am J Med, Vol.94, No.6 pp.646-650

stiffness, Anatomical Record, Vol.245, No.4 pp.633-644

Odontology, Vol.96, No.1 pp.1-11

Med, Vol.200, No.2 pp.165-170

Med, Vol.5, No.7 pp.753-759

International, Vol.58, No.6 pp.449-453

limb bud, Mech Dev, Vol.71, No.1-2 pp.143-150

transfer, J Immunol Methods, Vol.165, No.2 pp.149-156

527

476

68

pp.303-311

pp.142-150

morphogenetic protein-2, Biochem Biophys Res Commun, Vol.297, No.3 pp.523-

Histological assessment of bioengineered new bone in repairing osteoperiosteal mandibular defects in sheep using recombinant human bone morphogenetic

formation. Bone induction studied in rats, Acta Orthop Scand, Vol.60, No.4 pp.473-

proximal femur in rats: impact of ovariectomy on bone mass, structure, and

mammalian cells by calcium phosphate transfection, Gene, Vol.270, No.1-2 pp.61-

tissue and bone regeneration: morphology, attachment, proliferation, and migration of cells cultured on collagen barrier membranes. A systematic review,

Adenoviral-mediated transfer of human BMP-6 gene accelerates healing in a rabbit

delivery in vivo: prolonged therapy results in reproducible tissue regeneration, Nat

analysis of bone mineral density in the distal femur and proximal tibia using peripheral quantitative computed tomography in the rat In vivo, Calcified Tissue

effects of FGF4 on BMP induction of apoptosis and chondrogenesis in the chick

and macrophage primary cultures after particle bombardment-mediated gene

in an infected segmental defect in the rat femur, J Orthop Res, Vol.20, No.1


Osteoporosis and Bone Regeneration 799

Kuroda, S., Kondo, H., Ohya, K. & Kasugai, S. (2007). Bone increase in rat tibiae by local

Lieberman, J.R., Daluiski, A., Stevenson, S., Wu, L., McAllister, P., Lee, Y.P., Kabo, J.M.,

Loyter, A., Scangos, G.A. & Ruddle, F.H. (1982b). Mechanisms of DNA uptake by

May, H., Reader, R., Murphy, S. & Khaw, K.T. (1995). Self-reported tooth loss and bone mineral density in older men and women, Age Ageing, Vol.24, No.3 pp.217-221 Mayahara, H., Ito, T., Nagai, H., Miyajima, H., Tsukuda, R., Taketomi, S., Mizoguchi, J. &

Nakamura, T., Hanada, K., Tamura, M., Shibanushi, T., Nigi, H., Tagawa, M., Fukumoto, S.

Nakamura, T., Hara, Y., Tagawa, M., Tamura, M., Yuge, T., Fukuda, H. & Nigi, H. (1998).

Noda, M. & Camilliere, J.J. (1989). In vivo stimulation of bone formation by transforming

Noda, M. & Vogel, R. (1989). Fibroblast growth factor enhances type beta 1 transforming

Nyan, M., Miyahara, T., Noritake, K., Hao, J., Rodriguez, R., Kuroda, S. & Kasugai, S.

Ohuchi, H., Nakagawa, T., Yamauchi, M., Ohata, T., Yoshioka, H., Kuwana, T., Mima, T.,

Ono, I., Yamashita, T., Jin, H.Y., Ito, Y., Hamada, H., Akasaka, Y., Nakasu, M., Ogawa, T. &

growth factor-beta, Endocrinology, Vol.124, No.6 pp.2991-2994

Mater Res B Appl Biomater, Vol.93, No.1 pp.65-73

fluorescent dyes, Proc Natl Acad Sci U S A, Vol.79, No.2 pp.422-426

growth factor in rats, Growth Factors, Vol.9, No.1 pp.73-80

No.2 pp.415-422

Vol.136, No.3 pp.1276-1284

Vol.13, No.6 pp.942-949

pp.2529-2535

No.3 pp.809-816

234

administration of amino-terminally truncated rhFGF-4(73-206), Tissue Eng, Vol.13,

Finerman, G.A., Berk, A.J. & Witte, O.N. (1999). The effect of regional gene therapy with bone morphogenetic protein-2-producing bone-marrow cells on the repair of segmental femoral defects in rats, J Bone Joint Surg Am, Vol.81, No.7 pp.905-917 Loyter, A., Scangos, G., Juricek, D., Keene, D. & Ruddle, F.H. (1982a). Mechanisms of DNA

entry into mammalian cells. II. Phagocytosis of calcium phosphate DNA coprecipitate visualized by electron microscopy, Exp Cell Res, Vol.139, No.1 pp.223-

mammalian cells: fate of exogenously added DNA monitored by the use of

Kato, K. (1993). In vivo stimulation of endosteal bone formation by basic fibroblast

& Matsumoto, T. (1995). Stimulation of endosteal bone formation by systemic injections of recombinant basic fibroblast growth factor in rats, Endocrinology,

Recombinant human basic fibroblast growth factor accelerates fracture healing by enhancing callus remodeling in experimental dog tibial fracture, J Bone Miner Res,

growth factor gene expression in osteoblast-like cells, J Cell Biol, Vol.109, No.5

Molecular and tissue responses in the healing of rat calvarial defects after local application of simvastatin combined with alpha tricalcium phosphate, J Biomed

Mikawa, T., Nohno, T. & Noji, S. (1995). An additional limb can be induced from the flank of the chick embryo by FGF4, Biochem Biophys Res Commun, Vol.209,

Jimbow, K. (2004). Combination of porous hydroxyapatite and cationic liposomes as a vector for BMP-2 gene therapy, Biomaterials, Vol.25, No.19 pp.4709-4718


Globus, R.K., Plouet, J. & Gospodarowicz, D. (1989). Cultured bovine bone cells synthesize

Graham, C.F. (1973). The necessary conditions for gene expression during early mammalian

Hollinger, J.O. & Kleinschmidt, J.C. (1990). The critical size defect as an experimental model

Honma, K., Ochiya, T., Nagahara, S., Sano, A., Yamamoto, H., Hirai, K., Aso, Y. & Terada,

Idris, S.B., Arvidson, K., Plikk, P., Ibrahim, S., Finne-Wistrand, A., Albertsson, A.C.,

Jahangiri, L., Kim, A. & Nishimura, I. (1997). Effect of ovariectomy on the local residual ridge remodeling, Journal of Prosthetic Dentistry, Vol.77, No.4 pp.435-443 Joyce, M.E., Jingushi, S. & Bolander, M.E. (1990). Transforming growth factor-beta in the regulation of fracture repair, Orthop Clin North Am, Vol.21, No.1 pp.199-209 Kato, S., Anderson, R.A. & Camerini-Otero, R.D. (1986). Foreign DNA introduced by

Kawaguchi, H., Kurokawa, T., Hanada, K., Hiyama, Y., Tamura, M., Ogata, E. &

Klemetti, E. & Vainio, P. (1994). Effect of maxillary edentulousness on mandibular residual

Klemetti, E., Vainio, P. & Kroger, H. (1994). Muscle strength and mineral densities in the

Klemetti, E., Vainio, P., Lassila, V. & Alhava, E. (1993a). Cortical bone mineral density in the

Klemetti, E., Vainio, P., Lassila, V. & Alhava, E. (1993b). Trabecular bone mineral density of

Kuroda, S., Kasugai, S., Oida, S., Iimura, T., Ohya, K. & Ohyama, T. (1999). Anabolic effect of

Kuroda, S., Mukohyama, H., Kondo, H., Aoki, K., Ohya, K., Ohyama, T. & Kasugai, S.

to test bone repair materials, J Craniofac Surg, Vol.1, No.1 pp.60-68

Endocrinology, Vol.124, No.3 pp.1539-1547

pp.1075-1081

639

development, Symp Soc Dev Biol, Vol.31, pp.201-224

genome, Mol Cell Biol, Vol.6, No.5 pp.1787-1795

ridges, Scand J Dent Res, Vol.102, No.5 pp.309-312

mandible, Gerodontology, Vol.11, No.2 pp.76-79

tomography, Oral Dis, Vol.9, No.1 pp.24-28

Vol.135, No.2 pp.774-781

Vol.101, No.4 pp.219-223

Vol.101, No.3 pp.166-170

No.4 pp.431-437

basic fibroblast growth factor and store it in their extracellular matrix,

M. (2001). Atelocollagen-based gene transfer in cells allows high-throughput screening of gene functions, Biochem Biophys Res Commun, Vol.289, No.5

Bolstad, A.I. & Mustafa, K. Polyester copolymer scaffolds enhance expression of bone markers in osteoblast-like cells, J Biomed Mater Res A, Vol.94, No.2 pp.631-

calcium phosphate is integrated into repetitive DNA elements of the mouse L cell

Matsumoto, T. (1994). Stimulation of fracture repair by recombinant human basic fibroblast growth factor in normal and streptozotocin-diabetic rats, Endocrinology,

mandible and osteoporosis status in postmenopausal women, Scand J Dent Res,

mandible and alveolar height in postmenopausal women, Scand J Dent Res,

aminoterminally truncated fibroblast growth factor 4 (FGF4) on bone, Bone, Vol.25,

(2003). Bone mineral density of the mandible in ovariectomized rats: analyses using dual energy X-ray absorptiometry and peripheral quantitative computed


Osteoporosis and Bone Regeneration 801

Ulm, C.W., Solar, P., Ulm, M.R. & Matejka, M. (1994). Sex-related changes in the bone mineral content of atrophic mandibles, Calcif Tissue Int, Vol.54, No.3 pp.203-207

Watanabe, S.Y., Albsoul-Younes, A.M., Kawano, T., Itoh, H., Kaziro, Y., Nakajima, S. &

Welch, R.D., Jones, A.L., Bucholz, R.W., Reinert, C.M., Tjia, J.S., Pierce, W.A., Wozney, J.M.

Werner, M., Madreperla, S., Lieberman, P. & Adler, R. (1990). Expression of transfected

Wheeler, D.L., Chamberland, D.L., Schmitt, J.M., Buck, D.C., Brekke, J.H., Hollinger, J.O.,

Wozney, J.M., Rosen, V., Celeste, A.J., Mitsock, L.M., Whitters, M.J., Kriz, R.W., Hewick,

Wronski, T.J., Cintron, M. & Dann, L.M. (1988). Temporal relationship between bone loss

Wronski, T.J., Dann, L.M. & Horner, S.L. (1989a). Time course of vertebral osteopenia in

Wronski, T.J., Dann, L.M., Scott, K.S. & Cintron, M. (1989b). Long-term effects of

Wronski, T.J., Lowry, P.L., Walsh, C.C. & Ignaszewski, L.A. (1985). Skeletal alterations in

Yang, Y.W. & Yang, J.C. (1997). Calcium phosphate as a gene carrier: electron microscopy,

Yoshida, T., Miyagawa, K., Odagiri, H., Sakamoto, H., Little, P.F., Terada, M. & Sugimura, T.

Zegzula, H.D., Buck, D.C., Brekke, J., Wozney, J.M. & Hollinger, J.O. (1997). Bone formation

ovariectomized rats, Calcif Tissue Int, Vol.37, No.3 pp.324-328

ostectomy model, J Biomed Mater Res, Vol.43, No.4 pp.365-373

and activities, Science, Vol.242, No.4885 pp.1528-1534

ovariectomized rats, Bone, Vol.10, No.4 pp.295-301

Biomaterials, Vol.18, No.3 pp.213-217

Acad Sci U S A, Vol.84, No.20 pp.7305-7309

Joint Surg Am, Vol.79, No.12 pp.1778-1790

Aro, H.T. (2001). A metaphyseal defect model of the femur for studies of murine

Nakajima, Y. (1999). Calcium phosphate-mediated transfection of primary cultured brain neurons using GFP expression as a marker: application for single neuron

& Li, X.J. (1998). Effect of recombinant human bone morphogenetic protein-2 on fracture healing in a goat tibial fracture model, J Bone Miner Res, Vol.13, No.9

genes by differentiated, postmitotic neurons and photoreceptors in primary cell

Joh, S.P. & Suh, K.W. (1998). Radiomorphometry and biomechanical assessment of recombinant human bone morphogenetic protein 2 and polymer in rabbit radius

R.M. & Wang, E.A. (1988). Novel regulators of bone formation: molecular clones

and increased bone turnover in ovariectomized rats, Calcif Tissue Int, Vol.43, No.3

ovariectomy and aging on the rat skeleton, Calcified Tissue International, Vol.45,

(1987). Genomic sequence of hst, a transforming gene encoding a protein homologous to fibroblast growth factors and the int-2-encoded protein, Proc Natl

with use of rhBMP-2 (recombinant human bone morphogenetic protein-2), J Bone

Urist, M.R. (1965). Bone: formation by autoinduction, Science, Vol.150, No.698 pp.893-899 Uusitalo, H., Rantakokko, J., Ahonen, M., Jamsa, T., Tuukkanen, J., KaHari, V., Vuorio, E. &

bone healing, Bone, Vol.28, No.4 pp.423-429

cultures, J Neurosci Res, Vol.25, No.1 pp.50-57

pp.1483-1490

pp.179-183

No.6 pp.360-366

electrophysiology, Neurosci Res, Vol.33, No.1 pp.71-78


Orrantia, E. & Chang, P.L. (1990). Intracellular distribution of DNA internalized through calcium phosphate precipitation, Exp Cell Res, Vol.190, No.2 pp.170-174 Otawara, Y., Hosoya, N. & Moriuchi, S. (1983). Effect of aging and castration on the changes

Parfitt, A.M., Drezner, M.K., Glorieux, F.H., Kanis, J.A., Malluche, H., Meunier, P.J., Ott,

Rundle, C.H., Miyakoshi, N., Kasukawa, Y., Chen, S.T., Sheng, M.H., Wergedal, J.E., Lau,

Sanford, J.C., Smith, F.D. & Russell, J.A. (1993). Optimizing the biolistic process for different

Sano, A., Maeda, M., Nagahara, S., Ochiya, T., Honma, K., Itoh, H., Miyata, T. & Fujioka, K.

Schmitz, J.P. & Hollinger, J.O. (1986). The critical size defect as an experimental model for craniomandibulofacial nonunions, Clin Orthop Relat Res, No.205 pp.299-308 Seno, M., Sasada, R., Kurokawa, T. & Igarashi, K. (1990). Carboxyl-terminal structure of

Sikavitsas, V.I., Temenoff, J.S. & Mikos, A.G. (2001). Biomaterials and bone

Taira, M., Yoshida, T., Miyagawa, K., Sakamoto, H., Terada, M. & Sugimura, T. (1987).

Thompson, T.A., Gould, M.N., Burkholder, J.K. & Yang, N.S. (1993). Transient promoter

Tsuda, H., Wada, T., Ito, Y., Uchida, H., Dehari, H., Nakamura, K., Sasaki, K., Kobune, M.,

mechanotransduction, Biomaterials, Vol.22, No.19 pp.2581-2593

Nomenclature Committee, J Bone Miner Res, Vol.2, No.6 pp.595-610 Pastoureau, P., Chomel, A. & Bonnet, J. (1995). Specific evaluation of localized bone mass

analysis, Osteoporosis International, Vol.5, No.3 pp.143-149

biological applications, Methods Enzymol, Vol.217, pp.483-509

pp.249-260

Vol.32, No.6 pp.591-601

No.12 pp.1651-1677

pp.2980-2984

pp.1399-1406

Ther, Vol.7, No.3 pp.354-365

Eur J Biochem, Vol.188, No.2 pp.239-245

in the levels of bone gamma-carboxyglutamic acid-containing protein in bone and serum of female rat, Journal of Nutritional Science & Vitaminology, Vol.29, No.3

S.M. & Recker, R.R. (1987). Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry

and bone loss in the rat using dual-energy X-ray absorptiometry subregional

K.H. & Baylink, D.J. (2003). In vivo bone formation in fracture repair induced by direct retroviral-based gene therapy with bone morphogenetic protein-4, Bone,

(2003). Atelocollagen for protein and gene delivery, Adv Drug Deliv Rev, Vol.55,

basic fibroblast growth factor significantly contributes to its affinity for heparin,

cDNA sequence of human transforming gene hst and identification of the coding sequence required for transforming activity, Proc Natl Acad Sci U S A, Vol.84, No.9

activity in primary rat mammary epithelial cells evaluated using particle bombardment gene transfer, In Vitro Cell Dev Biol, Vol.29A, No.2 pp.165-170 Toolan, B.C., Shea, M., Myers, E.R., Borchers, R.E., Seedor, J.G., Quartuccio, H., Rodan, G. &

Hayes, W.C. (1992). Effects of 4-amino-1-hydroxybutylidene bisphosphonate on bone biomechanics in rats, Journal of Bone & Mineral Research, Vol.7, No.12

Yamashita, T. & Hamada, H. (2003). Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber-mutant adenoviral vector, Mol


**1. Introduction** 

**2. Lactoferrin** 

1999).

**39** 

**Lactoferrin – A Potential** 

*1University of Auckland* 

*New Zealand* 

Dorit Naot1, Kate Palmano2 and Jillian Cornish1

*2LactoPharma Consortium, Fonterra Research Centre* 

**Anabolic Intervention in Osteoporosis** 

Osteoporosis or porous bone was first described by Fuller Albright approximately 70 years ago as having "too little bone in the bone". Bone tissue is maintained throughout life by being continually replaced and in osteoporosis bone resorption exceeds bone formation resulting in bone loss. The majority of current treatments for osteoporosis are antiresorptive, decreasing osteoclast activity and preventing further bone loss. Therapeutic agents that activate osteoblasts and increase bone formation have the potential benefit of restoring bone rather than only preventing further deterioration, but only a small number of safe anabolic therapies are currently available. Milk is a rich biological fluid that contains many growth factors and provides nutrition at a time of very rapid skeletal growth and development in the neonate, and was therefore considered as a possible source of factors with anabolic effects on bone. Investigations of fractions of whey proteins extracted from milk identified lactoferrin as a bone-active factor. Lactoferrin is an iron-binding glycoprotein which as well as being present in milk is found in other epithelial secretions. It is a pleiotropic factor with potent antimicrobial and immunomodulatory activities, and shows anabolic effects in bone at physiological concentrations. In a number of recent studies in humans and experimental animals dietary lactoferrin supplementation improved bone mineral density, bone markers and bone strength. The current chapter discusses the structure and function of lactoferrin, the bone-effects of lactoferrin *in vitro* and *in vivo*, and

Lactoferrin is a multifunctional glycoprotein that was originally identified in bovine milk and first isolated from both human and bovine milk five decades ago (Groves et al. 1963). Lactoferrin is produced by mucosal epithelial cells and is present in very high concentrations in milk and colostrum, and in lower concentrations in mucosal secretions, including tears, saliva, nasal and bronchial secretions, bile and gastrointestinal fluids. Lactoferrin is also a major constituent of the secondary granules of neutrophilic leukocytes, and its serum level in healthy subjects is within the range of 1-10 g/mL (Caccavo et al.

the potential use of lactoferrin for the improvement of bone health.

Zelenin, A.V., Alimov, A.A., Titomirov, A.V., Kazansky, A.V., Gorodetsky, S.I. & Kolesnikov, V.A. (1991). High-velocity mechanical DNA transfer of the chloramphenicolacetyl transferase gene into rodent liver, kidney and mammary gland cells in organ explants and in vivo, FEBS Lett, Vol.280, No.1 pp.94-96

## **Lactoferrin – A Potential Anabolic Intervention in Osteoporosis**

Dorit Naot1, Kate Palmano2 and Jillian Cornish1 *1University of Auckland 2LactoPharma Consortium, Fonterra Research Centre New Zealand* 

### **1. Introduction**

802 Osteoporosis

Zelenin, A.V., Alimov, A.A., Titomirov, A.V., Kazansky, A.V., Gorodetsky, S.I. &

gland cells in organ explants and in vivo, FEBS Lett, Vol.280, No.1 pp.94-96

Kolesnikov, V.A. (1991). High-velocity mechanical DNA transfer of the chloramphenicolacetyl transferase gene into rodent liver, kidney and mammary

> Osteoporosis or porous bone was first described by Fuller Albright approximately 70 years ago as having "too little bone in the bone". Bone tissue is maintained throughout life by being continually replaced and in osteoporosis bone resorption exceeds bone formation resulting in bone loss. The majority of current treatments for osteoporosis are antiresorptive, decreasing osteoclast activity and preventing further bone loss. Therapeutic agents that activate osteoblasts and increase bone formation have the potential benefit of restoring bone rather than only preventing further deterioration, but only a small number of safe anabolic therapies are currently available. Milk is a rich biological fluid that contains many growth factors and provides nutrition at a time of very rapid skeletal growth and development in the neonate, and was therefore considered as a possible source of factors with anabolic effects on bone. Investigations of fractions of whey proteins extracted from milk identified lactoferrin as a bone-active factor. Lactoferrin is an iron-binding glycoprotein which as well as being present in milk is found in other epithelial secretions. It is a pleiotropic factor with potent antimicrobial and immunomodulatory activities, and shows anabolic effects in bone at physiological concentrations. In a number of recent studies in humans and experimental animals dietary lactoferrin supplementation improved bone mineral density, bone markers and bone strength. The current chapter discusses the structure and function of lactoferrin, the bone-effects of lactoferrin *in vitro* and *in vivo*, and the potential use of lactoferrin for the improvement of bone health.

### **2. Lactoferrin**

Lactoferrin is a multifunctional glycoprotein that was originally identified in bovine milk and first isolated from both human and bovine milk five decades ago (Groves et al. 1963). Lactoferrin is produced by mucosal epithelial cells and is present in very high concentrations in milk and colostrum, and in lower concentrations in mucosal secretions, including tears, saliva, nasal and bronchial secretions, bile and gastrointestinal fluids. Lactoferrin is also a major constituent of the secondary granules of neutrophilic leukocytes, and its serum level in healthy subjects is within the range of 1-10 g/mL (Caccavo et al. 1999).

Lactoferrin – A Potential Anabolic Intervention in Osteoporosis 805

Gram-positive bacteria lactoferrin damages the cell through direct interactions with lipoteichoic acid and other anionic surface molecules. Iron sequestration and interactions with anionic molecules are also the main mechanisms responsible for lactoferrin's activity against fungus and parasite infections. Lactoferrin has been described as an antiviral agent that affects a broad range of RNA and DNA viruses that infect humans and animals (Gonzalez-Chavez et al. 2009). Although the antiviral mechanisms of lactoferrin have not been well characterised yet, one of the most widely accepted hypothesis is that lactoferrin blocks viral receptors on the cell surface, and in particular heparin sulphate, preventing

Lactoferrin is a modulator of both the innate and acquired immune systems. Following the penetration of a microbe into a tissue, cells of the innate immune system release proinflammatory cytokines, including interleukins 1 and 6 (IL-1, IL-6) and tumor necrosis factor-alpha (TNF-α), which increase the permeability of blood vessels enabling the recruitment of circulating neutrophils to the site of infection. The release of neutrophil granule content creates very high local concentrations of lactoferrin. Apart from direct antimicrobial activity, lactoferrin interacts with cells of the innate immune systems as well as with cells of the adaptive immunity; regulating their recruitment, proliferation,

In different experimental systems, lactoferrin acts as either an anti-inflammatory or a proinflammatory factor. The anti-inflammatory activity of lactoferrin is attributed to its ability to bind free iron and exogenous proinflammatory bacterial components, such as LPS and their receptors (Legrand et al. 2005). Thus, lactoferrin activity as an iron scavenger prevents the formation of free radicals, which trigger oxidation processes and tissue damage, while binding to proinflammatory molecules inhibits the activation and recruitment of immune cells to the inflamed tissue. An additional mechanism implicated in the anti-inflammatory activity of lactoferrin has been recently described in apoptosis, the process of noninflammatory programmed cell death. Bournazou et al. (Bournazou et al. 2009; 2010) discovered that apoptotic cell of diverse lineages synthesize and secrete lactoferrin, which selectively inhibits the migration of granulocytes but not mononuclear phagocytes. This selective migration allows for the swift phagocytosis of the dying cells by the mononuclear cells without initiating an inflammatory response. Subsequently, lactoferrin was also found to have an inhibitory effect on eosinophil migration (Bournazou et al. 2010). A number of other studies describe the proinflammatory activities of lactoferrin. As a factor that induces inflammation, lactoferrin has been shown to promote cell motility, superoxide production, release of nitric oxide, release of the proinflammatory cytokines TNF-α and IL-8 and phagocytosis (Gahr et al. 1991; Legrand & Mazurier 2010; Shinoda et al. 1996; Sorimachi et

Although lactoferrin is an iron-binding protein and has been shown to influence iron status in pregnant women (Paesano et al. 2009) it is generally thought not to have a central role in iron-transport and homeostasis, unlike the transferrins. Nevertheless it does appear to have some role in iron regulation at local sites of inflammation and infection (Brock 2002) and

contact between the virus and the target cell.

al. 1997).

**2.2.3 Iron homeostasis and antioxidation** 

**2.2.2 Modulation of the immune response and inflammation** 

differentiation and activation (Legrand et al. 2006; Legrand & Mazurier 2010).

### **2.1 Molecular structure**

Lactoferrin is a non-haem iron-binding protein which belongs to the transferrin family of iron-transport proteins. It is a highly cationic monomeric glycoprotein with an isoelectric point of about 8.7 (Moguilevsky et al. 1985) and consists of about 690 amino acid residues folded into two homologous lobes, the so-called N-and C-lobes, which are linked by a 10-15 residue alpha helical peptide (Baker & Baker 2005). There is a high degree of homology between lactoferrin from various species with bovine and human lactoferrin sequence identity being 69% (Pierce et al. 1991).

### **2.1.1 Iron binding and glycosylation**

Each of the lactoferrin lobes contains a virtually identical iron-binding pocket, into which a trivalent iron cation (Fe3+) can be reversibly co-ordinated. The metal binding sites are highly conserved for all lactoferrins and transferrins thus far characterised (Baker & Baker 2009). Lactoferrin molecules can exist in several states whereby there is complete, partial (in either one of the two sites) or no occupancy of the two iron-binding sites. Lactoferrin isolated from both human and bovine milk has a low iron saturation, generally reported between 10-25% (Bezwoda & Mansoor 1989). In vitro, iron can be removed from lactoferrin to yield the ironfree or 'apo' form, or alternatively, lactoferrin can be loaded with iron to yield the fully ironbound or 'holo' form. Although other di- and trivalent transition metal ions such as Mn3+, Co3+, Cu2+ and Cr 3+ and even larger cations such as lanthanides (Smith et al. 1994) can be co-ordinated into the metal binding pocket, iron appears to be the natural ligand as it has optimal co-ordination and a very high binding affinity (Baker 1994). Nevertheless, lactoferrin may have a physiological role in binding trace amounts of other elements as manganese in milk is found exclusively associated with lactoferrin (Lonnerdal et al. 1985).

All lactoferrins are glycosylated, but the number and location of glycosylation sites varies from species to species, and is also tissue specific (Derisbourg et al. 1990). Differentially glycosylated lactoferrins appear to have similar biophysical and functional properties suggesting minimal structural impact of glycosylation (Moguilevsky et al. 1985).

### **2.1.2 Interactions with other molecules**

The highly basic nature of lactoferrin is contributed mainly by surface-exposed N-terminal domains containing clusters of highly basic residues which are capable of binding proteins such as ceruloplasmin (Vasilyev 2010) and osteopontin (Yamniuk et al. 2009). These cationic domains also confer on lactoferrin the ability to bind to many other anionic molecules including heparin, glycosaminoglycans, DNA, and various cell surface molecules (He & Furmanski 1995; Mann et al. 1994; van Berkel et al. 1997).

## **2.2 Physiological function**

### **2.2.1 Anti-microbial activity**

The highly cationic nature of lactoferrin and its high affinity iron binding are implicated in the anti-microbial function of this glycoprotein. Thus, iron sequestration in sites of bacterial infection deprives the bacteria of this essential nutrient, creating a bacteriostatic effect (Gonzalez-Chavez et al. 2009; Jenssen & Hancock 2009). Lactoferrin has bactericidal effect as well, as it interacts directly with anionic molecules on the cell surface, causing cell lysis. In Gram-negative bacteria, lactoferrin interacts directly with LPS, causing its release from the cell wall and increasing the external membrane permeability, which results in cell lysis. In

Lactoferrin is a non-haem iron-binding protein which belongs to the transferrin family of iron-transport proteins. It is a highly cationic monomeric glycoprotein with an isoelectric point of about 8.7 (Moguilevsky et al. 1985) and consists of about 690 amino acid residues folded into two homologous lobes, the so-called N-and C-lobes, which are linked by a 10-15 residue alpha helical peptide (Baker & Baker 2005). There is a high degree of homology between lactoferrin from various species with bovine and human lactoferrin sequence

Each of the lactoferrin lobes contains a virtually identical iron-binding pocket, into which a trivalent iron cation (Fe3+) can be reversibly co-ordinated. The metal binding sites are highly conserved for all lactoferrins and transferrins thus far characterised (Baker & Baker 2009). Lactoferrin molecules can exist in several states whereby there is complete, partial (in either one of the two sites) or no occupancy of the two iron-binding sites. Lactoferrin isolated from both human and bovine milk has a low iron saturation, generally reported between 10-25% (Bezwoda & Mansoor 1989). In vitro, iron can be removed from lactoferrin to yield the ironfree or 'apo' form, or alternatively, lactoferrin can be loaded with iron to yield the fully ironbound or 'holo' form. Although other di- and trivalent transition metal ions such as Mn3+, Co3+, Cu2+ and Cr 3+ and even larger cations such as lanthanides (Smith et al. 1994) can be co-ordinated into the metal binding pocket, iron appears to be the natural ligand as it has optimal co-ordination and a very high binding affinity (Baker 1994). Nevertheless, lactoferrin may have a physiological role in binding trace amounts of other elements as manganese in

All lactoferrins are glycosylated, but the number and location of glycosylation sites varies from species to species, and is also tissue specific (Derisbourg et al. 1990). Differentially glycosylated lactoferrins appear to have similar biophysical and functional properties

The highly basic nature of lactoferrin is contributed mainly by surface-exposed N-terminal domains containing clusters of highly basic residues which are capable of binding proteins such as ceruloplasmin (Vasilyev 2010) and osteopontin (Yamniuk et al. 2009). These cationic domains also confer on lactoferrin the ability to bind to many other anionic molecules including heparin, glycosaminoglycans, DNA, and various cell surface molecules (He &

The highly cationic nature of lactoferrin and its high affinity iron binding are implicated in the anti-microbial function of this glycoprotein. Thus, iron sequestration in sites of bacterial infection deprives the bacteria of this essential nutrient, creating a bacteriostatic effect (Gonzalez-Chavez et al. 2009; Jenssen & Hancock 2009). Lactoferrin has bactericidal effect as well, as it interacts directly with anionic molecules on the cell surface, causing cell lysis. In Gram-negative bacteria, lactoferrin interacts directly with LPS, causing its release from the cell wall and increasing the external membrane permeability, which results in cell lysis. In

milk is found exclusively associated with lactoferrin (Lonnerdal et al. 1985).

suggesting minimal structural impact of glycosylation (Moguilevsky et al. 1985).

**2.1 Molecular structure** 

identity being 69% (Pierce et al. 1991).

**2.1.1 Iron binding and glycosylation** 

**2.1.2 Interactions with other molecules** 

**2.2 Physiological function 2.2.1 Anti-microbial activity** 

Furmanski 1995; Mann et al. 1994; van Berkel et al. 1997).

Gram-positive bacteria lactoferrin damages the cell through direct interactions with lipoteichoic acid and other anionic surface molecules. Iron sequestration and interactions with anionic molecules are also the main mechanisms responsible for lactoferrin's activity against fungus and parasite infections. Lactoferrin has been described as an antiviral agent that affects a broad range of RNA and DNA viruses that infect humans and animals (Gonzalez-Chavez et al. 2009). Although the antiviral mechanisms of lactoferrin have not been well characterised yet, one of the most widely accepted hypothesis is that lactoferrin blocks viral receptors on the cell surface, and in particular heparin sulphate, preventing contact between the virus and the target cell.

### **2.2.2 Modulation of the immune response and inflammation**

Lactoferrin is a modulator of both the innate and acquired immune systems. Following the penetration of a microbe into a tissue, cells of the innate immune system release proinflammatory cytokines, including interleukins 1 and 6 (IL-1, IL-6) and tumor necrosis factor-alpha (TNF-α), which increase the permeability of blood vessels enabling the recruitment of circulating neutrophils to the site of infection. The release of neutrophil granule content creates very high local concentrations of lactoferrin. Apart from direct antimicrobial activity, lactoferrin interacts with cells of the innate immune systems as well as with cells of the adaptive immunity; regulating their recruitment, proliferation, differentiation and activation (Legrand et al. 2006; Legrand & Mazurier 2010).

In different experimental systems, lactoferrin acts as either an anti-inflammatory or a proinflammatory factor. The anti-inflammatory activity of lactoferrin is attributed to its ability to bind free iron and exogenous proinflammatory bacterial components, such as LPS and their receptors (Legrand et al. 2005). Thus, lactoferrin activity as an iron scavenger prevents the formation of free radicals, which trigger oxidation processes and tissue damage, while binding to proinflammatory molecules inhibits the activation and recruitment of immune cells to the inflamed tissue. An additional mechanism implicated in the anti-inflammatory activity of lactoferrin has been recently described in apoptosis, the process of noninflammatory programmed cell death. Bournazou et al. (Bournazou et al. 2009; 2010) discovered that apoptotic cell of diverse lineages synthesize and secrete lactoferrin, which selectively inhibits the migration of granulocytes but not mononuclear phagocytes. This selective migration allows for the swift phagocytosis of the dying cells by the mononuclear cells without initiating an inflammatory response. Subsequently, lactoferrin was also found to have an inhibitory effect on eosinophil migration (Bournazou et al. 2010). A number of other studies describe the proinflammatory activities of lactoferrin. As a factor that induces inflammation, lactoferrin has been shown to promote cell motility, superoxide production, release of nitric oxide, release of the proinflammatory cytokines TNF-α and IL-8 and phagocytosis (Gahr et al. 1991; Legrand & Mazurier 2010; Shinoda et al. 1996; Sorimachi et al. 1997).

#### **2.2.3 Iron homeostasis and antioxidation**

Although lactoferrin is an iron-binding protein and has been shown to influence iron status in pregnant women (Paesano et al. 2009) it is generally thought not to have a central role in iron-transport and homeostasis, unlike the transferrins. Nevertheless it does appear to have some role in iron regulation at local sites of inflammation and infection (Brock 2002) and

Lactoferrin – A Potential Anabolic Intervention in Osteoporosis 807

\*

**TUNEL Positive Nuclei**

**(per microscopic field)**

\*

0 1 10 100 **[bLF] (g/ml)** 0.1

\*

Fig. 1. Lactoferrin stimulates osteoblast proliferation, differentiation and survival *in vitro*

\*

**Number of Nodules**

A B C

**(per well)**

\*

\* \*

**[bLF](g/ml)** 0 0.1 1 10 100

\*

mineralization.

0

2

4

**Thymidine Incorporation**

**(treatment/control)**

6

**3.1.2 Local injection model** 

4374. Copyright 2004, The Endocrine Society.)

Thus, lactoferrin acts to expand the pool of pre-osteoblastic cells by exerting mitogenic and anti-apoptotic effects, as well as promoting differentiation of precursors to produce a more mature osteoblastic phenotype capable of promoting bone matrix deposition and

**[rhLF] (g/ml)** 0 10 100

The activities of lactoferrin on osteoblasts demonstrated *in vitro* are likely to contribute to the potent effects on bone formation seen *in vivo* after administration of lactoferrin, even with a very short-term exposure (Fig 2) (Cornish et al. 2004). The bone growth resulting from local lactoferrin injection is considerably greater than that found previously in response to factors such as insulin, amylin, adrenomedullin and C-terminal PTH-related peptide (Cornish et al. 1996; 1997a; 1997b). It is qualitatively different from the effects of PTH in this model, which produces a powerful stimulation of bone resorption in addition to its effect on formation (Cornish et al. 1995). This anabolic potency suggests that lactoferrin should be further explored as a therapy for osteoporosis that can restore skeletal strength.

A B Fig. 2. Photomicrographs of calvariae from animals treated with lactoferrin (A) and vehicle (B) for 5 days. Fluorochrome labels used: green, calcein; red, alizarin. Horizontal bar, 50 m. (Figure reproduced with kind permission. Cornish J., et al. Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo. Endocrinology 145(9): 2004, 4366-

iron sequestration is an important part of its role both as an antioxidant and antibacterial agent (Baldwin et al. 1984; Jenssen & Hancock 2009). In respect of iron-binding, lactoferrin is functionally different from transferrin as it retains iron to a much lower pH, giving it a more potent iron-withholding ability (Baker & Baker 2009). Although the antioxidant properties of lactoferrin are generally related to removal of free iron which otherwise reacts with reactive oxygen species (ROS) to form damaging hydroxyl radicals (Matsue et al. 1995; Raghuveer et al. 2002) a recent report suggests that apo- and holo bovine-lactoferrin have equal ability to act as antioxidants by scavenging ROS (Kanwar et al. 2011). This radical quenching ability, akin to antioxidant vitamins, is seemingly iron independent.

#### **2.2.4 Bioactive peptides derived from lactoferrin**

Functional cationic peptides with potent antibacterial activity, such as lactoferricin and lactoferrampin, can be derived from the N-terminal domain of lactoferrin by hydrolysis (Bellamy et al. 1992) or synthetic chemistry (van der Kraan et al. 2004), respectively. Lactoferrin can be degraded by digestive enzymes (Brock et al. 1976; Troost et al. 2001) and the functional peptide lactoferricin is likely to be formed in the gut by the action of pepsin. Lactoferrin 'half molecules' consisting of either the N-lobe or C-lobe can be generated by proteolysis or by recombinant technology (Baker & Baker 2005; Kim et al. 2006). These are useful as tools to probe for site-specific functionality or interactions. For example, the antiherpes virus activity of lactoferrin has been shown to be mediated mainly by the N-lobe (Siciliano et al. 1999) while simple sugars have been shown to interact with the C-lobe through a common recognition site (Mir et al. 2010).

### **3. The activity of lactoferrin in bone**

#### **3.1 Osteoblasts**

#### **3.1.1** *In vitro* **studies of lactoferrin activity in osteoblasts**

Lactoferrin potently induces proliferation of primary osteoblasts and osteoblastic-cell lines and increases osteoblast differentiation at physiological concentrations (Fig 1A) (Cornish et al. 2004; Takayama & Mizumachi 2008, 2009). In 3-week cultures of primary fetal rat osteoblasts lactoferrin dose-dependently increased osteoblast differentiation with increases in bone matrix deposition and the number of mineralized bone nodules formed (Fig 1B) (Cornish et al. 2004). In addition, lactoferrin decreased apoptosis induced by serum withdrawal in primary rat osteoblasts (Fig 1C) (Cornish et al. 2004) and in the human osteoblastic cell line SaOS2 (Grey et al. 2006). These effects on both the proliferation and survival of osteoblasts are profound, being far greater than those observed in response to several established osteoblast growth factors studied in the same *in vitro* assays, such as epidermal growth factor, transforming growth factor-β, parathyroid hormone, amylin or insulin. These factors increase thymidine incorporation in primary osteoblast cultures by only 20 – 30% (Cornish et al. 1999) whereas lactoferrin produces three- to five-fold increments (Cornish et al. 2004). This growth stimulating potency is complemented by the capacity of lactoferrin to substantially reduce osteoblast apoptosis, which again, is much more dramatic than the effects seen with other factors, such as insulin growth factor-1 (IGF-1) which maximally decreases apoptosis by 50% (Cornish et al. 2000) compared to 70% with lactoferrin (Cornish et al. 2004).

Fig. 1. Lactoferrin stimulates osteoblast proliferation, differentiation and survival *in vitro*

Thus, lactoferrin acts to expand the pool of pre-osteoblastic cells by exerting mitogenic and anti-apoptotic effects, as well as promoting differentiation of precursors to produce a more mature osteoblastic phenotype capable of promoting bone matrix deposition and mineralization.

### **3.1.2 Local injection model**

806 Osteoporosis

iron sequestration is an important part of its role both as an antioxidant and antibacterial agent (Baldwin et al. 1984; Jenssen & Hancock 2009). In respect of iron-binding, lactoferrin is functionally different from transferrin as it retains iron to a much lower pH, giving it a more potent iron-withholding ability (Baker & Baker 2009). Although the antioxidant properties of lactoferrin are generally related to removal of free iron which otherwise reacts with reactive oxygen species (ROS) to form damaging hydroxyl radicals (Matsue et al. 1995; Raghuveer et al. 2002) a recent report suggests that apo- and holo bovine-lactoferrin have equal ability to act as antioxidants by scavenging ROS (Kanwar et al. 2011). This radical

Functional cationic peptides with potent antibacterial activity, such as lactoferricin and lactoferrampin, can be derived from the N-terminal domain of lactoferrin by hydrolysis (Bellamy et al. 1992) or synthetic chemistry (van der Kraan et al. 2004), respectively. Lactoferrin can be degraded by digestive enzymes (Brock et al. 1976; Troost et al. 2001) and the functional peptide lactoferricin is likely to be formed in the gut by the action of pepsin. Lactoferrin 'half molecules' consisting of either the N-lobe or C-lobe can be generated by proteolysis or by recombinant technology (Baker & Baker 2005; Kim et al. 2006). These are useful as tools to probe for site-specific functionality or interactions. For example, the antiherpes virus activity of lactoferrin has been shown to be mediated mainly by the N-lobe (Siciliano et al. 1999) while simple sugars have been shown to interact with the C-lobe

Lactoferrin potently induces proliferation of primary osteoblasts and osteoblastic-cell lines and increases osteoblast differentiation at physiological concentrations (Fig 1A) (Cornish et al. 2004; Takayama & Mizumachi 2008, 2009). In 3-week cultures of primary fetal rat osteoblasts lactoferrin dose-dependently increased osteoblast differentiation with increases in bone matrix deposition and the number of mineralized bone nodules formed (Fig 1B) (Cornish et al. 2004). In addition, lactoferrin decreased apoptosis induced by serum withdrawal in primary rat osteoblasts (Fig 1C) (Cornish et al. 2004) and in the human osteoblastic cell line SaOS2 (Grey et al. 2006). These effects on both the proliferation and survival of osteoblasts are profound, being far greater than those observed in response to several established osteoblast growth factors studied in the same *in vitro* assays, such as epidermal growth factor, transforming growth factor-β, parathyroid hormone, amylin or insulin. These factors increase thymidine incorporation in primary osteoblast cultures by only 20 – 30% (Cornish et al. 1999) whereas lactoferrin produces three- to five-fold increments (Cornish et al. 2004). This growth stimulating potency is complemented by the capacity of lactoferrin to substantially reduce osteoblast apoptosis, which again, is much more dramatic than the effects seen with other factors, such as insulin growth factor-1 (IGF-1) which maximally decreases apoptosis by 50% (Cornish et al. 2000) compared to 70% with

quenching ability, akin to antioxidant vitamins, is seemingly iron independent.

**2.2.4 Bioactive peptides derived from lactoferrin** 

through a common recognition site (Mir et al. 2010).

**3.1.1** *In vitro* **studies of lactoferrin activity in osteoblasts** 

**3. The activity of lactoferrin in bone** 

lactoferrin (Cornish et al. 2004).

**3.1 Osteoblasts** 

The activities of lactoferrin on osteoblasts demonstrated *in vitro* are likely to contribute to the potent effects on bone formation seen *in vivo* after administration of lactoferrin, even with a very short-term exposure (Fig 2) (Cornish et al. 2004). The bone growth resulting from local lactoferrin injection is considerably greater than that found previously in response to factors such as insulin, amylin, adrenomedullin and C-terminal PTH-related peptide (Cornish et al. 1996; 1997a; 1997b). It is qualitatively different from the effects of PTH in this model, which produces a powerful stimulation of bone resorption in addition to its effect on formation (Cornish et al. 1995). This anabolic potency suggests that lactoferrin should be further explored as a therapy for osteoporosis that can restore skeletal strength.

Fig. 2. Photomicrographs of calvariae from animals treated with lactoferrin (A) and vehicle (B) for 5 days. Fluorochrome labels used: green, calcein; red, alizarin. Horizontal bar, 50 m. (Figure reproduced with kind permission. Cornish J., et al. Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo. Endocrinology 145(9): 2004, 4366- 4374. Copyright 2004, The Endocrine Society.)

A B

Lactoferrin – A Potential Anabolic Intervention in Osteoporosis 809

Lactoferrin supports osteogenic differentiation in mesenchymal pluripotent cells whilst reducing adipogenic differentiation. In the pluripotent mesenchymal cell line, C2C12, analyses of expression levels of mRNA and proteins indicated an induction of osteoblastic and chondroblastic differentiation markers and a reduction in myoblastic and adipocytic markers (Yagi et al. 2009). We have identified that lactoferrin reduces adipogenic differentiation in KUSA4b10 cells, a mouse mesenchymal progenitor cell-line capable of developing into adipogenic or osteogenic cell lineages (Fig 4). Lactoferrin has also been found to promote the proliferation and osteogenic differentiation of human adipose stem cells (Ying et al. 2011). The activity of lactoferrin to support osteogenic differentiation whilst reducing adipogenic differentiation could be a promising approach for enhancing

**\***

[bLF] (g/ml) 0 1 10 100

Fig. 4. Lactoferrin reduces adipogenic differentiation in KUSA4b10 cells as measured by Oil

In comparison with actions of lactoferrin on osteoblasts, its osteoclasts effects are strikingly different, in that it produces an almost total arrest of osteoclastogenesis in mouse bone marrow cultures (Fig 5) (Cornish et al. 2004). Reduced bone-resorbing activity was also demonstrated by Lorget et al, who used bovine lactoferrin in a rabbit mixed bone cell culture (Lorget et al. 2002). The mechanisms implicated in the inhibitory effect of lactoferrin on bone resorption are only partially understood. In the rabbit bone cell cultures, lactoferrin inhibited the development of mature osteoclasts by a mechanism independent of the receptor activator of NF-κB (RANK)/RANK-ligand (RANKL)/osteoprotegerin (OPG) system. In the mouse bone marrow cultures, lactoferrin reduced RANKL expression, which could in part explain the inhibition of osteoclastogenesis, although this was counterbalanced by the effects of lactoferrin to also inhibit expression of OPG (Cornish et al. 2004). As the RANK/RANKL/OPG pathway does not appear to play a major role in mediating the inhibitory effect of lactoferrin on osteoclast formation, the possibility of a direct effect on osteoclasts has been investigated. In RAW264.7 cells, which differentiate into osteoclasts in vitro in the absence of osteoblasts or stromal cells, lactoferrin inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, demonstrating a direct effect on osteoclasts (Cornish & Naot 2010). This effect of lactoferrin was not blocked by an inhibitor of LRP1, indicating that LRP1 is not the receptor that mediates the direct inhibition of

Red O release detected in a spectrophotometer at an optical density (OD) of 500nm.

**\***

**3.1.4 Lactoferrin effects on early stages of osteogenic differentiation** 

osteogenic capacity of cell-based construction in bone tissue engineering.

**0.00**

**0.05**

**0.10**

**OD (500nm)**

**3.2 Osteoclasts** 

**0.15**

**0.20**

#### **3.1.3 Signalling pathways activated by lactoferrin in osteoblasts**

The downstream pathways activated by lactoferrin are largely unknown, although a number of lactoferrin receptors have been described. A specific lactoferrin receptor was cloned from the human intestine (Kawakami & Lonnerdal 1991) but this receptor is not expressed in all cell types that respond to lactoferrin and we have been unable to detect the mRNA in osteoblastic cells (Naot, unpublished data). Proteins that can bind and induce endocytosis of lactoferrin are nucleolin (Legrand et al. 2004) as well as low-density lipoprotein receptor-related proteins 1 and 2 (LRP1 and LRP2) (Ji & Mahley 1994; Willnow et al. 1992). LRP1 and LRP2 are expressed in osteoblastic cells and LRP1 is at least partially responsible for lactoferrin's mitogenic effects in osteoblasts (Grey et al. 2004). As lactoferrin complexes with LRP1, extracellular signal-regulated kinase (ERK) signalling pathway is upregulated. In addition, lactoferrin upregulates phosphoinositide 3-kinase-dependent Akt signalling but this is in an LRP-independent manner. Lactoferrin's anti-apoptotic activity in osteoblasts is independent of both these two signalling pathways.

In primary osteoblasts, lactoferrin induces a transient, dose-dependent increases in the transcription levels of IL-6, IL-11, the pro-inflammatory factor prostaglandin-endoperoxide synthase 2 (Ptgs2, encoding for the enzyme cyclooxygenase-2, COX-2) and the transcription factor nuclear factor of activated T-cells (Nfatc1). The activity of COX-2 to produce and secrete prostaglandin E2 and the activity of NFATc1 to promote transcription from NFAT consensus elements are also induced by lactoferrin. Moreover, COX-2 and NFATc1 act as mediators of the proliferative effect of lactoferrin in osteoblasts, as inhibition of their activities significantly reduces lactoferrin-induced thymidine incorporation (Naot et al. 2011). Recently, Nakajima et al demonstrated that lactoferrin induces synthesis of angiogenic factors by osteoblasts. In murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts lactoferrin, purified from milk, increased mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in a p44/p42 MAP kinase-dependent manner (Nakajima et al. 2011). A summary of some of the signalling pathways'activated by lactoferrin in osteoblasts is presented in Figure 3.

Fig. 3. Mechanisms of action of lactoferrin in osteoblasts. Figure reproduced with kind permission from Springer Science+Business Media: (*Biometals,* Lactoferrin as an effector molecule in the skeleton, 23, 2010, 425-430, Cornish, J. & Naot, D. Figure 1.)

#### **3.1.4 Lactoferrin effects on early stages of osteogenic differentiation**

Lactoferrin supports osteogenic differentiation in mesenchymal pluripotent cells whilst reducing adipogenic differentiation. In the pluripotent mesenchymal cell line, C2C12, analyses of expression levels of mRNA and proteins indicated an induction of osteoblastic and chondroblastic differentiation markers and a reduction in myoblastic and adipocytic markers (Yagi et al. 2009). We have identified that lactoferrin reduces adipogenic differentiation in KUSA4b10 cells, a mouse mesenchymal progenitor cell-line capable of developing into adipogenic or osteogenic cell lineages (Fig 4). Lactoferrin has also been found to promote the proliferation and osteogenic differentiation of human adipose stem cells (Ying et al. 2011). The activity of lactoferrin to support osteogenic differentiation whilst reducing adipogenic differentiation could be a promising approach for enhancing osteogenic capacity of cell-based construction in bone tissue engineering.

Fig. 4. Lactoferrin reduces adipogenic differentiation in KUSA4b10 cells as measured by Oil Red O release detected in a spectrophotometer at an optical density (OD) of 500nm.

#### **3.2 Osteoclasts**

808 Osteoporosis

The downstream pathways activated by lactoferrin are largely unknown, although a number of lactoferrin receptors have been described. A specific lactoferrin receptor was cloned from the human intestine (Kawakami & Lonnerdal 1991) but this receptor is not expressed in all cell types that respond to lactoferrin and we have been unable to detect the mRNA in osteoblastic cells (Naot, unpublished data). Proteins that can bind and induce endocytosis of lactoferrin are nucleolin (Legrand et al. 2004) as well as low-density lipoprotein receptor-related proteins 1 and 2 (LRP1 and LRP2) (Ji & Mahley 1994; Willnow et al. 1992). LRP1 and LRP2 are expressed in osteoblastic cells and LRP1 is at least partially responsible for lactoferrin's mitogenic effects in osteoblasts (Grey et al. 2004). As lactoferrin complexes with LRP1, extracellular signal-regulated kinase (ERK) signalling pathway is upregulated. In addition, lactoferrin upregulates phosphoinositide 3-kinase-dependent Akt signalling but this is in an LRP-independent manner. Lactoferrin's anti-apoptotic activity in

In primary osteoblasts, lactoferrin induces a transient, dose-dependent increases in the transcription levels of IL-6, IL-11, the pro-inflammatory factor prostaglandin-endoperoxide synthase 2 (Ptgs2, encoding for the enzyme cyclooxygenase-2, COX-2) and the transcription factor nuclear factor of activated T-cells (Nfatc1). The activity of COX-2 to produce and secrete prostaglandin E2 and the activity of NFATc1 to promote transcription from NFAT consensus elements are also induced by lactoferrin. Moreover, COX-2 and NFATc1 act as mediators of the proliferative effect of lactoferrin in osteoblasts, as inhibition of their activities significantly reduces lactoferrin-induced thymidine incorporation (Naot et al. 2011). Recently, Nakajima et al demonstrated that lactoferrin induces synthesis of angiogenic factors by osteoblasts. In murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts lactoferrin, purified from milk, increased mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in a p44/p42 MAP kinase-dependent manner (Nakajima et al. 2011). A summary of some of the signalling

**3.1.3 Signalling pathways activated by lactoferrin in osteoblasts** 

osteoblasts is independent of both these two signalling pathways.

pathways'activated by lactoferrin in osteoblasts is presented in Figure 3.

Fig. 3. Mechanisms of action of lactoferrin in osteoblasts. Figure reproduced with kind permission from Springer Science+Business Media: (*Biometals,* Lactoferrin as an effector

molecule in the skeleton, 23, 2010, 425-430, Cornish, J. & Naot, D. Figure 1.)

In comparison with actions of lactoferrin on osteoblasts, its osteoclasts effects are strikingly different, in that it produces an almost total arrest of osteoclastogenesis in mouse bone marrow cultures (Fig 5) (Cornish et al. 2004). Reduced bone-resorbing activity was also demonstrated by Lorget et al, who used bovine lactoferrin in a rabbit mixed bone cell culture (Lorget et al. 2002). The mechanisms implicated in the inhibitory effect of lactoferrin on bone resorption are only partially understood. In the rabbit bone cell cultures, lactoferrin inhibited the development of mature osteoclasts by a mechanism independent of the receptor activator of NF-κB (RANK)/RANK-ligand (RANKL)/osteoprotegerin (OPG) system. In the mouse bone marrow cultures, lactoferrin reduced RANKL expression, which could in part explain the inhibition of osteoclastogenesis, although this was counterbalanced by the effects of lactoferrin to also inhibit expression of OPG (Cornish et al. 2004). As the RANK/RANKL/OPG pathway does not appear to play a major role in mediating the inhibitory effect of lactoferrin on osteoclast formation, the possibility of a direct effect on osteoclasts has been investigated. In RAW264.7 cells, which differentiate into osteoclasts in vitro in the absence of osteoblasts or stromal cells, lactoferrin inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, demonstrating a direct effect on osteoclasts (Cornish & Naot 2010). This effect of lactoferrin was not blocked by an inhibitor of LRP1, indicating that LRP1 is not the receptor that mediates the direct inhibition of

Lactoferrin – A Potential Anabolic Intervention in Osteoporosis 811

activity that appeared to be equivalent, on a molar basis, to that of intact recombinant human lactoferrin. In contrast, the C-lobe of bovine lactoferrin has only a weak effect in this assay. The ability of the various lactoferrin lobes and fragments to influence both osteoblast proliferation and osteoclast development suggests that several sites on the lactoferrin molecule might be involved in receptor recognition, binding and stabilisation, or alternatively, more than one receptor might be involved. On a molar basis, the activity of intact lactoferrin on bone cell proliferation was at least 10-fold greater than that of the part molecules, which suggests that a global structure is required for optimal activity. In contrast, the equivalent osteoclastogenic activities of the recombinant human lactoferrin and its N-lobe suggest that this activity might be largely located in the N-lobe. However, further structure/function studies are warranted, as from a therapeutic perspective small active synthetic peptides might present a more attractive option for drug development than the intact lactoferrin molecule.

\* \*

**LF**

Fig. 6. Thymidine incorporation in primary rat osteoblasts treated with lactoferrin

A number of recently published studies tested the potential use of lactoferrin for protection against bone loss. The effect of dietary supplementation of lactoferrin on bone was measured using ovariectomized (OVX) rodents as a model for post menopausal bone loss (Blais et al. 2009; Guo et al. 2009; Malet et al. 2011). C3H mice that were either OVX or sham operated, received a control diet or the same diet supplemented with different concentrations of bovine lactoferrin for 27 weeks. Lactoferrin supplementation improved bone mineral density and bone strength, measured as femoral failure load, in a dosedependent manner (Blais et al. 2009). A study in OVX rats produced similar results. Lactoferrin orally administered to OVX rats for 3 months protected them against the OVXinduced reduction of bone volume and bone mineral density and increased the parameters of mechanical strength. Measurements of biochemical markers of bone remodelling indicated greater bone formation and reduced bone resorption occurred in rats treated with lactoferrin (Guo et al. 2009). Yamano et al. (Yamano et al. 2010) studied the potential use of lactoferrin for the prevention of alveolar bone destruction associated with periodontitis in an LPS-induced periodontitis rat model. Lactoferrin or liposomal-lactoferrin, which

**Control**

**0.0**

**0.5**

**1.0**

**Thymidine Incorporation**

**(treatment/control)**

**1.5**

**2.0**

**LFC (20-30)**

**LFC (17-31)**

**Control**

\* \* \*

**0.1g/ml 10g/ml**

**LFC (20-30)**

**LFC (17-31)**

\*

**N-lobe**

**C-lobe**

**LF**

\* \*

**N-lobe**

**3.4 Lactoferrin's activity in bone** *in vivo*

**LF**

**C-lobe**

fragments. LF; intact lactoferrin, LFC; lactoferricin.

**10-8M 10-7M 10-6M**

**A B**

\* \*

**Thymidine Incorporation**

**(Treatment/Control)**

**N-lobe**

**C-lobe**

osteoclastogenesis by lactoferrin (Cornish & Naot 2010). It should be noted that lactoferrin has a capacity to inhibit the survival of progenitor cells in the bone marrow, implying that it might also act earlier in osteoclast development (Hangoc et al. 1991). In contrast to its inhibitory effect on osteoclast development, lactoferrin had no effect on bone resorption by isolated mature osteoclasts nor in organ cultures which also detect mature osteoclast activity (Cornish et al. 2004). Thus, lactoferrin inhibits bone resorption by reducing the number of osteoclasts formed from precursor cells.

Fig. 5. Inhibition of osteoclastogenesis by lactoferrin in mouse bone marrow cultures

#### **3.3 Structure/Function relationship of lactoferrin's bone activity**

Bovine, human and recombinant forms of lactoferrin have comparable ability in stimulating osteoblast proliferation (Cornish et al. 2004). This suggests that glycosylation is not critical to the mitogenic activity as these three forms of lactoferrin are differentially glycosylated. Furthermore, when carbohydrate chains were removed from bovine lactoferrin, the aglycoform was as potent as the glyco-form (Cornish et al. 2006), confirming that carbohydrate is not a major determinant in the mitogenic activity of lactoferrin in osteoblasts. The activity of lactoferrin on bone cells was also shown to be independent of iron-binding with apo-, native and holo- preparations of bovine lactoferrin giving similar levels of stimulation of proliferation (Cornish et al. 2006). Moreover, replacement of the iron with chromium and manganese, two transition metals of equivalent size, also had no effect. This suggests firstly that the conformational changes induced by iron-binding have no impact on lactoferrin activity, and secondly that bound iron is not essential to this activity.

Further structure/function studies were performed using the N-lobe and C-lobe of bovine lactoferrin (prepared by proteolysis), the N-lobe of human lactoferrin (prepared by recombinant technology), and synthetic bovine lactoferricin peptides (Cornish et al. 2006). The N-lobes of both human and bovine lactoferrin and the C-lobe of bovine lactoferrin all showed osteogenic activity as measured by proliferation of primary rat osteoblasts, but the magnitude of response was less than for the full length molecule (Fig 6A). Interestingly, the bovine C-lobe appeared to have a stronger effect on proliferation than the bovine N-lobe. The bovine lactoferricin peptides (17-31 & 20-30) were both mildly osteogenic (Fig 6B). The Nlobe of human lactoferrin decreased osteoclastogenesis in a dose-dependent manner with an

osteoclastogenesis by lactoferrin (Cornish & Naot 2010). It should be noted that lactoferrin has a capacity to inhibit the survival of progenitor cells in the bone marrow, implying that it might also act earlier in osteoclast development (Hangoc et al. 1991). In contrast to its inhibitory effect on osteoclast development, lactoferrin had no effect on bone resorption by isolated mature osteoclasts nor in organ cultures which also detect mature osteoclast activity (Cornish et al. 2004). Thus, lactoferrin inhibits bone resorption by reducing the number of

Fig. 5. Inhibition of osteoclastogenesis by lactoferrin in mouse bone marrow cultures

Bovine, human and recombinant forms of lactoferrin have comparable ability in stimulating osteoblast proliferation (Cornish et al. 2004). This suggests that glycosylation is not critical to the mitogenic activity as these three forms of lactoferrin are differentially glycosylated. Furthermore, when carbohydrate chains were removed from bovine lactoferrin, the aglycoform was as potent as the glyco-form (Cornish et al. 2006), confirming that carbohydrate is not a major determinant in the mitogenic activity of lactoferrin in osteoblasts. The activity of lactoferrin on bone cells was also shown to be independent of iron-binding with apo-, native and holo- preparations of bovine lactoferrin giving similar levels of stimulation of proliferation (Cornish et al. 2006). Moreover, replacement of the iron with chromium and manganese, two transition metals of equivalent size, also had no effect. This suggests firstly that the conformational changes induced by iron-binding have no impact on lactoferrin

Further structure/function studies were performed using the N-lobe and C-lobe of bovine lactoferrin (prepared by proteolysis), the N-lobe of human lactoferrin (prepared by recombinant technology), and synthetic bovine lactoferricin peptides (Cornish et al. 2006). The N-lobes of both human and bovine lactoferrin and the C-lobe of bovine lactoferrin all showed osteogenic activity as measured by proliferation of primary rat osteoblasts, but the magnitude of response was less than for the full length molecule (Fig 6A). Interestingly, the bovine C-lobe appeared to have a stronger effect on proliferation than the bovine N-lobe. The bovine lactoferricin peptides (17-31 & 20-30) were both mildly osteogenic (Fig 6B). The Nlobe of human lactoferrin decreased osteoclastogenesis in a dose-dependent manner with an

**3.3 Structure/Function relationship of lactoferrin's bone activity** 

activity, and secondly that bound iron is not essential to this activity.

osteoclasts formed from precursor cells.

activity that appeared to be equivalent, on a molar basis, to that of intact recombinant human lactoferrin. In contrast, the C-lobe of bovine lactoferrin has only a weak effect in this assay. The ability of the various lactoferrin lobes and fragments to influence both osteoblast proliferation and osteoclast development suggests that several sites on the lactoferrin molecule might be involved in receptor recognition, binding and stabilisation, or alternatively, more than one receptor might be involved. On a molar basis, the activity of intact lactoferrin on bone cell proliferation was at least 10-fold greater than that of the part molecules, which suggests that a global structure is required for optimal activity. In contrast, the equivalent osteoclastogenic activities of the recombinant human lactoferrin and its N-lobe suggest that this activity might be largely located in the N-lobe. However, further structure/function studies are warranted, as from a therapeutic perspective small active synthetic peptides might present a more attractive option for drug development than the intact lactoferrin molecule.

Fig. 6. Thymidine incorporation in primary rat osteoblasts treated with lactoferrin fragments. LF; intact lactoferrin, LFC; lactoferricin.

#### **3.4 Lactoferrin's activity in bone** *in vivo*

A number of recently published studies tested the potential use of lactoferrin for protection against bone loss. The effect of dietary supplementation of lactoferrin on bone was measured using ovariectomized (OVX) rodents as a model for post menopausal bone loss (Blais et al. 2009; Guo et al. 2009; Malet et al. 2011). C3H mice that were either OVX or sham operated, received a control diet or the same diet supplemented with different concentrations of bovine lactoferrin for 27 weeks. Lactoferrin supplementation improved bone mineral density and bone strength, measured as femoral failure load, in a dosedependent manner (Blais et al. 2009). A study in OVX rats produced similar results. Lactoferrin orally administered to OVX rats for 3 months protected them against the OVXinduced reduction of bone volume and bone mineral density and increased the parameters of mechanical strength. Measurements of biochemical markers of bone remodelling indicated greater bone formation and reduced bone resorption occurred in rats treated with lactoferrin (Guo et al. 2009). Yamano et al. (Yamano et al. 2010) studied the potential use of lactoferrin for the prevention of alveolar bone destruction associated with periodontitis in an LPS-induced periodontitis rat model. Lactoferrin or liposomal-lactoferrin, which

Lactoferrin – A Potential Anabolic Intervention in Osteoporosis 813

resulted in significantly stronger bone regeneration at the defect than was observed in either lactoferrin-free- or low-lactoferrin-treated rats (Takaoka et al. 2011). It was concluded that the sustained release from the gelatin hydrogels enabled lactoferrin to enhance the *in vivo* activity of bone regeneration. A titanium bone plate carrying lactoferrin for treatment of metaphyseal fracture has been patented, primarily as an implant for antibiosis, but such a device could conceivably be used for the promotion of bone repair (Fei et al. 2008). In the same context, a recent technology has been described whereby lactoferrin was coated in thin films onto inert substrates such as silica and biocompatibility assessed for use in

The potential use of lactoferrin as a food supplement that promotes bone health requires experimental evidence showing that it is active when administered orally. Most ingested proteins are degraded into oligopeptides and amino acids in the small intestine and then absorbed as nutrients. The digestion of lactoferrin was studied in adult mice and rats (Fischer et al. 2007; Kuwata et al. 2001). In mice, immunoreactive lactoferrin, measured by ELISA one hour following intragastric intubation of a single dose, was present at the highest concentrations in the stomach, and in lower concentrations in all segments of the intestine: proximal intestine, distal intestine, caecum and large bowel (Fischer et al. 2007). Oral administration of 125I-labelled lactoferrin in adult rats, followed by detection of multiple forms of degraded lactoferrin by surface-enhanced laser desorption/ionization (SELDI) affinity mass spectrometry showed that the bioactive fragment lactoferricin (17-42) could

Transport of intact lactoferrin from the gut lumen to the circulation has been shown in infants (Hutchens et al. 1989, 1991; Knapp & Hutchens 1994) young calves (Talukder et al. 2002, 2003) and piglets (Harada et al. 1999) suggesting that as the selective transport from the gut is not yet fully developed macromolecules can cross into the circulation. In addition, a number of recent studies demonstrated transport of intact lactoferrin in adult animals and in humans. Fischer et al. (Fischer et al. 2007) found that 10 minutes after the administration of 1mg lactoferrin to adult mice through intragastric intubation, the intact molecule could be detected in the peripheral blood as well as in the liver, kidneys, gall bladder, spleen and brain. Transport of lactoferrin into the circulation has also been shown in groups of Ovx mice that were fed different concentrations of bovine lactoferrin (1-20 g/kg) for 27 weeks. Blood concentrations of immunoreactive lactoferrin of mice that received the bovine lactoferrin-supplemented diets were significantly increased compared to controls and were

An important consideration for the use of food systems as vehicles for bioactive delivery is that the bioactive remains active throughout manufacture and shelf-life of the product. Recent work has shown that bovine lactoferrin dosed into stirred yoghurt remained structurally intact and retained its osteogenic activity on primary bone-forming cells up to 21 days after storage of the yoghurt at 4° C (Palmano et al. 2011). In many respects yoghurt is the ideal functional food matrix for bone as it is calcium rich and a popular consumer

survive proteolytic degradation in the small intestine (Kuwata et al. 2001).

correlated to the bovine lactoferrin concentration in the diet (Blais et al. 2009).

applications such as implants (Constantinescu et al. 2009).

**3.6.2 Oral delivery** 

**3.6.3 Functional foods** 

product.

improved the robustness of bovine lactoferrin to digestive enzymes, were added to the drinking water for 7 days. The study showed that bone resorption stimulated by LPS through activation of TNF-α production and modulation of RANKL/OPG balance in osteoblasts was inhibited by the orally administered lactoferrin. The researchers suggest that liposomal-lactoferrin could be a potent therapeutic and preventive agent for attenuating alveolar bone destruction in periodontitis patients.

In a small clinical study, 38 healthy postmenopausal women were randomized to receive placebo or a ribonuclease-enriched lactoferrin dietary supplement (Bharadwaj et al. 2009). In the lactoferrin-treated group there was a decrease in the bone resorption markers urine deoxypyridinoline (Dpd) crosslinks and serum N-telopeptides and an increase in the bone formation markers bone-specific alkaline phosphatase and osteocalcin, but the results are difficult to interpret due to differences in the levels of markers between the two groups before treatment.

#### **3.5 The expression of lactoferrin in bone and cartilage**

Investigations of the expression of lactoferrin in normal fetal and adult bone and cartilage by immunohistochemistry determined that fetal chondroblasts and osteoblasts are positive for lactoferrin immunoreactivity, whereas the corresponding adult cells are negative (Antonio et al. 2010; Ieni et al. 2009a; 2009b; 2011). Bone and cartilaginous specimens from fetuses at 8-34 weeks of gestation were studied. At the eighth gestational week, lactoferrin immunoreactivity was mainly present in the mesenchymal cells forming the periosteum and in chondroblasts; and a lactoferrin signal was also present in immature osteocytes and osteoblasts up to the 18th gestation week. The lactoferrin immunoreactivity decreased considerably by the 24th week, with no expression found in any bone area after the 30th week or in any samples from adult bone (Antonio et al. 2010; Ieni et al. 2011). The expression of lactoferrin in bone and cartilaginous tissue between 8 and 24 weeks of gestation suggests a possible role for lactoferrin as a bone growth regulator in the early phases of the human endochondral ossification.

The expression of lactoferrin was also studied by immunohistochemistry in a large number of tumors of bone and cartilage (Ieni et al. 2009a; 2009b; 2011). About half of all cases of osteocartilagineous tumors were positive, with lactoferrin expression in all giant cell tumors tested, all chondroblastomas, chondromyxoid fibromas and most osteoid osteomas. No lactoferrin immunoexpression was detected in osteosarcomas, chondrosarcomas, ossifying fibromas, osteochondroma and enchondromas. It is possible that lactoferrin expression reflects a less mature phenotype of these tumors, as lactoferrin is absent from normal adult bone and cartilage tissues.

#### **3.6 Lactoferrin as a therapeutic agent 3.6.1 Local delivery**

There is much interest in the potential use of lactoferrin as a factor that can act locally in topical applications for regenerative bone therapies and bone tissue engineering. Various biomaterials and biomedical devices have been used to improve delivery and enable sustained release of lactoferrin at the requisite site. Bovine lactoferrin incorporated into a type 1 collagen membrane promoted bone-like tissue formation by MG63 cells which were plated over the membrane (Takayama & Mizumachi 2009) and implantation of biodegradable gelatin hydrogels incorporating lactoferrin into a skull bone defect of rats resulted in significantly stronger bone regeneration at the defect than was observed in either lactoferrin-free- or low-lactoferrin-treated rats (Takaoka et al. 2011). It was concluded that the sustained release from the gelatin hydrogels enabled lactoferrin to enhance the *in vivo* activity of bone regeneration. A titanium bone plate carrying lactoferrin for treatment of metaphyseal fracture has been patented, primarily as an implant for antibiosis, but such a device could conceivably be used for the promotion of bone repair (Fei et al. 2008). In the same context, a recent technology has been described whereby lactoferrin was coated in thin films onto inert substrates such as silica and biocompatibility assessed for use in applications such as implants (Constantinescu et al. 2009).

#### **3.6.2 Oral delivery**

812 Osteoporosis

improved the robustness of bovine lactoferrin to digestive enzymes, were added to the drinking water for 7 days. The study showed that bone resorption stimulated by LPS through activation of TNF-α production and modulation of RANKL/OPG balance in osteoblasts was inhibited by the orally administered lactoferrin. The researchers suggest that liposomal-lactoferrin could be a potent therapeutic and preventive agent for attenuating

In a small clinical study, 38 healthy postmenopausal women were randomized to receive placebo or a ribonuclease-enriched lactoferrin dietary supplement (Bharadwaj et al. 2009). In the lactoferrin-treated group there was a decrease in the bone resorption markers urine deoxypyridinoline (Dpd) crosslinks and serum N-telopeptides and an increase in the bone formation markers bone-specific alkaline phosphatase and osteocalcin, but the results are difficult to interpret due to differences in the levels of markers between the two groups

Investigations of the expression of lactoferrin in normal fetal and adult bone and cartilage by immunohistochemistry determined that fetal chondroblasts and osteoblasts are positive for lactoferrin immunoreactivity, whereas the corresponding adult cells are negative (Antonio et al. 2010; Ieni et al. 2009a; 2009b; 2011). Bone and cartilaginous specimens from fetuses at 8-34 weeks of gestation were studied. At the eighth gestational week, lactoferrin immunoreactivity was mainly present in the mesenchymal cells forming the periosteum and in chondroblasts; and a lactoferrin signal was also present in immature osteocytes and osteoblasts up to the 18th gestation week. The lactoferrin immunoreactivity decreased considerably by the 24th week, with no expression found in any bone area after the 30th week or in any samples from adult bone (Antonio et al. 2010; Ieni et al. 2011). The expression of lactoferrin in bone and cartilaginous tissue between 8 and 24 weeks of gestation suggests a possible role for lactoferrin as a bone growth regulator in the early phases of the human

The expression of lactoferrin was also studied by immunohistochemistry in a large number of tumors of bone and cartilage (Ieni et al. 2009a; 2009b; 2011). About half of all cases of osteocartilagineous tumors were positive, with lactoferrin expression in all giant cell tumors tested, all chondroblastomas, chondromyxoid fibromas and most osteoid osteomas. No lactoferrin immunoexpression was detected in osteosarcomas, chondrosarcomas, ossifying fibromas, osteochondroma and enchondromas. It is possible that lactoferrin expression reflects a less mature phenotype of these tumors, as lactoferrin is absent from normal adult

There is much interest in the potential use of lactoferrin as a factor that can act locally in topical applications for regenerative bone therapies and bone tissue engineering. Various biomaterials and biomedical devices have been used to improve delivery and enable sustained release of lactoferrin at the requisite site. Bovine lactoferrin incorporated into a type 1 collagen membrane promoted bone-like tissue formation by MG63 cells which were plated over the membrane (Takayama & Mizumachi 2009) and implantation of biodegradable gelatin hydrogels incorporating lactoferrin into a skull bone defect of rats

alveolar bone destruction in periodontitis patients.

**3.5 The expression of lactoferrin in bone and cartilage** 

before treatment.

endochondral ossification.

bone and cartilage tissues.

**3.6.1 Local delivery** 

**3.6 Lactoferrin as a therapeutic agent** 

The potential use of lactoferrin as a food supplement that promotes bone health requires experimental evidence showing that it is active when administered orally. Most ingested proteins are degraded into oligopeptides and amino acids in the small intestine and then absorbed as nutrients. The digestion of lactoferrin was studied in adult mice and rats (Fischer et al. 2007; Kuwata et al. 2001). In mice, immunoreactive lactoferrin, measured by ELISA one hour following intragastric intubation of a single dose, was present at the highest concentrations in the stomach, and in lower concentrations in all segments of the intestine: proximal intestine, distal intestine, caecum and large bowel (Fischer et al. 2007). Oral administration of 125I-labelled lactoferrin in adult rats, followed by detection of multiple forms of degraded lactoferrin by surface-enhanced laser desorption/ionization (SELDI) affinity mass spectrometry showed that the bioactive fragment lactoferricin (17-42) could survive proteolytic degradation in the small intestine (Kuwata et al. 2001).

Transport of intact lactoferrin from the gut lumen to the circulation has been shown in infants (Hutchens et al. 1989, 1991; Knapp & Hutchens 1994) young calves (Talukder et al. 2002, 2003) and piglets (Harada et al. 1999) suggesting that as the selective transport from the gut is not yet fully developed macromolecules can cross into the circulation. In addition, a number of recent studies demonstrated transport of intact lactoferrin in adult animals and in humans. Fischer et al. (Fischer et al. 2007) found that 10 minutes after the administration of 1mg lactoferrin to adult mice through intragastric intubation, the intact molecule could be detected in the peripheral blood as well as in the liver, kidneys, gall bladder, spleen and brain. Transport of lactoferrin into the circulation has also been shown in groups of Ovx mice that were fed different concentrations of bovine lactoferrin (1-20 g/kg) for 27 weeks. Blood concentrations of immunoreactive lactoferrin of mice that received the bovine lactoferrin-supplemented diets were significantly increased compared to controls and were correlated to the bovine lactoferrin concentration in the diet (Blais et al. 2009).

#### **3.6.3 Functional foods**

An important consideration for the use of food systems as vehicles for bioactive delivery is that the bioactive remains active throughout manufacture and shelf-life of the product. Recent work has shown that bovine lactoferrin dosed into stirred yoghurt remained structurally intact and retained its osteogenic activity on primary bone-forming cells up to 21 days after storage of the yoghurt at 4° C (Palmano et al. 2011). In many respects yoghurt is the ideal functional food matrix for bone as it is calcium rich and a popular consumer product.

Lactoferrin – A Potential Anabolic Intervention in Osteoporosis 815

protects against bone loss associated with oestrogen deficiency. The molecular pathways activated by lactoferrin in bone cells are only partially understood, and it appears that a combination of direct and indirect physiological mechanisms is producing the overall anabolic effect of lactoferrin in bone. Pharmaceutical or nutriceutical use of lactoferrin would require the development of a preparation with assured safety and consistent quality of supply. A better understanding of lactoferrin's mechanism of action in bone would allow for the design of compounds that can mimic its anabolic bone activity, and would be useful

in pathological states of reduced bone quality in either systemic or local applications.

Baker, E. N. (1994). Structure and reactivity of transferrins. *Adv Inorg Chem* 41: 389-463. Baker, E. N. & Baker, H. M. (2005). Molecular structure, binding properties and dynamics of

multifunctional character of lactoferrin. *Biochimie* 91(1): 3-10.

in postmenopausal women. *Osteoporos Int* 20(9): 1603-1611.

granulocytes via release of lactoferrin. *J Clin Invest* 119(1): 20-32. Brock, J. H. (2002). The physiology of lactoferrin. *Biochem Cell Biol* 80(1): 1-6.

transferrin and lactoferrin. *Biochim Biophys Acta* 446(1): 214-225.

lactoferrin. *Cell Mol Life Sci* 62(22): 2531-2539.

peroxide. *J Biol Chem* 259(21): 13391-13394.

Antonio, I., Valeria, B., Maddalena, G. & Giovanni, T. (2010). Immunohistochemical

Baker, E. N. & Baker, H. M. (2009). A structural framework for understanding the

Baldwin, D. A., Jenny, E. R. & Aisen, P. (1984). The effect of human serum transferrin and

Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K. & Tomita, M. (1992).

Bethell, D., Cerven, D. & DeGeorge, G. (2008a). 28-day repeated dose oral toxicity of

Bethell, D., Cerven, D. & DeGeorge, G. (2008b). 28-Day repeated dose oral toxicity of recombinant human holo-lactoferrin in rats. *Regul Toxicol Pharm* 52(2): 174-179. Bezwoda, W. R. & Mansoor, N. (1989). Lactoferrin from human breast milk and from

Blais, A., Malet, A., Mikogami, T., Martin-Rouas, C. & Tome, D. (2009). Oral bovine

Bournazou, I., Mackenzie, K. J., Duffin, R., Rossi, A. G. & Gregory, C. D. (2010). Inhibition of eosinophil migration by lactoferrin. *Immunol Cell Biol* 88(2): 220-223. Bournazou, I., Pound, J. D., Duffin, R., Bournazos, S., Melville, L. A., Brown, S. B., Rossi, A.

Brock, J. H., Arzabe, F., Lampreave, F. & Pineiro, A. (1976). The effect of trypsin on bovine

evidence of lactoferrin in human embryo-fetal bone and cartilage tissues. *Cell Biol* 

milk lactoferrin on hydroxyl radical formation from superoxide and hydrogen

Identification of the bactericidal domain of lactoferrin. *Biochimt Biophys Acta* 1121(1-

recombinant human apo-lactoferrin or recombinant human lysozyme in rats. *Regul* 

neutrophil granulocytes. Comparative studies of isolation, quantitation, characterization and iron binding properties. *Biomed Chromatog* 3(3): 121-126. Bharadwaj, S., Naidu, A. G., Betageri, G. V., Prasadarao, N. V. & Naidu, A. S. (2009). Milk

ribonuclease-enriched lactoferrin induces positive effects on bone turnover markers

lactoferrin improves bone status of ovariectomized mice. *Am J Physiol Endocrinol* 

G. & Gregory, C. D. (2009). Apoptotic human cells inhibit migration of

**5. References** 

*Int* 34(8): 845-849.

2): 130-136.

*Toxicol Pharm* 51(2): 162-167.

*Metab* 296(6): E1281-1288.

### **3.6.4 Lactoferrin preparations with potential use for bone applications**

Bovine and human lactoferrin constitute the most studied of the lactoferrins. In general and in spite of some structural differences, bovine and human lactoferrin including recombinant forms appear to have comparable bioactivities. However, some differences between bovine lactoferrin and human lactoferrin have been noted with respect to intestinal receptor recognition (Kawakami & Lonnerdal 1991) and it cannot be assumed that activities are always interchangeable

The use of lactoferrin as a therapeutic agent requires not only proof of efficacy at the clinical level, but assured safety, consistent quality of supply and appropriate delivery mechanisms. Most studies on the effects of lactoferrin on bone, including clinical trials, have been performed using bovine lactoferrin. Bovine lactoferrin from milk has been available as a commercial isolate for many years (Tomita et al. 2009). It has a 'Generally Recognized As Safe' (GRAS) status from the United States Food and Drug Administration (FDA) and now has widespread acceptance for oral use in humans. Indeed, it has been available for a number of years in Japan and other countries as a dietary supplement and as a functional ingredient in foods such as yoghurt and fortified infant formulae (Wakabayashi & Tasaki 2006). Bovine lactoferrin has been administered orally at doses of 3g/day for one year in a cancer clinical trial, with some positive outcomes and no apparent adverse effects (Tomita et al. 2009).

Equally, recombinant human lactoferrin can be considered for oral application although to date there have been no oral efficacy clinical trials targeted specifically at bone. High expression levels can be achieved in rice (Nandi et al. 2005) and transgenic animals (van Berkel et al. 2002) and recombinant human lactoferrin expressed in both baby kidney hamster cells and rice has been shown to have comparable activity to human lactoferrin and bovine lactoferrin in stimulating proliferation of primary rat osteoblasts (Cornish et al. 2004; Huang et al. 2008). Rice recombinant human lactoferrin was shown to have no toxicity in rats when administered up to 1000 mg/kg body weight/day for 28 days (Bethell et al. 2008a, 2008b) and did not elicit an allergic response in plant-glycan sensitive humans in a limited clinical study (Mari et al. 2008). Moreover it was shown to have beneficial effects as an oral agent in a clinical trial targeted at reduction of diarrhoea in Peruvian children (Zavaleta et al. 2007). No adverse events were reported.

Another potential candidate for bone interventions is Talactoferrin alpha, a proprietary recombinant human lactoferrin expressed in the fungus *Aspergillus awamori* and produced at industrial scale by Aggenix AG (Sanchez et al. 2010). Talactoferrin is currently being evaluated for the oral treatment of several cancer types and Fast Track designation has been granted to Agennix by the FDA for treatment of non-small cell lung cancer (NSCLC) and first-line treatment of renal carcinoma in combination with sunitinib. Placebo-controlled Phase II clinical trials have been successfully completed for NSCLC (Jonasch et al. 2008) and at time of writing two Phase III trials evaluating Talactoferrin in NSCLC patients are ongoing. Talactoferrin appears to have no toxicity, is well tolerated and also appears to be safe for topical applications. It has shown efficacy in the local treatment of diabetic ulcers (Engelmayer et al. 2008) for which it also has Fast Track FDA approval (Sanchez et al. 2010).

#### **4. Conclusion**

The positive effects of lactoferrin in bone have been demonstrated *in vitro*; where lactoferrin induces osteoblast proliferation, survival and differentiation and inhibits osteoclast formation, and *in vivo*; where lactoferrin given as a dietary supplement to rat and mice protects against bone loss associated with oestrogen deficiency. The molecular pathways activated by lactoferrin in bone cells are only partially understood, and it appears that a combination of direct and indirect physiological mechanisms is producing the overall anabolic effect of lactoferrin in bone. Pharmaceutical or nutriceutical use of lactoferrin would require the development of a preparation with assured safety and consistent quality of supply. A better understanding of lactoferrin's mechanism of action in bone would allow for the design of compounds that can mimic its anabolic bone activity, and would be useful in pathological states of reduced bone quality in either systemic or local applications.

### **5. References**

814 Osteoporosis

Bovine and human lactoferrin constitute the most studied of the lactoferrins. In general and in spite of some structural differences, bovine and human lactoferrin including recombinant forms appear to have comparable bioactivities. However, some differences between bovine lactoferrin and human lactoferrin have been noted with respect to intestinal receptor recognition (Kawakami & Lonnerdal 1991) and it cannot be assumed that activities are

The use of lactoferrin as a therapeutic agent requires not only proof of efficacy at the clinical level, but assured safety, consistent quality of supply and appropriate delivery mechanisms. Most studies on the effects of lactoferrin on bone, including clinical trials, have been performed using bovine lactoferrin. Bovine lactoferrin from milk has been available as a commercial isolate for many years (Tomita et al. 2009). It has a 'Generally Recognized As Safe' (GRAS) status from the United States Food and Drug Administration (FDA) and now has widespread acceptance for oral use in humans. Indeed, it has been available for a number of years in Japan and other countries as a dietary supplement and as a functional ingredient in foods such as yoghurt and fortified infant formulae (Wakabayashi & Tasaki 2006). Bovine lactoferrin has been administered orally at doses of 3g/day for one year in a cancer clinical trial, with some

Equally, recombinant human lactoferrin can be considered for oral application although to date there have been no oral efficacy clinical trials targeted specifically at bone. High expression levels can be achieved in rice (Nandi et al. 2005) and transgenic animals (van Berkel et al. 2002) and recombinant human lactoferrin expressed in both baby kidney hamster cells and rice has been shown to have comparable activity to human lactoferrin and bovine lactoferrin in stimulating proliferation of primary rat osteoblasts (Cornish et al. 2004; Huang et al. 2008). Rice recombinant human lactoferrin was shown to have no toxicity in rats when administered up to 1000 mg/kg body weight/day for 28 days (Bethell et al. 2008a, 2008b) and did not elicit an allergic response in plant-glycan sensitive humans in a limited clinical study (Mari et al. 2008). Moreover it was shown to have beneficial effects as an oral agent in a clinical trial targeted at reduction of diarrhoea in Peruvian children (Zavaleta et

Another potential candidate for bone interventions is Talactoferrin alpha, a proprietary recombinant human lactoferrin expressed in the fungus *Aspergillus awamori* and produced at industrial scale by Aggenix AG (Sanchez et al. 2010). Talactoferrin is currently being evaluated for the oral treatment of several cancer types and Fast Track designation has been granted to Agennix by the FDA for treatment of non-small cell lung cancer (NSCLC) and first-line treatment of renal carcinoma in combination with sunitinib. Placebo-controlled Phase II clinical trials have been successfully completed for NSCLC (Jonasch et al. 2008) and at time of writing two Phase III trials evaluating Talactoferrin in NSCLC patients are ongoing. Talactoferrin appears to have no toxicity, is well tolerated and also appears to be safe for topical applications. It has shown efficacy in the local treatment of diabetic ulcers (Engelmayer et al. 2008) for which it also has Fast Track FDA approval (Sanchez et al. 2010).

The positive effects of lactoferrin in bone have been demonstrated *in vitro*; where lactoferrin induces osteoblast proliferation, survival and differentiation and inhibits osteoclast formation, and *in vivo*; where lactoferrin given as a dietary supplement to rat and mice

**3.6.4 Lactoferrin preparations with potential use for bone applications** 

positive outcomes and no apparent adverse effects (Tomita et al. 2009).

al. 2007). No adverse events were reported.

**4. Conclusion** 

always interchangeable


Lactoferrin – A Potential Anabolic Intervention in Osteoporosis 817

Gahr, M., Speer, C. P., Damerau, B. & Sawatzki, G. (1991). Influence of lactoferrin on the

Gonzalez-Chavez, S. A., Arevalo-Gallegos, S. & Rascon-Cruz, Q. (2009). Lactoferrin: structure, function and applications. *Int J Antimicrob Agents* 33(4): 301 e301-308. Grey, A., Banovic, T., Zhu, Q., Watson, M., Callon, K., Palmano, K., Ross, J., Naot, D., Reid, I. R.

Grey, A., Zhu, Q., Watson, M., Callon, K. & Cornish, J. (2006). Lactoferrin potently inhibits

Groves, M. L., Basch, J. J. & Gordon, W. G. (1963). Isolation, Characterization, and Amino Acid

Guo, H. Y., Jiang, L., Ibrahim, S. A., Zhang, L., Zhang, H., Zhang, M. & Ren, F. Z. (2009).

Hangoc, G., Falkenburg, J. H. & Broxmeyer, H. E. (1991). Influence of T-lymphocytes and

He, J. & Furmanski, P. (1995). Sequence specificity and transcriptional activation in the

Huang, N., Bethell, D., Card, C., Cornish, J., Marchbank, T., Wyatt, D., Mabery, K. &

Hutchens, T. W., Henry, J. F. & Yip, T. T. (1991). Structurally intact (78-kDa) forms of

result in fragment dissociation. *Proc Natl Acad Sci U S A* 88(8): 2994-2998. Ieni, A., Barresi, V., Grosso, M., Rosa, M. A. & Tuccari, G. (2009a). Immunolocalization of lactoferrin in cartilage-forming neoplasms. *J Orthop Sci* 14(6): 732-737. Ieni, A., Barresi, V., Grosso, M., Rosa, M. A. & Tuccari, G. (2009b). Lactoferrin immuno-

Ieni, A., Barresi, V., Grosso, M., Speciale, G., Rosa, M. A. & Tuccari, G. (2011). Does

Jenssen, H. & Hancock, R. E. (2009). Antimicrobial properties of lactoferrin. *Biochimie* 91(1): 19-29. Ji, Z. S. & Mahley, R. W. (1994). Lactoferrin binding to heparan sulfate proteoglycans and the

cartilage human neoplasms? *Pathol Oncol Res* 17(2): 287-293.

piglets. *Comp Biochem Physiol A Mol Integr Physiol* 124(3): 321-327.

binding of lactoferrin to DNA. *Nature* 373(6516): 721-724.

ovariectomized rats. *J Nutr* 139(5): 958-964.

49(5): 427-433.

2): 96-102.

1928-1933.

364-371.

function of human polymorphonuclear leukocytes and monocytes. *J Leukoc Biol*

& Cornish, J. (2004). The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells. *Mol Endocrinol* 18(9): 2268-2278.

osteoblast apoptosis, via an LRP1-independent pathway. *Mol Cell Endocrinol* 251(1-

Composition of a New Crystalline Protein, Lactollin, from Milk. *Biochemistry* 2: 814-817.

Orally administered lactoferrin preserves bone mass and microarchitecture in

lactoferrin on the survival-promoting effects of IL-1 and IL-6 on human bone marrow granulocyte-macrophage and erythroid progenitor cells. *Exp Hematol* 19(7): 697-703. Harada, E., Itoh, Y., Sitizyo, K., Takeuchi, T., Araki, Y. & Kitagawa, H. (1999). Characteristic

transport of lactoferrin from the intestinal lumen into the bile via the blood in

Playford, R. (2008). Bioactive recombinant human lactoferrin, derived from rice, stimulates mammalian cell growth. *In Vitro Cell Dev Biol Anim* 44(10): 464-471. Hutchens, T. W., Henry, J. F. & Yip, T. T. (1989). Purification and characterization of intact

lactoferrin found in the urine of human milk-fed preterm infants. *Clin Chem* 35(9):

maternal lactoferrin purified from urine of preterm infants fed human milk: identification of a trypsin-like proteolytic cleavage event in vivo that does not

expression in human normal and neoplastic bone tissue. *J Bone Miner Metab* 27(3):

lactoferrin behave as an immunohistochemical oncofetal marker in bone and

LDL receptor-related protein. Further evidence supporting the importance of direct


Caccavo, D., Sebastiani, G. D., Di Monaco, C., Guido, F., Galeazzi, M., Ferri, G. M., Bonomo,

Cornish, J., Balchin, L. M., Callon, K. E., Grey, A. B., Cooper, G. J. & Reid, I. R. (2000).

Cornish, J., Callon, K. E., Coy, D. H., Jiang, N. Y., Xiao, L., Cooper, G. J. & Reid, I. R. (1997a).

Cornish, J., Callon, K. E., Lin, C. Q., Xiao, C. L., Gamble, G. D., Cooper, G. J. & Reid, I. R.

Cornish, J., Callon, K. E., Naot, D., Palmano, K. P., Banovic, T., Bava, U., Watson, M., Lin, J.

Cornish, J., Callon, K. E., Nicholson, G. C. & Reid, I. R. (1997b). Parathyroid hormone-related protein-(107-139) inhibits bone resorption in vivo. *Endocrinology* 138(3): 1299-1304. Cornish, J., Callon, K. E. & Reid, I. R. (1995). An in vivo model for the rapid assessment of

Cornish, J., Callon, K. E. & Reid, I. R. (1996). Insulin increases histomorphometric indices of

Cornish, J. & Naot, D. (2010). Lactoferrin as an effector molecule in the skeleton. *Biometals*

Cornish, J., Palmano, K., Callon, K. E., Watson, M., Lin, J. M., Valenti, P., Naot, D., Grey, A.

Derisbourg, P., Wieruszeski, J. M., Montreuil, J. & Spik, G. (1990). Primary Structure of Glycans

Fischer, R., Debbabi, H., Blais, A., Dubarry, M., Rautureau, M., Boyaka, P. N. & Tome, D.

bone formation In vivo. *Calcif Tissue Int* 59(6): 492-495.

*Sci* 255(10): 5491-5495.

apoptosis. *Bone* 27: 19S.

*Am J Physiol* 273(6 Pt 1): E1113-1120.

*Endocrinology* 145(9): 4366-4374.

*Biochem Cell Biol* 84(3): 297-302.

defensins or lactoferrin.

tissues. *Int Immunopharmacol* 7(10): 1387-1393.

Suppl): 249S-254S.

23(3): 425-430.

osteoblasts. *J Bone Miner Res* 14(8): 1302-1309.

L. & Afeltra, A. (1999). Increased levels of lactoferrin in synovial fluid but not in serum from patients with rheumatoid arthritis. *Int J Clin Lab Res* 29(1): 30-35. Constantinescu, C., Palla-Papavlu, A., Rotaru, A., Florian, P., Chelu, F., Icriverzi, M.,

Nedelcea, A., Dinca, V., Roseanu, A. & Dinescu, M. (2009). Multifunctional thin films of lactoferrin for biochemical use deposited by MAPLE technique. *Appl Surf* 

Amylin and adrenomedullin - novel promotors of osteoblast survival from

Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo.

(1999). Comparison of the effects of calcitonin gene-related peptide and amylin on

M., Tong, P. C., Chen, Q., Chan, V. A., Reid, H. E., Fazzalari, N., Baker, H. M., Baker, E. N., Haggarty, N. W., Grey, A. B. & Reid, I. R. (2004). Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo.

the local effects of parathyroid hormone on bone histomorphometry. *Bone* 17(4

B. & Reid, I. R. (2006). Lactoferrin and bone; structure-activity relationships.

Isolated from Human-Leukocyte Lactotransferrin - Absence of Fucose Residues Questions the Proposed Mechanism of Hyposideremia. *Biochem J* 269(3): 821-825. Engelmayer, J., Blezinger, P. & Varadhachary, A. (2008). Talactoferrin stimulates wound healing with modulation of inflammation. *J Surg Res* 149(2): 278-286. Fei, J., Yu, H., Wang, A., Zhao, C. & Pan, Z. (2008). Bone plate for carrying and releasing

antibacterial peptide comprises titanium or titanium alloy material having hydroxy phosphorite coating on its surface, where outer surface of coating has beta-

(2007). Uptake of ingested bovine lactoferrin and its accumulation in adult mouse


Lactoferrin – A Potential Anabolic Intervention in Osteoporosis 819

Mir, R., Kumar, R. P., Singh, N., Vikram, G. P., Sinha, M., Bhushan, A., Kaur, P., Srinivasan,

Moguilevsky, N., Retegui, L. A. & Masson, P. L. (1985). Comparison of human lactoferrins

iron-binding properties and uptake by the liver. *Biochem J* 229(2): 353-359. Nakajima, K. I., Kanno, Y., Nakamura, M., Gao, X. D., Kawamura, A., Itoh, F. & Ishisaki, A.

and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway. *Biometals*. Nandi, S., Yalda, D., Lu, S., Nikolov, Z., Misaki, R., Fujiyama, K. & Huang, N. (2005). Process

Naot, D., Chhana, A., Matthews, B. G., Callon, K. E., Tong, P. C., Lin, J. M., Costa, J. L.,

Paesano, R., Pietropaoli, M., Gessani, S. & Valenti, P. (2009). The influence of lactoferrin,

Palmano, K. P., Ramos, R., Watson, M., Callon, K. E. & Cornish, J. (2011). Survival and bone-

Pierce, A., Colavizza, D., Benaissa, M., Maes, P., Tartar, A., Montreuil, J. & Spik, G. (1991).

Raghuveer, T. S., McGuire, E. M., Martin, S. M., Wagner, B. A., Rebouche, C. J., Buettner, G.

Sanchez, L., Conesa, C. & Calvo, M. (2010). Recombinant human lactoferrin: A valuable protein for pharmaceutical products and functional foods. *Biotechnol Adv* 28(6): 831-838. Shinoda, I., Takase, M., Fukuwatari, Y., Shimamura, S., Koller, M. & Konig, W. (1996).

polymorphonuclear leukocytes. *Biosci Biotechnol Biochem* 60(3): 521-523. Siciliano, R., Rega, B., Marchetti, M., Seganti, L., Antonini, G. & Valenti, P. (1999). Bovine

Smith, C. A., Ainscough, E. W., Baker, H. M., Brodie, A. M. & Baker, E. N. (1994). Specific

Sorimachi, K., Akimoto, K., Hattori, Y., Ieiri, T. & Niwa, A. (1997). Activation of macrophages by lactoferrin: secretion of TNF-alpha, IL-8 and NO. *Biochem Mol Biol Int* 43(1): 79-87. Takaoka, R., Hikasa, Y., Hayashi, K. & Tabata, Y. (2011). Bone Regeneration by Lactoferrin Released from a Gelatin Hydrogel. *J Biomater Sci Polym Ed* 22(12): 1581-1589. Takayama, Y. & Mizumachi, K. (2008). Effect of bovine lactoferrin on extracellular matrix calcification by human osteoblast-like cells. *Biosci Biotechnol Biochem* 72(1): 226-230.

mitogenic effect of lactoferrin in osteoblasts. *Bone* 49(2): 217-224.

iron deficiency and iron deficiency anaemia. *Biochimie* 91(1): 44-51.

implications on diabetes. *Int J Biol Macromol* 47(1): 50-59.

expressed in rice grain. *Transgenic Res* 14(3): 237-249.

induced oxidation products. *Pediatr Res* 52(6): 964-972.

infection. *Biochem Biophys Res Commun* 264(1): 19-23.

Ce4+. . *J Am Chem Soc* 116: 7889-7890.

*J* 21: 477-483.

196(1): 177-184.

A., Sharma, S. & Singh, T. P. (2010). Specific interactions of C-terminal half (C-lobe) of lactoferrin protein with edible sugars: binding and structural studies with

from milk and neutrophilic leucocytes. Relative molecular mass, isoelectric point,

(2011). Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF

development and economic evaluation of recombinant human lactoferrin

Watson, M., Grey, A. B. & Cornish, J. (2011). Molecular mechanisms involved in the

orally administered, on systemic iron homeostasis in pregnant women suffering of

active properties of bovine lactoferrin supplemented into stirred yoghurt. *Int Dairy* 

Molecular cloning and sequence analysis of bovine lactotransferrin. *Eur J Biochem*

R. & Widness, J. A. (2002). Lactoferrin in the preterm infants' diet attenuates iron-

Effects of lactoferrin and lactoferricin on the release of interleukin 8 from human

lactoferrin peptidic fragments involved in inhibition of herpes simplex virus type 1

Binding of Cerium by Human Lactoferrin Stimulates the Oxidation of Ce3+ to

binding of remnant lipoproteins to HSPG. *Arteriosclerosis and Thrombosis* 14(12): 2025-2031.


Jonasch, E., Stadler, W. M., Bukowski, R. M., Hayes, T. G., Varadhachary, A., Malik, R.,

Kanwar, J. R., Burrow, H. & Kanwar, R. K. (2011). Antioxidant Enzyme Activities of Iron-

Kawakami, H. & Lonnerdal, B. (1991). Isolation and function of a receptor for human lactoferrin in human fetal intestinal brush-border membranes. *Am J Physiol* 261(5 Pt 1): G841-846. Kim, W. S., Shimazaki, K. & Tamura, T. (2006). Expression of bovine lactoferrin C-lobe in

Knapp, R. D. & Hutchens, T. W. (1994). Maternal lactoferrin in the urine of preterm infants. Evidence for retention of structure and function. *Adv Exp Med Biol* 357: 177-181. Kuwata, H., Yamauchi, K., Teraguchi, S., Ushida, Y., Shimokawa, Y., Toida, T. & Hayasawa,

cells involved in immune function. *Biochem Cell Biol* 84(3): 282-290.

Legrand, D. & Mazurier, J. (2010). A critical review of the roles of host lactoferrin in

Legrand, D., Vigie, K., Said, E. A., Elass, E., Masson, M., Slomianny, M. C., Carpentier, M.,

Lonnerdal, B., Keen, C. L. & Hurley, L. S. (1985). Manganese binding proteins in human and

Lorget, F., Clough, J., Oliveira, M., Daury, M. C., Sabokbar, A. & Offord, E. (2002).

Malet, A., Bournaud, E., Lan, A., Mikogami, T., Tome, D. & Blais, A. (2011). Bovine

Mann, D. M., Romm, E. & Migliorini, M. (1994). Delineation of the glycosaminoglycan-

Mari, A., Ooievaar-de Heer, P., Scala, E., Giani, M., Pirrotta, L., Zuidmeer, L., Bethell, D. &

Matsue, M., Matsuyama, J. & Kiyosawa, I. (1995). Interaction of lactoferrin with ascorbate

Briand, J. P., Mazurier, J. & Hovanessian, A. G. (2004). Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells. *Eur J* 

Lactoferrin reduces in vitro osteoclast differentiation and resorbing activity.

lactoferrin improves bone status of ovariectomized mice via immune function

binding site in the human inflammatory response protein lactoferrin. *J Biol Chem*

van Ree, R. (2008). Evaluation by double-blind placebo-controlled oral challenge of the clinical relevance of IgE antibodies against plant glycans. *Allergy* 63(7): 891-896.

and the relationship with bleomycin-dependent DNA damage. *Biosci Biotechnol* 

patients with metastatic renal cell carcinoma. *Cancer* 113(1): 72-77.

Oxidative Stress. *Med Chem* 7(3): 224-230.

*Biochem* 70(11): 2641-2645.

immunity. *Biometals* 23(3): 365-376.

cow's milk. *Am J Clin Nutr* 41(3): 550-559.

*Biochem Biophys Res Commun* 296(2): 261-266.

modulation. *Bone* 48(5): 1028-1035.

*Biochem* 271(2): 303-317.

269(38): 23661-23667.

*Biochem* 59(3): 358-362.

2025-2031.

binding of remnant lipoproteins to HSPG. *Arteriosclerosis and Thrombosis* 14(12):

Figlin, R. A. & Srinivas, S. (2008). Phase 2 trial of talactoferrin in previously treated

Saturated Bovine Lactoferrin (Fe-bLf) in Human Gut Epithelial Cells Under

Rhodococcus erythropolis and its purification and characterization. *Biosci Biotechnol* 

H. (2001). Functional fragments of ingested lactoferrin are resistant to proteolytic degradation in the gastrointestinal tract of adult rats. *J Nutr* 131(8): 2121-2127. Legrand, D., Elass, E., Carpentier, M. & Mazurier, J. (2005). Lactoferrin: a modulator of immune and inflammatory responses. *Cell Mol Life Sci* 62(22): 2549-2559. Legrand, D., Elass, E., Carpentier, M. & Mazurier, J. (2006). Interactions of lactoferrin with


**40** 

*Brazil* 

**How Dentistry Can** 

**Help Fight Osteoporosis** 

Plauto Christopher Aranha Watanabe,

*1São Paulo University, Ribeirão Preto Dental School* 

Marlivia Gonçalves de Carvalho Watanabe and Rodrigo Tiossi

The late 20th century has brought to many patients a promising horizon: the lust of aging. Implicit in this "present" in longevity, however, is the known adverse changes in body tissues. The consequences of aging often involve the risk of osteoporosis, leading to an impaired quality of life of the elderly patients. Morphological and functional oral sequelae of aging are well documented in dental literature, but not those resulting from osteoporosis. Many authors have cited the possible correlation between age, systemic osteoporosis, periodontal disease, tooth loss, and changes in quantity and quality of bone of the maxilla and mandible. The restoration of occlusion for partially and totally edentulous patients often requires adequate bone therapy. Consequently, the frequent use of implant-supported prostheses for elderly patients who are routinely or potentially osteoporotic demand a better understanding of the relationship between osteoporosis, the stomatognathic system and muscle insertions. The jaw is constantly subjected to masticatory forces, movements during speech, breathing, and

Panoramic radiography produces an image that displays both maxilla and mandible, the teeth, their supporting structures, and other important structures such as the nasal cavity, maxillary sinuses, temporomandibular joint, styloid process, and often to the bone hyoid. Although dentists routinely focus interest on the teeth and alveolar ridges when examining some panoramic radiograph, they should also be able to consider major changes in other structures that appear in the image (WHITE et al, 2004; FARMAN et al, 1993; WATANABE et al, 2004). "The Selection of Patients for X-Ray Examination", US Food And Drug Administration Center for Devices and Radiological Health (FDA/CDRH), or "GUIDELINES FOR PRESCRIBING DENTAL RADIOGRAPHS", GPR, was first published in 1987 using the American population (USA), considering the total exposure to ionizing radiation arising from any source. In 2004, the GPR has been updated and published once more after hard work of a board of experts from the "American Dental Association and Food Drugs Administration" (ADA, 2004). The actual GPR expanded the use of panoramic radiography, proposing this technique as the first alternative for supplementary examination for dental diagnosis, recognizing the great technological advancement and improvement of the

While the panoramic radiograph should not be prescribed primarily for the detection of the systemic conditions affecting the maxillomandibular region, we must recognize that in the

swallowing and is therefore an adequate structure for studying bone quality.

equipment for panoramic radiographs.

**1. Introduction** 


## **How Dentistry Can Help Fight Osteoporosis**

Plauto Christopher Aranha Watanabe, Marlivia Gonçalves de Carvalho Watanabe and Rodrigo Tiossi *1São Paulo University, Ribeirão Preto Dental School Brazil* 

### **1. Introduction**

820 Osteoporosis

Takayama, Y. & Mizumachi, K. (2009). Effect of lactoferrin-embedded collagen membrane on osteogenic differentiation of human osteoblast-like cells. *J Biosci Bioeng* 107(2): 191-195. Talukder, M. J., Takeuchi, T. & Harada, E. (2002). Transport of colostral macromolecules into the cerebrospinal fluid via plasma in newborn calves. *J Dairy Sci* 85(3): 514-524. Talukder, M. J., Takeuchi, T. & Harada, E. (2003). Receptor-mediated transport of lactoferrin

Tomita, M., Wakabayashi, H., Shin, K., Yamauchi, K., Yaeshima, T. & Iwatsuki, K. (2009). Twenty-five years of research on bovine lactoferrin applications. *Biochimie* 91(1): 52-57. Troost, F. J., Steijns, J., Saris, W. H. & Brummer, R. J. (2001). Gastric digestion of bovine

van Berkel, P. H., Geerts, M. E., van Veen, H. A., Mericskay, M., de Boer, H. A. & Nuijens, J.

van Berkel, P. H., Welling, M. M., Geerts, M., van Veen, H. A., Ravensbergen, B.,

van der Kraan, M. I., Groenink, J., Nazmi, K., Veerman, E. C., Bolscher, J. G. & Nieuw

Vasilyev, V. B. (2010). Interactions of caeruloplasmin with other proteins participating in

Wakabayashi, H. Y., K. & Tasaki, M. (2006). Lactoferrin research, technology and

Willnow, T. E., Goldstein, J. L., Orth, K., Brown, M. S. & Herz, J. (1992). Low density

Yagi, M., Suzuki, N., Takayama, T., Arisue, M., Kodama, T., Yoda, Y., Otsuka, K. & Ito, K.

Yamano, E., Miyauchi, M., Furusyo, H., Kawazoe, A., Ishikado, A., Makino, T., Tanne, K.,

Ying, X., Cheng, S., Wang, W., Lin, Z., Chen, Q., Zhang, W., Kou, D., Shen, Y., Cheng, X.,

Zavaleta, N., Figueroa, D., Rivera, J., Sanchez, J., Alfaro, S. & Lonnerdal, B. (2007). Efficacy of

differentiation of human adipose stem cells. *Int Orthop*.

chylomicron remnant clearance. *J Biol Chem* 267(36): 26172-26180.

lactoferrin in vivo in adults. *J Nutr* 131(8): 2101-2104.

domain of bovine lactoferrin. *Peptides* 25(2): 177-183.

inflammation. *Biochem Soc Trans* 38(4): 947-951.

DNA. *Biochem J* 328 ( Pt 1): 145-151.

cows. *Nat Biotechnol* 20(5): 484-487.

applications. *Int Dairy J* 16: 1241-1252.

*Cell Biol Int* 33(3): 283-289.

44(2): 258-264.

*Mol Immunol* 46(11-12): 2395-2402.

into the cerebrospinal fluid via plasma in young calves. *J Vet Med Sci* 65(9): 957-964.

H. (1997). N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and

Salaheddine, M., Pauwels, E. K., Pieper, F., Nuijens, J. H. & Nibbering, P. H. (2002). Large scale production of recombinant human lactoferrin in the milk of transgenic

Amerongen, A. V. (2004). Lactoferrampin: a novel antimicrobial peptide in the N1-

lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of

(2009). Effects of lactoferrin on the differentiation of pluripotent mesenchymal cells.

Tanaka, E. & Takata, T. (2010). Inhibitory effects of orally administrated liposomal bovine lactoferrin on the LPS-induced osteoclastogenesis. *Lab Invest* 90(8): 1236-1246. Yamniuk, A. P., Burling, H. & Vogel, H. J. (2009). Thermodynamic characterization of the

interactions between the immunoregulatory proteins osteopontin and lactoferrin.

Peng, L., Zi Xu, H. & Zhu Lu, C. (2011). Effect of lactoferrin on osteogenic

rice-based oral rehydration solution containing recombinant human lactoferrin and lysozyme in Peruvian children with acute diarrhea. *J Pediatr Gastroenterol Nutr*

The late 20th century has brought to many patients a promising horizon: the lust of aging. Implicit in this "present" in longevity, however, is the known adverse changes in body tissues. The consequences of aging often involve the risk of osteoporosis, leading to an impaired quality of life of the elderly patients. Morphological and functional oral sequelae of aging are well documented in dental literature, but not those resulting from osteoporosis. Many authors have cited the possible correlation between age, systemic osteoporosis, periodontal disease, tooth loss, and changes in quantity and quality of bone of the maxilla and mandible. The restoration of occlusion for partially and totally edentulous patients often requires adequate bone therapy. Consequently, the frequent use of implant-supported prostheses for elderly patients who are routinely or potentially osteoporotic demand a better understanding of the relationship between osteoporosis, the stomatognathic system and muscle insertions. The jaw is constantly subjected to masticatory forces, movements during speech, breathing, and swallowing and is therefore an adequate structure for studying bone quality.

Panoramic radiography produces an image that displays both maxilla and mandible, the teeth, their supporting structures, and other important structures such as the nasal cavity, maxillary sinuses, temporomandibular joint, styloid process, and often to the bone hyoid. Although dentists routinely focus interest on the teeth and alveolar ridges when examining some panoramic radiograph, they should also be able to consider major changes in other structures that appear in the image (WHITE et al, 2004; FARMAN et al, 1993; WATANABE et al, 2004).

"The Selection of Patients for X-Ray Examination", US Food And Drug Administration Center for Devices and Radiological Health (FDA/CDRH), or "GUIDELINES FOR PRESCRIBING DENTAL RADIOGRAPHS", GPR, was first published in 1987 using the American population (USA), considering the total exposure to ionizing radiation arising from any source. In 2004, the GPR has been updated and published once more after hard work of a board of experts from the "American Dental Association and Food Drugs Administration" (ADA, 2004). The actual GPR expanded the use of panoramic radiography, proposing this technique as the first alternative for supplementary examination for dental diagnosis, recognizing the great technological advancement and improvement of the equipment for panoramic radiographs.

While the panoramic radiograph should not be prescribed primarily for the detection of the systemic conditions affecting the maxillomandibular region, we must recognize that in the

How Dentistry Can Help Fight Osteoporosis 823

and estimated cost of seven to ten billion dollars. In Brazil, although the fragility of our statistics do not allow further information, we can say that the problem worsens each year, mostly by increasing the relative population of menopausal women and increased life expectancy of this group. So, osteoporosis is a public health problem, affecting millions of people worldwide. Like any other disease, osteoporosis affects the self-esteem of the patient and leads to complications in the family routine, generating costs for the public health system in the patient treatment. These concepts resonate across the world, and especially in

Asymptomatic progression of osteoporosis, in conjunction with the possibility of catastrophic disability, this disorder is the biggest public health priority in many countries. Osteoporosis can progress asymptomatically until a bone fracture or a dental loss. One in two women and one in eight men over age 50 will develop osteoporosis. If the disease occurs, 15% and 20% of women will need special care for long periods due to loss of the ability to manage basic activities at home. Half of the persons who suffered a hip fracture lose their ability to live independently, and around 20% of this persons will die within a

Osteoporosis is not only a woman's disease. Not as many men have it as women do—maybe because most men have more bone density. As they age, men lose bone density slowly than women. However, men also need to be aware of the risks of osteoporosis and men have

Brazil, a developing country with explicit socio-economic-cultural deficit.

year as a result of the fracture (COSMAN & LINDSAY, 2004), (Fig.2).

Fig. 2. Some data alarming epidemiologists on osteoporosis in the world.

treatment (ARAUJO et al, 2006; MARQUES NETO & LEDERMAN, 1995).

In Brazil, the prevalence of osteoporosis is poorly understood (SS-HSPE, 1995); however, in 2001 the osteoporosis clinic at UNIFESP measured the use of public resources and the annual cost for patients with postmenopausal osteoporosis. The average annual cost for the patient was approximately \$442.00 per patient. However, some authors9 assessed the direct cost during hospitalization for an osteoporotic hip fracture in the private health system, such as health insurance coverage, and the authors concluded that the cost was approximately \$ 6.900/patient (KOWALSKI et al, 2001). The study, "Osteoporosis - 2000 of Brazil", developed by 300 medical experts, estimated that less than one-third of Brazilians who have osteoporosis diagnosis of disease, and that only 20% of those are receiving

more fractured femurs than women.

routine of health professionals, dentists, is used for initial assessment of dento-alveolar conditions, and it would be important that these dentists recognize certain conditions in these radiographic images that indicate the presence of systemic diseases. It should be understood that "systemic disease" means conditions that are spread inside the body, rather than localized primarily or only in tissues of the oral cavity. Below we will mention and describe some conditions on the images on panoramic radiographs that suggest significant disease extent, enough to affect quality of life and longevity of patients. A major disease that afflicts mainly men and women in old age is osteoporosis.

"Osteo" is Latin for bone. "Pores" means "full of pores or holes." Thus, osteoporosis means "bones that are full of holes." The bone mass reflects the balance between formation by osteoblasts and resorption by osteoclasts. Around the third decade of life the peak bone mass is reached, and then begins a slow process more continuous bone loss progresses with age. Osteoporosis is a multifactorial metabolic bone disease characterized by low bone mineral density (BMD), the deterioration of the microarchitecture of cancellous or trabecular bone, and changes in the physical properties of bone, leading to increased bone fragility with a consequent increase in fracture risk mainly of bones like the femur, forearm and spine (Fig.1). In the case of the oral cavity, the biggest consequence of this damage is the resorption of the alveolar ridge and possibly teeth loss as well as providing poor quality bone for the installation of oral implants. Nevertheless, it may also lead to mandibular fracture.

Fig. 1. Schematically drawing of the normal bone trabecular tissue, showing a trabecular net of thick and linked. Already to the right we see sharpening trabecular tissue and a net with bigger medullar spaces.

Osteoporosis is the process of quantitative loss of bone density per unit volume, with maintenance and reduction of the qualitative properties of mineralized bone. This change is responsible for the imbalance of the mechanics of the skeleton, increased number of fractures, notably in the spine, femoral neck and distal segment of the radio. The most common structural changes are the reduction of trabecular bone in size and number, and the thinning of the cortical region, with greater involvement of trabecular structure. This can also be seen in the maxillomandibular region, most obviously in the jaw, with decreased cortical thinning and inferior mandibular body.

Osteoporosis in postmenopausal women in the United States constitutes a public health problem because it affects 25 million women, with an annual average of 1.3 million fractures

routine of health professionals, dentists, is used for initial assessment of dento-alveolar conditions, and it would be important that these dentists recognize certain conditions in these radiographic images that indicate the presence of systemic diseases. It should be understood that "systemic disease" means conditions that are spread inside the body, rather than localized primarily or only in tissues of the oral cavity. Below we will mention and describe some conditions on the images on panoramic radiographs that suggest significant disease extent, enough to affect quality of life and longevity of patients. A major disease that

"Osteo" is Latin for bone. "Pores" means "full of pores or holes." Thus, osteoporosis means "bones that are full of holes." The bone mass reflects the balance between formation by osteoblasts and resorption by osteoclasts. Around the third decade of life the peak bone mass is reached, and then begins a slow process more continuous bone loss progresses with age. Osteoporosis is a multifactorial metabolic bone disease characterized by low bone mineral density (BMD), the deterioration of the microarchitecture of cancellous or trabecular bone, and changes in the physical properties of bone, leading to increased bone fragility with a consequent increase in fracture risk mainly of bones like the femur, forearm and spine (Fig.1). In the case of the oral cavity, the biggest consequence of this damage is the resorption of the alveolar ridge and possibly teeth loss as well as providing poor quality bone for the

installation of oral implants. Nevertheless, it may also lead to mandibular fracture.

Fig. 1. Schematically drawing of the normal bone trabecular tissue, showing a trabecular net of thick and linked. Already to the right we see sharpening trabecular tissue and a net with

Osteoporosis is the process of quantitative loss of bone density per unit volume, with maintenance and reduction of the qualitative properties of mineralized bone. This change is responsible for the imbalance of the mechanics of the skeleton, increased number of fractures, notably in the spine, femoral neck and distal segment of the radio. The most common structural changes are the reduction of trabecular bone in size and number, and the thinning of the cortical region, with greater involvement of trabecular structure. This can also be seen in the maxillomandibular region, most obviously in the jaw, with decreased

Osteoporosis in postmenopausal women in the United States constitutes a public health problem because it affects 25 million women, with an annual average of 1.3 million fractures

afflicts mainly men and women in old age is osteoporosis.

bigger medullar spaces.

cortical thinning and inferior mandibular body.

and estimated cost of seven to ten billion dollars. In Brazil, although the fragility of our statistics do not allow further information, we can say that the problem worsens each year, mostly by increasing the relative population of menopausal women and increased life expectancy of this group. So, osteoporosis is a public health problem, affecting millions of people worldwide. Like any other disease, osteoporosis affects the self-esteem of the patient and leads to complications in the family routine, generating costs for the public health system in the patient treatment. These concepts resonate across the world, and especially in Brazil, a developing country with explicit socio-economic-cultural deficit.

Asymptomatic progression of osteoporosis, in conjunction with the possibility of catastrophic disability, this disorder is the biggest public health priority in many countries. Osteoporosis can progress asymptomatically until a bone fracture or a dental loss. One in two women and one in eight men over age 50 will develop osteoporosis. If the disease occurs, 15% and 20% of women will need special care for long periods due to loss of the ability to manage basic activities at home. Half of the persons who suffered a hip fracture lose their ability to live independently, and around 20% of this persons will die within a year as a result of the fracture (COSMAN & LINDSAY, 2004), (Fig.2).

Osteoporosis is not only a woman's disease. Not as many men have it as women do—maybe because most men have more bone density. As they age, men lose bone density slowly than women. However, men also need to be aware of the risks of osteoporosis and men have more fractured femurs than women.

Fig. 2. Some data alarming epidemiologists on osteoporosis in the world.

In Brazil, the prevalence of osteoporosis is poorly understood (SS-HSPE, 1995); however, in 2001 the osteoporosis clinic at UNIFESP measured the use of public resources and the annual cost for patients with postmenopausal osteoporosis. The average annual cost for the patient was approximately \$442.00 per patient. However, some authors9 assessed the direct cost during hospitalization for an osteoporotic hip fracture in the private health system, such as health insurance coverage, and the authors concluded that the cost was approximately \$ 6.900/patient (KOWALSKI et al, 2001). The study, "Osteoporosis - 2000 of Brazil", developed by 300 medical experts, estimated that less than one-third of Brazilians who have osteoporosis diagnosis of disease, and that only 20% of those are receiving treatment (ARAUJO et al, 2006; MARQUES NETO & LEDERMAN, 1995).

How Dentistry Can Help Fight Osteoporosis 825

Beyond the influences aboriginal, Portuguese and black, the Northeast region received contributions from dutches, Frenchmen and English that had invaded the territory had dominated and it during a time. The result is a rich and varied culinary, that came to characterize the food of the region. With exception of the blacks, all the other peoples are group of risk

In the region Southeastern they are the states richest of the country. Its food received diverse influences, that follow the history of the settling: of the indians; Jesuits; tamers (bandeirantes); Italian immigrants, of the Spaniard and Arabs in *Rio de Janeiro*, and of the Germans and Italians. Here also influences we see it sociological of these European, caucasianos peoples, groups of osteoporosis risk. The South region was the one that received greater influence from immigrants. This because the tempering climate of the region was more similar to the European climate, facilitating the adaptation of the Italians, Germans, Poles and ucranianos, that if had established preferential in agricultural activities.

Table 1. Brazilian geographic regions and the main alimentary characteristics. Demographic

The region Center-West, possesss innumerable families of colonists of the states of the South. These colonists had wide experience in cattle agriculture and modern. Of this form, its culinary was conditional to the resources of the environment, especially of fishes and the hunting, as: "pacu", "piranha", "golden", "painted", "anta", "cotia", "paca", "capivara", deer and alligator. Thus, this feeding

is positive in relation osteoporosis.

osteoporosis.

The native indians of the Region North had as basic food the "mandioca". The fish also represent an important parcel of the feeding, being the most consumed "tambaqui", "traíra", "piranha", fished, sardine of river,

"tucunaré", "pacu" and "pirarucu" (called "cod the Amazônia"). All rich ones in sodium, potassium and calcium.

census. Brazil, 2000.

### **2. Singular characteristics in geographical location**

### **2.1 Emergent country**

Food and / or nutrition are some of the main factors that affect bone quality as a whole, and significantly influence the osteoporosis disease in the world.

As to the geographical aspect, we should mention that Brazil, for example is between the equator and temperate zone just below the Tropic of Capricorn, which clearly favors the protection against the deleterious effects of osteoporosis, because fortunately, as a tropical country, received the largest part of the year the sunlight that is essential to activate vitamin D. Vitamin D (or calciferol) is a vitamin that promotes the absorption of calcium (after exposure to sunlight), essential for normal development of bones and teeth; it also acts as newly discovered immune system, heart, brain and in insulin secretion by the pancreas. This has primary function in the absorption of calcium in the body.

Among the environmental factors involved in osteoporosis, nutrition, particularly with respect to consumption of protein, dairy products and vegetables, has been named as a participant in the formation of bone mass (ANDERSSON 1999; RIZZOLI & BONJOUR, 1999; ROUSSEAU, 1997). The calcium and vitamin D during childhood seem to play an important role in the health of bones (WARDLAW, 1993). Retrospective studies in adults suggest that calcium intake in the first phase of development are associated with the risk of developing osteoporosis and fractures during adulthood (STALLINGS, 1997; VON MULHEN et al, 1999). However, we must consider the sensitivity of this nutrient absorption varies depending on the genetic constitution of the individual (MAY et al, 1994).

This privileged geographic location facilitates the cultivation of a variety of foods (from temperate and tropical climates). Fishing, and therefore the habit of eating fish is extremely encouraged by the fact that our country has an extensive Atlantic coast, more than 8000 km, and thus favors the fishery, which is quite diversified. Including food fish in the daily diet can greatly contribute to bone quality, mainly due to calcium that food provides. Fish is a major source of calcium along with other foods such as milk, yogurt, vegetables, nuts and cereals (Table 1).

This geographical location, yet is related to the sociological aspect, therefore, should remember the mixture among Indians, Portuguese and African blacks and among immigrants who came to Brazil from the nineteenth century, attracted by the opening of the immigration movement. Italian families, German, Portuguese, Spanish, Polish, Japanese and Arabs introduced their eating habits in the regions where they settled. These people, mainly Asians and Caucasians, are the main osteoporosis risk groups, owing much to the genetic characteristics. Even with respect to indigenous peoples, one of the most accepted theories of his presence in the Americas, is the migration of peoples from Asia across the Bering Strait (Bering Strait is a strait between Cape Dezhnev, the easternmost point of mainland Asian and Cape Prince of Wales, the westernmost of the American continent). During the last glacial era, with the recession of ocean water, the area of the Straits has become a natural bridge between Asia and the Americas, now called the Bering Land Bridge , where they could have reached America the people who first colonized). So you can see how confusing it is analyzing the DXA scans, which has standard tables to compare the bone mass values determined by tests of X-ray absorption, mainly due to the intense miscegenation of the population in Brazil (Fig. 3).

But we can go further in this confused analysis in our country, commenting on the various geographic regions of Brazil and food characteristics.

Food and / or nutrition are some of the main factors that affect bone quality as a whole, and

As to the geographical aspect, we should mention that Brazil, for example is between the equator and temperate zone just below the Tropic of Capricorn, which clearly favors the protection against the deleterious effects of osteoporosis, because fortunately, as a tropical country, received the largest part of the year the sunlight that is essential to activate vitamin D. Vitamin D (or calciferol) is a vitamin that promotes the absorption of calcium (after exposure to sunlight), essential for normal development of bones and teeth; it also acts as newly discovered immune system, heart, brain and in insulin secretion by the pancreas. This

Among the environmental factors involved in osteoporosis, nutrition, particularly with respect to consumption of protein, dairy products and vegetables, has been named as a participant in the formation of bone mass (ANDERSSON 1999; RIZZOLI & BONJOUR, 1999; ROUSSEAU, 1997). The calcium and vitamin D during childhood seem to play an important role in the health of bones (WARDLAW, 1993). Retrospective studies in adults suggest that calcium intake in the first phase of development are associated with the risk of developing osteoporosis and fractures during adulthood (STALLINGS, 1997; VON MULHEN et al, 1999). However, we must consider the sensitivity of this nutrient absorption varies

This privileged geographic location facilitates the cultivation of a variety of foods (from temperate and tropical climates). Fishing, and therefore the habit of eating fish is extremely encouraged by the fact that our country has an extensive Atlantic coast, more than 8000 km, and thus favors the fishery, which is quite diversified. Including food fish in the daily diet can greatly contribute to bone quality, mainly due to calcium that food provides. Fish is a major source of calcium along with other foods such as milk, yogurt, vegetables, nuts and

This geographical location, yet is related to the sociological aspect, therefore, should remember the mixture among Indians, Portuguese and African blacks and among immigrants who came to Brazil from the nineteenth century, attracted by the opening of the immigration movement. Italian families, German, Portuguese, Spanish, Polish, Japanese and Arabs introduced their eating habits in the regions where they settled. These people, mainly Asians and Caucasians, are the main osteoporosis risk groups, owing much to the genetic characteristics. Even with respect to indigenous peoples, one of the most accepted theories of his presence in the Americas, is the migration of peoples from Asia across the Bering Strait (Bering Strait is a strait between Cape Dezhnev, the easternmost point of mainland Asian and Cape Prince of Wales, the westernmost of the American continent). During the last glacial era, with the recession of ocean water, the area of the Straits has become a natural bridge between Asia and the Americas, now called the Bering Land Bridge , where they could have reached America the people who first colonized). So you can see how confusing it is analyzing the DXA scans, which has standard tables to compare the bone mass values determined by tests of X-ray absorption, mainly due to the intense miscegenation of the

But we can go further in this confused analysis in our country, commenting on the various

**2. Singular characteristics in geographical location** 

significantly influence the osteoporosis disease in the world.

has primary function in the absorption of calcium in the body.

depending on the genetic constitution of the individual (MAY et al, 1994).

**2.1 Emergent country** 

cereals (Table 1).

population in Brazil (Fig. 3).

geographic regions of Brazil and food characteristics.

The native indians of the Region North had as basic food the "mandioca". The fish also represent an important parcel of the feeding, being the most consumed "tambaqui", "traíra", "piranha", fished, sardine of river, "tucunaré", "pacu" and "pirarucu" (called "cod the Amazônia"). All rich ones in sodium, potassium and calcium.

Beyond the influences aboriginal, Portuguese and black, the Northeast region received contributions from dutches, Frenchmen and English that had invaded the territory had dominated and it during a time. The result is a rich and varied culinary, that came to characterize the food of the region. With exception of the blacks, all the other peoples are group of risk osteoporosis.

In the region Southeastern they are the states richest of the country. Its food received diverse influences, that follow the history of the settling: of the indians; Jesuits; tamers (bandeirantes); Italian immigrants, of the Spaniard and Arabs in *Rio de Janeiro*, and of the Germans and Italians. Here also influences we see it sociological of these European, caucasianos peoples, groups of osteoporosis risk. The South region was the one that received greater influence from immigrants. This because the tempering climate of the region was more similar to the European climate, facilitating the adaptation of the Italians, Germans, Poles and ucranianos, that if had established preferential in agricultural activities.

The region Center-West, possesss innumerable families of colonists of the states of the South. These colonists had wide experience in cattle agriculture and modern. Of this form, its culinary was conditional to the resources of the environment, especially of fishes and the hunting, as: "pacu", "piranha", "golden", "painted", "anta", "cotia", "paca", "capivara", deer and alligator. Thus, this feeding is positive in relation osteoporosis.

Table 1. Brazilian geographic regions and the main alimentary characteristics. Demographic census. Brazil, 2000.

(Figure 4)

severe kyphosis.

How Dentistry Can Help Fight Osteoporosis 827

women aged 40-60 years. They found that there was good evidence that low body weight and Postmenopausal (PM) status are risk factors for low bone mineral density, and also found that there is good evidence that ingestion of alcohol and caffeine, and reproductive history were not factors risk. The results of a recent study (VONDRACEK et al, 2009), however, contradict these results. Changes in the lifestyle and healthy habits to favor the bones, such as calcium and vitamin D nutrition, regular exercise, limiting consumption of caffeine and alcohol, and the fact that tobacco smoking is not essential to the management of risk of osteoporosis.

Fig. 4. Relative illustration to the health of the Brazilian (FIOCRUZ/WHO, 2008.)

Radiographic factors of osteoporosis in the skeleton include generalized osteopenia which is always more prominent in the column, cortical thinning, and accentuation of primary trabeculation and loss of the secondary trabeculation. Osteoporosis can be linked to pain, especially in the lower back. It can also result in pathological fracture, loss of stature, and

Radiological factors of osteoporosis in the mandible include relative radiolucency of the jaws and jaw and defining the reduced cortical, and erosions (Fig. 5 and 6). At the early stages of the disease is possible to find a sharp contrast from the oblique line of mandible,

Fig. 3. Composition of the Brazilian population for race, 1991/2000 (IBGE-Brazilian Institute of the Geography and Statistic)

Caffeine, for example, is a food consumed by millions of Brazilians and their effect has been studied on bone quality. In 2002, Heaney conducted a literature review and concluded that "there is no evidence that caffeine has the harmful effect on bone status or calcium deposition in individuals who eat the recommended amount of calcium per day, or more than 3 cups of coffee. In our studies we have observed that there is a significant increase in the circulating calcium in rats that ingested caffeine daily, causing obvious radiolucency of the mandibular bone, the tibia and femur.

There are at least 31 recent cross-sectional studies, case control and cohort studies (observational studies are where individuals are selected or classified) according to exposure status, being followed to evaluate the incidence of disease, combining caffeine intake and bone health involving many thousands of patients (PINTO NETO, 2002). Aspects of bone health measured include BMD, its changes, the rate of fracture and osteoporosis. These observational studies could demonstrate only associations and not relationships of cause and effect.

Although the reviewed evidence is contradictory, the weight of evidence does not support the idea that beverages containing caffeine adversely affect bone health. The reason for the contradictory results is unclear. Taking as an example study, the inverse association observed before adjustment for obscure aspects between intake of caffeinated beverages and bone mass, disappeared after adjusting for other risk factors (JOHANSSON et al, 1992). It would also be possible that the intake of caffeinated beverages is acting as a marker for a true causal factor. It is known that there is an inverse relationship between milk intake and consumption of beverages containing cafeína (BAUER et al, 1993). It is possible, therefore, that a low intake of milk instead of a high intake of caffeinated beverages is the true cause of ill health óssea (HALLSTON et al, 2006). In 2009, some authors (WAUGH et al, 2009) published a systematic review of checking the risk factors for low bone mass in healthy

Ethnic

Fig. 3. Composition of the Brazilian population for race, 1991/2000 (IBGE-Brazilian Institute

Caffeine, for example, is a food consumed by millions of Brazilians and their effect has been studied on bone quality. In 2002, Heaney conducted a literature review and concluded that "there is no evidence that caffeine has the harmful effect on bone status or calcium deposition in individuals who eat the recommended amount of calcium per day, or more than 3 cups of coffee. In our studies we have observed that there is a significant increase in the circulating calcium in rats that ingested caffeine daily, causing obvious radiolucency of

There are at least 31 recent cross-sectional studies, case control and cohort studies (observational studies are where individuals are selected or classified) according to exposure status, being followed to evaluate the incidence of disease, combining caffeine intake and bone health involving many thousands of patients (PINTO NETO, 2002). Aspects of bone health measured include BMD, its changes, the rate of fracture and osteoporosis. These observational

Although the reviewed evidence is contradictory, the weight of evidence does not support the idea that beverages containing caffeine adversely affect bone health. The reason for the contradictory results is unclear. Taking as an example study, the inverse association observed before adjustment for obscure aspects between intake of caffeinated beverages and bone mass, disappeared after adjusting for other risk factors (JOHANSSON et al, 1992). It would also be possible that the intake of caffeinated beverages is acting as a marker for a true causal factor. It is known that there is an inverse relationship between milk intake and consumption of beverages containing cafeína (BAUER et al, 1993). It is possible, therefore, that a low intake of milk instead of a high intake of caffeinated beverages is the true cause of ill health óssea (HALLSTON et al, 2006). In 2009, some authors (WAUGH et al, 2009) published a systematic review of checking the risk factors for low bone mass in healthy

studies could demonstrate only associations and not relationships of cause and effect.

of the Geography and Statistic)

the mandibular bone, the tibia and femur.

women aged 40-60 years. They found that there was good evidence that low body weight and Postmenopausal (PM) status are risk factors for low bone mineral density, and also found that there is good evidence that ingestion of alcohol and caffeine, and reproductive history were not factors risk. The results of a recent study (VONDRACEK et al, 2009), however, contradict these results. Changes in the lifestyle and healthy habits to favor the bones, such as calcium and vitamin D nutrition, regular exercise, limiting consumption of caffeine and alcohol, and the fact that tobacco smoking is not essential to the management of risk of osteoporosis. (Figure 4)

Fig. 4. Relative illustration to the health of the Brazilian (FIOCRUZ/WHO, 2008.)

Radiographic factors of osteoporosis in the skeleton include generalized osteopenia which is always more prominent in the column, cortical thinning, and accentuation of primary trabeculation and loss of the secondary trabeculation. Osteoporosis can be linked to pain, especially in the lower back. It can also result in pathological fracture, loss of stature, and severe kyphosis.

Radiological factors of osteoporosis in the mandible include relative radiolucency of the jaws and jaw and defining the reduced cortical, and erosions (Fig. 5 and 6). At the early stages of the disease is possible to find a sharp contrast from the oblique line of mandible,

How Dentistry Can Help Fight Osteoporosis 829

Institute of Geography and Statistics (IBGE), the group 30 to 59 rose from 25% of the population (in 1940) to 32.9% (1998), and is expected to reach 40.2% in 2020. The elderly above 60 years, amounted to 12.4 million people in 1998 and may be 25 million over the next 21 years. We must emphasize that life expectancy measured in the last IBGE Sense is

In Europe, every 30 seconds someone suffers a fracture due to osteoporosis. It is estimated that in 2050, Latin America and Asia, the incidence of hip fractures due to bone disease should be for one in two cases. In Asia, according to medical records, is considered more dramatic the expected increase in cases of hip fractures in the coming decades. Other studies show that in the Middle East will triple the number of hip fractures caused by disease in the

The major consequence of bone loss (PO) in our aging society is fracture. In the oral cavity could be considered tooth loss, affecting 26 million people in Brazil, according to the IBGE. Oral health of Brazilian social inequality is indicative of the country. According to the Brazilian part of the World Health Survey, released by Fiocruz (Oswaldo Cruz Foundation) and held last year at the WHO (World Health Organization), 14.4% of Americans have lost

Taking into account that the IBGE estimates 179 million in the current population in Brazil, that means about 26 million no longer have any natural teeth. The comparison between the social classes shows that, among the poorest, this percentage reaches 17.5%, and among the richest, is 5.9%. Fiocruz made the separation between classes by the number of consumer goods (televisions, refrigerators, etc.). The worst situation was found among women over 50 in poor families: 55.9%. That is, postmenopausal women are main group at risk for osteoporosis. The researchers compared risk factors for health. The data show that 10.1% of the population can be considered obese, according to the WHO standards, and that 28.5% of Americans are overweight. The percentage of underweight people is 5%. These people probably have very brittle bones, and thus risk of systemic osteoporosis. The rate of those who said they had drunk at least five servings of alcoholic beverages in the previous week is about 14.8%, a percentage that is higher in the younger age group (18-34 years), which reaches 17.4%. Daily smokers represent 18.1%, and the habit is lower among those between 18 and 34 -15.2%. It is a fact that excessive alcohol and smoking affects bone mass and quality of oral health. The poll found that 24% of the population is sedentary. This is a major

Many physicians believe that an X-ray may also be appropriate after experiencing a loss in height or a change in posture, or some alteration in the skeleton. This is an old concept that still exists. One should remember that this is a concept of more than 50 years, when radiology still worked with slow films, with technology of grains in emulsions, ecrans that emitted blue light, 16 times more radiation and chemical processing. Nowadays, radiology works with little radiation, X-ray equipments are more accurate, conventional films have the technology of tabular grains, ecrans that emit green light (more sensitive), and still digital

73 years on average.

next 20 years. Cosman & Lindsay, 2004.

**4. Bone loss: Clinical implication** 

all their teeth (SZWARCWALD & VIACAVA, 2005).

factor influencing the quality of the bone skeleton.

**4.1 Measurements based on radiographs** 

mainly due to loss of trabecular bone mass, which leaves the body more mandibular radiolucent, accentuating the contrast effect in relation to the oblique line. The precision with which the panoramic radiographs can be used to assess the likelihood of a person having osteoporosis is still debated, with evidence being divided, polarized rather than for or against.

Fig. 5. Interest Region . Normal Mandibular inferior cortex – Cropped panoramics images

Fig. 6. Osteoporosis – Cropped panoramics images shows a relative radiolucency of both jaws with reduced definition and mandibular inferior cortex moderately eroded, evidence of lacunar resorption (right-D) or cortex severely eroded (left-E),

### **3. Population in developing countries will be the most affected**

Currently, according to WHO, the majority of hip fractures due to osteoporosis happens in the countries of Europe and North America. This projection is based on the fact that the demographic changes that must occur within the next 50 years will significantly increase the number of elderly in Asia, Africa and South America. According to the Brazilian

mainly due to loss of trabecular bone mass, which leaves the body more mandibular radiolucent, accentuating the contrast effect in relation to the oblique line. The precision with which the panoramic radiographs can be used to assess the likelihood of a person having osteoporosis is still debated, with evidence being divided, polarized rather than for

Fig. 5. Interest Region . Normal Mandibular inferior cortex – Cropped panoramics images

Fig. 6. Osteoporosis – Cropped panoramics images shows a relative radiolucency of both jaws with reduced definition and mandibular inferior cortex moderately eroded, evidence of

Currently, according to WHO, the majority of hip fractures due to osteoporosis happens in the countries of Europe and North America. This projection is based on the fact that the demographic changes that must occur within the next 50 years will significantly increase the number of elderly in Asia, Africa and South America. According to the Brazilian

lacunar resorption (right-D) or cortex severely eroded (left-E),

**3. Population in developing countries will be the most affected** 

or against.

Institute of Geography and Statistics (IBGE), the group 30 to 59 rose from 25% of the population (in 1940) to 32.9% (1998), and is expected to reach 40.2% in 2020. The elderly above 60 years, amounted to 12.4 million people in 1998 and may be 25 million over the next 21 years. We must emphasize that life expectancy measured in the last IBGE Sense is 73 years on average.

In Europe, every 30 seconds someone suffers a fracture due to osteoporosis. It is estimated that in 2050, Latin America and Asia, the incidence of hip fractures due to bone disease should be for one in two cases. In Asia, according to medical records, is considered more dramatic the expected increase in cases of hip fractures in the coming decades. Other studies show that in the Middle East will triple the number of hip fractures caused by disease in the next 20 years. Cosman & Lindsay, 2004.

### **4. Bone loss: Clinical implication**

The major consequence of bone loss (PO) in our aging society is fracture. In the oral cavity could be considered tooth loss, affecting 26 million people in Brazil, according to the IBGE. Oral health of Brazilian social inequality is indicative of the country. According to the Brazilian part of the World Health Survey, released by Fiocruz (Oswaldo Cruz Foundation) and held last year at the WHO (World Health Organization), 14.4% of Americans have lost all their teeth (SZWARCWALD & VIACAVA, 2005).

Taking into account that the IBGE estimates 179 million in the current population in Brazil, that means about 26 million no longer have any natural teeth. The comparison between the social classes shows that, among the poorest, this percentage reaches 17.5%, and among the richest, is 5.9%. Fiocruz made the separation between classes by the number of consumer goods (televisions, refrigerators, etc.). The worst situation was found among women over 50 in poor families: 55.9%. That is, postmenopausal women are main group at risk for osteoporosis. The researchers compared risk factors for health. The data show that 10.1% of the population can be considered obese, according to the WHO standards, and that 28.5% of Americans are overweight. The percentage of underweight people is 5%. These people probably have very brittle bones, and thus risk of systemic osteoporosis. The rate of those who said they had drunk at least five servings of alcoholic beverages in the previous week is about 14.8%, a percentage that is higher in the younger age group (18-34 years), which reaches 17.4%. Daily smokers represent 18.1%, and the habit is lower among those between 18 and 34 -15.2%. It is a fact that excessive alcohol and smoking affects bone mass and quality of oral health. The poll found that 24% of the population is sedentary. This is a major factor influencing the quality of the bone skeleton.

#### **4.1 Measurements based on radiographs**

Many physicians believe that an X-ray may also be appropriate after experiencing a loss in height or a change in posture, or some alteration in the skeleton. This is an old concept that still exists. One should remember that this is a concept of more than 50 years, when radiology still worked with slow films, with technology of grains in emulsions, ecrans that emitted blue light, 16 times more radiation and chemical processing. Nowadays, radiology works with little radiation, X-ray equipments are more accurate, conventional films have the technology of tabular grains, ecrans that emit green light (more sensitive), and still digital

How Dentistry Can Help Fight Osteoporosis 831

Fig. 8. Densitometric exam of the mandible in the Software Cromox

**5.1 Panoramic and oral radiography** 

the inferior border. (Fig. 5)

**5. Radiographic signs of osteoporosis in dental radiography** 

Dentists are in a potentially valuable position for patient screening for signs of osteoporosis; significant portion of the population visits their dentist annually and dental radiographs are prescribed for many. In the last four decades, numerous researchers have reported that osteoporosis can be diagnosed through oral radiographs; panoramic radiography is widely used for routine dental examinations and it would be very useful to determine if radiographic changes in the mandible could show skeletal osteopenia and have an important role in detection of osteoporosis. Thickness of the inferior border of the mandible below the mental foramen has often been measured as the panoramic mandibular index (PMI) either directly or as a ratio of the thickness to the distance of the mental foramen from

The use of panoramic radiography is common in a dental setting and is also advocated by the International Guide to Prescription Radiographs4. Digital radiographs are an increasingly popular option in the clinic. Such images are composed of pixels with a specific numerical value for each one. Two important methods of evaluating the pixels in these images are Fractal dimension (FD) and Pixel Intensity (PI) analyses. FD is expressed numerically and consists in describing complex shapes and structural patterns in the bone. PI is a grayscale measure, ranging from zero (black) to 256 (white) in a 8-bit digital image (VON MULHEN et al, 1999). Because the panoramic radiograph is an exam more common and affordable than DXA, its application in the early detection of low bone mass would

bring significant benefits for the treatment of osteoporosis (VON WOWERN, 1986).

The cardinal radiographic signs of osteoporosis in the skeleton include osteopenia generalized thinning and the accentuation of corticosteroids in the bones, and the accentuation of the trabecular bone. The factors include spontaneous fracture, and

images, with ample possibility in computers that increase the diagnostic capacity. This has led professionals to change their thoughts in relation to the older concept.

#### **4.2 X-ray absorptiometry: Dental radiography**

X-ray absorptiometry is a technique widely utilized for measuring bone mineral density (BMD). The low correlation among densitometric results obtained for distinct bone sites valuation imposes the development of a technique for accurate mineral density assessment on maxillary and mandibular bones specifically, adequate for dentistry procedures. For maxillary and mandibular bone mineral density measurements by single energy x-ray computed absorptiometry, periapical intraoral radiographs can be taken using an aluminum densitometric scale. Watanabe et al, 2008 mesured the density values on 55 adult patients that were arranged in four population groups, with distinction among maxillary and mandibular bone and patient gender. The measurements were statistically evaluated, resulting in the determination of the population reference data based on the average density and respective standard deviation for each group. Assuming one standard deviation as the confidence interval, the lower threshold for normality corresponds to bone mineral density of 2.00 mm for women maxillary bone, 3.28 mm for women mandibular bone, 3.88 mm for men maxillary bone and 5.45 mm for men mandibular bone. These thresholds were implemented on Cromox ® DOMM 3.2.2 system, for normal and osteopenia distinct diagnosis, associated to low effective radiation dose on a technique with more comprehensive use in the population (WATANABE et al, 2008 ). (Figure 7 and 8)

Fig. 7. Prototypes III and IV of the densitometry scale (coins are used as a size reference).

images, with ample possibility in computers that increase the diagnostic capacity. This has

X-ray absorptiometry is a technique widely utilized for measuring bone mineral density (BMD). The low correlation among densitometric results obtained for distinct bone sites valuation imposes the development of a technique for accurate mineral density assessment on maxillary and mandibular bones specifically, adequate for dentistry procedures. For maxillary and mandibular bone mineral density measurements by single energy x-ray computed absorptiometry, periapical intraoral radiographs can be taken using an aluminum densitometric scale. Watanabe et al, 2008 mesured the density values on 55 adult patients that were arranged in four population groups, with distinction among maxillary and mandibular bone and patient gender. The measurements were statistically evaluated, resulting in the determination of the population reference data based on the average density and respective standard deviation for each group. Assuming one standard deviation as the confidence interval, the lower threshold for normality corresponds to bone mineral density of 2.00 mm for women maxillary bone, 3.28 mm for women mandibular bone, 3.88 mm for men maxillary bone and 5.45 mm for men mandibular bone. These thresholds were implemented on Cromox ® DOMM 3.2.2 system, for normal and osteopenia distinct diagnosis, associated to low effective radiation dose on a technique with more

led professionals to change their thoughts in relation to the older concept.

comprehensive use in the population (WATANABE et al, 2008 ). (Figure 7 and 8)

Fig. 7. Prototypes III and IV of the densitometry scale (coins are used as a size reference).

**4.2 X-ray absorptiometry: Dental radiography** 

Fig. 8. Densitometric exam of the mandible in the Software Cromox

### **5. Radiographic signs of osteoporosis in dental radiography**

### **5.1 Panoramic and oral radiography**

Dentists are in a potentially valuable position for patient screening for signs of osteoporosis; significant portion of the population visits their dentist annually and dental radiographs are prescribed for many. In the last four decades, numerous researchers have reported that osteoporosis can be diagnosed through oral radiographs; panoramic radiography is widely used for routine dental examinations and it would be very useful to determine if radiographic changes in the mandible could show skeletal osteopenia and have an important role in detection of osteoporosis. Thickness of the inferior border of the mandible below the mental foramen has often been measured as the panoramic mandibular index (PMI) either directly or as a ratio of the thickness to the distance of the mental foramen from the inferior border. (Fig. 5)

The use of panoramic radiography is common in a dental setting and is also advocated by the International Guide to Prescription Radiographs4. Digital radiographs are an increasingly popular option in the clinic. Such images are composed of pixels with a specific numerical value for each one. Two important methods of evaluating the pixels in these images are Fractal dimension (FD) and Pixel Intensity (PI) analyses. FD is expressed numerically and consists in describing complex shapes and structural patterns in the bone. PI is a grayscale measure, ranging from zero (black) to 256 (white) in a 8-bit digital image (VON MULHEN et al, 1999). Because the panoramic radiograph is an exam more common and affordable than DXA, its application in the early detection of low bone mass would bring significant benefits for the treatment of osteoporosis (VON WOWERN, 1986).

The cardinal radiographic signs of osteoporosis in the skeleton include osteopenia generalized thinning and the accentuation of corticosteroids in the bones, and the accentuation of the trabecular bone. The factors include spontaneous fracture, and

How Dentistry Can Help Fight Osteoporosis 833



The measurements can be made in the panoramic radiographs in the mental foramen area, with the aid of the RADIOIMP software (RADIO MEMORY LTDA, version 2.0). When opening the image of the panoramic radiograph, the program requests the calibration of the

After the simultaneous alteration of the brightness and contrast tool of the images for better visualization of the mental foramen area, the measurements can be initiated, according to the technique proposed by Benson et al, 1991 based in the technique of the Wical & Swoope (1974). The first step is the identification and the tracing of the mental foramen unilaterally; then, a parallel line will be drawn down the long axis of the mandibular body and tangent to the inferior border of each side. Later, the mandibular cortex will be measured, through a line drawn perpendicularly from the first and afterwards the height of the mental foramen will be also measured, given by the distance of the inferior border of the foramen to the base of the mandible. All the measured lines presented a 90o degree format. For better identification of the measured area, the zoom tool will be also used to facilitate the the

The thickness of the mandibular cortex is divided by the distance between the mental

Fig. 9. Measurement of the Mentual and Antigoniac indices. Radioimp-RADIO MEMORY

measurements, as the presented values were around 0,3 cm (Figure 9 and 10).

foremen and the inferior mandibular cortex to obtain the PMI (BENSON et al, 1991).

**6.1 Radio-morphometric indices (Table 2)** 

same, where the following information should be inserted: 1. Type of equipment used: e.g. SuperVeraviewscope.

mm) (Fig. 6-7);

2. Image Resolution: 300 dpi. 3. Equipment Magnification: 30%

(Fig. 6-7)

LTDA, version 2.0

traumatic, especially the spine, wrist, hip or spine, invagination at the base of the skull and bones of grainy skull (VON WOWERN, 1986). The main radiographic signs of osteoporosis in the maxilla and mandible (Figure 2) include a generalized radiolucency on both the maxilla and mandible, as evidenced by defining the cortical or accentuation of the maxillary sinus, nasal cavity, oblique line, and others. Where you can see some cervical vertebrae in panoramic radiography, the appearance of the "frame" of the bones can also be observed. A morphometric analysis of bone in cross section (VON WOWERN, 1986) showed that the structure of the jaw bones and jaw in dentate elderly, is characterized by cortical porous, relatively thin, with demineralization of the bone endosteum, as in other skeletal bones, and these changes age-related cortical tend to be more common in women than in men. The bones of the jaw and jaw variations between individuals and regional structures and density of trabecular bone may mask the decrease in bone mass that is related with gender and age, as seen in other trabecular bones of the skeleton. The methods for evaluating these agerelated changes in the maxilla and mandible were listed by Bras et al, 1982.

### **6. Panoramic radiography**

The integrity of the bone microarchitecture is an important element of the bone quality and contributes for the mechanical abilities of the bone ( FARMAN *et al*., 1993; TAGUCHI, 2004; BOUXSEIN, 2003; SEEMAN, 2003; WOWERN, 2001).


Table 2. Original methods for evaluation of the changes of the bone of the jaw *in vivo*

traumatic, especially the spine, wrist, hip or spine, invagination at the base of the skull and bones of grainy skull (VON WOWERN, 1986). The main radiographic signs of osteoporosis in the maxilla and mandible (Figure 2) include a generalized radiolucency on both the maxilla and mandible, as evidenced by defining the cortical or accentuation of the maxillary sinus, nasal cavity, oblique line, and others. Where you can see some cervical vertebrae in panoramic radiography, the appearance of the "frame" of the bones can also be observed. A morphometric analysis of bone in cross section (VON WOWERN, 1986) showed that the structure of the jaw bones and jaw in dentate elderly, is characterized by cortical porous, relatively thin, with demineralization of the bone endosteum, as in other skeletal bones, and these changes age-related cortical tend to be more common in women than in men. The bones of the jaw and jaw variations between individuals and regional structures and density of trabecular bone may mask the decrease in bone mass that is related with gender and age, as seen in other trabecular bones of the skeleton. The methods for evaluating these age-

The integrity of the bone microarchitecture is an important element of the bone quality and contributes for the mechanical abilities of the bone ( FARMAN *et al*., 1993; TAGUCHI, 2004;

> Gonio, mentual forame, and mandibular molar

region.

region

Basal of the mandibular molar

Edentulus

Ratio: width of the cortical one with in the distance of the inferior edge of mentual forame for the inferior edge of

the jaw

Basal Cortical, classification: C1; C2

endosteals edge.

Bony density by microdensitometer.

(BMC), g/cm

sides .

Mandible BMC em g/cm

Bone Mineral Content

Bone Mineral Density – cortical and trabecular bony, separately horizontal in mg/cm3

2 .

> 3 , both

; C3,

**RESEARCHERS THECNICS Regions Measures**  Bras *et al*. (1982) Panoramic Radiography ------------------------- Cortical width

related changes in the maxilla and mandible were listed by Bras et al, 1982.

**6. Panoramic radiography** 

BOUXSEIN, 2003; SEEMAN, 2003; WOWERN, 2001).

Benson *et al.* (1991) Panoramic Radiography -------------------------

Klemetti *et al.* (1994) Panoramic Radiography ----------------------

Aluminun penetrometer

absorciometria (DPA),

X-rays Dual Emission

CTQC Dual or Single

Energy. Mandible

Table 2. Original methods for evaluation of the changes of the bone of the jaw *in vivo*

Kribbs *et al.* (1992) Intramural film with

Dual-Foton

mandible.

(DXA)

Wowern (1993)

Corten (1995) Horner *et al.* (1998)

Bassi *et al.*(1999) Klemetti *et al*.(1995) Lindth *et a*l.(1996) Taguchi *et al*.(1996)

### **6.1 Radio-morphometric indices (Table 2)**


The measurements can be made in the panoramic radiographs in the mental foramen area, with the aid of the RADIOIMP software (RADIO MEMORY LTDA, version 2.0). When opening the image of the panoramic radiograph, the program requests the calibration of the same, where the following information should be inserted:


After the simultaneous alteration of the brightness and contrast tool of the images for better visualization of the mental foramen area, the measurements can be initiated, according to the technique proposed by Benson et al, 1991 based in the technique of the Wical & Swoope (1974). The first step is the identification and the tracing of the mental foramen unilaterally; then, a parallel line will be drawn down the long axis of the mandibular body and tangent to the inferior border of each side. Later, the mandibular cortex will be measured, through a line drawn perpendicularly from the first and afterwards the height of the mental foramen will be also measured, given by the distance of the inferior border of the foramen to the base of the mandible. All the measured lines presented a 90o degree format. For better identification of the measured area, the zoom tool will be also used to facilitate the the measurements, as the presented values were around 0,3 cm (Figure 9 and 10).

The thickness of the mandibular cortex is divided by the distance between the mental foremen and the inferior mandibular cortex to obtain the PMI (BENSON et al, 1991).

Fig. 9. Measurement of the Mentual and Antigoniac indices. Radioimp-RADIO MEMORY LTDA, version 2.0

How Dentistry Can Help Fight Osteoporosis 835


In the study of indices proposed in radio-morphometric mandible (TAGUCHI et al, 1995) it was found that the indices evaluated were reproducible; PMI and MI showed the highest sensitivity for detecting osteopenia / osteoporosis, but the specificity of the panoramic mandibular index was low, all the indices evaluated were able to identify low bone mass, however, only PMI and MI could differentiate patients with osteopenia / osteoporosis.

If persons at risk of osteoporosis can be screened using panoramic radiographs, screening of persons without subjective symptoms that are difficult to diagnose or persons without concern for osteoporosis, and instruction for only persons potentially having osteoporosis to undergo closer examination such as DXA or referral to a facility equipped with that apparatus allow early detection and early treatment of patients suffering from osteoporosis, and also reduce costs of the examination. Furthermore, the method used for this screening must be simple and usable even without having any special skills or requiring complicated

The relationship between osteoporosis and oral signals was investigated to evaluate the possibility of using this as an indicator of osteoporosis. Some authors Taguchi et al 1995 studied 64 postmenopausal women aged between 50 and 70 years. Signals consisted of osteoporotic fracture of the thoracic spine seen on lateral radiographs of the lung. Oral signs were the number of teeth present, cortical thickness, alveolar bone resorption, and

**7. Evidence to support panoramic radiography for the diagnosis of** 

the mandibular base (normal greater than or equal to 1.0 mm). (Figure 12)

Fig. 12. Method of the measured Goniac Index (IG)

**osteoporosis** 

operations.

### **Klemetti Classification**

The mandibular cortical shape is classified into one of three groups according to the method of Klemetti et al, which considers qualitatively the endosteal margin of mandibular cortical (KLEMETTI et al, 1994): C1—the endosteal cortical margin is even and sharp on both sides, normal cortex (Figure 11); C2—the endosteal margin has semi-lunar defects (lacunar resorption) or endosteal cortical residues on one or both sides (Figure 11), mild to moderate cortex erosion; C3—the cortical layer forms heavy endosteal cortical residues and is clearly porous, severely eroded cortex (Figure 11).

Fig. 11. Klemetti classification


The mandibular cortical shape is classified into one of three groups according to the method of Klemetti et al, which considers qualitatively the endosteal margin of mandibular cortical (KLEMETTI et al, 1994): C1—the endosteal cortical margin is even and sharp on both sides, normal cortex (Figure 11); C2—the endosteal margin has semi-lunar defects (lacunar resorption) or endosteal cortical residues on one or both sides (Figure 11), mild to moderate cortex erosion; C3—the cortical layer forms heavy endosteal cortical residues and is clearly


Fig. 10. Mentual index in detail.

Fig. 11. Klemetti classification

equal to 3.2 mm);

porous, severely eroded cortex (Figure 11).

**Klemetti Classification** 


Fig. 12. Method of the measured Goniac Index (IG)

In the study of indices proposed in radio-morphometric mandible (TAGUCHI et al, 1995) it was found that the indices evaluated were reproducible; PMI and MI showed the highest sensitivity for detecting osteopenia / osteoporosis, but the specificity of the panoramic mandibular index was low, all the indices evaluated were able to identify low bone mass, however, only PMI and MI could differentiate patients with osteopenia / osteoporosis.

### **7. Evidence to support panoramic radiography for the diagnosis of osteoporosis**

If persons at risk of osteoporosis can be screened using panoramic radiographs, screening of persons without subjective symptoms that are difficult to diagnose or persons without concern for osteoporosis, and instruction for only persons potentially having osteoporosis to undergo closer examination such as DXA or referral to a facility equipped with that apparatus allow early detection and early treatment of patients suffering from osteoporosis, and also reduce costs of the examination. Furthermore, the method used for this screening must be simple and usable even without having any special skills or requiring complicated operations.

The relationship between osteoporosis and oral signals was investigated to evaluate the possibility of using this as an indicator of osteoporosis. Some authors Taguchi et al 1995 studied 64 postmenopausal women aged between 50 and 70 years. Signals consisted of osteoporotic fracture of the thoracic spine seen on lateral radiographs of the lung. Oral signs were the number of teeth present, cortical thickness, alveolar bone resorption, and

How Dentistry Can Help Fight Osteoporosis 837

comprised the standard. Nineteen of the 21 untrained general dental practice showed a moderate to almost perfect intra-observer agreement. We conclude that dental panoramic radiographs could be used in clinical dental practice to identify postmenopausal women

The frequency of osteoporosis was evaluated according to bone sites using a cross-sectional clinical study. The authors evaluated 610 densitometric examinations in relation to frequency of osteoporosis / osteopenia and agreement of the diagnosis according to the bone site. Despite the high correlation of BMD between the different bone sites, the frequency of osteoporosis varied with the site assessed. This study demonstrated that there is discordance in the BMD results according to the study area, affecting the occurrence of osteoporosis. Clinical trial for fracture risk assessment, the use of two different bone sites is the most appropriate procedure. For routine clinical dental surgeon, which includes the panoramic radiography in the care protocol and the jaws could be used for that purpose, and to request carpal radiography, which can add information of bone quality, especially in view of the proportions of trabecular and cortical bone of the phalanges of the hand and also

In 2005, Klein conducted a study conducted with the objective to modify the skeletonization algorithm to quantify and create other radiographic images in panoramic radiographs. According to the study of observations and the evaluations that were made, it can be concluded that: 1) the part of the experiment related to the use of radiographic images by means of skeletonization on panoramic radiographs was effective because it increased the radius of the visual perception of the architecture in the trabecular bone and observed the trabeculae, the marrow spaces, such as micro-damage, or micro fractures, 2) despite the agreement between the examiners who have not reached recorded levels above 80%, a high significance in the overall proportion of black points and end points with the odds of a diagnosis concerning the existence or not of bone fragility. 3) results confirmed that the greater bone fragility actually revealed to be a loss of lamellae of trabecular bone

When studying osteoporosis, there is consensus that inexpensive methods of screening for osteoporosis are needed. The results of this study (WHITE et al, 2005) suggest that dentists have sufficient information to routinely identify people with low BMD using the images of panoramic radiography in dental practice. Radiographs with low doses of radiation, comparable to 4 bitewing radiographs, and the patients identified as having risk of

The literature on oral radiographic signs of osteoporosis was revised in 2002, including alveolar bone resorption, and decreased inferior cortical mandibular (ICM). The authors concluded that the panoramic radiograph is an important tool that displays enough information to diagnose osteoporosis (WATANABE et al, 2002). Also in 2002, HORNER et al made a study evaluating the relative utility of clinical indices and radiographic diagnosis in patients with low skeletal bone mass between 135 on healthy pre-menopausal women, aged 45-55 years who sought dental treatment. The DOM was measured in the spine and hip using DXA and classified according to the WHO criteria for Caucasian women. In each patient the (ICM) was measured on panoramic radiographs. The body mass index (BMI) is a simple calculation of the estimated risk of osteoporosis (SCORE). All indexes, (ICM), BMI and SCORE showed significant correlation with skeletal bone density. Thus, the authors concluded that the thinning of the ICM <3mm in peri-menopausal healthy women is

osteoporosis should be referred to a primary health care for further evaluation.

who have low DOM undetected.

in the cortical distal radio (ZANETTE et al, 2003).

architecture and its fairly large marrow spaces.

associated with low skeletal bone mass.

morphological classification of the cortex in the panoramic radiograph. The number of teeth (N) was significantly correlated with the probability of fracture in the thoracic spine and was used to derive equation and the probability for the presence of fractures of the thoracic spine: probability value = 1 / (1 + z), where Z age = 18.68 to 0.29 - 0.27 N. The probability value greater than 0.5 suggests the possibility of fractures in the thoracic vertebrae. It can be concluded that this equation combined with the findings in the panoramic radiograph could serve as a simple and useful tool for the dentist to evaluate the possibility of latent osteoporosis.

Panoramic radiographs are routinely used in most radiographic indications for the various types of dental patients. Such use as the primary complementary diagnostic exam is endorsed by Nº. 453 Law of the Health Ministry - ANVISA – Brazil, a recommendation supported by the principle of radioprotection known as ALARA (As Low As Reasonably Achievable), i.e. we should always use the least amount of radiation possible, for better diagnostic information and for the well-being of the patient (SVS-MS, 1998).

In 1991 an index of bone mass radiomorphometric cortical (BENSON et al, 1991) the panoramic mandibular index (PMI). The MPI was obtained as the result of the ratio between the thickness of the mandibular inferior cortex (ECM) and the distance between the bottom of the mental foramen and the lower limit of the mandibular inferior cortex (DMC). Being that the higher the value of IPM, the lower jaw bone resorption. The differences in the index in a population of 353 adults, evenly divided by gender, age (30 to 90), and racial groups (blacks, Hispanics and whites) were evaluated with respect to the side, race, gender, and age, and combinations of these variables. Blacks were found in average IPM higher than in Hispanics or whites, who were demographically similar. Age-related changes comparing younger and older groups within each sex and racial group showed a significant decrease in average IPM with increasing age in black and Hispanic women. The average PMI in whites increases with advancing age.

The precision of the panoramic mandibular index in detecting patients with osteopenia and osteoporosis was studied and the authors concluded that the action taken in panoramic radiographs (IPM) of the patients studied was able to identify low bone mass and is able to differentiate patients with osteopenia and osteoporosis. Thus, IPM can be used by dentists to make an early approach that osteoporosis is a systemic condition that affects almost half the female population and brings many risks and damage their health (KNEZOVIC-ZLATARIC & CELEBIC, 2005).

Other authors (NAKAMOTO et al, 2003) assessed whether untrained dental practice would be able to determine whether panoramic radiographs women have low bone mineral density (OD). The researchers studied the concordance of the observer and the diagnostic efficiency in detecting low DOM in women. This was done when the appearance (normal or eroded) of the mandibular inferior cortex on dental panoramic radiographs of 100 postmenopausal women who had carried out assessments DOM lumbar spine and femoral neck. The intra-and inter-observer was assessed with kappa statistics. The diagnostic efficiency (sensitivity, specificity, and predictive values) was analyzed by comparing the two groups classified by the mandibular inferior cortex (women with normal cortex and women with eroded mandibular inferior cortex) with those classified by DOM (DOM women with normal and women with osteopenia or osteoporosis). The average sensitivity and specificity were 77% and 40%, respectively, when the DOM of the lumbar spine was used as the default, and 75% and 39%, respectively, when the DOM of the femoral neck

morphological classification of the cortex in the panoramic radiograph. The number of teeth (N) was significantly correlated with the probability of fracture in the thoracic spine and was used to derive equation and the probability for the presence of fractures of the thoracic spine: probability value = 1 / (1 + z), where Z age = 18.68 to 0.29 - 0.27 N. The probability value greater than 0.5 suggests the possibility of fractures in the thoracic vertebrae. It can be concluded that this equation combined with the findings in the panoramic radiograph could serve as a simple and useful tool for the dentist to evaluate the possibility of latent

Panoramic radiographs are routinely used in most radiographic indications for the various types of dental patients. Such use as the primary complementary diagnostic exam is endorsed by Nº. 453 Law of the Health Ministry - ANVISA – Brazil, a recommendation supported by the principle of radioprotection known as ALARA (As Low As Reasonably Achievable), i.e. we should always use the least amount of radiation possible, for better

In 1991 an index of bone mass radiomorphometric cortical (BENSON et al, 1991) the panoramic mandibular index (PMI). The MPI was obtained as the result of the ratio between the thickness of the mandibular inferior cortex (ECM) and the distance between the bottom of the mental foramen and the lower limit of the mandibular inferior cortex (DMC). Being that the higher the value of IPM, the lower jaw bone resorption. The differences in the index in a population of 353 adults, evenly divided by gender, age (30 to 90), and racial groups (blacks, Hispanics and whites) were evaluated with respect to the side, race, gender, and age, and combinations of these variables. Blacks were found in average IPM higher than in Hispanics or whites, who were demographically similar. Age-related changes comparing younger and older groups within each sex and racial group showed a significant decrease in average IPM with increasing age in black and Hispanic women. The average PMI in whites

The precision of the panoramic mandibular index in detecting patients with osteopenia and osteoporosis was studied and the authors concluded that the action taken in panoramic radiographs (IPM) of the patients studied was able to identify low bone mass and is able to differentiate patients with osteopenia and osteoporosis. Thus, IPM can be used by dentists to make an early approach that osteoporosis is a systemic condition that affects almost half the female population and brings many risks and damage their health (KNEZOVIC-

Other authors (NAKAMOTO et al, 2003) assessed whether untrained dental practice would be able to determine whether panoramic radiographs women have low bone mineral density (OD). The researchers studied the concordance of the observer and the diagnostic efficiency in detecting low DOM in women. This was done when the appearance (normal or eroded) of the mandibular inferior cortex on dental panoramic radiographs of 100 postmenopausal women who had carried out assessments DOM lumbar spine and femoral neck. The intra-and inter-observer was assessed with kappa statistics. The diagnostic efficiency (sensitivity, specificity, and predictive values) was analyzed by comparing the two groups classified by the mandibular inferior cortex (women with normal cortex and women with eroded mandibular inferior cortex) with those classified by DOM (DOM women with normal and women with osteopenia or osteoporosis). The average sensitivity and specificity were 77% and 40%, respectively, when the DOM of the lumbar spine was used as the default, and 75% and 39%, respectively, when the DOM of the femoral neck

diagnostic information and for the well-being of the patient (SVS-MS, 1998).

osteoporosis.

increases with advancing age.

ZLATARIC & CELEBIC, 2005).

comprised the standard. Nineteen of the 21 untrained general dental practice showed a moderate to almost perfect intra-observer agreement. We conclude that dental panoramic radiographs could be used in clinical dental practice to identify postmenopausal women who have low DOM undetected.

The frequency of osteoporosis was evaluated according to bone sites using a cross-sectional clinical study. The authors evaluated 610 densitometric examinations in relation to frequency of osteoporosis / osteopenia and agreement of the diagnosis according to the bone site. Despite the high correlation of BMD between the different bone sites, the frequency of osteoporosis varied with the site assessed. This study demonstrated that there is discordance in the BMD results according to the study area, affecting the occurrence of osteoporosis. Clinical trial for fracture risk assessment, the use of two different bone sites is the most appropriate procedure. For routine clinical dental surgeon, which includes the panoramic radiography in the care protocol and the jaws could be used for that purpose, and to request carpal radiography, which can add information of bone quality, especially in view of the proportions of trabecular and cortical bone of the phalanges of the hand and also in the cortical distal radio (ZANETTE et al, 2003).

In 2005, Klein conducted a study conducted with the objective to modify the skeletonization algorithm to quantify and create other radiographic images in panoramic radiographs. According to the study of observations and the evaluations that were made, it can be concluded that: 1) the part of the experiment related to the use of radiographic images by means of skeletonization on panoramic radiographs was effective because it increased the radius of the visual perception of the architecture in the trabecular bone and observed the trabeculae, the marrow spaces, such as micro-damage, or micro fractures, 2) despite the agreement between the examiners who have not reached recorded levels above 80%, a high significance in the overall proportion of black points and end points with the odds of a diagnosis concerning the existence or not of bone fragility. 3) results confirmed that the greater bone fragility actually revealed to be a loss of lamellae of trabecular bone architecture and its fairly large marrow spaces.

When studying osteoporosis, there is consensus that inexpensive methods of screening for osteoporosis are needed. The results of this study (WHITE et al, 2005) suggest that dentists have sufficient information to routinely identify people with low BMD using the images of panoramic radiography in dental practice. Radiographs with low doses of radiation, comparable to 4 bitewing radiographs, and the patients identified as having risk of osteoporosis should be referred to a primary health care for further evaluation.

The literature on oral radiographic signs of osteoporosis was revised in 2002, including alveolar bone resorption, and decreased inferior cortical mandibular (ICM). The authors concluded that the panoramic radiograph is an important tool that displays enough information to diagnose osteoporosis (WATANABE et al, 2002). Also in 2002, HORNER et al made a study evaluating the relative utility of clinical indices and radiographic diagnosis in patients with low skeletal bone mass between 135 on healthy pre-menopausal women, aged 45-55 years who sought dental treatment. The DOM was measured in the spine and hip using DXA and classified according to the WHO criteria for Caucasian women. In each patient the (ICM) was measured on panoramic radiographs. The body mass index (BMI) is a simple calculation of the estimated risk of osteoporosis (SCORE). All indexes, (ICM), BMI and SCORE showed significant correlation with skeletal bone density. Thus, the authors concluded that the thinning of the ICM <3mm in peri-menopausal healthy women is associated with low skeletal bone mass.

Fig. 7.11

Fig. 7.13

Fig. 7.15

connectivity.

Fig. 7.17 - Image of the Figure 7.1

How Dentistry Can Help Fight Osteoporosis 839

Fig. 7.12 Histogram of the image Figure 7.11

Fig. 7.14 Histogram of the image Figure 7.13

Fig. 7.16 Histogram of the image Figure 7.15

Result of the dilate process of the image Figure

Result of the 7.11 image inversion

Result of the image

7.13

esqueletonizing action Figure

conclusão

7.1 e 7.15

Fig. 13. Representation of the process of esqueletonized of region of mandibular interest, panoramic x-ray, for study of the percentage of trabeculae, fractal dimension and bony

Fig. 7.18 –Fusion Images of the Figures

7.9

Three indicators of bone quality on panoramic radiographs were studied to determine the correlation with low DOM using DXA in a Brazilian population (Watanabe, 2003). Examination of the trabecular bone and ICM in the panoramic radiograph showed early signs of osteoporosis. There was significant correlation of these factors with the parameters measured as the percentage of trabeculae, fractal dimension and trabecular connectivity (Figures. 13 and 14).


Three indicators of bone quality on panoramic radiographs were studied to determine the correlation with low DOM using DXA in a Brazilian population (Watanabe, 2003). Examination of the trabecular bone and ICM in the panoramic radiograph showed early signs of osteoporosis. There was significant correlation of these factors with the parameters measured as the percentage of trabeculae, fractal dimension and trabecular connectivity

Fig. 7.2

Image copy of the Figure 7.1, with "Gaussian blurr" of 33 radius (pixels)

Fig. 7.4 Histogram 7.3 image

Fig. 7.6 Histogram 7.5 image

Fig. 7.8 Histogram 7.7 image

Fig. 7.10 Histogram of the image Figure 7.9

Image interest area (original), 230 X 130 *pixels* 

Result of subtraction Figures 7.1 and 7.2 images process

Result of addition of the constant, 128 to 7.3 image

Result of binary transformation (threshold) of the 7.5 image, with 128 brightness

value

7.7

Result of the erode process of the image Figure

(Figures. 13 and 14).

Fig. 7.1

Fig. 7.3

Fig.7.5

Fig. 7.7

Fig. 7.9


Fig. 13. Representation of the process of esqueletonized of region of mandibular interest, panoramic x-ray, for study of the percentage of trabeculae, fractal dimension and bony connectivity.

How Dentistry Can Help Fight Osteoporosis 841

sharp contrast between the branch / mandibular body and strengthening of structures,

Fig. 15. Detail of the pseudoperoartrite in the mandibular inferior cortical and accented

It is possible to correlate Bone Mineral Index (BMI)with mandibular bone quality (MBQ). The authors studied the correlation between body mass index and mandibular bone quality in Brazilians of both sexes. According to the methodology employed in this study, not all patients with poor MBQ had low BMI, but the majority who had low BMI, had bad MBQ

> Normal 22,0 - 24,9

Lee et al (2005) conducted a study in the visual cortical lower mandible on panoramic radiographs to identify postmenopausal women with low BMD. The authors concluded that the visual analysis of the mandibular inferior cortex on panoramic radiographs may be

Dutra et al studied the radiomorphometric indices and their relationships with gender, age and dental status, using the antigoniac index (AI) and chin index (MI) in patients with and without teeth. It was concluded that there is a renovation in the mandibular inferior cortex (MIC) with age and that would be influenced by gender and dental status. The difficulty in measuring the AI in a reproducible way, and their interaction with dental status and low correlation with MI in younger patients would discourage its use for the purpose of

An image analysis software that can accurately measure the thickness of the mandibular inferior cortex (MIC) in PR has been developed as an indicator of low BMD. The authors found that the action taken by the software had significant correlation with the BMD and could contribute to the identification of osteopenia. The study was supported by the European Commission FP5 "Quality of Life and Management of Living Resources"(Report

Overweight 25,0 – 29,9

Obese ≥ 30,0

such as the oblique line (Figure 15).

oblique line in the mandible.

( ) ²( ²)

*Heigth m* Underweight <22

useful in identifying women with low DOM PM (LEE et al, 2005).

identifying patients at risk of osteoporosis ((DUTRA et al, 2005).

(DUTRA et al, 2005).

*Weigth kg IMC*

Fig. 14. Detail of the erosion in the mandibular inferior cortical

The diagnostic performance of measurements on panoramic radiographs (PR) and a selfassessment tool for osteoporosis (OST) that identifies women with spinal osteoporosis (WHO) on 159 pre-menopausal (PrM) and 157 post-menopausal (PsM) women with a history of hysterectomy, ovariectomy, or use estrogen were compared. The morphology of the ICM and its thickness were evaluated in the panoramic radiographs. The authors concluded that clinicians can refer women with suspected osteoporosis (PsM) in the column to undergo DXA based on tests conducted on panoramic radiographs with similar performance in the OST (TAGUCHI et al, 2004). The correlation of Klemetti rating for the PR using digital panoramic radiographs of Brazilian women was also studied in 2004 by TAGUCHI et al. Significant correlation was found with DXA of the forearm DOM, indicating that the panoramic radiograph is valuable in the identification of patients at risk for osteoporosis.

DOM and radio-morphometric linear parameters in elderly patients with different types of dentures were studied (DUTRA et al, 2005). Three parameters were measured: Mandibular inferior córtex thickness (CIMT), the Antegoniac Index (IA) and Goniac Index (GA). The DOM was measured in the jaw with the use of a copper penetrometer. The results showed that there was a statistically significant difference between patients with all teeth and those with denture for all the measured radio-morphometric indices (p <0.001). Also in 2005, LEE et al suggested that dentists had sufficient radiographic and clinical information to identify patients with osteoporosis. The author concluded that the changes found in the trabecular structures on panoramic radiographs supplemented with clinical information is an indicative of risk for hip fracture in elderly women.

It can be concluded that the dentist may suspect systemic risk of osteoporosis when the patient presents the following radiographic signs found in panoramic and periapical radiographs (WATANABE et al, 2004):


The diagnostic performance of measurements on panoramic radiographs (PR) and a selfassessment tool for osteoporosis (OST) that identifies women with spinal osteoporosis (WHO) on 159 pre-menopausal (PrM) and 157 post-menopausal (PsM) women with a history of hysterectomy, ovariectomy, or use estrogen were compared. The morphology of the ICM and its thickness were evaluated in the panoramic radiographs. The authors concluded that clinicians can refer women with suspected osteoporosis (PsM) in the column to undergo DXA based on tests conducted on panoramic radiographs with similar performance in the OST (TAGUCHI et al, 2004). The correlation of Klemetti rating for the PR using digital panoramic radiographs of Brazilian women was also studied in 2004 by TAGUCHI et al. Significant correlation was found with DXA of the forearm DOM, indicating that the panoramic

DOM and radio-morphometric linear parameters in elderly patients with different types of dentures were studied (DUTRA et al, 2005). Three parameters were measured: Mandibular inferior córtex thickness (CIMT), the Antegoniac Index (IA) and Goniac Index (GA). The DOM was measured in the jaw with the use of a copper penetrometer. The results showed that there was a statistically significant difference between patients with all teeth and those with denture for all the measured radio-morphometric indices (p <0.001). Also in 2005, LEE et al suggested that dentists had sufficient radiographic and clinical information to identify patients with osteoporosis. The author concluded that the changes found in the trabecular structures on panoramic radiographs supplemented with clinical information is an

It can be concluded that the dentist may suspect systemic risk of osteoporosis when the patient presents the following radiographic signs found in panoramic and periapical

Klemetti Class II or III, radiolucent spaces in the mandibular inferior cortical (Fig. 10);

Disorganization of the basal mandibular trabecular bone with low numbers and low

Fig. 14. Detail of the erosion in the mandibular inferior cortical

radiograph is valuable in the identification of patients at risk for osteoporosis.

indicative of risk for hip fracture in elderly women.

radiographs (WATANABE et al, 2004):

CIM thickness less than 3.0 mm;

connectivity;

 sharp contrast between the branch / mandibular body and strengthening of structures, such as the oblique line (Figure 15).

Fig. 15. Detail of the pseudoperoartrite in the mandibular inferior cortical and accented oblique line in the mandible.

It is possible to correlate Bone Mineral Index (BMI)with mandibular bone quality (MBQ). The authors studied the correlation between body mass index and mandibular bone quality in Brazilians of both sexes. According to the methodology employed in this study, not all patients with poor MBQ had low BMI, but the majority who had low BMI, had bad MBQ (DUTRA et al, 2005).


Lee et al (2005) conducted a study in the visual cortical lower mandible on panoramic radiographs to identify postmenopausal women with low BMD. The authors concluded that the visual analysis of the mandibular inferior cortex on panoramic radiographs may be useful in identifying women with low DOM PM (LEE et al, 2005).

Dutra et al studied the radiomorphometric indices and their relationships with gender, age and dental status, using the antigoniac index (AI) and chin index (MI) in patients with and without teeth. It was concluded that there is a renovation in the mandibular inferior cortex (MIC) with age and that would be influenced by gender and dental status. The difficulty in measuring the AI in a reproducible way, and their interaction with dental status and low correlation with MI in younger patients would discourage its use for the purpose of identifying patients at risk of osteoporosis ((DUTRA et al, 2005).

An image analysis software that can accurately measure the thickness of the mandibular inferior cortex (MIC) in PR has been developed as an indicator of low BMD. The authors found that the action taken by the software had significant correlation with the BMD and could contribute to the identification of osteopenia. The study was supported by the European Commission FP5 "Quality of Life and Management of Living Resources"(Report

How Dentistry Can Help Fight Osteoporosis 843

Fig. 16. Measure of the Elongated styloid process in the panoramic radiographic

Fig. 17. The same interest region to prepare the image to skeletonized

Some studies have suggested that osteoporosis and periodontitis are associated (PERSSON et al, 2002): (1) the prevalence of self-reported history of osteoporosis in an older population, ethnically diverse, (2) the concordance between panoramic mandibular index (PMI) and self-reported osteoporosis, and (3) the probability of having a self-

**8. Periodontal disease and osteoporosis** 

of the 10th European Congress of Dentomaxillofacial Radiology, 2006). Another study investigated the OSTEODENT trabecular pattern in intraoral radiographs, and concluded that this factor would serve for the diagnosis of osteoporosis. Other authors stated that ICM is effective in the diagnosis of osteoporosis because it had high specificity and thus could be used in primary health care. Analyzing the densitometric measurements in intraoral radiographs to detect osteoporosis was the proposal of some authors who concluded that bone density in the region of premolars, expressed in millimeters of aluminum would be favorable for showing the presence of systemic osteoporosis.

In 2007, Ishii et al evaluated the diagnostic efficiency in identifying postmenopausal women with osteoporosis by analyzing femoral bone loss in the jaw. It is known that cortical thickness measurements in lower jaw is useful to that purpose. The results suggest that the assessment of alveolar bone resorption was not as effective in detecting postmenopausal women with osteoporosis compared to femoral cortical thickness measures lower jaw.

The detection of cortical erosions in lower mandible on panoramic radiographs and tools based on questionnaires were studied in 2008, and it was found similar diagnostic efficacy in identifying postmenopausal osteoporotic women. Furthermore the authors evaluated the diagnostic performance to identify osteoporosis and biochemical markers of bone turnover for high risk of fracture. The analysis of urine and blood plasma were measured for bone mineral density (OD) of the spine and hip by DXA. The results suggest that panoramic radiography was superior to questionnaire-based tool to identify women with high risk of fractures (TAGUCHI et al, 2008).

In 2009, Elsubeihi & Heersche studied the effects of ovariectomy on the toothed jaws and mandible of rats were investigated and compared to changes in relation to the tibia and femur using DXA scans (dual X-ray Absorptiometry) and histomorphometric measurements. The results showed that the loss of bone in the jaws without teeth in ovaryectomized animals was similar to what occurred in the tibia and femur, while the lack of significant effects of ovariectomy on bone mass in toothed mandibles suggests that the functional load on the force bite prevents bone loss in the jaws with teeth. Evaluating the quality of mandibular bone in edentulous persons, persons with less than 21 teeth and those over 21 teeth, it was found significant differences in the bone quality, measured by the mandibular inferior cortical thickness, indicating that the person with more than 21 teeth in the oral cavity has a better quality of mandibular bone.

It was also studied in 2010, by Watanabe et al, the correlation of the elongated styloid process with low BMD diagnosed by DXA. The authors could verify the existence of a strong correlation between women with osteopenia and osteoporosis, and women with fracture risk presented calcification of the stylohyoid ligament (Figure 16). In another article of the same year, Watanabe et al, 2009 studied the morphological pattern trabecular digitally comparing the same regions of interest in different radiographs, periapical and panoramic (Figure 17). The authors could verify that when the analysis of skeletonized images of certain regions was performed, significant differences between the measurements were found and such comparison should therefore be carefull. Khojastehpour et al. analyzed the usefulness of the Panoramic Mandibular Index (PMI) on panoramic radiographs for the diagnosis of osteoporosis in women and concluded that dental panoramic radiographs could be used in the clinical practice to assist identifying individuals with low bone mass through PMI (KHOJASTEHPOUR et al, 2009).

of the 10th European Congress of Dentomaxillofacial Radiology, 2006). Another study investigated the OSTEODENT trabecular pattern in intraoral radiographs, and concluded that this factor would serve for the diagnosis of osteoporosis. Other authors stated that ICM is effective in the diagnosis of osteoporosis because it had high specificity and thus could be used in primary health care. Analyzing the densitometric measurements in intraoral radiographs to detect osteoporosis was the proposal of some authors who concluded that bone density in the region of premolars, expressed in millimeters of aluminum would be

In 2007, Ishii et al evaluated the diagnostic efficiency in identifying postmenopausal women with osteoporosis by analyzing femoral bone loss in the jaw. It is known that cortical thickness measurements in lower jaw is useful to that purpose. The results suggest that the assessment of alveolar bone resorption was not as effective in detecting postmenopausal women with osteoporosis compared to femoral cortical thickness

The detection of cortical erosions in lower mandible on panoramic radiographs and tools based on questionnaires were studied in 2008, and it was found similar diagnostic efficacy in identifying postmenopausal osteoporotic women. Furthermore the authors evaluated the diagnostic performance to identify osteoporosis and biochemical markers of bone turnover for high risk of fracture. The analysis of urine and blood plasma were measured for bone mineral density (OD) of the spine and hip by DXA. The results suggest that panoramic radiography was superior to questionnaire-based tool to identify women with high risk of

In 2009, Elsubeihi & Heersche studied the effects of ovariectomy on the toothed jaws and mandible of rats were investigated and compared to changes in relation to the tibia and femur using DXA scans (dual X-ray Absorptiometry) and histomorphometric measurements. The results showed that the loss of bone in the jaws without teeth in ovaryectomized animals was similar to what occurred in the tibia and femur, while the lack of significant effects of ovariectomy on bone mass in toothed mandibles suggests that the functional load on the force bite prevents bone loss in the jaws with teeth. Evaluating the quality of mandibular bone in edentulous persons, persons with less than 21 teeth and those over 21 teeth, it was found significant differences in the bone quality, measured by the mandibular inferior cortical thickness, indicating that the person with more than 21 teeth in

It was also studied in 2010, by Watanabe et al, the correlation of the elongated styloid process with low BMD diagnosed by DXA. The authors could verify the existence of a strong correlation between women with osteopenia and osteoporosis, and women with fracture risk presented calcification of the stylohyoid ligament (Figure 16). In another article of the same year, Watanabe et al, 2009 studied the morphological pattern trabecular digitally comparing the same regions of interest in different radiographs, periapical and panoramic (Figure 17). The authors could verify that when the analysis of skeletonized images of certain regions was performed, significant differences between the measurements were found and such comparison should therefore be carefull. Khojastehpour et al. analyzed the usefulness of the Panoramic Mandibular Index (PMI) on panoramic radiographs for the diagnosis of osteoporosis in women and concluded that dental panoramic radiographs could be used in the clinical practice to assist identifying individuals with low bone mass

favorable for showing the presence of systemic osteoporosis.

the oral cavity has a better quality of mandibular bone.

through PMI (KHOJASTEHPOUR et al, 2009).

measures lower jaw.

fractures (TAGUCHI et al, 2008).

Fig. 16. Measure of the Elongated styloid process in the panoramic radiographic

Fig. 17. The same interest region to prepare the image to skeletonized

### **8. Periodontal disease and osteoporosis**

Some studies have suggested that osteoporosis and periodontitis are associated (PERSSON et al, 2002): (1) the prevalence of self-reported history of osteoporosis in an older population, ethnically diverse, (2) the concordance between panoramic mandibular index (PMI) and self-reported osteoporosis, and (3) the probability of having a self-

How Dentistry Can Help Fight Osteoporosis 845

within 8 weeks after the identification by a healthcare provider in a patient who is receiving or has been exposed to a bisphosphonate and has not had radiation therapy in the craniofacial region" (KHOSLA et al, 2007) . The American Association of Oral and Maxillofacial Surgeons (AAOMS) (WOO et al) has revised its 2006 landmark position paper on Bisphosphonate-Related Osteonecrosis of the Jaw to reflect the most current research on this condition. BRONJ appears as a non-healing exposed bone in the maxillofacial region and may affect patients undergoing intravenous cancer-related bisphosphonate therapy or more rarely, patients treated with oral or IV bisphosphonates for osteoporosis. Despite its low prevalence, the potential risk of BRONJ occurring after the use of oral bisphosphonate

Also, the European Society on Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) has developed some standards for the prevention, recognition and management of BRONJ (PHAL et al, 2007). In osteoporosis patients, no specific interventions prior to starting bisphosphonate therapy are required except to encourage

The radiographic aspects associated with osteochemonecrosis of the jaws related with the oral use of bisphosphonate (BP) should be identified. The most important radiographic features are: Osteosclerosis limited to the alveolar process, widening of the lamina dura, expansion of the periodontal ligament space, bony sequestra, jaw expansion, radiolucency, and periosteal new bone formation. Osteosclerosis is frequent. It is usually found in the presence of periodontal disease, probably because it is attributed to the fact that BP accumulates preferentially in sites of high bone turnover or remodeling (KHAN et

Radiographic changes are not evident until there is extensive bone involvement. Therefore, panoramic radiographs may not reveal significant changes in the early stages of osteonecrosis as numerous different patologies. When there is extensive bone involvement, regions of mottled bone similar to diffuse osteomyelitis or postirradiation osteoradionecrosis are noted (KUNCHUR & GOSS, 2008). After prolonged exposure to intravenous bisphosphonates, osteosclerosis of the bone, especially osteosclerotic lamina, may be noted radiographically Edwards et al, 2008 (Figures 18-19). The patient in these images received the diagnosis from breast cancer IIIa - AP, and began chemotherapy in July 2003, and gone through bilateral mastectomy. In 2007 it had been diagnosised metastasis in the liver and in the column when the x-ray of the column was made and the monthly use of zometa. The first requested x-ray to the Radiologic Service was in 2008 for the Dental Service of the hospital for diagnostic purpose. It was observed the absence of 7 teeth and horizontal resorption of the alveolar Crests and sclerosis of the horny lamina of teeth 16; 15; 14; 13; 25; 27; 36; 35; 45 and 48. These alterations are indicative of possible bone exposition, suggesting the presence of a sub-clinic degree of osteonecrosis, "the zero" in accordance with AAOMS. In April of 2009, new panoramic x-ray was made and served as base for dentists to consider the interruption of the administration of zometa. After the evaluation of the patient, physicians had substituted zometa by 70mg of alendronate sodium, in an attempt to prevent the occurrence of osteonecrosis in the maxillaries. The jaws are mainly affected because of the teeth, that are embedded in bone. This active bone has fast turnover, principally the horny lamina (alveolar bone also). Most of the times, dentists need to proceed with invasive

dental procedures that injures the bone. Hence, the osteonecrosis is viable.

for osteoporosis should never be neglected.

regular dental care.

al, 2008).

reported history of osteoporosis and a diagnosis of periodontitis. Panoramic radiographs and medical histories were obtained from 1084 Chinese women aged 60-75 years (mean ± 68.5 years). Patients were classified as having or not periodontitis or within three grades of severity. The PMI was found positive in 39% of patients, in contrast to self-reported osteoporosis (8%). The intra-class correlation between the PMI and self-reported osteoporosis was 0.20 (p <0.01). The probability of an association between osteoporosis and IPM was of 3%. Patients with osteoporosis and self-reported a positive PMI had worse periodontal conditions (p <0.01).

The prevalence dominance PMI positive was high and consistent with the epidemiological studies however, only partly consistent with a self-reported history of osteoporosis, with a higher prevalence of positive PMI. The loss of horizontal alveolar bone was associated with osteoporosis and self-reported positive results of PMI. Contradictory findings were found by authors (LUNDSTROM et al, 2001) who examined periodontal conditions in a cohort of women aged 70 years compared with an osteoporotic flu control with a normal BMD (210 women, 70 years). Hip radiographs were measured with DXA. The examination included a PR and intraoral radiographs. In conclusion, the study found no statistical significance in the periodontal conditions or marginal bone level between the two groups, although the results should be interpreted with extreme caution as the study sample was small.

We studied the correlation between periodontal disease and osteoporosis, comparing age, parameters of the panoramic radiographic and clinical periodontal disease. The panoramic radiographic parameters evaluated were: mandibular cortical thickness (MCT), patients were not treated, adults who had no other systemic disease and should have more than 20 teeth. They were evaluated by panoramic radiography with respect to alveolar bone loss (ABL). The mandibular bone mass was assessed by measuring the mandibular inferior cortical thickness (MICT). The POA was significantly higher CIMT and significantly lower for patients PMs (> 6 years after menopause). The number of teeth was significantly lower in the group PM (> 11 years after menopause). The age and ABL had positive correlation in men and women. Women in which MICT was lower than the average (- 2 SD) should be diagnosed as osteoporosis. The results showed that periodontal disease has correlation with osteoporosis, and thus the MICT could be useful in detecting signs of osteoporosis in women with periodontal disease (OTOGOTO & OTA, 2003).

Some authors (JAGELAVICIENE & KUBILIUS, 2006) evaluated the relationship between systemic osteoporosis and periodontal disease. Radiology provides information in determining the type and degree of alveolar resorption, periodontal condition, and the number of teeth. These parameters provide valuable information when the corresponding data correlation study was searched.

#### **8.1 Osteonecrosis of the jaw after oral bisphosphonate for osteoporosis**

Although all the benefits of the therapy with bisphosphonate, mainly for the treatment of osteoporosis, this drug is commonly associated with osteonecrosis of the jaw (ONJ). The use of bisphosphonate was first reported in 2003 (MARX, 2003), and other case series reported similar findings, usually in patients undergoing parenteral treatment for malignancies. Osteonecrosis of the jaw associated with the use of oral bisphosphonate for the treatment of osteoporosis is much less frequently reported (PAZIANAS et al, 2007; YARON et al, 2007). The American Society for Bone and Mineral Research defines bisphosphonate-associated ONJ or BRONJ as "an area of exposed bone in the maxillofacial region that has not healed

reported history of osteoporosis and a diagnosis of periodontitis. Panoramic radiographs and medical histories were obtained from 1084 Chinese women aged 60-75 years (mean ± 68.5 years). Patients were classified as having or not periodontitis or within three grades of severity. The PMI was found positive in 39% of patients, in contrast to self-reported osteoporosis (8%). The intra-class correlation between the PMI and self-reported osteoporosis was 0.20 (p <0.01). The probability of an association between osteoporosis and IPM was of 3%. Patients with osteoporosis and self-reported a positive PMI had

The prevalence dominance PMI positive was high and consistent with the epidemiological studies however, only partly consistent with a self-reported history of osteoporosis, with a higher prevalence of positive PMI. The loss of horizontal alveolar bone was associated with osteoporosis and self-reported positive results of PMI. Contradictory findings were found by authors (LUNDSTROM et al, 2001) who examined periodontal conditions in a cohort of women aged 70 years compared with an osteoporotic flu control with a normal BMD (210 women, 70 years). Hip radiographs were measured with DXA. The examination included a PR and intraoral radiographs. In conclusion, the study found no statistical significance in the periodontal conditions or marginal bone level between the two groups, although the results

We studied the correlation between periodontal disease and osteoporosis, comparing age, parameters of the panoramic radiographic and clinical periodontal disease. The panoramic radiographic parameters evaluated were: mandibular cortical thickness (MCT), patients were not treated, adults who had no other systemic disease and should have more than 20 teeth. They were evaluated by panoramic radiography with respect to alveolar bone loss (ABL). The mandibular bone mass was assessed by measuring the mandibular inferior cortical thickness (MICT). The POA was significantly higher CIMT and significantly lower for patients PMs (> 6 years after menopause). The number of teeth was significantly lower in the group PM (> 11 years after menopause). The age and ABL had positive correlation in men and women. Women in which MICT was lower than the average (- 2 SD) should be diagnosed as osteoporosis. The results showed that periodontal disease has correlation with osteoporosis, and thus the MICT could be useful in detecting signs of osteoporosis in

Some authors (JAGELAVICIENE & KUBILIUS, 2006) evaluated the relationship between systemic osteoporosis and periodontal disease. Radiology provides information in determining the type and degree of alveolar resorption, periodontal condition, and the number of teeth. These parameters provide valuable information when the corresponding

Although all the benefits of the therapy with bisphosphonate, mainly for the treatment of osteoporosis, this drug is commonly associated with osteonecrosis of the jaw (ONJ). The use of bisphosphonate was first reported in 2003 (MARX, 2003), and other case series reported similar findings, usually in patients undergoing parenteral treatment for malignancies. Osteonecrosis of the jaw associated with the use of oral bisphosphonate for the treatment of osteoporosis is much less frequently reported (PAZIANAS et al, 2007; YARON et al, 2007). The American Society for Bone and Mineral Research defines bisphosphonate-associated ONJ or BRONJ as "an area of exposed bone in the maxillofacial region that has not healed

**8.1 Osteonecrosis of the jaw after oral bisphosphonate for osteoporosis** 

should be interpreted with extreme caution as the study sample was small.

women with periodontal disease (OTOGOTO & OTA, 2003).

data correlation study was searched.

worse periodontal conditions (p <0.01).

within 8 weeks after the identification by a healthcare provider in a patient who is receiving or has been exposed to a bisphosphonate and has not had radiation therapy in the craniofacial region" (KHOSLA et al, 2007) . The American Association of Oral and Maxillofacial Surgeons (AAOMS) (WOO et al) has revised its 2006 landmark position paper on Bisphosphonate-Related Osteonecrosis of the Jaw to reflect the most current research on this condition. BRONJ appears as a non-healing exposed bone in the maxillofacial region and may affect patients undergoing intravenous cancer-related bisphosphonate therapy or more rarely, patients treated with oral or IV bisphosphonates for osteoporosis. Despite its low prevalence, the potential risk of BRONJ occurring after the use of oral bisphosphonate for osteoporosis should never be neglected.

Also, the European Society on Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) has developed some standards for the prevention, recognition and management of BRONJ (PHAL et al, 2007). In osteoporosis patients, no specific interventions prior to starting bisphosphonate therapy are required except to encourage regular dental care.

The radiographic aspects associated with osteochemonecrosis of the jaws related with the oral use of bisphosphonate (BP) should be identified. The most important radiographic features are: Osteosclerosis limited to the alveolar process, widening of the lamina dura, expansion of the periodontal ligament space, bony sequestra, jaw expansion, radiolucency, and periosteal new bone formation. Osteosclerosis is frequent. It is usually found in the presence of periodontal disease, probably because it is attributed to the fact that BP accumulates preferentially in sites of high bone turnover or remodeling (KHAN et al, 2008).

Radiographic changes are not evident until there is extensive bone involvement. Therefore, panoramic radiographs may not reveal significant changes in the early stages of osteonecrosis as numerous different patologies. When there is extensive bone involvement, regions of mottled bone similar to diffuse osteomyelitis or postirradiation osteoradionecrosis are noted (KUNCHUR & GOSS, 2008). After prolonged exposure to intravenous bisphosphonates, osteosclerosis of the bone, especially osteosclerotic lamina, may be noted radiographically Edwards et al, 2008 (Figures 18-19). The patient in these images received the diagnosis from breast cancer IIIa - AP, and began chemotherapy in July 2003, and gone through bilateral mastectomy. In 2007 it had been diagnosised metastasis in the liver and in the column when the x-ray of the column was made and the monthly use of zometa. The first requested x-ray to the Radiologic Service was in 2008 for the Dental Service of the hospital for diagnostic purpose. It was observed the absence of 7 teeth and horizontal resorption of the alveolar Crests and sclerosis of the horny lamina of teeth 16; 15; 14; 13; 25; 27; 36; 35; 45 and 48. These alterations are indicative of possible bone exposition, suggesting the presence of a sub-clinic degree of osteonecrosis, "the zero" in accordance with AAOMS. In April of 2009, new panoramic x-ray was made and served as base for dentists to consider the interruption of the administration of zometa. After the evaluation of the patient, physicians had substituted zometa by 70mg of alendronate sodium, in an attempt to prevent the occurrence of osteonecrosis in the maxillaries. The jaws are mainly affected because of the teeth, that are embedded in bone. This active bone has fast turnover, principally the horny lamina (alveolar bone also). Most of the times, dentists need to proceed with invasive dental procedures that injures the bone. Hence, the osteonecrosis is viable.

How Dentistry Can Help Fight Osteoporosis 847

Dentist are healthcare professionals and currently graduate with a different vision for prevention. Modern dentistry has been made responsible for important technical and socioeconomic status. New technologies and treatments developed brought great advances in improving the health of the population. Visits to the dentist are much more frequent, and it is routinely visited by patients who have never had cavities. Elucidation of the population with respect to dental care has turned dentists in a professional for oral diagnosis,

This expanded the role of dentistry, including the Family Health Program (FHP) in Brazil that is the most significant advancement of the profession, which leads us to think about how these professionals can contribute to the improvement of health as a whole, enabling

The dentist is who examines the mouth of the population. The teeth are only a portion of the mouth. There are a huge range of other elements that require constant care and observation. Thus, the saying "dentistry beyond the teeth" reinforces, and aims to modernize dentistry career on several fronts, giving a broader professional training and creating conditions so that they can increasingly contribute in improving the health of the population. This modernization involves different aspects ranging from a reformulation, modification of certain areas of research within the faculties, until a fight over a new aspect of insertion of

We feel that there is sufficient evidence that the radiographic images that the dentist routinely uses, particularly the panoramic radiograph can provide important signals related to poor bone quality, and thus we suspect that the involvement of other bone sites such as spine, hip and forearm, and sites that increase the risk of osteoporotic fracture. We therefore endorse the patients with poor bone quality diagnosed by the oral physician to search for other skeletal sites for poor bone quality. Early detection can lead to appropriate treatment and relief of adversities. This is an area where the dentist can greatly contribute in reducing the morbidity and even the mortality, thus enhancing its performance as a health

As osteoporosis is a global epidemic with enormous social costs, with high morbidity and mortality; the Global Forum 2005 indicated osteoporosis as a neglected disease. These are the diseases that, despite having a high incidence in the developing countries, do not receive investments in Research & Development in proportion to their epidemiological importance. Neglected diseases can be defined as a group of diseases associated with poverty. The precarious living conditions and health inequities are major factors responsible for the incidence of neglected diseases. So it is important that the Dentist be prepared for the possibility of evaluating and interpreting the morfometric indices on panoramic radiographs, which could allow the interaction with other health professionals in assessing

American Dental Association, U.S. Food & Drug Administration. (2004). The Selection of

Patients For Dental Radiograph examinations. GUIDELINES FOR PRESCRIBING

monitoring, prevention and oral aesthetics, rather than a curative professional.

**9. Conclusions** 

the dentist to act more widely.

the dental professional in the job market.

professional, understanding the patient as a whole.

and preventing the risk for osteoporosis.

DENTAL RADIOGRAPHS.

**10. References** 

### **8.2 Incidence**

	-

Molar Superior Right Region Molar Superior Left Region

Molar Inferior Right Region Molar Inferior Left Region

0.01% -0.04% (oral) 0.8% -12% (i.v.)

Fig. 18. Periapical radiographs showed widening of the lamina dura in all regions in the mouth

Fig. 19. Panoramic radiographs showed widening of the lamina dura in all regions in the mouth in four differents periods.

### **9. Conclusions**

846 Osteoporosis

0.01% -0.04% (oral) 0.8% -12% (i.v.)

Molar Superior Right Region Molar Superior Left Region

Molar Inferior Right Region Molar Inferior Left Region

04/2008 10/2008

04/2009 11/2010

Fig. 19. Panoramic radiographs showed widening of the lamina dura in all regions in the

Fig. 18. Periapical radiographs showed widening of the lamina dura in all regions in the

0.8% -1.6% (industry-sponsored) 8% -12% (independent)

**8.2 Incidence** 

AAOMS

mouth

mouth in four differents periods.

Dentist are healthcare professionals and currently graduate with a different vision for prevention. Modern dentistry has been made responsible for important technical and socioeconomic status. New technologies and treatments developed brought great advances in improving the health of the population. Visits to the dentist are much more frequent, and it is routinely visited by patients who have never had cavities. Elucidation of the population with respect to dental care has turned dentists in a professional for oral diagnosis, monitoring, prevention and oral aesthetics, rather than a curative professional.

This expanded the role of dentistry, including the Family Health Program (FHP) in Brazil that is the most significant advancement of the profession, which leads us to think about how these professionals can contribute to the improvement of health as a whole, enabling the dentist to act more widely.

The dentist is who examines the mouth of the population. The teeth are only a portion of the mouth. There are a huge range of other elements that require constant care and observation. Thus, the saying "dentistry beyond the teeth" reinforces, and aims to modernize dentistry career on several fronts, giving a broader professional training and creating conditions so that they can increasingly contribute in improving the health of the population. This modernization involves different aspects ranging from a reformulation, modification of certain areas of research within the faculties, until a fight over a new aspect of insertion of the dental professional in the job market.

We feel that there is sufficient evidence that the radiographic images that the dentist routinely uses, particularly the panoramic radiograph can provide important signals related to poor bone quality, and thus we suspect that the involvement of other bone sites such as spine, hip and forearm, and sites that increase the risk of osteoporotic fracture. We therefore endorse the patients with poor bone quality diagnosed by the oral physician to search for other skeletal sites for poor bone quality. Early detection can lead to appropriate treatment and relief of adversities. This is an area where the dentist can greatly contribute in reducing the morbidity and even the mortality, thus enhancing its performance as a health professional, understanding the patient as a whole.

As osteoporosis is a global epidemic with enormous social costs, with high morbidity and mortality; the Global Forum 2005 indicated osteoporosis as a neglected disease. These are the diseases that, despite having a high incidence in the developing countries, do not receive investments in Research & Development in proportion to their epidemiological importance. Neglected diseases can be defined as a group of diseases associated with poverty. The precarious living conditions and health inequities are major factors responsible for the incidence of neglected diseases. So it is important that the Dentist be prepared for the possibility of evaluating and interpreting the morfometric indices on panoramic radiographs, which could allow the interaction with other health professionals in assessing and preventing the risk for osteoporosis.

### **10. References**

American Dental Association, U.S. Food & Drug Administration. (2004). The Selection of Patients For Dental Radiograph examinations. GUIDELINES FOR PRESCRIBING DENTAL RADIOGRAPHS.

How Dentistry Can Help Fight Osteoporosis 849

Jagelavičienė, E., & Kubilius, R. (2006). The relationship between general osteoporosis of the organism and periodontal diseases. *Medicina* (Kaunas), Vol.42, Nº(8), pp. 613-618.

Khan, A.A., Sándor, G.K., & Dore, E. (2008). Canadian consensus practice guidelines for

Khojastehpour, L., Shahidi, S.H., Barghan, S., & Aflaki, E.L. (2009). Efficacy of Panoramic

Khosla, S., Burr, D., & Cauley, J. (2007). Bisphosphonate-associated osteonecrosis of the jaw:

Klein, C.G. (2005). *Evaluation of the bone architecture to trabecular by means of digital image* 

Klemetti, E., Kolmakov, S., & Kroger, H. (1994). Pantomography in assessment of the

Knezović-Zlatarić, D., & Čelebić, A. (2005). Comparison of mandibular bone density and

Kowalski, S.C., Sjenzeld, V.L., & Ferraz, M.B. (2001). Utilização de recursos em osteoporose.

Kunchur, R., & Goss, A.N. (2008). The oral health status of patients on oral bisphosphonates

Ledgerton, D., Horner, K., & Devlin, H. (1997). Panoramic mandibular index as a

Ledgerton, D., Horner, K., & Devlin, H. (1999). Radiomorphometric índices of the mandible in a British female population. Dentomaxillofac Radiol., Vol.28, pp.173–81. Lee, K., Taguchi, A., Ishii, K., Suei, Y., Fujita, M., Nakamoto, T., Ohtsuka, M., Sanada, M.,

Lundstrom, A., Jendlo, J., Stenstrom, B., Toss, G., & Ravald, N. (2001). Periodontal conditions in 70-year-old women with osteoporosis. *Swed Dent J*., Vol.25, pp.89-96. Mahl, C.R.W., Licks, R., & Fontanella, V.R.C. (2008). Comparison of morphometric indices

osteoporosis/osteopenia *Radiol Bras*. Mai/Jun, Vol.41, Nº(3), pp.183–187. Marques Neto, J.F., & Lederman, R. (1995). Osteoporose: Brasil ano 2000. 1st ed. São Paulo:

Marx, R.E.. (2003). Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic. J Oral Maxillofac Surg, Vol.61, pp.115-7.

osteoporosis risk group. *Scand J Dent Res*.,Vol.102, pp.68–72.

Nº(4), pp.479-84.

pp.11-15,.

university

pp.95–100.

Limay,.

Johansson, C. et al. (*1992*). *Age and Ageing*, Vol.21, pp.20-26,.

in: J Rheumatol, Vol.35, pp.2084.

Bone Miner Res, Vol.22, pp.1479-91.

*Oral Radiol*., Vol.21, Nº(2), pp51 - 55 .

Aug, Vol.100, Nº(2), pp.:226-31 .

*Ver Ass Méd Brasil*., Vol.47, Nº(4), pp. 352-7.

for osteoporosis. *Aust Dent J*, Vol.53, pp354-7.

alveolar bone loss of the mandible for identifying postmenopausal women with femoral osteoporosis. *Dentomaxillofac Radiol* (England), Jan, Vol.36, Nº(1), p28-33,

bisphosphonate associated osteonecrosis of the jaw. *J Rheumatol*,35:1391-7. Erratum

Mandibular Index in Diagnosing Osteoporosis in Women. *J. Dent.,*Vol. 6, No (1200),

report of a task force of the American Society for Bone and Mineral Research. J

*processing in panoramic x-rays.* Master thesis. Paraná Federal Technological

radiomorphometric índices in wearers of complete or removable partial dentures.

radiomorphometric tool an assessment of precision. *Dentomaxillofac Radiol*., Vol.26,

Tsuda, M., Ohama, K., Tanimoto, K., & White, S.C. (2005). Visual assessment of the mandibular cortex on panoramic radiographs to identify postmenopausal women with low bone mineral densities. *Oral Surg Oral Med Oral Pathol Oral Radiol Endod*

obtained from dental panoramic radiography for identifying individuals with


Anderson, J. J. (1999). Plant- based diets and bone health: nutritional implications. *Am J Clin* 

Andrade, K.M., Mazzetto, M.O., & Watanabe, P.C.A. (2009). A precisão do Índice

Araújo, D.V., Oliveira, J.H.A., & Bracco, O.L. (2006). Custo da fratura osteoporótica de fêmur

Benson, B.W., Prihoda, T.J., & Glass, B,J. (1991). Variations in adult cortical bone mass as

Bras, J., van Ooij, C.P., Abraham-Inpijn, K., Kusen, G.J., & Wilmink, J.M. (1982).

Cosman, R., & Lindsay, R. (2004). Therapeutic potential of parathyroid hormone. (2004).

Watanabe, P.C.A, Dias, F. C. , Issa, J. P. M., Monteiro, S. A. C., Paula, F. J. A., & Tiossi, R.

Dutra, V., Yang, J., Devlin, H., & Susin, C. (2005). Radiomorphometric índices and their

Edwards, B.J., Hellstein, J.W., Jacobsen, P.L., Kaltman, S., Mariotti, A., & Migliorati, C.A.,

Elsubeihi, E.S., & Heersche, J.N. (2009). Comparison of the effect of ovariectomy on bone

FIOCRUZ/WHO. A Saúde do Brasileiro. 2008 FUNDAÇÃO INSTITUTO BRASILEIRO DE

Farman, A.G., Nortjé, C.J., & Wood, R.E. (1993). *Oral and maxillofadal diagnostic imaging*. St

Global Forum. (2005). Poverty, equity e health research: *A Report on Forum 9*, Mumbai.

Homer, K., Devlin, H., & Harvey, L. (2002). Detecting patients with low skeletal bone mass. *J* 

Ishii, K., Taguchi, A., Nakamoto, T., Ohtsuka, M., Sutthiprapaporn, P., Tsuda, M., Kodama,

I., Kudo, Y., Sumida, H., Suei, Y., & Tanimoto, K. (2007). Diagnostic efficacy of

Bauer, D.C. et al. (*1993*). *Annals of Internal Medicine*, Vol.118, pp.657-665.

Panorâmico Mandibular na detecção de pacientes com osteopenia e osteoporose.

no sistema suplementar de saúde brasileiro. *Arq Bras Endocrinol Metab*., Vol. 49, No.

measured by a panoramic mandibular index. *Oral Surg Oral Med Oral Pathol*.,

Interpretation of the mandibular angular cortex: a diagnostic tool in metabolic bone loss. Part I. Normal state and postmenopausal osteoporosis. *Oral Surg*., Vol.53,

(2010). Elongated styloid process and atheroma in panoramic radiography and its relationship with systemic osteoporosis and osteopenia. *Osteoporosis International*,

relation to gender, age, and dental status. *Oral Surg Oral Med Oral Pathol Oral Radiol* 

(2008). American Dental Association Council on Scientific Affairs Expert Panel on Bisphosphonate-Associated Osteonecrosis of the Jaw. Updated recommendations for managing the care of patients receiving oral bisphosphonate therapy*. J Am Dent* 

mass in dentate and edentulous mandibles of adult rats. *Eur J Prosthodont Restor* 

GEOGRAFIA E ESTATÍSTICA - IBGE. Anuário Estatístico do Brasil, 1995. Rio de

*Nutr*, Vol. Vol.70, pp.539-542.

*Anais da 31ª JORP*, FORP/USP.

*Curr Osteoporos Rep*., Vol.2, pp.5-11.

May; Vol.21, Nº(5), pp.831-6 p. 177-181.

*Endod*, Vol. 99, Nº(4), pp.479-84 Apr.

*Assoc*, Vol .139, Nº(12), pp.1674-1677.

*Dent* (England), Mar, Vol.17, Nº(1), pp.9-21.

Hallstrom, H. et al, (2006). *Osteoporosis International,* Online Edition, May*.*  Heaney, R.P. (2002). Food and Chemical Toxicology, Vol.40, pp. 1263-1270.

6, pp. 897-901.

Vol.71, pp.617–21.

pp.541–545.

Janeiro, 1996.

Geneva, p.63,.

Louis: Mosby-Year Book.

*Dent.*, Vol.30, pp.171-175.

alveolar bone loss of the mandible for identifying postmenopausal women with femoral osteoporosis. *Dentomaxillofac Radiol* (England), Jan, Vol.36, Nº(1), p28-33, Nº(4), pp.479-84.


How Dentistry Can Help Fight Osteoporosis 851

Taguchi, A., Tanimoto, K., Suei, Y., Otani, K. & Wada, T. (1995). Oral signs as indicators of

Von Muhlen, D., Visby, L., Barret-Connor, E., & Bettencourt, R. (1999). Evaluation of the

Vondracek, S.F., Hansen, L.B., McDermott, M.T. (2009). Osteoporosis risk in premenopausal

Wardlaw, G M. (1993). Putting osteoporosis in perspective. *J Am Diet Assoc*, Vol.93, pp1000-

Watanabe, P.C.A., Arita, E.S., Monteiro, S.A.C., Oliveira, T.M., & Taguchi A. (2004). The

panoramic radiographic. *Osteoporos Int.*, Vol.15(Suppl 1), S.67, pp255MO. Watanabe, P.C.A., Bóscolo, F.N., Nishioka, R.S., Shitomi, C.I.H., & Menezes, W.M.M. (2008).

radiografias periapicais. *Rev. ABRO*, Vol. 09, Nº 1 JANEIRO/JULHO. Watanabe P.C.A., Faria, L.M.L., Issa J.PM., & MONTEIRO, S.A.C. (2009). Morphodigital

Dentomaxillofac Radiol (England), Dec, Vol.37, Nº(8), p433-7.

the Rancho Bernardo Study. *Osteoporos Int*, Vol.10, pp.79-84.

*Oral Radiology and Endodontics*, Vol.80, pp.612–616.

women. *Pharmacotherapy,* Vol29, Nº(3). pp305-17.

Título de Livre-docente da USP, Ribeirão Preto,.

*J. Prosthet. Dent.,* St.Louis, Vol.32, Nº(1), p.7-12.

National osteoporosis Foundation.

Vol.16, Nº(3), pp.339-46.

1006.

206.

1-21.

Nº.4, p. 379-382,.

elevated biochemical markers of bone turnover by panoramic radiographs.

possible osteoporosis in elderly women. *Oral Surgery, Oral Medicine, Oral Pathology,* 

simple calculated osteoporosis risk estimation (SCORE) in older caucasian women:

relationship among three indicators of bone quality in the osteoporosis research on

Avaliação da densidade mineral óssea na maxila e mandíbula utilizando-se

evaluation of the trabecular bone pattern in the mandible using digitized panoramic an periapical radiographs. *Minerva Stomatologica*, Vol. 58, p. 73-80. Watanabe P.C.A., Monteiro S.A.C., Lacerda, S.A., Taguchi A., & Arita, E.S. (2004).

Interrelation of two quality indicators for osteoporosis research in panoramic X-Rays of Brazilian Women. *Rev Int Estomatol*. Curitiba, Vol.1, Nº(3), pp.200-

de estudo na odontologia. RPG. *Revista de Pós-Graduação* (USP), São Paulo, Vol. 9,

*Osteoporose em Radiografias Panorâmicas*" – Tese de apresentada para a obtenção do

a systematic review of the literature. *Osteoporosis International,* Vol.20, Nº1, pp.

*predictors of femur bone mineral density.* International Osteoporosis Foundation,

panoramic predictors of femur bone mineral density. *Osteoporos Int.,* Londres,

panoramic radiographs for evaluation and classification of mandibular resorption.

Watanabe, P. C. A. , Silva, A. B. M. , Pardini, L. .C., & Arita E.S. (2002). Osteoporose: um foco

Watanabe, P.C.A. (2003). – "*Relação de Três Indicadores de Qualidade Óssea na Pesquisa da* 

Waugh, E.J. et al*,* (2009). Risk factors for low bone mass in healthy 40-60 year old women:

White, S.C., Taguchi A., Kao D., Wu, S., Susan K., & Yoon D. (2004). *Clinical and panoramic* 

White, S.C., Taguchi, A., Kao, D., Wu, S., Service, S.K., & Yoon, D. (2005). Clinical and

Wical, K.E., & Swoope, C.C. (1974). Studies of residual ridge resorption. Part I. Use of

Woo S.B., Hellstein J.W., & Kalmer J.R. (2006). Narrative review: bisphosphonates and

osteonecrosis of the jaws. *Ann Intern Med*, Vol. 144. Pp.753-61.


May, H., Murphy, S., & Khaw, K. T. (1994). Cigarette smoking and bone mineral density in

Nakamoto, T., Taguchi, A., Ohtsuka, M., Suei, Y., Fujiya, M., & Tanimoto, K. (2003). Dental

American Dental Association Council on Scientific Affairs. (2008). Osteonecrosis of the Jaw.

Otogoto, J, & Ota, N. (2003). Correlation between periodontal disease and osteoporosis

Pazianas, M., Miller, P., Blumentals, W.A., Bernal, M., & Kothawala, P. (2007). A review of

Persson, R.E., Hollender, L.G., Powell, L.V., MacEntee, M.I., Wyatt, C.C., & Kiyak, H.A., et

Pinto Neto A.M., et al. (2002). Consenso Brasileiro em Densitometria. *Rev. Bras. Reumatol*.,

Rousseau, M. E. (). *Dietary prevention of osteoporosis*. Lippincotts Prim Care Pract, 1997, 1:307-

Secretaria da Saúde. Hospital do Servidor Público Estadual. (1995). Osteoporosis: *Basic* 

Secretaria de Vigilância Sanitária, Ministério da Saúde Brasil. Portaria 453 - (1998).

Stallings, V. A. (1997). Calcium and bone health in children: a review. Am J Ther, Vol.4,

Szwarcwald, C.L., & Viacava, F. (2005). *Pesquisa Mundial de Saúde (PMS).* Cad. Saúde

Taguchi A., Suei Y., Sanada M., Ohtsuka M., Nakamoto T., & Sumida H.. (2004). Validation

Taguchi, A., Ohtsuka, M., Nakamoto, T., Suei, Y., Kudo, Y., Tanimoto, K., & Bollen, A.M..

Women with Spinal *Osteoporosis AJR*., Vol.183, pp1755-1760.

*diagnosis and therapeutic elements for a National Consensus Proposal.* São Paulo (São

"*Diretrizes de proteção radiológica e radiodiagnóstico médico e odontológico*" Brasília

of Dental Panoramic Radiography Measures for Identifying postmenopausal

(2008). Detection of post-menopausal women with low bone mineral density and

Report of the 10th European Congress of Dentomaxillofacial Radiology, may-june, 2006. Rizzoli, R., & Bonjour, J. P. (1999). *Malnutrition and osteoporosis.* Z. Gerontol Geriatr, Vol.32,

panoramic radiograph as a tool to detect postmenopausal women with low bone mineral density: untrained general dental practitioners diagnostic performance.

Updated recommendations for managing the care of patients receiving oral bisphosphonate therapy: an advisory statement from the. *J Am Dent Assoc*, Vol.139,

using panoramic radiographic parameters for diagnosed osteoporosis in dental

the literature on osteonecrosis of the jaw in patients with osteoporosis treated with oral bisphosphonates: prevalence, risk factors, and clinical characteristics. *Clin* 

al. (2002). Assessment of periodontal conditions and systemic disease in older subjects. I. Focus on osteoporosis. *J Clin Pedodontol*., Vol.29, Nº(9), pp.796-802. Phal, P.M., Myall, R.W., Assael, L.A., & ,.Weissman, J.L. (2007). Imaging findings of

bisphosphonateassociated ostenecrosis of the jaws. *AJNR Am J Neuroradiol*.Vol.28,

older men. *Q J M*, Vol.87, pp.625-630.

*Osteoporos Int.*, Vol.14, pp.659-664.

clinic. *Clin Calcium.* May, Vol.13, Nº(5), pp.582-6.

pp.1674-7.

*Ther*,Vol.29, pp.1548-58.

Nº(6), pp.1139-1145.

pp.31-37.

(Brasil).

pp.259-273.

319.

Vol.42, Nº(6), pp.343-354, nov/dez.

Paulo). Med J., Vol.113, Suppl, pp.1-64.

Pública, Rio de Janeiro, Vol.21 Sup:S4-S5.

elevated biochemical markers of bone turnover by panoramic radiographs. Dentomaxillofac Radiol (England), Dec, Vol.37, Nº(8), p433-7.


**41** 

*Spain*

**Effect of Bisphosphonates on** 

Maria A. Günther Sillero2 and Antonio Sillero2 *1Centro de Investigaciones Biológicas, CSIC, Madrid 2Departamento de Bioquímica, Instituto de Investigaciones* 

**Root Growth and on Chlorophyll** 

**Formation in** *Arabidopsis thaliana* **Seedlings** 

Bisphosphonates (BPs) are analogues of pyrophosphate in which the oxygen bridge between the two phosphates is replaced by a methylene group (–CH2–). Substitution of one or both hydrogen atoms of this group by radicals generates a variety of bisphosphonates; usually they are grouped into two types, namely non-N-BP and N-BP, depending on the absence or presence of a nitrogen atom in its molecule, respectively (Russell, 2011). Examples of BPs used in this report are: etidronate and clodronate (non-N-BPs), and pamidronate and

Bisphosphonates are the leading drug class for the treatment of osteoporosis; as an indication of their usefulness and the spread out of the disease the combined sales of drugs to treat osteoporosis reached \$ 6.2 billions in 2004. Besides their application in humans, bisphosphonates are also used for other purposes, mainly as herbicides in plants (Cromartie et al., 1999; Oberhauser et al., 1998), as chemotherapeutic agents (Artz et al., 2008; Docampo & Moreno, 2001; Leon et al., 2006; Moreno & Li, 2008) and in basic research (Rogers et al.,

The usefulness of bisphosphonates is due to their mechanism of action when they are supplied to the living organism. In fact, they have the ability of interfering with metabolic pathways located at the crossroads of essential processes for life. Some important examples

a) Bisphosphonates may act as analogs of PP in many of the reactions catalyzed by ligases and some transferases (reaction 1) in which derivatives of the type NRpp-CH2-p are synthesized in the reverse reaction (reaction 2) (Günther Sillero et al., 2008; Günther Sillero

X + NRPPP ----> X-NRP + PP (1)

X-NRP + p-CH2-p ---> NRpp-CH2-p (2)

X-NRP + mev-pp --->mevpppN (3)

**1. Introduction** 

alendronate (N-BPs).

2010; Russell, 2011).

of this crucial role are as follows:

et al., 2006; Rogers et al., 1996; Russell, 2011).

Ana I. Manzano1, F. Javier Medina1, Francisco J. Pérez-Zuñiga2,

*Biomédicas Alberto Sols, UAM/CSIC, Facultad de Medicina, Madrid* 


## **Effect of Bisphosphonates on Root Growth and on Chlorophyll Formation in** *Arabidopsis thaliana* **Seedlings**

Ana I. Manzano1, F. Javier Medina1, Francisco J. Pérez-Zuñiga2, Maria A. Günther Sillero2 and Antonio Sillero2 *1Centro de Investigaciones Biológicas, CSIC, Madrid 2Departamento de Bioquímica, Instituto de Investigaciones Biomédicas Alberto Sols, UAM/CSIC, Facultad de Medicina, Madrid Spain*

### **1. Introduction**

852 Osteoporosis

von Wowern N. (). Bone mass of mandibles. In vitro and in vivo analyses (Thesis). Danish

Yarom N., Yahalom R., Shoshani Y., Hamed W., Regev E., & Elad S. (2007).Osteonecrosis of

Zanette E., Stringari F.F., Machado F., Marroni B.J., Ng D.P.K., & Canani L.H. (2003).

Sítio Ósseo. *Arq Bras Endocrinol Metab*, Vol.47. Nº(1). pp30-36.

the jaw induced by orally administrated bisphosphonates: incidence, clinical features, predisposing factors and treatment outcome. *Osteoporos Int*, Vol.18.

Avaliação do Diagnóstico Densitométrico de Osteoporose/Osteopenia Conforme o

Med Bull. Vol.33. pp23–44.

pp1363-70.

Bisphosphonates (BPs) are analogues of pyrophosphate in which the oxygen bridge between the two phosphates is replaced by a methylene group (–CH2–). Substitution of one or both hydrogen atoms of this group by radicals generates a variety of bisphosphonates; usually they are grouped into two types, namely non-N-BP and N-BP, depending on the absence or presence of a nitrogen atom in its molecule, respectively (Russell, 2011). Examples of BPs used in this report are: etidronate and clodronate (non-N-BPs), and pamidronate and alendronate (N-BPs).

Bisphosphonates are the leading drug class for the treatment of osteoporosis; as an indication of their usefulness and the spread out of the disease the combined sales of drugs to treat osteoporosis reached \$ 6.2 billions in 2004. Besides their application in humans, bisphosphonates are also used for other purposes, mainly as herbicides in plants (Cromartie et al., 1999; Oberhauser et al., 1998), as chemotherapeutic agents (Artz et al., 2008; Docampo & Moreno, 2001; Leon et al., 2006; Moreno & Li, 2008) and in basic research (Rogers et al., 2010; Russell, 2011).

The usefulness of bisphosphonates is due to their mechanism of action when they are supplied to the living organism. In fact, they have the ability of interfering with metabolic pathways located at the crossroads of essential processes for life. Some important examples of this crucial role are as follows:

a) Bisphosphonates may act as analogs of PP in many of the reactions catalyzed by ligases and some transferases (reaction 1) in which derivatives of the type NRpp-CH2-p are synthesized in the reverse reaction (reaction 2) (Günther Sillero et al., 2008; Günther Sillero et al., 2006; Rogers et al., 1996; Russell, 2011).

X + NRPPP ----> X-NRP + PP (1)

X-NRP + p-CH2-p ---> NRpp-CH2-p (2)

X-NRP + mev-pp --->mevpppN (3)

Effect of Bisphosphonates on Root Growth

known metabolic pathway (Günther Sillero et al., 2009) (Fig. 1).

and on Chlorophyll Formation in *Arabidopsis thaliana* Seedling 855

this biosynthetic pathway. We have focused our attention on this pathway as a good target to examine the effect of BP given the two metabolic peculiarities present only in the mevalonate cycle: occurrence of both three consecutive enzymes requiring ATP as a cosubstrate and the major pool of compounds containing a terminal pyrophosphate in any

The metabolic pathway from acetyl-CoA to mevalonate-5-phospahte, and the implicated enzymes (1-5) are

hydroxymethylglutaryl-CoA synthase (EC 4.1.3.5); 3, 3-hydromethylglutaryl-CoA reductase, (EC 1.1.1.34); 4, mevalonate kinase (EC 2.7.1.36); 5, phosphomevalonate kinase (EC 2.7.4.2). Note that the three consecutive steps catalyzed by enzymes (4), (5) and (A), consume three moles of ATP. The inset in the figure comprises the compounds of the mevalonate pathway and the enzymes (A-F) involved: A, diphosphomevalonate

dimethylallyl*trans*transferase, (EC 2.5.1.1); D, geranyl*trans*transferase (farnesyl-diphosphate synthase), (EC 2.5.1.10); E, farnesyl*trans*transferase, (EC 2.5.1.29); F, farnesyl-diphosphate farnesyltransferase, (EC 2.5.1.21). To emphasize the occurrence of pyrophosphate in all the compounds of the cycle, this moiety has been marked out (). Some of the great variety of compounds synthesized from those metabolites, and essential for plant functions, is annotated. The mevalonate independent pathway is not represented in the Figure.

Fig. 1. Components of the mevalonate pathway, its precursors and some metabolic

indicated in the figure. The enzymes are: 1, acetyl-CoA acetyltransferase (EC 2.3.1.9); 2,

decarboxylase, (EC 4.1.1.33); B, isopentenyl-diphosphate D-isomerase, (EC 5.3.3.2); C,

connections in plants.

Where X is a potential substrate of the reaction; NRP and NRPPP are nucleosides mono- and triphosphates, respectively; NRpp-CH2-p is a nucleoside 5'( methylene triphosphate) and mev-pp a compound of the mevalonate pathway with a terminal pyrophosphate (see below). As the more abundant nucleoside triphosphate is ATP, the more common derivative from bisphosphonate, would be ARpp-CH2-p; given that ATP is a co-substrate of at least 150 enzymes with transferase activity and more than 90 with ligase activity (see Enzyme Nomenclature), bisphosphonates may indirectly interfere with multiple cellular processes through the formation of bisphosphonate derivatives of ATP or of any nucleoside triphosphate. As in these reactions BPs act as analogs of PP it would be expected that the non-N-BP (the smallest type of BP) were the preferred substrates for these reactions.

b) BPs may be inhibitors of enzymes having substrates with a terminal PP (R-PP) (see below and Günther Sillero et al., 2009). Although the inhibition of these enzymes could take place at any step of the pathway, specific inhibitions have been reported on isolated enzymes from bacteria, yeast or plants: pyrophosphatase (Baykov et al., 1993; Cromartie et al., 1999; Drozdowicz et al., 2003; Gordon-Weeks et al., 1999; Kim et al.,1994; Kuo et al., 2005; Rodrígues et al., 2000; Szabo & Oldfield, 2001; Zhen et al., 1994 ); geranyl diphospho synthetase (Burke et al., 2004; Oberhauser et al., 1998); isopentenyl pyrophosphate synthase (Cromartie et al., 1999) and P5C reductase (Forlani et al., 2008).

c) The synthesis of isopentenyl triphosphoadenosine (iso-pppA) was previously described (Monkkonen et al., 2006). Following this finding, the synthesis of derivatives of a variety of compounds of the mevalonate pathway capped with an adenosine moiety catalyzed by several ligases, was later reported (mev-pppA and mev-ppppA) (Günther Sillero et al., 2009). Increase in the concentration of metabolites upstream the inhibited step could stimulate synthesis of the corresponding mevalonate derivative (reaction 3) (Günther Sillero et al., 2009; Rogers et al., 2010).

Related to the use of bisphosphonates in humans for the treatment of osteoporosis, Paget's disease and bone tumour metastasis it can be stressed that upon their oral or intravenous administration BP are partially eliminated by kidney and partially fixed in bones, with very little amount in the systemic circulation (Cremers et al., 2005). Being otherwise bisphosphonates could have general toxic effects. After their capture by osteoclasts from the bone they exert noxious effects on these cells by some of the above-mentioned mechanisms. The basic approach, necessary to explore the effect of BPs, is not sufficient to envisage their clinical effects. These studies, although mandatory, are cumbersome, costly and requiring a cohort of patients as each patient has distinct characteristics and each bisphosphonate may present special pharmacokinetic properties (Cremers et al., 2005).

Based on the above we thought of interest to approach the effect of bisphosphonates in plants: plants are a whole living entity, of cheaper handling, where BPs present a quite different pharmacokinetics; they are easily assimilated by the roots, are not fixed in a structure similar to bones and probably circulate through the vegetal tissues more easily than in animal tissues. As an initial approach to the problem, we have used germinating seeds and early seedlings of the model plant species *Arabidopsis thaliana* to investigate the alteration of a few phenotypic characters, considered of high relevance for key functions of the plant, namely the seed germination rate, the early development of primary and secondary roots (essential for establishing the plant developmental pattern) and the presence and content of chlorophyll in the first leaves. This later character, used as an indicator of the ability of plants for performing the fundamental process of photosynthesis, also indicates the alteration of the mevalonate pathway, since chlorophyll is a product of

Where X is a potential substrate of the reaction; NRP and NRPPP are nucleosides mono- and triphosphates, respectively; NRpp-CH2-p is a nucleoside 5'( methylene triphosphate) and mev-pp a compound of the mevalonate pathway with a terminal pyrophosphate (see below). As the more abundant nucleoside triphosphate is ATP, the more common derivative from bisphosphonate, would be ARpp-CH2-p; given that ATP is a co-substrate of at least 150 enzymes with transferase activity and more than 90 with ligase activity (see Enzyme Nomenclature), bisphosphonates may indirectly interfere with multiple cellular processes through the formation of bisphosphonate derivatives of ATP or of any nucleoside triphosphate. As in these reactions BPs act as analogs of PP it would be expected that the

non-N-BP (the smallest type of BP) were the preferred substrates for these reactions.

(Cromartie et al., 1999) and P5C reductase (Forlani et al., 2008).

present special pharmacokinetic properties (Cremers et al., 2005).

et al., 2009; Rogers et al., 2010).

b) BPs may be inhibitors of enzymes having substrates with a terminal PP (R-PP) (see below and Günther Sillero et al., 2009). Although the inhibition of these enzymes could take place at any step of the pathway, specific inhibitions have been reported on isolated enzymes from bacteria, yeast or plants: pyrophosphatase (Baykov et al., 1993; Cromartie et al., 1999; Drozdowicz et al., 2003; Gordon-Weeks et al., 1999; Kim et al.,1994; Kuo et al., 2005; Rodrígues et al., 2000; Szabo & Oldfield, 2001; Zhen et al., 1994 ); geranyl diphospho synthetase (Burke et al., 2004; Oberhauser et al., 1998); isopentenyl pyrophosphate synthase

c) The synthesis of isopentenyl triphosphoadenosine (iso-pppA) was previously described (Monkkonen et al., 2006). Following this finding, the synthesis of derivatives of a variety of compounds of the mevalonate pathway capped with an adenosine moiety catalyzed by several ligases, was later reported (mev-pppA and mev-ppppA) (Günther Sillero et al., 2009). Increase in the concentration of metabolites upstream the inhibited step could stimulate synthesis of the corresponding mevalonate derivative (reaction 3) (Günther Sillero

Related to the use of bisphosphonates in humans for the treatment of osteoporosis, Paget's disease and bone tumour metastasis it can be stressed that upon their oral or intravenous administration BP are partially eliminated by kidney and partially fixed in bones, with very little amount in the systemic circulation (Cremers et al., 2005). Being otherwise bisphosphonates could have general toxic effects. After their capture by osteoclasts from the bone they exert noxious effects on these cells by some of the above-mentioned mechanisms. The basic approach, necessary to explore the effect of BPs, is not sufficient to envisage their clinical effects. These studies, although mandatory, are cumbersome, costly and requiring a cohort of patients as each patient has distinct characteristics and each bisphosphonate may

Based on the above we thought of interest to approach the effect of bisphosphonates in plants: plants are a whole living entity, of cheaper handling, where BPs present a quite different pharmacokinetics; they are easily assimilated by the roots, are not fixed in a structure similar to bones and probably circulate through the vegetal tissues more easily than in animal tissues. As an initial approach to the problem, we have used germinating seeds and early seedlings of the model plant species *Arabidopsis thaliana* to investigate the alteration of a few phenotypic characters, considered of high relevance for key functions of the plant, namely the seed germination rate, the early development of primary and secondary roots (essential for establishing the plant developmental pattern) and the presence and content of chlorophyll in the first leaves. This later character, used as an indicator of the ability of plants for performing the fundamental process of photosynthesis, also indicates the alteration of the mevalonate pathway, since chlorophyll is a product of this biosynthetic pathway. We have focused our attention on this pathway as a good target to examine the effect of BP given the two metabolic peculiarities present only in the mevalonate cycle: occurrence of both three consecutive enzymes requiring ATP as a cosubstrate and the major pool of compounds containing a terminal pyrophosphate in any known metabolic pathway (Günther Sillero et al., 2009) (Fig. 1).

The metabolic pathway from acetyl-CoA to mevalonate-5-phospahte, and the implicated enzymes (1-5) are indicated in the figure. The enzymes are: 1, acetyl-CoA acetyltransferase (EC 2.3.1.9); 2, hydroxymethylglutaryl-CoA synthase (EC 4.1.3.5); 3, 3-hydromethylglutaryl-CoA reductase, (EC 1.1.1.34); 4, mevalonate kinase (EC 2.7.1.36); 5, phosphomevalonate kinase (EC 2.7.4.2). Note that the three consecutive steps catalyzed by enzymes (4), (5) and (A), consume three moles of ATP. The inset in the figure comprises the compounds of the mevalonate pathway and the enzymes (A-F) involved: A, diphosphomevalonate decarboxylase, (EC 4.1.1.33); B, isopentenyl-diphosphate D-isomerase, (EC 5.3.3.2); C, dimethylallyl*trans*transferase, (EC 2.5.1.1); D, geranyl*trans*transferase (farnesyl-diphosphate synthase), (EC 2.5.1.10); E, farnesyl*trans*transferase, (EC 2.5.1.29); F, farnesyl-diphosphate farnesyltransferase, (EC 2.5.1.21). To emphasize the occurrence of pyrophosphate in all the compounds of the cycle, this moiety has been marked out (). Some of the great variety of compounds synthesized from those metabolites, and essential for plant functions, is annotated. The mevalonate independent pathway is not represented in the Figure.

Fig. 1. Components of the mevalonate pathway, its precursors and some metabolic connections in plants.

Effect of Bisphosphonates on Root Growth

of results.

each bisphosphonate.

and on Chlorophyll Formation in *Arabidopsis thaliana* Seedling 857

The expressed results are referred to the control experiment performed with standard culture medium, without the addition of any other substance. Number of (+) indicates deviation with respect to the control experiment for the particular parameter evaluated. (): Similar to the control experiment. na: Not applicable, because secondary roots were not initiated in the corresponding control samples. #: Reference number of the experiment, attributed for the sake of clarity in the description and discussion

Table 1. Summary of results obtained on *Arabidopsis thaliana* seedlings with the four bisphosphonates analyzed, the three parameters evaluated and the nine conditions used, resulting from the combination of three concentrations and three times of incubation for

The cytosol-located mevalonate pathway (starting with 2 moles of acetyl-CoA and ending in geranygeranyl pyrophosphate (Fig. 1)) is the starting point for the synthesis of a great variety of compounds in bacteria, plants and animals, embracing more than 23,000 currently identified compounds (Wendt & Schulz, 1998). A different additional route for the synthesis of isopentenyl pyrophosphate is localized in plant plastids. This mevalonate-independent pathway starts with the condensation of pyruvate and D-glyceraldehyde 3-phosphate and, through the action of seven additional enzyme activities, the synthesis of isopentenyl pyrophosphate is obtained (see (Hunter, 2007) for a review).

This short work is an initial report of this study, showing that the plant growth and development, as well as the photosynthetic efficiency, are indeed affected by the presence of BPs in the culture medium, in some cases in such an extent that the actual viability of the plant is compromised. Apart from its basic interest, this finding could be of some help to understand the role of bisphosphonates in animal tissues.

### **2. Materials and methods**

The following bisphosphonates were used in this study: alendronate, pC(OH)((CH2)3-NH2)p (Ref. A-4978) and etidronate, pC(OH)(CH3)p (Ref. P-5248) were from Sigma; clodronate, pCCl2p (Ref. 233183), and pamidronate, pC(OH)((CH2)2-NH2)p, (Ref. 506600) were from Calbiochem. In addition, sodium pyrophosphate and tripolyphosphate (P3), used in controls, were from Sigma (Refs P9146 and T-5633, respectively).

Seeds of *Arabidopsis thaliana* ecotype Columbia were placed in Petri dishes containing Murashige and Skoog's (MS) medium (Duchefa) in agar supplemented with one of the four bisphosphonates (BPs) indicated, at a concentration of 0.05 mM, 0.1 mM or 0.5 mM. Controls consisted of culture medium alone, or supplemented with pyrophosphate (0.1 mM or 0.5 mM), or tripolyphosphate (0.1 mM or 0.5 mM). Plates were placed vertically and incubated at 22ºC under illumination with a flux of photo synthetically active photons of 150-µE m-2 s-1, with a photoperiod of 16 hours of light and 8 hours of darkness. Samples were photographed at 4, 8 and 11 days of incubation and analyzed for the following parameters: a) rate of germination; b) length of the primary root; c) time of appearance, number and length of secondary roots; and d) colour of leaves.

#### **3. Results and discussion**

The plants were analyzed by the rate of seed germination and, in germinated seedlings, by the occurrence and aspect of three visible phenotypic characters: growth (length) of the primary root, development of secondary roots (time of appearance, position, number and length) and colour of the leaves, indicative of the presence of chlorophyll. The full set of results obtained for every evaluated parameter with the four BPs, at the three concentrations and in the three time points of sampling assayed, is shown in Table 1.

In order to facilitate the presentation of the results, the experimental conditions have been numbered from 1-12 (#column at the right in Table 1). Furthermore, representative results corresponding to *A. thaliana* seedlings grown during 4, 8 and 11 days in the presence of 0.1 mM etidronate; 0.05 mM clodronate; 0.5 mM pamidronate and 0.5 mM alendronate are shown in Fig. 2. Controls grown without any addition to the culture medium (a-c), or in the absence of BPs, but in the presence of pyrophosphate (d-f) or tripolyphosphate (g-i), were also included in Fig. 2.

The cytosol-located mevalonate pathway (starting with 2 moles of acetyl-CoA and ending in geranygeranyl pyrophosphate (Fig. 1)) is the starting point for the synthesis of a great variety of compounds in bacteria, plants and animals, embracing more than 23,000 currently identified compounds (Wendt & Schulz, 1998). A different additional route for the synthesis of isopentenyl pyrophosphate is localized in plant plastids. This mevalonate-independent pathway starts with the condensation of pyruvate and D-glyceraldehyde 3-phosphate and, through the action of seven additional enzyme activities, the synthesis of isopentenyl

This short work is an initial report of this study, showing that the plant growth and development, as well as the photosynthetic efficiency, are indeed affected by the presence of BPs in the culture medium, in some cases in such an extent that the actual viability of the plant is compromised. Apart from its basic interest, this finding could be of some help to

The following bisphosphonates were used in this study: alendronate, pC(OH)((CH2)3-NH2)p (Ref. A-4978) and etidronate, pC(OH)(CH3)p (Ref. P-5248) were from Sigma; clodronate, pCCl2p (Ref. 233183), and pamidronate, pC(OH)((CH2)2-NH2)p, (Ref. 506600) were from Calbiochem. In addition, sodium pyrophosphate and tripolyphosphate (P3), used in

Seeds of *Arabidopsis thaliana* ecotype Columbia were placed in Petri dishes containing Murashige and Skoog's (MS) medium (Duchefa) in agar supplemented with one of the four bisphosphonates (BPs) indicated, at a concentration of 0.05 mM, 0.1 mM or 0.5 mM. Controls consisted of culture medium alone, or supplemented with pyrophosphate (0.1 mM or 0.5 mM), or tripolyphosphate (0.1 mM or 0.5 mM). Plates were placed vertically and incubated at 22ºC under illumination with a flux of photo synthetically active photons of 150-µE m-2 s-1, with a photoperiod of 16 hours of light and 8 hours of darkness. Samples were photographed at 4, 8 and 11 days of incubation and analyzed for the following parameters: a) rate of germination; b) length of the primary root; c) time of appearance, number and

The plants were analyzed by the rate of seed germination and, in germinated seedlings, by the occurrence and aspect of three visible phenotypic characters: growth (length) of the primary root, development of secondary roots (time of appearance, position, number and length) and colour of the leaves, indicative of the presence of chlorophyll. The full set of results obtained for every evaluated parameter with the four BPs, at the three concentrations

In order to facilitate the presentation of the results, the experimental conditions have been numbered from 1-12 (#column at the right in Table 1). Furthermore, representative results corresponding to *A. thaliana* seedlings grown during 4, 8 and 11 days in the presence of 0.1 mM etidronate; 0.05 mM clodronate; 0.5 mM pamidronate and 0.5 mM alendronate are shown in Fig. 2. Controls grown without any addition to the culture medium (a-c), or in the absence of BPs, but in the presence of pyrophosphate (d-f) or tripolyphosphate (g-i), were

pyrophosphate is obtained (see (Hunter, 2007) for a review).

understand the role of bisphosphonates in animal tissues.

controls, were from Sigma (Refs P9146 and T-5633, respectively).

and in the three time points of sampling assayed, is shown in Table 1.

length of secondary roots; and d) colour of leaves.

**2. Materials and methods** 

**3. Results and discussion** 

also included in Fig. 2.


The expressed results are referred to the control experiment performed with standard culture medium, without the addition of any other substance. Number of (+) indicates deviation with respect to the control experiment for the particular parameter evaluated. (): Similar to the control experiment. na: Not applicable, because secondary roots were not initiated in the corresponding control samples. #: Reference number of the experiment, attributed for the sake of clarity in the description and discussion of results.

Table 1. Summary of results obtained on *Arabidopsis thaliana* seedlings with the four bisphosphonates analyzed, the three parameters evaluated and the nine conditions used, resulting from the combination of three concentrations and three times of incubation for each bisphosphonate.

Effect of Bisphosphonates on Root Growth

and on Chlorophyll Formation in *Arabidopsis thaliana* Seedling 859

Fig. 2. Selected images of the results obtained after the germination and growth of *Arabidopsis thaliana* seedlings in the presence of four different bisphosphonates.

pamidronate for 4, 8 and 11 days, respectively; at the 4th day of growth, primary roots are shorter (p) than the controls (a, d, g); the root growth does not progress further (q to r); leaves show a pale yellowish colour in all cases, but this effect is more intense as the culture

In this initial approach, the estimation of the effects of BPs on the characters investigated has been performed in a semi-quantitative way, by recording the differences that can be appreciated in these parameters by a simple visual inspection. The purpose of this type of analysis was, firstly, to determine whether or not BPs are potent inhibitors of essential functions of the plant; secondly, to discriminate differential effects between the analyzed BPs, the concentrations used and the times of development, in order to select targets for a more detailed and deep mechanistic analysis that may provide relevant information on the specific biochemical reactions that become affected during the inhibitory process.

From the full set of data presented in Table 1, and the selected images shown in Fig. 2, the most relevant results of our study are as follows:

a) None of the tested BPs, at any of the concentrations assayed, affected the seed germination rate. b) Clodronate, pamidronate and alendronate affected both the growth of the primary root, resulting in its shortening, and the number and length of secondary roots (Table 1 and Fig. 2, m-o; p-r; s-u). c) Two BPs, belonging to the same group of non-N-BP (etidronate and clodronate) showed effects of quite different intensity on root development, and might have a different degree of action (Fig. 2, j-l; m-o) (Table 1, compare 1-3 and 4-6). d) The effect of BPs is time dependent, and the highest effect was obtained after 11 days in culture, irrespective of the concentration used. In few cases we observed that the maximum effect was already reached after 8 days and then it persisted after longer time in culture (Fig. 2, n-o; t-u) (Table 1, line 4, 0.05 mM; line 7, 0.5 mM; line 10, 0.5 mM). e) The effect of BPs regarding the alteration of primary root growth was concentration dependent. This feature was more clearly observed with N-BPs rather than with non-N-BPs. However, in the particular case of etidronate, concentrations of 0.05 mM seemed to be more effective than 0.1 mM (Table 1, line 1), a striking result of difficult interpretation. f) There are visible differences on the influence of BPs on chlorophyll loss: non-N-BPs either did not affect visibly that parameter, even at 0.5 mM and during 11 days in culture (etidronate; Table 1, line 3), or affected only slightly at the highest concentration (clodronate; Table 1, line 6); on the contrary, N-BPs, such as alendronate (Table 1, line 12) and to a lesser extent pamidronate, greatly affected the colour of the leaves (Table 1, line 9) (Fig. 2, p-r; s-u). In general, conspicuous effect on chlorophyll loss was only detected at the highest BP concentrations. g) Alendronate was the most effective BP acting on *Arabidopsis thaliana*  development, affecting the primary and secondary roots and chlorophyll loss, producing a drastic inhibition of seedling growth, with total loss of chlorophyll, which was observed at a concentration of 0.5 mM, already in the intermediate time point of sampling (Fig. 2, t-u) (Table 1, lines 10, 11 and 12).

Images from a to i correspond to controls, either with the standard MS culture medium alone (a to c), or with the addition of pyrophosphate (d to f), or with the addition of tripolyphosphate (g to i): the analysis of the features of these control samples (a to i) does not reveal significant differences among them. From j to l: culture in the presence of 0.1 mM etidronate for 4, 8 and 11 days, respectively; images j and k are practically unaltered with respect to controls; root length similar to controls, but secondary roots are much less developed (l). From m to o: culture in the presence of 0.05 mM clodronate for 4, 8 and 11 days, respectively; at the 4th day of growth, primary roots are shorter (m) than the controls (a, d and g) and the root growth appears practically arrested in successive samples (n to o); secondary roots are totally absent (o). From p to r: culture in the presence of 0.5 mM

In this initial approach, the estimation of the effects of BPs on the characters investigated has been performed in a semi-quantitative way, by recording the differences that can be appreciated in these parameters by a simple visual inspection. The purpose of this type of analysis was, firstly, to determine whether or not BPs are potent inhibitors of essential functions of the plant; secondly, to discriminate differential effects between the analyzed BPs, the concentrations used and the times of development, in order to select targets for a more detailed and deep mechanistic analysis that may provide relevant information on the

From the full set of data presented in Table 1, and the selected images shown in Fig. 2, the

a) None of the tested BPs, at any of the concentrations assayed, affected the seed germination rate. b) Clodronate, pamidronate and alendronate affected both the growth of the primary root, resulting in its shortening, and the number and length of secondary roots (Table 1 and Fig. 2, m-o; p-r; s-u). c) Two BPs, belonging to the same group of non-N-BP (etidronate and clodronate) showed effects of quite different intensity on root development, and might have a different degree of action (Fig. 2, j-l; m-o) (Table 1, compare 1-3 and 4-6). d) The effect of BPs is time dependent, and the highest effect was obtained after 11 days in culture, irrespective of the concentration used. In few cases we observed that the maximum effect was already reached after 8 days and then it persisted after longer time in culture (Fig. 2, n-o; t-u) (Table 1, line 4, 0.05 mM; line 7, 0.5 mM; line 10, 0.5 mM). e) The effect of BPs regarding the alteration of primary root growth was concentration dependent. This feature was more clearly observed with N-BPs rather than with non-N-BPs. However, in the particular case of etidronate, concentrations of 0.05 mM seemed to be more effective than 0.1 mM (Table 1, line 1), a striking result of difficult interpretation. f) There are visible differences on the influence of BPs on chlorophyll loss: non-N-BPs either did not affect visibly that parameter, even at 0.5 mM and during 11 days in culture (etidronate; Table 1, line 3), or affected only slightly at the highest concentration (clodronate; Table 1, line 6); on the contrary, N-BPs, such as alendronate (Table 1, line 12) and to a lesser extent pamidronate, greatly affected the colour of the leaves (Table 1, line 9) (Fig. 2, p-r; s-u). In general, conspicuous effect on chlorophyll loss was only detected at the highest BP concentrations. g) Alendronate was the most effective BP acting on *Arabidopsis thaliana*  development, affecting the primary and secondary roots and chlorophyll loss, producing a drastic inhibition of seedling growth, with total loss of chlorophyll, which was observed at a concentration of 0.5 mM, already in the intermediate time point of sampling (Fig. 2, t-u)

Images from a to i correspond to controls, either with the standard MS culture medium alone (a to c), or with the addition of pyrophosphate (d to f), or with the addition of tripolyphosphate (g to i): the analysis of the features of these control samples (a to i) does not reveal significant differences among them. From j to l: culture in the presence of 0.1 mM etidronate for 4, 8 and 11 days, respectively; images j and k are practically unaltered with respect to controls; root length similar to controls, but secondary roots are much less developed (l). From m to o: culture in the presence of 0.05 mM clodronate for 4, 8 and 11 days, respectively; at the 4th day of growth, primary roots are shorter (m) than the controls (a, d and g) and the root growth appears practically arrested in successive samples (n to o); secondary roots are totally absent (o). From p to r: culture in the presence of 0.5 mM

specific biochemical reactions that become affected during the inhibitory process.

most relevant results of our study are as follows:

(Table 1, lines 10, 11 and 12).

Fig. 2. Selected images of the results obtained after the germination and growth of *Arabidopsis thaliana* seedlings in the presence of four different bisphosphonates.

pamidronate for 4, 8 and 11 days, respectively; at the 4th day of growth, primary roots are shorter (p) than the controls (a, d, g); the root growth does not progress further (q to r); leaves show a pale yellowish colour in all cases, but this effect is more intense as the culture

Effect of Bisphosphonates on Root Growth

**4. Conclusions** 

bisphosphonates is suggested.

pp. 883-893, ISSN 0305-7364

**5. Acknowledgments** 

technical assistance.

1074-5521

**6. References** 

interconnecting all the biological cycles on earth.

and on Chlorophyll Formation in *Arabidopsis thaliana* Seedling 861

present in the hypothetical Last Universal Common Ancestor (LUCA) (Ranea et al., 2006). All the above points could be also considered as examples of the multiple relationships

Bisphosphonates are widely used in the treatment of osteoporosis and hence their great importance in clinical investigation. Upon their administration they bind to the bone tissues where they exert a noxious effect mainly on the mevalonate pathway of the osteoclasts. Given the universality of this pathway, present in bacteria, plants and animals, it seemed to us of interest to analyze the effect of etidronate, clodronate (non containing nitrogen or non-N-BPs), pamidronate and alendronate (N-BPs) on some visible traits of the model plant *Arabidopsis thaliana*. The seeds were grown in a medium containing three concentrations (0.05 mM, 0.1 mM or 0.5 mM) of each bisphosphonate and the growth of primary and secondary roots and formation of chlorophyll were observed during early development of the seedlings. Each bisphosphonate showed a different pattern of influence on those parameters. In general, the inhibitory effects were, in increasing order: etidronate, pamidronate, clodronate, and alendronate. Specific effects on the evaluated parameters ranged from simple reduction in the number and length of secondary roots caused by 0.1 mM etidronate, only after 11 days of culture, until a drastic inhibition of seedling growth, with total loss of chlorophyll observed with 0.5 mM alendronate already in the intermediate time point of sampling. The utility of the use of plants to analyze the action of

This work was supported by grants from the Spanish National Plan for Research, Development and Innovation (BFU 2008-00666/BMC, BFU 2009-08977 and AYA2009- 07952). We thank Mercedes Carnota, Javier Pérez and Anabel de Diego for very helpful

Aloni, R., Aloni, E., Langhans, M. & Ullrich, C. I. (2006). Role of cytokinin and auxin in

Artz, J. D., Dunford, J. E., Arrowood, M. J., Dong, A., Chruszcz, M., Kavanagh, K. L., Minor,

Baykov, A. A., Dubnova, E. B., Bakuleva, N. P., Evtushenko, O. A., Zhen, R. G. & Rea, P. A.

*Lett,* Vol.327, No.2, (July 1993), pp. 199-202, ISSN 0014-5793

shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism. *Ann Bot,* Vol.97, No.5, (May 2006),

W., Russell, R. G., Ebetino, F. H., Oppermann, U. & Hui, R. (2008). Targeting a uniquely nonspecific prenyl synthase with bisphosphonates to combat cryptosporidiosis. *Chem Biol,* Vol.15, No.12, (December, 2008), pp. 1296-1306, ISSN

(1993). Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzymes. *FEBS* 

in the presence of the drug progresses in time. From s to u: culture in the presence of 0.5 mM alendronate for 4, 8 and 11 days, respectively; the root growth is severely hampered in these conditions; 8 - and 11-day old seedlings show an almost white colour of leaves, indicative of a very serious loss of chlorophyll.

The above results can be interpreted as a consequence of the toxic effects of BPs on the mevalonate pathway of *Arabidopsis thaliana* (Fig. 1). Isoprenoids, the most diverse group of natural products, are synthesized in prokaryotes and eukaryotes, animals and plants by condensation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. They play important and diverse roles in the synthesis of quinones, sterols, prenylation of proteins, photosynthetic pigments, hormones, attractants for pollinators in plants, and others. Certainly, most of the studies on the synthesis of isoprenoids were carried out in animal tissues; however, after the more recent discovery of the synthesis of isoprenoids by the mevalonate independent pathway operating in plants, new approaches on their metabolism and function have been undertaken in other biological model systems (Eisenreich et al., 2004; Lange et al., 2000; Rodriguez-Concepcion & Boronat, 2002; Rohmer, 1999). The plastid mevalonate independent pathway involves the synthesis of 1-deoxy-D.xilulose from pyruvate and glyceraldehydes-3-phosphate. There is a cross talk between the cytosolic and plastidial pathway for the synthesis of isoprenoids in *Arabidopsis thaliana* (Laule et al., 2003). In relation with the postulated inhibitory role of bisphosphonates on the synthesis of isoprenoids, it could be noted that the two last compounds in that synthesis by the mevalonate independent pathway (2-C-methyl-D-erythritol 2, 4, cyclo pyrophosphate and hydroxymethybutenyl-4 pyrophosphate) both contain a terminal pyrophosphate; in addition, isopentenyl and dimethylallyl pyrophosphate, common to the two pathways, also contain a terminal pyrophosphate (Fig. 1). All these points could be raised in favour of the inhibitory effects of bisphosphonates on the synthesis of isoprenoids and hence on the development of *Arabidopsis thaliana.*

The mevalonate pathway is responsible, in plants, of the production of different phytohormones, among which cytokinins (Letham & Palni, 1983). Cytokinins have been shown to reverse the effects of lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the enzyme catalyzing the synthesis of mevalonate (Crowell & Salaz 1992). Although root growth and lateral growth formation are preferentially determined by auxins, a role of cytokinins in root development has been described, coherent with their role in promoting cell division, specifically in root meristematic cells. Cytokinins, synthesized in the root cap, promote cytokinesis, vascular differentiation, root apical dominance and gravitropism (Aloni et al., 2006). Therefore, the observed effects of BPs on root growth and development may, in principle, be attributed to an inhibitory effect of BPs on cytokinins, although an indirect effect on the synthesis and/or transport of auxins cannot be excluded either. Whereas the differential effect of the various assayed BPs on root growth and development is unequivocal, additional work is necessary to understand the reasons for this differential effect and the mechanisms of the inhibition throughout the action of one or more phytohormones.

Finally, the importance of the mevalonate pathway in the biological systems can also be contemplated from an evolutionary viewpoint: isoprenoids are the oldest known biomolecules recovered from sediments, as old as 2.5 billion years (Summons et al., 2006); the mevalonate pathway is germane to archaebacteria (Lange et al., 2000) and finally, farnesyl diphosphate synthase and polypropenyl synthase activities have been reported as present in the hypothetical Last Universal Common Ancestor (LUCA) (Ranea et al., 2006). All the above points could be also considered as examples of the multiple relationships interconnecting all the biological cycles on earth.

## **4. Conclusions**

860 Osteoporosis

in the presence of the drug progresses in time. From s to u: culture in the presence of 0.5 mM alendronate for 4, 8 and 11 days, respectively; the root growth is severely hampered in these conditions; 8 - and 11-day old seedlings show an almost white colour of leaves, indicative of

The above results can be interpreted as a consequence of the toxic effects of BPs on the mevalonate pathway of *Arabidopsis thaliana* (Fig. 1). Isoprenoids, the most diverse group of natural products, are synthesized in prokaryotes and eukaryotes, animals and plants by condensation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. They play important and diverse roles in the synthesis of quinones, sterols, prenylation of proteins, photosynthetic pigments, hormones, attractants for pollinators in plants, and others. Certainly, most of the studies on the synthesis of isoprenoids were carried out in animal tissues; however, after the more recent discovery of the synthesis of isoprenoids by the mevalonate independent pathway operating in plants, new approaches on their metabolism and function have been undertaken in other biological model systems (Eisenreich et al., 2004; Lange et al., 2000; Rodriguez-Concepcion & Boronat, 2002; Rohmer, 1999). The plastid mevalonate independent pathway involves the synthesis of 1-deoxy-D.xilulose from pyruvate and glyceraldehydes-3-phosphate. There is a cross talk between the cytosolic and plastidial pathway for the synthesis of isoprenoids in *Arabidopsis thaliana* (Laule et al., 2003). In relation with the postulated inhibitory role of bisphosphonates on the synthesis of isoprenoids, it could be noted that the two last compounds in that synthesis by the mevalonate independent pathway (2-C-methyl-D-erythritol 2, 4, cyclo pyrophosphate and hydroxymethybutenyl-4 pyrophosphate) both contain a terminal pyrophosphate; in addition, isopentenyl and dimethylallyl pyrophosphate, common to the two pathways, also contain a terminal pyrophosphate (Fig. 1). All these points could be raised in favour of the inhibitory effects of bisphosphonates on the synthesis of isoprenoids and hence on the

The mevalonate pathway is responsible, in plants, of the production of different phytohormones, among which cytokinins (Letham & Palni, 1983). Cytokinins have been shown to reverse the effects of lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the enzyme catalyzing the synthesis of mevalonate (Crowell & Salaz 1992). Although root growth and lateral growth formation are preferentially determined by auxins, a role of cytokinins in root development has been described, coherent with their role in promoting cell division, specifically in root meristematic cells. Cytokinins, synthesized in the root cap, promote cytokinesis, vascular differentiation, root apical dominance and gravitropism (Aloni et al., 2006). Therefore, the observed effects of BPs on root growth and development may, in principle, be attributed to an inhibitory effect of BPs on cytokinins, although an indirect effect on the synthesis and/or transport of auxins cannot be excluded either. Whereas the differential effect of the various assayed BPs on root growth and development is unequivocal, additional work is necessary to understand the reasons for this differential effect and the mechanisms of the inhibition throughout the action of one or more

Finally, the importance of the mevalonate pathway in the biological systems can also be contemplated from an evolutionary viewpoint: isoprenoids are the oldest known biomolecules recovered from sediments, as old as 2.5 billion years (Summons et al., 2006); the mevalonate pathway is germane to archaebacteria (Lange et al., 2000) and finally, farnesyl diphosphate synthase and polypropenyl synthase activities have been reported as

a very serious loss of chlorophyll.

development of *Arabidopsis thaliana.*

phytohormones.

Bisphosphonates are widely used in the treatment of osteoporosis and hence their great importance in clinical investigation. Upon their administration they bind to the bone tissues where they exert a noxious effect mainly on the mevalonate pathway of the osteoclasts. Given the universality of this pathway, present in bacteria, plants and animals, it seemed to us of interest to analyze the effect of etidronate, clodronate (non containing nitrogen or non-N-BPs), pamidronate and alendronate (N-BPs) on some visible traits of the model plant *Arabidopsis thaliana*. The seeds were grown in a medium containing three concentrations (0.05 mM, 0.1 mM or 0.5 mM) of each bisphosphonate and the growth of primary and secondary roots and formation of chlorophyll were observed during early development of the seedlings. Each bisphosphonate showed a different pattern of influence on those parameters. In general, the inhibitory effects were, in increasing order: etidronate, pamidronate, clodronate, and alendronate. Specific effects on the evaluated parameters ranged from simple reduction in the number and length of secondary roots caused by 0.1 mM etidronate, only after 11 days of culture, until a drastic inhibition of seedling growth, with total loss of chlorophyll observed with 0.5 mM alendronate already in the intermediate time point of sampling. The utility of the use of plants to analyze the action of bisphosphonates is suggested.

### **5. Acknowledgments**

This work was supported by grants from the Spanish National Plan for Research, Development and Innovation (BFU 2008-00666/BMC, BFU 2009-08977 and AYA2009- 07952). We thank Mercedes Carnota, Javier Pérez and Anabel de Diego for very helpful technical assistance.

### **6. References**


Effect of Bisphosphonates on Root Growth

pp. 6128-6132, ISSN 0027-8424

(February 2005), pp. 129-138, ISSN 0176-1617

2003), pp. 6866-6871, ISSN 0027-8424

*Rev Plant Physiol,* Vol*.* 34, pp. 163-197.

263, ISSN 1744-7631

2004), pp. 513-525.

0264-6021.

*Phys*, Vol.60, pp. 111-117.

No.4, (Fefruary 2006), pp. 437-445, ISSN 0007-1188

(November 2002), pp. 1079-1089, ISSN 0032-0889

and on Chlorophyll Formation in *Arabidopsis thaliana* Seedling 863

Kim, E. J., Zhen, R. G. & Rea, P. A. (1994). Heterologous expression of plant vacuolar

Kuo, S. Y., Chien, L. F., Hsiao, Y. Y., Van Ru, C., Yan, K. H., Liu, P. F., Mao, S. J. & Pan, R. L.

Lange, B. M., Rujan, T., Martin, W. & Croteau, R. (2000). Isoprenoid biosynthesis: the

Leon, A., Liu, L., Yang, Y., Hudock, M. P., Hall, P., Yin, F., Studer, D., Puan, K. J., Morita, C.

*Med Chem,* Vol*.*49, No.25, (December 2006), pp. 7331-7341, ISSN 0022-2623 Letham, D. S. & Palni, L. M. S. (1983). The biosynthesis and metaabolism of cytokinins. *Annu* 

Monkkonen, H., Auriola, S., Lehenkari, P., Kellinsalmi, M., Hassinen, I. E., Vepsalainen, J. &

Moreno, S. N. & Li, Z. H. (2008). Anti-infectives targeting the isoprenoid pathway of

Oberhauser, V., Gaudin, J., Fonne-Pfister, R. & Schar, H.-P. (1998). New target enzyme(s) for

Ranea, J. A., Sillero , A., Thornton, J. & Orengo, C. (2006). Protein superfamilies evolution

Rodrígues, C. O., Scott, D. A., Bailey, B. N., De Souza, W., Benchimol, M., Moreno, B.,

Rodriguez-Concepcion, M. & Boronat, A. (2002). Elucidation of the methylerythritol

Rogers, M. J., Brown, R. J., Hodkin, V., Blackburn, G. M., Russell, R. G. & Watts, D. J. (1996)

*U S A,* Vol.97, No.24, (November 2000), pp. 13172-13177. ISSN 0027-8424 Laule, O., Furholz, A., Chang, H. S., Zhu, T., Wang, X., Heifetz, P. B., Gruissem, W. & Lange,

pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport. *Proc Natl Acad Sci U S A,* Vol.91, No.13, (June 1994),

(2005). Proton pumping inorganic pyrophosphatase of endoplasmic reticulumenriched vesicles from etiolated mung bean seedlings. *J Plant Physiol,* Vol.162, No.2,

evolution of two ancient and distinct pathways across genomes. *Proc Natl Acad Sci* 

M. (2003). Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in *Arabidopsis thaliana*. *Proc Natl Acad Sci U S A,* Vol.100, No.11, (May

T. & Oldfield, E. (2006). Isoprenoid biosynthesis as a drug target: bisphosphonate inhibition of *Escherichia coli* K12 growth and synergistic effects of fosmidomycin. *J* 

Monkkonen, J. (2006). A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates. *Br J Pharmacol,* Vol.147,

Toxoplasma gondii. *Expert Opin Ther Targets,* Vol*.*12, No.3, (March 2008), pp. 253-

bisphosphonates: Inhibition of geranylgeranyl diphosphate synthase. *Pestic Biochem* 

and the last universal common ancestor (LUCA). *J. Mol. Evol.* Vol.65, No.4, (October

Urbina, J. A., Oldfield, E. & Moreno, S. N. (2000). Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets. *Biochem J,* Vol*.*349 Pt 3, (August 2000), pp. 737-745, ISSN

phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. *Plant Physiol,* Vol.130, No.3,

Bisphosphonates are incorporated into adenine nucleotides by human aminoacyl-


Burke, C., Klettke, K. & Croteau, R. (2004). Heteromeric geranyl diphosphate synthase from

Cremers, S. C., Pillai, G. & Papapoulos, S. E. (2005). Pharmacokinetics/pharmacodynamics

*Clin Pharmacokinet,* Vol.44, No.6, (June 2005), pp. 551-570, ISSN 0312-5963 Cromartie, T. H., Fisher, K. J. & Grossman, J. N. (1999). The discovery of a novel site of action for herbicidal bisphosphonates. *Pestic Biochem Phys,* Vol.63, pp. 114-126. Crowell, D. N. & Salaz, M. S. (1992). Inhibition of Growth of Cultured Tobacco Cells at Low

Docampo, R. & Moreno, S. N. (2001). Bisphosphonates as chemotherapeutic agents against

Drozdowicz, Y. M., Shaw, M., Nishi, M., Striepen, B., Liwinski, H. A., Roos, D. S. & Rea, P.

Eisenreich, W., Bacher, A., Arigoni, D. & Rohdich, F. (2004). Biosynthesis of isoprenoids via

Forlani, G., Occhipinti, A., Berlicki, L., Dziedziola, G., Wieczorek, A. & Kafarski, P. (2008).

*Agric Food Chem*, Vol.56, No.9, (May 2008), pp. 3193-3199, ISSN 0021-8561 Gordon-Weeks, R., Parmar, S., Davies, T. G. & Leigh, R. A. (1999). Structural aspects of the

Günther Sillero, M. A., de Diego, A., Pérez-Zuñiga, F. J. & Sillero, A. (2008). Synthesis of

Günther Sillero, M. A., de Diego, A., Silles, E., Pérez-Zúñiga, F. & Sillero, A. (2006).

Günther Sillero, M. A., de Diego, A., Tavares, J. E., Silva, J. A., Pérez-Zúñiga, F. J. & Sillero,

Hunter, W. N. (2007). The non-mevalonate pathway of isoprenoid precursor biosynthesis. *J Biol Chem,* Vol.282, No.30, (July 2007), pp. 21573-21577, ISSN 0021-9258

*Chem,* Vol.278, No.2, (January 2003), pp. 1075-1085, ISSN 0021-9258

(December 1992), pp. 2090-2095, ISSN 0032-0889

No.1, (May 2001), pp. 51-61, ISSN 1568-0053

Vol.580, No.24, (October 2006), pp. 5723-5727

ISSN 0003-9861

1426, ISSN 1420-682X

373-377, ISSN 0264-6021

ISSN 0006-2952

2968

mint: construction of a functional fusion protein and inhibition by bisphosphonate substrate analogs. *Arch Biochem Biophys,* Vol.422, No.1, (February 2004), pp. 52-60,

of bisphosphonates: use for optimisation of intermittent therapy for osteoporosis.

Concentrations of Lovastatin Is Reversed by Cytokinin. *Plant Physiol,* Vol.100, No.4,

trypanosomatid and apicomplexan parasites. *Curr Drug Targets Infect Disord* Vol.1,

A. (2003). Isolation and characterization of TgVP1, a type I vacuolar H+ translocating pyrophosphatase from Toxoplasma gondii. The dynamics of its subcellular localization and the cellular effects of a diphosphonate inhibitor. *J Biol* 

the non-mevalonate pathway. *Cell Mol Life Sci*, Vol.61, No.12, (June 2004), pp. 1401-

Tailoring the structure of aminobisphosphonates to target plant P5C reductase. *J* 

effectiveness of bisphosphonates as competitive inhibitors of the plant vacuolar proton-pumping pyrophosphatase. *Biochem J*, Vol.337( Pt 3), (February 1999), pp.

bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases. *Biochem Pharmacol,* Vol.75, No.10, (May 2008), pp. 1959-1965,

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase. *FEBS Lett,*

A. (2009). Synthesis of ATP derivatives of compounds of the mevalonate pathway (isopentenyl di- and triphosphate; geranyl di- and triphosphate, farnesyl di- and triphosphate, and dimethylallyl diphosphate) catalyzed by T4 RNA ligase, T4 DNA ligase and other ligases Potential relationship with the effect of bisphosphonates on osteoclasts. *Biochem Pharmacol,* Vol.78, No.4, (August 2009), pp. 335-343, ISSN 1873-


tRNA synthetase enzymes. *Biochem Biophys Res Commun,* Vol.224, No.3, (July 1996) pp. 863-869.


Rogers, M. J., Crockett, J. C., Coxon, F. P. & Monkkonen, J. (2010). Biochemical and

Rohmer, M. (1999). The discovery of a mevalonate-independent pathway for isoprenoid

Russell, R. G. (2011). Bisphosphonates: The first 40 years. *Bone,* PubMed, in process. ISSN

Summons, R. E., Bradley, A. S., Jahnke, L. L. & Waldbauer, J. R. (2006). Steroids,

Szabo, C. M. & Oldfield, E. (2001). An investigation of bisphosphonate inhibition of a

Wendt, K. U. & Schulz, G. E. 1998 Isoprenoid biosynthesis: manifold chemistry catalyzed by

Zhen, R. G., Baykov, A. A., Bakuleva, N. P. & Rea, P. A. (1994) Aminomethylenediphosphonate:

pp. 863-869.

1873-2763

2126

0889

(November 2010), ISSN 1873-2763

(October 1999), pp. 565-574, ISSN 0265-0568

No.1470, (June 2006), pp. 951-968, ISSN 0962-8436

No.2, (September 2001), pp. 468-473, ISSN 0006-291X

tRNA synthetase enzymes. *Biochem Biophys Res Commun,* Vol.224, No.3, (July 1996)

molecular mechanisms of action of bisphosphonates. *Bone*, PubMed, in process.

biosynthesis in bacteria, algae and higher plants. *Nat Prod Rep,* Vol.16, No.5,

triterpenoids and molecular oxygen. *Philos Trans R Soc Lond B Biol Sci,* Vol.361,

vacuolar proton-pumping pyrophosphatase. *Biochem Biophys Res Commun,* Vol.287,

similar enzymes. *Structure,* Vol.6, No.2, (February 1998), pp. 127-133, ISSN 0969-

A Potent Type-Specific Inhibitor of Both Plant and Phototrophic Bacterial H+- Pyrophosphatases. *Plant Physiol,* Vol.104, No.1, (January 1994), pp. 153-159, ISSN 0032-

## *Edited by Yannis Dionyssiotis*

Osteoporosis is a public health issue worldwide. During the last few years, progress has been made concerning the knowledge of the pathophysiological mechanism of the disease. Sophisticated technologies have added important information in bone mineral density measurements and, additionally, geometrical and mechanical properties of bone. New bone indices have been developed from biochemical and hormonal measurements in order to investigate bone metabolism. Although it is clear that drugs are an essential element of the therapy, beyond medication there are other interventions in the management of the disease. Prevention of osteoporosis starts in young ages and continues during aging in order to prevent fractures associated with impaired quality of life, physical decline, mortality, and high cost for the health system. A number of different specialties are holding the scientific knowledge in osteoporosis. For this reason, we have collected papers from scientific departments all over the world for this book. The book includes up-to-date information about basics of bones, epidemiological data, diagnosis and assessment of osteoporosis, secondary osteoporosis, pediatric issues, prevention and treatment strategies, and research papers from osteoporotic fields.

Osteoporosis

Osteoporosis

*Edited by Yannis Dionyssiotis*

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