**Meet the editor**

Dr Yannis Dionyssiotis studied Medicine at the University of Athens, and specialized in Physical Medicine and Rehabilitation in the National Rehabilitation Center EIAA in Athens. He was a Research Fellow in the Laboratory for Research of the Musculoskeletal System at the University of Athens, where he completed his thesis in osteoporosis. From 2005, he has been

Fellow of the European Board of Rehabilitation. He worked in the Rehabilitation Department of KAT Hospital in Athens, as Head of the Physical Medicine and Rehabilitation Department in Rhodes General Hospital, and is currently the Medical Director of the Physical and Social Rehabilitation Center Amyntaeo in Florina, Greece. He is an elected member of the Board of the International Society of Musculoskeletal and Neuronal Interactions (ISMNI), has been elected twice onto the Board of Hellenic Osteoporosis Foundation (HELIOS), has written medical books and chapters in the English and Greek language, and many papers in international journals. Additionally, Dr. Yannis Dionyssiotis won the Heim Ring Silver award in October 2010, and the European Board award in June 2008. He is a reviewer in many PubMed journals and also the editor of the online rehabilitation magazine medreha.com.

Contents

**Preface XI** 

**Part 1 The Basics of Bone 1** 

Chapter 1 **Bone Mineral Quality 3** 

Chapter 2 **Genetics and Osteoporosis 33** 

Gholamreza Rouhi

Chapter 4 **Self-Reported Prevalence of** 

Naohisa Miyakoshi,

Frank Bonura

Chapter 7 **Evolutionary Pathways of** 

**Part 3 The Diagnosis and Assessment** 

Delphine Farlay and Georges Boivin

Chapter 3 **Biomechanics of Osteoporosis: The Importance** 

**Part 2 Public Health Data of Osteoporosis 79** 

**Osteoporosis in Australia 81**  Tiffany K. Gill, Anne W. Taylor, Julie Black and Catherine L. Hill

Michio Hongo and Yoichi Shimada

Chapter 6 **The Diagnosis and Workup of Patients for** 

**Diagnosis in Osteoporosis 133**  Antonio Bazarra-Fernández

Margarita Valdés-Flores, Leonora Casas-Avila,

Valeria Ponce de León-Suárez and Edith Falcón-Ramírez

**of Bone Resorption and Remodeling Processes 59** 

Chapter 5 **Prevalence of Back Pain in Postmenopausal Osteoporosis and Associations with Multiple Spinal Factors 103** 

**of Osteoporosis and Fractures Risk 115** 

**Osteoporosis or Osteopenia (Low Bone Mass) 117** 

## Contents

### **Preface XV**

#### **Part 1 The Basics of Bone 1**

	- **Part 2 Public Health Data of Osteoporosis 79**
	- **Part 3 The Diagnosis and Assessment of Osteoporosis and Fractures Risk 115**

Contents VII

Chapter 20 **Physical Activity Interactions with** 

Chapter 22 **Rehabilitation in Osteoporosis 435**  Yannis Dionyssiotis

Chapter 23 **Physical Exercise for** 

**Bone Accrual in Children and Adolescents 379** 

Izabella A. Ludwa and Panagiota Klentrou

Chapter 21 **Osteoporosis, Nutrition and Adolescence 411** 

**Prevention of Falls and Fractures 467** Lucas Teixeira, Joelma Magalhães, Stella Peccin, Rebeca Teixeira, Kelson Silva, Tiago Teixeira,

Jander Souza and Virgínia Trevisani

Chapter 25 **Impaired Ability to Perform the Sit-to-Stand Task in Osteoporotic Women 517** 

Daniela Cristina Carvalho de Abreu

**Effects of Exercise Training 529**

Chapter 28 **Pharmacological Treatment of Osteoporosis 555** 

Chapter 29 **The Role of Hormone Replacement Therapy (HRT)** 

**Postmenopausal Osteoporosis 609** 

Maria Panaś, Małgorzata Zaleska and

Jorge Malouf-Sierra and Roberto Güerri-Fernández

**and Tibolone in the Prevention and Treatment of** 

**Bisphosphonate Treatment for Osteoporosis 625** 

Chapter 26 **Osteoporosis and Arterial Stiffness:** 

Sumihisa Orita, Seiji Ohtori, Gen Inoue and Kazuhisa Takahashi

Takanobu Okamoto

Chapter 27 **Osteoporotic Pain 541** 

Marta Lamarca

Chapter 30 **Osteonecrosis of the Jaw Involving** 

Tomasz Kaczmarzyk

Chapter 24 **The Effect of Exercise on Bone Mineral Density, Bone Markers and Postural Stability in Subjects with Osteoporosis 493** 

M. Janura, Z. Krhutová, Z. Svoboda and P. Novosad

Deborah Colucci Trevisan, Francisco José Albuquerque de Paula, Júlia Guimarães Reis, Gustavo de Carvalho da Costa and

**Part 6 Prevention and Management of Osteoporosis 409** 

Isabel Seiquer, Marta Mesías and M. Pilar Navarro

	- **Part 4 Secondary Osteoporosis 247**
	- **Part 5 Pediatric Issues in Osteoporosis 367**



VI Contents

Chapter 8 **Approach to the Screening and** 

Chapter 10 **Sophisticated Imaging Technology** 

Zohreh Hamidi

Zohreh Hamidi

Chapter 13 **Patchy Osteoporosis in**

**Part 4 Secondary Osteoporosis 247** 

Yannis Dionyssiotis

Choi H.J.

**Diagnosis of Osteoporosis 151** 

Chapter 11 **What We Learn from Bone Complications in**

Chapter 9 **Early Detection Techniques for Osteoporosis 165**  Kanika Singh and Kyung Chun Kim

> **in the Assessment of Osteoporosis Risk 181**  Huayue Chen, Tatsuro Hayashi, Xiangrong Zhou, Hiroshi Fujita, Minoru Onozuka and Kin-ya Kubo

Chapter 12 **What's BMD and What We Do in a BMD Centre? 225**

**Complex Regional Pain Syndrome 249**  Geun-Young Park, Sun Im and Seong Hoon Lim

Chapter 14 **Osteoporosis in Microgravity Environments 265** 

Chapter 15 **Neurological Osteoporosis in Disabilities 277** 

Federico G. Hawkins, Sonsoles Guadalix, Raquel Sanchez and Guillermo Martínez

**with Neurofibromatosis Type 1: Important Consequences of Abnormal Gene Function 323** 

**Patients and Its Clinical Management 369** 

**in Slovakia – Experience from Single Institute 343**

Marek W. Karwacki and Wojciech Wozniak

Chapter 16 **Post-Transplantation Bone Disease 299** 

Chapter 17 **The Skeleton Abnormalities in Patients** 

Chapter 18 **Studies of Osteoporosis in Cancer Patients**

**Part 5 Pediatric Issues in Osteoporosis 367** 

Emilio González Jiménez

Chapter 19 **Osteoporosis in Pediatric** 

Beata Spanikova and Stanislav Spanik

**Congenital Diseases? Thalassemia, an Example 195** 

Bradley K. Weiner, Scott E. Parazynski and Ennio Tasciotti



Contents IX

Chapter 41 **Effect of Bisphosphonates on Root** 

**Growth and on Chlorophyll Formation in**  *Arabidopsis thaliana* **Seedlings 853** 

A. Günther Sillero and Antonio Sillero

Ana I. Manzano, F. Javier Medina, Francisco J. Pérez-Zuñiga, Maria

### Chapter 41 **Effect of Bisphosphonates on Root Growth and on Chlorophyll Formation in**  *Arabidopsis thaliana* **Seedlings 853**

VIII Contents

Chapter 31 **Balloon Kyphoplasty for** 

Chapter 33 **Osteoporosis:**

Chapter 32 **Minimally Invasive Treatment** 

**Osteoporosis: Technical Notes 637**  Antoine Nachanakian, Antonios El Helou, Sami Salem and Moussa Alaywan

**of Vertebral Body Fractures 649**  Pasquale De Negri and Tiziana Tirri

Cyril Popov and Margarita D. Apostolova

Roberto Monetti, Jan Bauer, Thomas Baum, Maiko Matsuura, Philippe Zysset and Felix Eckstein

**in Preventing/Recovering Bone Loss:** 

Branko Filipović and Branka Šošić-Jurjević

Chapter 37 **Nutrition for Enhancing Bone Volume in Mice 765** 

Masahide Motokawa and Kazuo Tanne

Chapter 38 **Osteoporosis and Bone Regeneration 781** 

Kanako Noritake and Shohei Kasugai

**Intervention in Osteoporosis 803**  Dorit Naot, Kate Palmano and Jillian Cornish

Chapter 40 **How Dentistry Can Help Fight Osteoporosis 821**  Plauto Christopher Aranha Watanabe,

**Thyroid Hormones: Effects on Bone Tissue 733** 

R.A. Marquez Hernandez, Toshitsugu Kawata, Masato Kaku,

Marlivia Gonçalves de Carvalho Watanabe and Rodrigo Tiossi

Carla Palumbo, Francesco Cavani, Laura Bertoni and Marzia Ferretti

Chapter 34 **Simulating Bone Atrophy and Its Effects on**

Christoph Räth, Irina Sidorenko,

Chapter 35 **Role of Phytoestrogen Ferutinin** 

Chapter 36 **The Phytoestrogens, Calcitonin and** 

Junji Ohtani, Fujita Tadashi,

Shinji Kuroda,

Chapter 39 **Lactoferrin – A Potential Anabolic**

**A Look at the Future 667** Iliyan Kolev, Lyudmila Ivanova, Leni Markova, Anelia Dimitrova,

**Part 7 Research and New Challenges in Osteoporosis 665** 

**the Structure and Stability of the Trabecular Bone 695** 

**Results from Experimental Ovariectomized Rat Models 710** 

Ana I. Manzano, F. Javier Medina, Francisco J. Pérez-Zuñiga, Maria A. Günther Sillero and Antonio Sillero

Preface

papers from osteoporotic fields.

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

**Yannis Dionyssiotis, MD, PhD,**

Greece

Physical and Social Rehabilitation Center Amyntæo

University of Athens, Laboratory for Research of the Musculoskeletal System

## Preface

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.

**Yannis Dionyssiotis, MD, PhD,** 

Physical and Social Rehabilitation Center Amyntæo University of Athens, Laboratory for Research of the Musculoskeletal System Greece

**Part 1** 

**The Basics of Bone** 

**Part 1** 

**The Basics of Bone** 

**1** 

*France* 

**Bone Mineral Quality** 

*1INSERM, UMR 1033, F-69372 Lyon, 2Université de Lyon, F-69008 Lyon,* 

Delphine Farlay1,2 and Georges Boivin1,2

The main function of bone is to promote locomotion and protection of vital organs. Bone is also an important mineral ions reservoir, essential to maintain phosphocalcic homeostasis. Bone mineral is a calcium phosphate named "apatite", which form naturally in the Earth's crust (Wopenka & Pasteris 2005). Compared to others minerals, apatite is more "tolerant" and is very accommodating to chemical substitutions. This ability to easily absorb ions confers to bone a detoxification property, with some ions normally absent of bone and which are captured by bone mineral. But the substitutions in bone mineral change the structure of apatite, conferring to bone several properties such as solubility, morphology,

Thanks to those remarkable properties, bone has the ability to continually adapt to changes to its mechanical environment (Bouxsein 2005). Bone is an anisotropic composite material tissue, and highly hierarchical viscoelastic (Bouxsein 2005). When a load is applied to bone, this produces energy, and as this energy can not be destroy, the bone has to absorbed it (Seeman & Delmas 2006). The elastic properties of bone allow to absorb this energy by deforming reversibly. But if the load exceeds the ability of the bone to carry this load, it can deform permanently by plastic deformation (Fig.1). This produces microcracks allowing

Fig. 1. Stress-strain curve divided into the elastic and plastic regions. The fracture occurs at the end of the curve (marked with **X**). Reprinted from Turner & Burr, 1993, with permission

**1. Introduction** 

hardness, strain etc.

from Elsevier.

## **Bone Mineral Quality**

Delphine Farlay1,2 and Georges Boivin1,2 *1INSERM, UMR 1033, F-69372 Lyon, 2Université de Lyon, F-69008 Lyon, France* 

### **1. Introduction**

The main function of bone is to promote locomotion and protection of vital organs. Bone is also an important mineral ions reservoir, essential to maintain phosphocalcic homeostasis. Bone mineral is a calcium phosphate named "apatite", which form naturally in the Earth's crust (Wopenka & Pasteris 2005). Compared to others minerals, apatite is more "tolerant" and is very accommodating to chemical substitutions. This ability to easily absorb ions confers to bone a detoxification property, with some ions normally absent of bone and which are captured by bone mineral. But the substitutions in bone mineral change the structure of apatite, conferring to bone several properties such as solubility, morphology, hardness, strain etc.

Thanks to those remarkable properties, bone has the ability to continually adapt to changes to its mechanical environment (Bouxsein 2005). Bone is an anisotropic composite material tissue, and highly hierarchical viscoelastic (Bouxsein 2005). When a load is applied to bone, this produces energy, and as this energy can not be destroy, the bone has to absorbed it (Seeman & Delmas 2006). The elastic properties of bone allow to absorb this energy by deforming reversibly. But if the load exceeds the ability of the bone to carry this load, it can deform permanently by plastic deformation (Fig.1). This produces microcracks allowing

Fig. 1. Stress-strain curve divided into the elastic and plastic regions. The fracture occurs at the end of the curve (marked with **X**). Reprinted from Turner & Burr, 1993, with permission from Elsevier.

Bone Mineral Quality 5

bone fragility (Rauch & Glorieux 2004). Among these determinants, microcracks are normally present in bone, and permit to dissipate energy when bone is submitted to a load. However, the presence of too long microcracks is not good for bone. Thus, all together those

extrinsic and intrinsic determinants are involved in bone strength.

Fig. 2. Description of determinants of bone strength (INSERM UMR 1033)

mineralization process, mineral crystals and mineral composition.

**3. Mineralization process: A dynamic process** 

mechanisms of bone fragility.

crystals.

Bone mineral properties are important determinants of bone strength. Those properties include material (degree of mineralization, hardness…) and crystalline (crystallinity, mineral maturity, ionic substitutions...) characteristics. The importance of the components of bone quality is thus evident and the relationships between the determinants of bone quality are essential to maintain an overall mechanical competent bone. Indeed, bone mineral is complex and knowledge of its composition is important to better understand the

The main purpose of this present chapter is to bring a best approach of bone mineral quality,

As bone is submitted to a constant remodeling during all the adult life, old Bone Structural Units (BSUs, named the osteons in cortical bone and the trabecular packets in cancellous bone) will be resorbed by osteoclasts, and replaced by new formed bone. Thus, the recently formed BSUs will be less mineralized than the older BSUs present in interstitial bone and not already resorbed. This heterogeneity of mineralization in the different BSUs can be easily visualized on a X rays microradiograph (Fig. 3). This mineralization process related to bone remodeling can be decomposed into two steps: a primary mineralization which corresponds to a very rapid deposition of first crystals, and a secondary mineralization which is much longer, with a slow and gradual increase in size, perfection and number of

energy release. If the microcracks remain small, this has no impact on bone. However, if the microcracks become numerous and/or too long, the bone fractures. Thus, to resist to a fracture, the bone need to find the best compromise between *stiffness* and *flexibility* (to resist deformation) (Seeman & Delmas 2006). A high mineral content increases *stiffness* reducing *flexibility,* and if the bone is too flexible, it will deform beyond its peak strain and crack. Several studies showed that mineral part is involved in the elastic properties of bone, whereas organic part is rather involved in the plastic deformation (Bala et al., 2011b; Bouxsein 2005; Currey 2003; Follet et al., 2004).

### **2. Determinants of bone quality**

Bone is constituted by three major components which are the organic matrix (30%, mainly type I collagen), mineral (60%, carbonated apatite) and water (10%). Organic matrix is essentially constituted by ~ 90% of a network of type I collagen fibrils, and ~10% of noncollagenous proteins. Type I collagen molecules are formed by three polypeptides α chains [2 α1(I) and 1 α2(I)] forming a tight triple helix structure with a repetition of Gly-X-Y triplets (Myllyharju & Kivirikko 2004). To provide the stability of type I collagen fibrils, several mechanisms of maturation and ageing of bone collagen occurs, including enzymatic collagen cross-linking and non-enzymatic modifications (Saito & Marumo 2010; Viguet-Carrin et al., 2006). In parallel, the organic matrix mineralizes, and the organization of type I collagen network determines the specific arrangement of mineral crystals (Höhling et al., 1990; Riggs et al., 1993). The crystals first grow in length, typically plate-like (Landis, 1995; Roschger et al., 1998), then they become thicker but stay relatively thin (Roschger et al., 1998). Concomitantly, the crystals number increases up to physiologic limits of mineralization (Bala et al., 2010; Boivin et al., 2008; Boivin & Meunier 2002).

The quantity of bone mineral (assimilated to the quantity of total bone) is usually measured by bone mineral density (BMD), using dual X-ray absorptiometry (DXA). However, about one-half of fractures occur in women having a T-Score above the World Health Organization (WHO) diagnosis threshold of osteoporosis (-2.5) (Siris et al., 2004; Sornay-Rendu et al., 2005) suggesting that other factors than bone quantity are involved in the apparition of fractures. These factors, involved in bone quality, are called intrinsic determinants. Both extrinsic determinants (including bone mass, macro/microarchitecture) and intrinsic determinants are involved in bone strength, and are directly dependent of bone remodeling activity (Fig.2).

DXA measurement gives information on bone mineral mass but not on its mineral quality. For example, when fluoride salts was used to treat post menopausal osteoporosis, an increase in bone mineral density was measured by DXA, but the bones of patients treated with fluoride salts were much more brittle than untreated patients. In fact, fluoride ions in bone mineral impaired the bone mineral quality increasing the size of bone (large crystals), and thus reducing the contact area with collagen matrix, despite a higher amount of bone mineral density.

The determinants of bone quality, called "intrinsic determinants", are thus essential in bone strength. Those determinants include mineral quality, collagen quality, and presence of microcracks. A good collagen quality is required to an optimal bone strength. For example, osteogenesis imperfecta, which is an heritable brittle-bone disease, is characterized by a type-I collagen mutation, leading to collagen fibrils abnormally thin, and to an excessive

energy release. If the microcracks remain small, this has no impact on bone. However, if the microcracks become numerous and/or too long, the bone fractures. Thus, to resist to a fracture, the bone need to find the best compromise between *stiffness* and *flexibility* (to resist deformation) (Seeman & Delmas 2006). A high mineral content increases *stiffness* reducing *flexibility,* and if the bone is too flexible, it will deform beyond its peak strain and crack. Several studies showed that mineral part is involved in the elastic properties of bone, whereas organic part is rather involved in the plastic deformation (Bala et al., 2011b;

Bone is constituted by three major components which are the organic matrix (30%, mainly type I collagen), mineral (60%, carbonated apatite) and water (10%). Organic matrix is essentially constituted by ~ 90% of a network of type I collagen fibrils, and ~10% of noncollagenous proteins. Type I collagen molecules are formed by three polypeptides α chains [2 α1(I) and 1 α2(I)] forming a tight triple helix structure with a repetition of Gly-X-Y triplets (Myllyharju & Kivirikko 2004). To provide the stability of type I collagen fibrils, several mechanisms of maturation and ageing of bone collagen occurs, including enzymatic collagen cross-linking and non-enzymatic modifications (Saito & Marumo 2010; Viguet-Carrin et al., 2006). In parallel, the organic matrix mineralizes, and the organization of type I collagen network determines the specific arrangement of mineral crystals (Höhling et al., 1990; Riggs et al., 1993). The crystals first grow in length, typically plate-like (Landis, 1995; Roschger et al., 1998), then they become thicker but stay relatively thin (Roschger et al., 1998). Concomitantly, the crystals number increases up to physiologic limits of

The quantity of bone mineral (assimilated to the quantity of total bone) is usually measured by bone mineral density (BMD), using dual X-ray absorptiometry (DXA). However, about one-half of fractures occur in women having a T-Score above the World Health Organization (WHO) diagnosis threshold of osteoporosis (-2.5) (Siris et al., 2004; Sornay-Rendu et al., 2005) suggesting that other factors than bone quantity are involved in the apparition of fractures. These factors, involved in bone quality, are called intrinsic determinants. Both extrinsic determinants (including bone mass, macro/microarchitecture) and intrinsic determinants are involved in bone strength, and are directly dependent of

DXA measurement gives information on bone mineral mass but not on its mineral quality. For example, when fluoride salts was used to treat post menopausal osteoporosis, an increase in bone mineral density was measured by DXA, but the bones of patients treated with fluoride salts were much more brittle than untreated patients. In fact, fluoride ions in bone mineral impaired the bone mineral quality increasing the size of bone (large crystals), and thus reducing the contact area with collagen matrix, despite a higher amount of bone

The determinants of bone quality, called "intrinsic determinants", are thus essential in bone strength. Those determinants include mineral quality, collagen quality, and presence of microcracks. A good collagen quality is required to an optimal bone strength. For example, osteogenesis imperfecta, which is an heritable brittle-bone disease, is characterized by a type-I collagen mutation, leading to collagen fibrils abnormally thin, and to an excessive

mineralization (Bala et al., 2010; Boivin et al., 2008; Boivin & Meunier 2002).

Bouxsein 2005; Currey 2003; Follet et al., 2004).

**2. Determinants of bone quality** 

bone remodeling activity (Fig.2).

mineral density.

bone fragility (Rauch & Glorieux 2004). Among these determinants, microcracks are normally present in bone, and permit to dissipate energy when bone is submitted to a load. However, the presence of too long microcracks is not good for bone. Thus, all together those extrinsic and intrinsic determinants are involved in bone strength.

Fig. 2. Description of determinants of bone strength (INSERM UMR 1033)

Bone mineral properties are important determinants of bone strength. Those properties include material (degree of mineralization, hardness…) and crystalline (crystallinity, mineral maturity, ionic substitutions...) characteristics. The importance of the components of bone quality is thus evident and the relationships between the determinants of bone quality are essential to maintain an overall mechanical competent bone. Indeed, bone mineral is complex and knowledge of its composition is important to better understand the mechanisms of bone fragility.

The main purpose of this present chapter is to bring a best approach of bone mineral quality, mineralization process, mineral crystals and mineral composition.

### **3. Mineralization process: A dynamic process**

As bone is submitted to a constant remodeling during all the adult life, old Bone Structural Units (BSUs, named the osteons in cortical bone and the trabecular packets in cancellous bone) will be resorbed by osteoclasts, and replaced by new formed bone. Thus, the recently formed BSUs will be less mineralized than the older BSUs present in interstitial bone and not already resorbed. This heterogeneity of mineralization in the different BSUs can be easily visualized on a X rays microradiograph (Fig. 3). This mineralization process related to bone remodeling can be decomposed into two steps: a primary mineralization which corresponds to a very rapid deposition of first crystals, and a secondary mineralization which is much longer, with a slow and gradual increase in size, perfection and number of crystals.

Bone Mineral Quality 7

In adult, MAR varies from 0.60 to 0.80 µm/day whatever the age and the sex (Vedi et al., 1983). MAR is slightly increased in young children, reaching 1 µm/day (Glorieux et al., 2000). During the process of primary mineralization, the first depositions of mineral correspond to about 50 to 60 % of the maximal mineral charge in bone tissue (Meunier & Boivin 1997). This process is extremely rapid, and the first depositions of mineral are used as nucleator for the secondary mineralization. The secondary mineralization corresponds to a slow and gradual increase in both crystal size and number. This process increases until a physiological limit: once the maximum number of crystals attained in a given volume, it is not possible to exceed this limit. Thus in bone remodeling, there is no process of "hypermineralization",

The duration of the secondary mineralization is unknown in humans. This duration has been reported in rabbits (Fuchs et al., 2008) and more recently in an animal model (ewes) having a remodeling activity close to the Humans (Bala et al., 2010). The chronology of secondary mineralization has been identify by injection of different fluorescent labels every six months, in order to date the "age" of the BSUs (Bala et al., 2010). In this study, it has been shown that the secondary mineralization lasts approximatively for 24 to 30 months,

 Fig. 5. Left: Degree of mineralization measured by quantitative microradiography in ewes in cortical and cancellous bone, every 6 months (on 512 BSUs; Reprinted from Bala et al., 2010, with permission from Elsevier). Right: Schematic representation of the duration of primary

This duration of mineralization should be taking into account into anti-resorptive treatment of post menopausal osteoporosis, because once that all the BSUs have attained their

Several methods are used to measure the degree of mineralization of bone. The technique used in the laboratory is the quantitative microradiography, which is a computerized microdensitometric method based on the X-rays absorption (Boivin & Meunier 2002). Others methods are used to measure the degree of mineralization, as the quantitative

and secondary mineralizations.

maximal mineralization, no gain in term of DMB will occurs.

backscattering electron or synchrotron infrared microspectroscopy.

**3.2 Methods to measure degree of mineralization** 

because a given BSU can not contain more crystals than its physiological capacity.

suggesting that after this time, no increase in degree of mineralization occurs (Fig. 5).

Fig. 3. Microradiograph of human femur illustrating the heterogeneity of the mineralization (cortical bone of a man, 48 year-old) (INSERM UMR 1033)

### **3.1 Primary and secondary mineralization processes**

The process of primary mineralization is a very rapid process, starting in the unmineralized bone matrix (osteoid) deposited by the osteoblasts; in humans, the new matrix begins to mineralize after 5 to 10 days after the deposition of osteoid. The primary mineralization can be measured using double tetracycline labeling (Frost 1969) (Fig. 4). The double labeling involves the administration of two short courses of tetracycline which is deposited along the calcification front as two distinct lines visualized on bone sections under ultraviolet (UV) light. This allows the measurement of the mineral apposition rate (MAR). Usually, a labeling procedure is 10 mg/kg/day demethylchlortetracycline or tetracycline hydrochloride orally for 2 days, 12 days off, 4 days on. The bone biopsy is then taken 4-6 days later (Frost 1969).

Fig. 4. Histological slide of bone tissue observed under UV light and illustrating a double tetracycline labelling (yellow lines) in trabecular bone and showing the front of mineralization (INSERM UMR 1033).

Fig. 3. Microradiograph of human femur illustrating the heterogeneity of the mineralization

The process of primary mineralization is a very rapid process, starting in the unmineralized bone matrix (osteoid) deposited by the osteoblasts; in humans, the new matrix begins to mineralize after 5 to 10 days after the deposition of osteoid. The primary mineralization can be measured using double tetracycline labeling (Frost 1969) (Fig. 4). The double labeling involves the administration of two short courses of tetracycline which is deposited along the calcification front as two distinct lines visualized on bone sections under ultraviolet (UV) light. This allows the measurement of the mineral apposition rate (MAR). Usually, a labeling procedure is 10 mg/kg/day demethylchlortetracycline or tetracycline hydrochloride orally for 2 days, 12 days off, 4 days on. The bone biopsy is then taken 4-6 days later (Frost 1969).

Fig. 4. Histological slide of bone tissue observed under UV light and illustrating a double

tetracycline labelling (yellow lines) in trabecular bone and showing the front of

mineralization (INSERM UMR 1033).

(cortical bone of a man, 48 year-old) (INSERM UMR 1033)

**3.1 Primary and secondary mineralization processes** 

In adult, MAR varies from 0.60 to 0.80 µm/day whatever the age and the sex (Vedi et al., 1983). MAR is slightly increased in young children, reaching 1 µm/day (Glorieux et al., 2000). During the process of primary mineralization, the first depositions of mineral correspond to about 50 to 60 % of the maximal mineral charge in bone tissue (Meunier & Boivin 1997). This process is extremely rapid, and the first depositions of mineral are used as nucleator for the secondary mineralization. The secondary mineralization corresponds to a slow and gradual increase in both crystal size and number. This process increases until a physiological limit: once the maximum number of crystals attained in a given volume, it is not possible to exceed this limit. Thus in bone remodeling, there is no process of "hypermineralization", because a given BSU can not contain more crystals than its physiological capacity.

The duration of the secondary mineralization is unknown in humans. This duration has been reported in rabbits (Fuchs et al., 2008) and more recently in an animal model (ewes) having a remodeling activity close to the Humans (Bala et al., 2010). The chronology of secondary mineralization has been identify by injection of different fluorescent labels every six months, in order to date the "age" of the BSUs (Bala et al., 2010). In this study, it has been shown that the secondary mineralization lasts approximatively for 24 to 30 months, suggesting that after this time, no increase in degree of mineralization occurs (Fig. 5).

Fig. 5. Left: Degree of mineralization measured by quantitative microradiography in ewes in cortical and cancellous bone, every 6 months (on 512 BSUs; Reprinted from Bala et al., 2010, with permission from Elsevier). Right: Schematic representation of the duration of primary and secondary mineralizations.

This duration of mineralization should be taking into account into anti-resorptive treatment of post menopausal osteoporosis, because once that all the BSUs have attained their maximal mineralization, no gain in term of DMB will occurs.

#### **3.2 Methods to measure degree of mineralization**

Several methods are used to measure the degree of mineralization of bone. The technique used in the laboratory is the quantitative microradiography, which is a computerized microdensitometric method based on the X-rays absorption (Boivin & Meunier 2002). Others methods are used to measure the degree of mineralization, as the quantitative backscattering electron or synchrotron infrared microspectroscopy.

Bone Mineral Quality 9

and cancellous bone. The main parameters, extracted from the DMB measurements, are the mean DMB, the mean highest and most frequent DMB (DMB Freq. Max) and the mean index of heterogeneity of the distribution of DMB expressed as the mean of the widths at

The mineral content of bone samples has also been evaluated (Roschger et al., 1995, 1998, 2003; Ruffoni et al. 2007) by quantitative Backscattered Electron Imaging (qBEI). This method, based on the detection of electrons backscattered (BSE) on the surface of the bone specimen, is generally used on the same type of bone biopsy fixed in alcohol and embedded in MMA. As the intensity of the BSE signal is strongly related to the atomic number (Z) of the specimen, BSE images provide information about the distribution of different elements in the sample. A calibration of the BE signal with carbon and aluminium as references was performed. Osteoid and hydroxyapatite were also employed as references to convert gray level values into calcium weight % values. In bone, the main signal is related to Ca (Z=20) and P (Z=15) which are the main mineral elements (Roschger et al., 1998). These authors have correlated BE gray levels of bone with calcium content (in weight percent Ca) based on the Ca K-line intensities detected from identical bone areas (Roschger et al., 1995). The intensity of the backscattered electron signal from the sample is directly proportional to the bone calcium concentration and can therefore be used for the generation of bone mineralization density distribution (BMDD). BMDDs display the frequency of certain calcium concentrations and are analyzed for the weighted mean calcium concentration (Ca mean), the most frequent calcium concentration (Ca peak) and the homogeneity of mineralization (Ca width). The BMDD of trabecular bone from healthy, adult individuals was shown to be nearly constant over several biological factors (gender, age, ethnicity, skeletal site). Technical and biological variations showed that it is a method sensitive for

Aside from the difficulty of access to synchrotron radiation facilities, a main advantage of this technique is the use of a mono-energetic synchrotron beam, thus avoiding beamhardening effects. Indeed, the reconstructed gray levels of tomographic images correspond directly to a map of a linear attenuation coefficient within the sample. The SRµCT method has been tested on human bone tissue (Borah et al., 2005, 2006; Nuzzo et al., 2002) but it is still an equipment with difficulty accessible. The availability of a three-dimensional (3D) measuring technique coupled to specific image processing method opens new possibilities. SRµCT may provide 3D images with spatial resolution as high as one micrometer. The acquisition of 3D bone samples images at high spatial resolution using SRµCT has proved to be very accurate for quantifying human bone micro-architecture. Moreover SRµCT is a non destructive, fast, and very precise procedure to determine the DMB in 3D, simultaneously to the micro-architecture. The calibration procedure used homogeneous phantoms of water solutions at different concentrations of K2HPO4 (Nuzzo et al., 2002). This method was compared with the quantitative microradiography technique on the same bone samples, and showed that the values of the DMB are both in the range 0.5-1.6 g/cm3 of bone, both in cortical and cancellous bone, with a mean difference around 4.7%, slightly higher in

half-maximum measured on the individual DMB curves.

subtle changes in mineralization.

trabecular region (Nuzzo et al., 2002).

**3.2.3 Synchrotron radiation microtomography (SRµCT)** 

**3.2.2 Quantitative backscattering electron imaging (qBEI)** 

#### **3.2.1 Quantitative microradiography**

#### **3.2.1.1 Specimen preparation**

Undecalcified iliac bone samples were generally used in humans, fixed in 70% alcohol for ten days or more (depending on the size of the samples), and then specimens are placed two days in absolute alcohol to complete dehydration. Alcohol baths are changed every day and specimens are substituted in methylcyclohexane again for two days, before embedding in methyl methacrylate (MMA). The latter is a transparent and hard plastic having a very low X-ray absorption power. Samples are kept for two days in MMA monomer alone, at 4°C, two days in MMA with 1% of catalyst (anhydrous dibenzoyl peroxide) and 2 days in MMA with 2% of catalyst. Then the specimens are placed in an oven (30°C) for final polymerization to obtain hard blocks. After polymerization, thick sections are cut from the embedded bone samples with a precision diamond wire saw, progressively ground to a thickness of 100µm and polished with an alumina suspension. The thickness of the section was measured with an accuracy of 1 µm using a precision micrometer. After ultrasonic cleaning in demineralised water, the bone sections were microradiographed. If orientation of the blocks is possible before sectioning, the cutting plane perpendicular to the haversian canals of cortical bone is preferred.

#### **3.2.1.2 Measurement of degree of mineralization (DMB) (Boivin & Meunier 2002)**

Soft X-rays are produced in a X-ray generator (Philips compact PW1830/40 X-ray diffraction generator, Limeil Brévannes, France), equipped with a diffraction tube PW 2273/20. A monochromatic X-ray beam is employed, i.e., nickel-filtered copper K radiation with a wavelength of 1.54 Å for which the ratio of the mass- absorption coefficients of aluminium to apatite is 0.561. The distance between the X-ray source and the specimen is about 25-30 cm. In a dark room, the 100 µm-thick bone sections are placed on a photographic emulsion covered by a thin polyester (mylar) film transparent to X-rays, and placed in a specimen holder. An aluminium step-wedge is also exposed on each microradiography. Aluminium was chosen because it is convenient material having an atomic number not far from the effective atomic number of hydroxyapatite. The section is firmly pressed flat by tightening the specimen holder cap and evacuating the air situated between the mylar and the emulsion by means of a vacuum pump, thus bringing the section in direct contact with the emulsion. The specimen holder is placed in a camera perpendicular to the X-ray beam and locked into position during X-ray exposure, during 20 min at 25 kV and 25 mA.

After X-ray exposure, the film (VRP-M green sensitive emulsion from Geola, Slavich International Wholesale Office, Vilnius, Lithuania) is developed for 5 min in Kodak D19 at 20°C, rinsed and then fixed for 5 min in Ilford Hypam. The film is washed and dried, then mounted between two slides. The DMB is quantified using an automatic program for analyzing grayness levels (MorphoExpert and Mineralization, ExploraNova, La Rochelle, France). A digital camera (resolution: 1600 x 1200 pixels or 800 x 600 after binning), captures the microscopic image of the microradiograph. After calibration with the aluminium reference system, the measured regions of bone tissue are automatically selected, and the gray levels are segmented after bone thresholding. The values of the gray levels are then obtained at pixel level (for a magnification x2.5, the size of the pixel is 2.82 µm). Finally, gray-level are converted into DMB measurements with the construction of a calibration curve based on the measurements obtained on the aluminium step-wedge. DMB is finally expressed in gram of mineral over cm3 of bone (g/cm3) and measured separately in cortical

Undecalcified iliac bone samples were generally used in humans, fixed in 70% alcohol for ten days or more (depending on the size of the samples), and then specimens are placed two days in absolute alcohol to complete dehydration. Alcohol baths are changed every day and specimens are substituted in methylcyclohexane again for two days, before embedding in methyl methacrylate (MMA). The latter is a transparent and hard plastic having a very low X-ray absorption power. Samples are kept for two days in MMA monomer alone, at 4°C, two days in MMA with 1% of catalyst (anhydrous dibenzoyl peroxide) and 2 days in MMA with 2% of catalyst. Then the specimens are placed in an oven (30°C) for final polymerization to obtain hard blocks. After polymerization, thick sections are cut from the embedded bone samples with a precision diamond wire saw, progressively ground to a thickness of 100µm and polished with an alumina suspension. The thickness of the section was measured with an accuracy of 1 µm using a precision micrometer. After ultrasonic cleaning in demineralised water, the bone sections were microradiographed. If orientation of the blocks is possible before sectioning, the cutting plane perpendicular to the haversian

**3.2.1.2 Measurement of degree of mineralization (DMB) (Boivin & Meunier 2002)** 

locked into position during X-ray exposure, during 20 min at 25 kV and 25 mA.

After X-ray exposure, the film (VRP-M green sensitive emulsion from Geola, Slavich International Wholesale Office, Vilnius, Lithuania) is developed for 5 min in Kodak D19 at 20°C, rinsed and then fixed for 5 min in Ilford Hypam. The film is washed and dried, then mounted between two slides. The DMB is quantified using an automatic program for analyzing grayness levels (MorphoExpert and Mineralization, ExploraNova, La Rochelle, France). A digital camera (resolution: 1600 x 1200 pixels or 800 x 600 after binning), captures the microscopic image of the microradiograph. After calibration with the aluminium reference system, the measured regions of bone tissue are automatically selected, and the gray levels are segmented after bone thresholding. The values of the gray levels are then obtained at pixel level (for a magnification x2.5, the size of the pixel is 2.82 µm). Finally, gray-level are converted into DMB measurements with the construction of a calibration curve based on the measurements obtained on the aluminium step-wedge. DMB is finally expressed in gram of mineral over cm3 of bone (g/cm3) and measured separately in cortical

Soft X-rays are produced in a X-ray generator (Philips compact PW1830/40 X-ray diffraction generator, Limeil Brévannes, France), equipped with a diffraction tube PW 2273/20. A monochromatic X-ray beam is employed, i.e., nickel-filtered copper K radiation with a wavelength of 1.54 Å for which the ratio of the mass- absorption coefficients of aluminium to apatite is 0.561. The distance between the X-ray source and the specimen is about 25-30 cm. In a dark room, the 100 µm-thick bone sections are placed on a photographic emulsion covered by a thin polyester (mylar) film transparent to X-rays, and placed in a specimen holder. An aluminium step-wedge is also exposed on each microradiography. Aluminium was chosen because it is convenient material having an atomic number not far from the effective atomic number of hydroxyapatite. The section is firmly pressed flat by tightening the specimen holder cap and evacuating the air situated between the mylar and the emulsion by means of a vacuum pump, thus bringing the section in direct contact with the emulsion. The specimen holder is placed in a camera perpendicular to the X-ray beam and

**3.2.1 Quantitative microradiography** 

canals of cortical bone is preferred.

**3.2.1.1 Specimen preparation** 

and cancellous bone. The main parameters, extracted from the DMB measurements, are the mean DMB, the mean highest and most frequent DMB (DMB Freq. Max) and the mean index of heterogeneity of the distribution of DMB expressed as the mean of the widths at half-maximum measured on the individual DMB curves.

#### **3.2.2 Quantitative backscattering electron imaging (qBEI)**

The mineral content of bone samples has also been evaluated (Roschger et al., 1995, 1998, 2003; Ruffoni et al. 2007) by quantitative Backscattered Electron Imaging (qBEI). This method, based on the detection of electrons backscattered (BSE) on the surface of the bone specimen, is generally used on the same type of bone biopsy fixed in alcohol and embedded in MMA. As the intensity of the BSE signal is strongly related to the atomic number (Z) of the specimen, BSE images provide information about the distribution of different elements in the sample. A calibration of the BE signal with carbon and aluminium as references was performed. Osteoid and hydroxyapatite were also employed as references to convert gray level values into calcium weight % values. In bone, the main signal is related to Ca (Z=20) and P (Z=15) which are the main mineral elements (Roschger et al., 1998). These authors have correlated BE gray levels of bone with calcium content (in weight percent Ca) based on the Ca K-line intensities detected from identical bone areas (Roschger et al., 1995). The intensity of the backscattered electron signal from the sample is directly proportional to the bone calcium concentration and can therefore be used for the generation of bone mineralization density distribution (BMDD). BMDDs display the frequency of certain calcium concentrations and are analyzed for the weighted mean calcium concentration (Ca mean), the most frequent calcium concentration (Ca peak) and the homogeneity of mineralization (Ca width). The BMDD of trabecular bone from healthy, adult individuals was shown to be nearly constant over several biological factors (gender, age, ethnicity, skeletal site). Technical and biological variations showed that it is a method sensitive for subtle changes in mineralization.

#### **3.2.3 Synchrotron radiation microtomography (SRµCT)**

Aside from the difficulty of access to synchrotron radiation facilities, a main advantage of this technique is the use of a mono-energetic synchrotron beam, thus avoiding beamhardening effects. Indeed, the reconstructed gray levels of tomographic images correspond directly to a map of a linear attenuation coefficient within the sample. The SRµCT method has been tested on human bone tissue (Borah et al., 2005, 2006; Nuzzo et al., 2002) but it is still an equipment with difficulty accessible. The availability of a three-dimensional (3D) measuring technique coupled to specific image processing method opens new possibilities. SRµCT may provide 3D images with spatial resolution as high as one micrometer. The acquisition of 3D bone samples images at high spatial resolution using SRµCT has proved to be very accurate for quantifying human bone micro-architecture. Moreover SRµCT is a non destructive, fast, and very precise procedure to determine the DMB in 3D, simultaneously to the micro-architecture. The calibration procedure used homogeneous phantoms of water solutions at different concentrations of K2HPO4 (Nuzzo et al., 2002). This method was compared with the quantitative microradiography technique on the same bone samples, and showed that the values of the DMB are both in the range 0.5-1.6 g/cm3 of bone, both in cortical and cancellous bone, with a mean difference around 4.7%, slightly higher in trabecular region (Nuzzo et al., 2002).

Bone Mineral Quality 11

Human bone mineral is a non-stoichiometric and poorly crystallized apatite. Bone apatite structure is hexagonal with space group P63/*m,* with lattice parameters a=9.42Å and c=6.88Å. It is a calcium (Ca)-deficient apatite analog, contains major elements like calcium, [Ca2+ (40 wt

magnesium (Mg2+) or sodium (Na2+), and trace elements (LeGeros & LeGeros 1983; LeGeros et al., 1968). Bone mineral also contains ions normally absent from body fluids (lead, fluoride, aluminium etc). Indeed, the apatite lattice is very tolerant to substitutions and vacancies. Compared to dental enamel, the bone mineral contains much more vacancies. In fact, apatite is able to incorporate itself, in its atomic structure, the half of the elements in the periodic chart (Wopenka & Pasteris 2005). Apatite lattice contains about 40 ions, and the unit cell is the smallest basic unit which is a sample of the entire lattice array (Glimcher 1998). In the apatite unit cell, four different types of crystallographic positions (or "sites") have been identified (Fig.7): (1) tetrahedral sites for six P5+-ions, each in 4-fold coordination with oxygen, (2) Ca[1] sites for four of the Ca2+ ions, (3) Ca[II] sites for the six other Ca2+ ions, and (4) the channel site,

, F-

2005). The small ions (Cd2+, Zn2+, Mg2+) are preferentially incorporated into Ca[I], whereas bigger ions (Sr2+, Ba2+, Pb2+) are incorporated into Ca[II] (Fig. 7). The reason why apatite is the mineral component of vertebral skeleton is not known, but it was shown that apatite is the only calcium-phosphate mineral phase that is stable at both a neutral and basic pH (Glimcher 2006; Omelon et al., 2009). Another explanation is coming from the presence of denses granules containing polyphosphates near the mineralizing cartilage and resorbing bone (Omelon et al., 2009). Indeed, when the mineral apatite is dissolved after acidification and resorption by osteoclasts, there is no reprecipitation within the resorption pits, even the return to a neutral pH. The hypothesis of the authors was that polyphosphates formation provides a mechanism for accumulating phosphate, controlling the apatite at locations sites previously mentioned. Enzymatic action can thus control apatite supersaturation at neutral pH, directly

Fig. 7. (Left) Hexagonal system of apatite lattice, showing the disposition of atoms. (Right) The Ca ions occupy two crystallographic non-equivalent sites (Ca I and Ca II). From (Reprinted from Li et al., 2007, with permission from Elsevier). Small ions (Cd2+, Zn2+ and Mg2+) are preferentially incorporated into Ca[I], whereas bigger ions (Sr2+, Ba2+ and Pb2+) are

and/or Cl-

2- (6-7 wt %)], minor elements such as

) (Posner 1969; Wopenka & Pasteris

3-(18 wt %)], carbonates [CO3

**4. Characteristics of bone mineral crystals** 

occupied by two monovalent anions (OH-

by controlling orthophosphate ion activity (Omelon et al., 2009).

%)], phosphate [PO4

incorporated into Ca[II].

#### **3.3 Bone microhardness at the tissue level**

Another important characteristic of bone mineral is its hardness (Currey 2003; Nalla et al., 2003). Thanks to indentation techniques, it has been shown that microhardness of bone osteon was strongly related to its mineral content (Amprino 1958; Bala et al., 2010; Boivin et al., 2008; Carlstrom 1954; Weaver 1966). From a mechanical point of view, microhardness parameter is related to both elastic and plastic deformations, and an indentation technique has been developed to measure directly both elastic modulus (*E*) and contact hardness (*Hc*) on small area of bone tissue (Oliver & Pharr 1992). However, this technique has been developed for isotropic materials. While it is known that the bone tissue is complex with an anisotropic structure, the measurements of *E* is usually performed with a defined Poisson's ratio (=0.3). It has been shown that contact hardness was linearly interdependent with elastic modulus (Oyen 2006). Therefore, contact hardness can give an evaluation of bone stiffness which is directly related to its brittleness. Contact hardness can be evaluated at the microstructural (BSU) or nanostructural (lamellar) levels. At the microstructural level in bone, the pyramidal square-based Vickers indenter is often used (Fig. 6) (Boivin et al., 2008), and nanoindentation is rather used at the lamellar level using Berckovich indenter (Ammann & Rizzoli 2003). Very recently, it has been shown, by instrumented nanoindentation, that contact hardness was correlated both to DMB and collagen maturity (Bala et al., 2011b). Mineralization is a major determinant of microhardness, with about two-thirds of the variance, and one-third being explained by the organic matrix (Boivin et al., 2008). In human control bone, the microhardness does not vary with age and sex, in cortical and cancellous bone, as for the degree of mineralization. In 19 human control bones, the hardness in cortical bone is about 49.30 2.16 kg/mm2 and in cancellous bone about 48.92 1.57 kg/mm2.

Fig. 6. Iliac bone from ewe showing a Bone Structural Units (BSU) with 4 Vickers indents (INSERM UMR 1033).

Another important characteristic of bone mineral is its hardness (Currey 2003; Nalla et al., 2003). Thanks to indentation techniques, it has been shown that microhardness of bone osteon was strongly related to its mineral content (Amprino 1958; Bala et al., 2010; Boivin et al., 2008; Carlstrom 1954; Weaver 1966). From a mechanical point of view, microhardness parameter is related to both elastic and plastic deformations, and an indentation technique has been developed to measure directly both elastic modulus (*E*) and contact hardness (*Hc*) on small area of bone tissue (Oliver & Pharr 1992). However, this technique has been developed for isotropic materials. While it is known that the bone tissue is complex with an anisotropic structure, the measurements of *E* is usually performed with a defined Poisson's ratio (=0.3). It has been shown that contact hardness was linearly interdependent with elastic modulus (Oyen 2006). Therefore, contact hardness can give an evaluation of bone stiffness which is directly related to its brittleness. Contact hardness can be evaluated at the microstructural (BSU) or nanostructural (lamellar) levels. At the microstructural level in bone, the pyramidal square-based Vickers indenter is often used (Fig. 6) (Boivin et al., 2008), and nanoindentation is rather used at the lamellar level using Berckovich indenter (Ammann & Rizzoli 2003). Very recently, it has been shown, by instrumented nanoindentation, that contact hardness was correlated both to DMB and collagen maturity (Bala et al., 2011b). Mineralization is a major determinant of microhardness, with about two-thirds of the variance, and one-third being explained by the organic matrix (Boivin et al., 2008). In human control bone, the microhardness does not vary with age and sex, in cortical and cancellous bone, as for the degree of mineralization. In 19 human control bones, the hardness in cortical bone is about

49.30 2.16 kg/mm2 and in cancellous bone about 48.92 1.57 kg/mm2.

Fig. 6. Iliac bone from ewe showing a Bone Structural Units (BSU) with 4 Vickers indents

(INSERM UMR 1033).

**3.3 Bone microhardness at the tissue level** 

### **4. Characteristics of bone mineral crystals**

Human bone mineral is a non-stoichiometric and poorly crystallized apatite. Bone apatite structure is hexagonal with space group P63/*m,* with lattice parameters a=9.42Å and c=6.88Å. It is a calcium (Ca)-deficient apatite analog, contains major elements like calcium, [Ca2+ (40 wt %)], phosphate [PO4 3-(18 wt %)], carbonates [CO3 2- (6-7 wt %)], minor elements such as magnesium (Mg2+) or sodium (Na2+), and trace elements (LeGeros & LeGeros 1983; LeGeros et al., 1968). Bone mineral also contains ions normally absent from body fluids (lead, fluoride, aluminium etc). Indeed, the apatite lattice is very tolerant to substitutions and vacancies. Compared to dental enamel, the bone mineral contains much more vacancies. In fact, apatite is able to incorporate itself, in its atomic structure, the half of the elements in the periodic chart (Wopenka & Pasteris 2005). Apatite lattice contains about 40 ions, and the unit cell is the smallest basic unit which is a sample of the entire lattice array (Glimcher 1998). In the apatite unit cell, four different types of crystallographic positions (or "sites") have been identified (Fig.7): (1) tetrahedral sites for six P5+-ions, each in 4-fold coordination with oxygen, (2) Ca[1] sites for four of the Ca2+ ions, (3) Ca[II] sites for the six other Ca2+ ions, and (4) the channel site, occupied by two monovalent anions (OH- , F and/or Cl- ) (Posner 1969; Wopenka & Pasteris 2005). The small ions (Cd2+, Zn2+, Mg2+) are preferentially incorporated into Ca[I], whereas bigger ions (Sr2+, Ba2+, Pb2+) are incorporated into Ca[II] (Fig. 7). The reason why apatite is the mineral component of vertebral skeleton is not known, but it was shown that apatite is the only calcium-phosphate mineral phase that is stable at both a neutral and basic pH (Glimcher 2006; Omelon et al., 2009). Another explanation is coming from the presence of denses granules containing polyphosphates near the mineralizing cartilage and resorbing bone (Omelon et al., 2009). Indeed, when the mineral apatite is dissolved after acidification and resorption by osteoclasts, there is no reprecipitation within the resorption pits, even the return to a neutral pH. The hypothesis of the authors was that polyphosphates formation provides a mechanism for accumulating phosphate, controlling the apatite at locations sites previously mentioned. Enzymatic action can thus control apatite supersaturation at neutral pH, directly by controlling orthophosphate ion activity (Omelon et al., 2009).

Fig. 7. (Left) Hexagonal system of apatite lattice, showing the disposition of atoms. (Right) The Ca ions occupy two crystallographic non-equivalent sites (Ca I and Ca II). From (Reprinted from Li et al., 2007, with permission from Elsevier). Small ions (Cd2+, Zn2+ and Mg2+) are preferentially incorporated into Ca[I], whereas bigger ions (Sr2+, Ba2+ and Pb2+) are incorporated into Ca[II].

Bone Mineral Quality 13

crystals replaced the water within the fibrils (Fratzl et al., 1993). The expansion of mineral crystals compressed the collagen molecule packing, thus decreasing the molecular spacing. This indicated the close relationship between water and the mineral deposition process. Modification of the collagen packing probably influences secondary structure of organic matrix. More recently, in intact bovine bone, the effects of dehydration have been studied using solid-state NMR spectroscopy (Zhu et al., 2009). Interestingly, well-resolved peaks broadened with dehydration, suggesting a conformational disorder and structural changes of bone matrix. This is in agreement with other studies showing a collagen conformational

Fig. 9. Electron micrograph of human cancellous bone (woman, 80 year-old) illustrating the

It was suggested that water could play a role in the mechanical behavior of cortical bone (Nyman et al., 2006) and the removal of water alter crystallographic structure of synthetic apatites (LeGeros et al., 1978). As the water content decreases in bone with age (Jonsson et al., 1985; Mueller et al., 1966), it was suggested that water could be involved in bone fragility. Three main types of water exist in bone: the freely mobile water located into vascular lacunar canalicular spaces, the water bound to the collagen network, and the water bound to the mineral. More precisely, there is two type of water bound to the mineral: (a) the water bound to the surface of bone crystals, and (b) the water located within the apatite lattice. Water bound to the collagen fibrils provides post-yield toughness to bone and when water was removed, the strength and stiffness was increased whereas the toughness was decreased (Nyman et al., 2006). The role of the loss of water bound to the mineral on bone

bone crystals within the type I collagen (INSERM UMR 1033).

change with dehydration (Naito et al, 1994 ; Saito et al, 1984, 1992).

#### **4.1 Bone crystal size and shape**

The crystal structure and morphology of bone minerals have often been controversial, mainly due to the different techniques used to characterize bone mineral. Today, with the use of more accurate method (atomic force microscopy, high resolution transmission electron microscopy), it is clear that the bone mineral crystals are very small and plateletshaped (length 200-600 Å, width 100-200 Å, thickness 20-50 Å, Figs. 8 and 9). Compared to bone crystals, enamel crystals are needle-shape and much bigger. This small bone crystal size has several advantages. First, it permits an extended surface area (100-200 m2/g). Two factors are involved in the surface activity: the surface area expressed in m2/g and the physical and chemical properties of the surface. These properties determine the type of reactions, while the surface area determines the number of reactions. The combination of both factors makes the bone mineral substance metabolically very active; consequently, crystals have a very large interface with extracellular fluids. For example, the crystals contain in a small lumbar vertebra (L1 or L2) having a dry wet of 30 g, have a specific surface comparable to that of the playing field of soccer. Bone mineral is metabolically active, various and numerous interactions between ions from the extracellular fluid and ions constituting apatite crystals, are thus possible. Second, another interest of the small crystal sizes is mechanic. Indeed, the highly ordered location and orientation of very small crystals within the collagen fibrils permits an acceptable range of flexibility without fracture or disruption of the bone substance (Glimcher 1998; Landis 1995).

Fig. 8. Schematic representation and crystal size of bone apatite with the 3 axis (INSERM UMR 1033)

#### **4.2 Relationships between water, organic matrix and mineral**

In bone, the process of crystal nucleation in bone matrix is heterogenous, and is formed within the "hole" band of type I collagen (670 Å) (Glimcher et al., 1957). During the process of mineralization, the apatite crystals replaced some of the molecules of water so their content is inversely proportional to that of water (Elliott & Robinson 1957; Robinson 1975). Once deposited, the mineral phase induces compaction of the collagen fibril structure. Neutron and X-ray diffraction have shown that the Bragg-spacing of collagen strongly decreases with increasing mineral content (Lees 1987). Computer modeling and SAXS confirmed the process of closer packing of the collagen molecules when clusters of mineral

The crystal structure and morphology of bone minerals have often been controversial, mainly due to the different techniques used to characterize bone mineral. Today, with the use of more accurate method (atomic force microscopy, high resolution transmission electron microscopy), it is clear that the bone mineral crystals are very small and plateletshaped (length 200-600 Å, width 100-200 Å, thickness 20-50 Å, Figs. 8 and 9). Compared to bone crystals, enamel crystals are needle-shape and much bigger. This small bone crystal size has several advantages. First, it permits an extended surface area (100-200 m2/g). Two factors are involved in the surface activity: the surface area expressed in m2/g and the physical and chemical properties of the surface. These properties determine the type of reactions, while the surface area determines the number of reactions. The combination of both factors makes the bone mineral substance metabolically very active; consequently, crystals have a very large interface with extracellular fluids. For example, the crystals contain in a small lumbar vertebra (L1 or L2) having a dry wet of 30 g, have a specific surface comparable to that of the playing field of soccer. Bone mineral is metabolically active, various and numerous interactions between ions from the extracellular fluid and ions constituting apatite crystals, are thus possible. Second, another interest of the small crystal sizes is mechanic. Indeed, the highly ordered location and orientation of very small crystals within the collagen fibrils permits an acceptable range of flexibility without fracture or

Fig. 8. Schematic representation and crystal size of bone apatite with the 3 axis (INSERM

In bone, the process of crystal nucleation in bone matrix is heterogenous, and is formed within the "hole" band of type I collagen (670 Å) (Glimcher et al., 1957). During the process of mineralization, the apatite crystals replaced some of the molecules of water so their content is inversely proportional to that of water (Elliott & Robinson 1957; Robinson 1975). Once deposited, the mineral phase induces compaction of the collagen fibril structure. Neutron and X-ray diffraction have shown that the Bragg-spacing of collagen strongly decreases with increasing mineral content (Lees 1987). Computer modeling and SAXS confirmed the process of closer packing of the collagen molecules when clusters of mineral

**4.1 Bone crystal size and shape** 

UMR 1033)

disruption of the bone substance (Glimcher 1998; Landis 1995).

**4.2 Relationships between water, organic matrix and mineral** 

crystals replaced the water within the fibrils (Fratzl et al., 1993). The expansion of mineral crystals compressed the collagen molecule packing, thus decreasing the molecular spacing. This indicated the close relationship between water and the mineral deposition process. Modification of the collagen packing probably influences secondary structure of organic matrix. More recently, in intact bovine bone, the effects of dehydration have been studied using solid-state NMR spectroscopy (Zhu et al., 2009). Interestingly, well-resolved peaks broadened with dehydration, suggesting a conformational disorder and structural changes of bone matrix. This is in agreement with other studies showing a collagen conformational change with dehydration (Naito et al, 1994 ; Saito et al, 1984, 1992).

Fig. 9. Electron micrograph of human cancellous bone (woman, 80 year-old) illustrating the bone crystals within the type I collagen (INSERM UMR 1033).

It was suggested that water could play a role in the mechanical behavior of cortical bone (Nyman et al., 2006) and the removal of water alter crystallographic structure of synthetic apatites (LeGeros et al., 1978). As the water content decreases in bone with age (Jonsson et al., 1985; Mueller et al., 1966), it was suggested that water could be involved in bone fragility. Three main types of water exist in bone: the freely mobile water located into vascular lacunar canalicular spaces, the water bound to the collagen network, and the water bound to the mineral. More precisely, there is two type of water bound to the mineral: (a) the water bound to the surface of bone crystals, and (b) the water located within the apatite lattice. Water bound to the collagen fibrils provides post-yield toughness to bone and when water was removed, the strength and stiffness was increased whereas the toughness was decreased (Nyman et al., 2006). The role of the loss of water bound to the mineral on bone

Bone Mineral Quality 15

significantly larger ions, but less frequently by smaller ions (Blumenthal 1990). An ion can only be substituted to another if its ionic radius is less than 10% higher than the radius of the ion

are stronger than the ones between Ca2+ and OH-

apatite is the fluoroapatite [chemical formula: Ca10(PO4)6F2]. To be resorbed by osteoclasts, the bone mineral have to be more soluble than hydroxyapatite, and the vacancies present in bone mineral enable this dissolution. Carbonates can be found in apatite crystals as CO3

Some foreign ions can increase or decrease the bone crystal size. In the case of Mg2+, *in vitro* studies have shown that Mg2+ bound to the hydroxyapatite crystals retarded nucleation and growth of the crystal. In vivo studies show a decrease in crystal size in Mg-deficient rats, thus Mg interferes with the mineralization process (Bigi et al., 1992; Blumenthal et al., 1977;

Others ions, as Fe3+ ions, have a direct effect on hydroxyapatite, inhibiting the growth and changing the quality of crystals (decrease in crystallinity and increase in carbonate substitution) (Guggenbuhl et al., 2008). Aluminium also affects bone mineralization, and osteomalacia renal osteodystrophy has been associated, in patients on long-term hemodialysis, with Al3+ accumulation in bone (Blumenthal & Posner 1984). A recent study on rats showed that a long-term Al3+ exposure reduces the levels of mineral and trace elements in bone (Zn, Fe, Cu, Mn, Se, B, and Sr) (Li et al., 2010b). This is accompagnied by a decrease in BMD especially in cancellous bone. An high amount of ions which are normally present in small proportion in bone mineral can cause alteration of bone substance. As previously mentioned, fluoride ions at high doses cause osteomalacia and defects of mineralization (Balena et al., 1998). On the other hand, small doses of some ions can have positive effect on bone strength. For example, Sr2+ (Strontium ranelate is an osteoporosis treatment) reduces both the vertebral and non vertebral fractures (Meunier et al., 2004; Reginster et al., 2005). Besides the effect of Sr2+ on bone cells (stimulating bone formation and decreasing bone resorption) (Grynpas & Marie 1990; Marie et al., 1993), the presence of Sr2+ is shown in the bone mineral formed during treatment, in osteoporotic women treated with strontium ranelate for 3-5 years (Boivin et al., 2010; Doublier et al, 2011a, b). The concentration of Sr2+ is very low, and do not exceed in human a maximum of 0.5 ions Sr2+ for 10 Ca2+ (Li et al., 2010a). Moreover, the thickness and length of the plate-shaped bone mineral crystals were not affected by the strontium ranelate treatment (Li et al., 2010a). Presence of Sr2+ causes no osteomalacia, no modification in the mineralization process or crystal size. However, the Sr2+ increases the bone strength, thus the presence of Sr2+ in bone

) around the crystal surface is also well known. The

. The more insoluble

2- ions increases,

2- ions,

ions can not be reversed because it leads to the formation of a more

ions. When the volume of CO3

ions are more similar to Ca2+ ions than OH- ions and the electrostatic links

3-, F-

3- or OH-

the *a* parameter of the crystal unit cell decreases, while the *c* parameter increases.

mineral has certainly positive effects, but this mechanism is to date unknown.

**4.4 Bone apatite: A particular structure, with a hydrated layer around an apatitic core**  The surface of bone crystals, formed in the water of extracellular fluid, exhibits a "hydrated layer" (Fig. 10). Ions in this layer are very labile and reactive, and constitute the non-apatitic domain, surrounding the relatively inert and more stable apatite domain of the bone crystal (Cazalbou et al., 2004; Termine et al., 1973). Newly deposited bone mineral contains many labile non-apatitic domains [HPO4, PO4, and CO3], located in the

replaced. The exchange of anions (PO4

which can substituted for either PO4

with OH-

substitution of F-

stable compound: F-

between Ca2+ and F-

Boskey et al., 1992).

strength was not clear in this study, while it was suggested that the loss of water located in apatite lattice could change the size of the bone mineral crystals (Nyman et al., 2006), since it was already observed in dehydrated enamel or precipitated apatites (LeGeros et al., 1978). Consequently, the decrease in bone strength and toughness related to age could be due to a change in water distribution.

### **4.3 Chemical composition of bone mineral**

The composition of bone mineral was for a long time assimilated to hydroxyapatite, but it is not. Several studies showed the lack of OH- , by inelastic neutron scattering, Raman of infrared spectroscopies (Loong et al., 2000; Pasteris et al., 2004; Rey et al., 1995b). Solid state-NMR shows that the percentage of OH– does not exceed 20% of the amount expected in stoichiometric apatite in human cortical bone (Cho et al., 2003). In fact, the small crystal size could be one of the reasons for the absence of OH ions in the bone apatite. Indeed, Wopenka and Pasteris have suggested that the small crystal size and the great the atomic disorder within the unit cells of the crystal was not energetically favourable for apatite for incorporate OH- into its channel (Pasteris et al., 2004). Another reason for the lack of OHions in bone apatite could be due to the type–B substitution of PO4 by CO3, creating a vacancy in the channel site to maintain electrostatic equilibrium.


Table 1. Chemical formula of different apatites (: vacancy) (adapted from Cazalbou et al., 2004a)

When an ion with the same electric charge is substituted within the bone apatite, no effect is produced on the structure lattice. If ions have a different electric charge (CO32- substituted for PO43-), a vacancy is created to maintained electrostatic equilibrium. Some ions can be replaced by other ions of almost identical radius. These ions induce only minor changes in shape at crystal level and do not affect the structure of the crystal. Such substitutions occur during formation of the crystal or through ionic exchanges with the existing crystals. In vitro, some interactions between mineral substance and the solution lead to the diffusion of ions within the hydrated shell, the exchanges at the crystal surface and the exchanges inside the crystal. Similar mechanisms are likely to occur in vivo during remodeling of bone tissue (Blumenthal 1990; LeGeros 1981; Posner 1985). Some cations of similar size and charge as the Ca2+ (Sr2+, Na+), as well as others that cannot substitute for the Ca2+ in the apatite structure (Ba2+, Ra2+, Mg2+, K+), are easily exchangeable from the solution to the surface Ca2+ ions. Such substitutions lead to modifications in the *a* and *c* parameters of the apatite unit cell. Substitutions with Mg2+ are only partial. In biomimetic apatite nanocrystal, the incorporation of Mg has been recently studied to analyze the effect of substitution with Ca2+ on particule morphology (Bertinetti et al., 2009). Incorporation of Mg2+ does not affect apatitic nature, nonetheless, a lower degree of crystallinity was observed by XRD (Bertinetti et al., 2009). Moreover, it was shown that apatites enriched with Mg2+ retain more water at their surface than Mg2+-free apatites (Bertinetti et al., 2009). Calcium can be easily substituted by

strength was not clear in this study, while it was suggested that the loss of water located in apatite lattice could change the size of the bone mineral crystals (Nyman et al., 2006), since it was already observed in dehydrated enamel or precipitated apatites (LeGeros et al., 1978). Consequently, the decrease in bone strength and toughness related to age could be due to a

The composition of bone mineral was for a long time assimilated to hydroxyapatite, but it is

infrared spectroscopies (Loong et al., 2000; Pasteris et al., 2004; Rey et al., 1995b). Solid state-NMR shows that the percentage of OH– does not exceed 20% of the amount expected in stoichiometric apatite in human cortical bone (Cho et al., 2003). In fact, the small crystal size could be one of the reasons for the absence of OH- ions in the bone apatite. Indeed, Wopenka and Pasteris have suggested that the small crystal size and the great the atomic disorder within the unit cells of the crystal was not energetically favourable for apatite for incorporate OH- into its channel (Pasteris et al., 2004). Another reason for the lack of OHions in bone apatite could be due to the type–B substitution of PO4 by CO3, creating a

> **Nature of apatite Chemical formula**  Hydroxyapatite Ca10 (PO4)6 (OH)2

Table 1. Chemical formula of different apatites (: vacancy) (adapted from Cazalbou et al.,

When an ion with the same electric charge is substituted within the bone apatite, no effect is produced on the structure lattice. If ions have a different electric charge (CO32- substituted for PO43-), a vacancy is created to maintained electrostatic equilibrium. Some ions can be replaced by other ions of almost identical radius. These ions induce only minor changes in shape at crystal level and do not affect the structure of the crystal. Such substitutions occur during formation of the crystal or through ionic exchanges with the existing crystals. In vitro, some interactions between mineral substance and the solution lead to the diffusion of ions within the hydrated shell, the exchanges at the crystal surface and the exchanges inside the crystal. Similar mechanisms are likely to occur in vivo during remodeling of bone tissue (Blumenthal 1990; LeGeros 1981; Posner 1985). Some cations of similar size and charge as the Ca2+ (Sr2+, Na+), as well as others that cannot substitute for the Ca2+ in the apatite structure (Ba2+, Ra2+, Mg2+, K+), are easily exchangeable from the solution to the surface Ca2+ ions. Such substitutions lead to modifications in the *a* and *c* parameters of the apatite unit cell. Substitutions with Mg2+ are only partial. In biomimetic apatite nanocrystal, the incorporation of Mg has been recently studied to analyze the effect of substitution with Ca2+ on particule morphology (Bertinetti et al., 2009). Incorporation of Mg2+ does not affect apatitic nature, nonetheless, a lower degree of crystallinity was observed by XRD (Bertinetti et al., 2009). Moreover, it was shown that apatites enriched with Mg2+ retain more water at their surface than Mg2+-free apatites (Bertinetti et al., 2009). Calcium can be easily substituted by

Bone mineral Ca8.31.7 (PO4)4.3 (HPO4) (CO3)1.7 (OH)0.31.7 Dental enamel Ca9.40.6 (PO4)5.4 (HPO4) (CO3)0.6 (OH)1.40.6

, by inelastic neutron scattering, Raman of

change in water distribution.

2004a)

**4.3 Chemical composition of bone mineral** 

not. Several studies showed the lack of OH-

vacancy in the channel site to maintain electrostatic equilibrium.

significantly larger ions, but less frequently by smaller ions (Blumenthal 1990). An ion can only be substituted to another if its ionic radius is less than 10% higher than the radius of the ion replaced. The exchange of anions (PO4 3-, F- ) around the crystal surface is also well known. The substitution of F with OH ions can not be reversed because it leads to the formation of a more stable compound: F ions are more similar to Ca2+ ions than OH- ions and the electrostatic links between Ca2+ and F are stronger than the ones between Ca2+ and OH- . The more insoluble apatite is the fluoroapatite [chemical formula: Ca10(PO4)6F2]. To be resorbed by osteoclasts, the bone mineral have to be more soluble than hydroxyapatite, and the vacancies present in bone mineral enable this dissolution. Carbonates can be found in apatite crystals as CO3 2- ions, which can substituted for either PO4 3- or OH ions. When the volume of CO3 2- ions increases, the *a* parameter of the crystal unit cell decreases, while the *c* parameter increases.

Some foreign ions can increase or decrease the bone crystal size. In the case of Mg2+, *in vitro* studies have shown that Mg2+ bound to the hydroxyapatite crystals retarded nucleation and growth of the crystal. In vivo studies show a decrease in crystal size in Mg-deficient rats, thus Mg interferes with the mineralization process (Bigi et al., 1992; Blumenthal et al., 1977; Boskey et al., 1992).

Others ions, as Fe3+ ions, have a direct effect on hydroxyapatite, inhibiting the growth and changing the quality of crystals (decrease in crystallinity and increase in carbonate substitution) (Guggenbuhl et al., 2008). Aluminium also affects bone mineralization, and osteomalacia renal osteodystrophy has been associated, in patients on long-term hemodialysis, with Al3+ accumulation in bone (Blumenthal & Posner 1984). A recent study on rats showed that a long-term Al3+ exposure reduces the levels of mineral and trace elements in bone (Zn, Fe, Cu, Mn, Se, B, and Sr) (Li et al., 2010b). This is accompagnied by a decrease in BMD especially in cancellous bone. An high amount of ions which are normally present in small proportion in bone mineral can cause alteration of bone substance. As previously mentioned, fluoride ions at high doses cause osteomalacia and defects of mineralization (Balena et al., 1998). On the other hand, small doses of some ions can have positive effect on bone strength. For example, Sr2+ (Strontium ranelate is an osteoporosis treatment) reduces both the vertebral and non vertebral fractures (Meunier et al., 2004; Reginster et al., 2005). Besides the effect of Sr2+ on bone cells (stimulating bone formation and decreasing bone resorption) (Grynpas & Marie 1990; Marie et al., 1993), the presence of Sr2+ is shown in the bone mineral formed during treatment, in osteoporotic women treated with strontium ranelate for 3-5 years (Boivin et al., 2010; Doublier et al, 2011a, b). The concentration of Sr2+ is very low, and do not exceed in human a maximum of 0.5 ions Sr2+ for 10 Ca2+ (Li et al., 2010a). Moreover, the thickness and length of the plate-shaped bone mineral crystals were not affected by the strontium ranelate treatment (Li et al., 2010a). Presence of Sr2+ causes no osteomalacia, no modification in the mineralization process or crystal size. However, the Sr2+ increases the bone strength, thus the presence of Sr2+ in bone mineral has certainly positive effects, but this mechanism is to date unknown.

#### **4.4 Bone apatite: A particular structure, with a hydrated layer around an apatitic core**

The surface of bone crystals, formed in the water of extracellular fluid, exhibits a "hydrated layer" (Fig. 10). Ions in this layer are very labile and reactive, and constitute the non-apatitic domain, surrounding the relatively inert and more stable apatite domain of the bone crystal (Cazalbou et al., 2004; Termine et al., 1973). Newly deposited bone mineral contains many labile non-apatitic domains [HPO4, PO4, and CO3], located in the

Bone Mineral Quality 17

Crystallinity is defined as the degree of structural order in a crystal. The atoms in the crystal are arranged in a regular and periodic manner. The referent method to measure *absolute crystallinity* is the X-rays diffraction, and is based on the elastic scattering of the X-rays. This technique can be used to determine the crystal structure (Le Bail & Loüer 1978; Rietveld 1969) or chemical composition of a sample through the power diffraction bank data file (Powder diffraction file). The WAXS (wide angle X-rays scattering) is based on scattering angles 2θ larger than 5°, and SAXS (small X-ray scattering) gives informations on angles 2θ close to 0°. This method gives informations on the size, strain and orientation of crystals. A peak broadening can be due the small crystal size or microstrains. The crystal size can be

Vibrational spectroscopy techniques, such as Fourier Transform InfraRed Spectroscopy (FTIRS), Synchrotron InfraRed or Raman Spectroscopy, have been extensively used to study calcified tissues (Ager et al., 2005; Akkus et al., 2003; Boskey et al., 2005; Cazalbou et al., 2004; Farlay et al., 2010b; LeGeros 1981; Miller et al., 2001; Paschalis et al., 1996; Pleshko et al., 1991; Rey et al., 1990, 1991). Spectroscopic techniques allow assessment of physicochemical modifications of mineral induced by mechanical tests (Ager et al., 2005; Akkus et al., 2003; Carden et al., 2003; Morris & Mandair 2011; Tarnowski et al., 2004), agerelated modifications (Ager et al., 2005; Akkus et al., 2003; Miller et al., 2007), and pathologic or treatment-related changes (Boskey et al., 2005; Carden & Morris 2000; Fratzl 2004; Huang et al., 2003; Siris et al., 2004). The application of Fourier Transform InfraRed Microspectroscopy and Imaging (FTIRM, FTIRI) for bone allows in situ analysis of embedded bone samples at the BSU level. These techniques are based on the vibrations of the atoms of a molecule and give complementary informations. The functional groups present in the sample absorb infrared light (or scatter light for Raman) at different wavelengths. In bone, infrared and Raman spectroscopies were used to obtain information on bone mineral and organic matrix, the two major components of bone. The main advantage of these techniques is that they can be used on embedded bone biopsies, on thin sections (2µm-thick MMA sections), keeping the integrity of the bone microarchitecture. By infrared spectroscopy, different vibrations can be analyzed in bone mineral: the phosphate mode vibrations with the 13PO4 corresponding to an antisymmetric stretch, the 4PO4 (bending stretch), the carbonate vibrations 2CO3 and 3CO3. Organic matrix can be analyzed through the Amides I, II and III vibrations. Different parameters can be deduced from these vibrations, as the mineral maturity, crystallinity, carbonate substitution, mineral to matrix ratio, providing informations on bone mineral quality (Boskey et al., 1998, 2005; Miller et al., 2007; Paschalis et al., 1996, 1997a; 1997b; Paschalis & Mendelsohn 2000, Pleshko et al., 1991, Rey et al., 1989, 1995a). By Raman spectroscopy, crystallinity, carbonate type B substitution, and mineral to matrix ratio can be determined (Akkus et al., 2003; Morris & Mandair 2011). Others techniques as vibrational methods have been extensively used to obtain informations on the structural and compound identification, especially on the relative crystallinity (also

As mentioned previously, with maturation, stable apatitic domains grow, whereas hydrated layer decreases. Thus, when bone crystals mature, their crystallinity also increases. These two variables can be assessed separately in infrared spectroscopy (Fig.11). Mineral maturity and mineral crystallinity are two parameters temporally linked and often well correlated in

**4.5 Measurement of mineral crystallinity** 

determined by the *Scherrer* equation.

called crystallinity index).

**4.6 Maturity and crystallinity of bone crystals** 

well-developed hydrated layer involved in the high surface reactivity of mineral (Cazalbou et al., 2004). Labile PO4 and CO3 groups are easily and reversibly exchangeable with other ions in the hydrated layer. During maturation, the decrease in labile nonapatitic environments is associated with an increase in stable apatitic environments (Cazalbou et al., 2004). A particularity of the bone mineral is its non-stoichiometry, leading to the presence of numerous vacancies in the apatite crystal. Consequently, bone crystal is mainly maintained by electrostatic cohesion, thus bone crystals are easily soluble relative to stoichiometric apatite (Barry et al., 2002) . As bone becomes more mature, both the size and number of crystals increase.

Fig. 10. Evolution of the hydrated layer and apatite core from bone crystal. During the maturation and growth of the crystal, the hydrated layer, involved in a high surface reactivity, progressively decreases and led to a stable apatitic domain. The structure of the hydrated layer constitutes a pool of loosely bound ions which can be incorporated in the growing apatite domains and can be exchanged by foreign ions in the solution and charged groups of proteins (Pr) (Adapted from Rey et al., 2009).

During mineral maturation, the hydrated layer decreases while the stable apatite domain grow, corresponding to the evolution of non-apatitic environments into apatitic environments detected by Fourier Transform InfraRed spectroscopy (FTIR). This hydrated layer, different from a hydration layer (Stern double layer), corresponds to the mode of formation of apatite crystals in physiologic conditions. The existence of these two domains (hydrated layer and apatite core) in biomimetic nanocrystals, has been recently confirmed by solid-state NMR (Jager et al., 2006). The hydrated surface layer contains loosely bound ions, which are easily exchangeable, and determine the surface properties of the nanocrystalline apatites (Cazalbou et al., 2004; Termine et al., 1973). In bone, those loosely bound ions can be also exchanged with charged groups of proteins present in collagen and non-collagenous proteins. The role of charged proteins on mineralization is well known (Boskey et al, 1989, 1998; Boskey 1989; Georges & Veis 2008; Landis et al., 1993; Malaval et al., 2008; Traub et al., 1992).

well-developed hydrated layer involved in the high surface reactivity of mineral (Cazalbou et al., 2004). Labile PO4 and CO3 groups are easily and reversibly exchangeable with other ions in the hydrated layer. During maturation, the decrease in labile nonapatitic environments is associated with an increase in stable apatitic environments (Cazalbou et al., 2004). A particularity of the bone mineral is its non-stoichiometry, leading to the presence of numerous vacancies in the apatite crystal. Consequently, bone crystal is mainly maintained by electrostatic cohesion, thus bone crystals are easily soluble relative to stoichiometric apatite (Barry et al., 2002) . As bone becomes more mature, both

Fig. 10. Evolution of the hydrated layer and apatite core from bone crystal. During the maturation and growth of the crystal, the hydrated layer, involved in a high surface reactivity, progressively decreases and led to a stable apatitic domain. The structure of the hydrated layer constitutes a pool of loosely bound ions which can be incorporated in the growing apatite domains and can be exchanged by foreign ions in the solution and charged

During mineral maturation, the hydrated layer decreases while the stable apatite domain grow, corresponding to the evolution of non-apatitic environments into apatitic environments detected by Fourier Transform InfraRed spectroscopy (FTIR). This hydrated layer, different from a hydration layer (Stern double layer), corresponds to the mode of formation of apatite crystals in physiologic conditions. The existence of these two domains (hydrated layer and apatite core) in biomimetic nanocrystals, has been recently confirmed by solid-state NMR (Jager et al., 2006). The hydrated surface layer contains loosely bound ions, which are easily exchangeable, and determine the surface properties of the nanocrystalline apatites (Cazalbou et al., 2004; Termine et al., 1973). In bone, those loosely bound ions can be also exchanged with charged groups of proteins present in collagen and non-collagenous proteins. The role of charged proteins on mineralization is well known (Boskey et al, 1989, 1998; Boskey 1989; Georges & Veis 2008; Landis et al., 1993; Malaval et

groups of proteins (Pr) (Adapted from Rey et al., 2009).

al., 2008; Traub et al., 1992).

the size and number of crystals increase.

#### **4.5 Measurement of mineral crystallinity**

Crystallinity is defined as the degree of structural order in a crystal. The atoms in the crystal are arranged in a regular and periodic manner. The referent method to measure *absolute crystallinity* is the X-rays diffraction, and is based on the elastic scattering of the X-rays. This technique can be used to determine the crystal structure (Le Bail & Loüer 1978; Rietveld 1969) or chemical composition of a sample through the power diffraction bank data file (Powder diffraction file). The WAXS (wide angle X-rays scattering) is based on scattering angles 2θ larger than 5°, and SAXS (small X-ray scattering) gives informations on angles 2θ close to 0°. This method gives informations on the size, strain and orientation of crystals. A peak broadening can be due the small crystal size or microstrains. The crystal size can be determined by the *Scherrer* equation.

Vibrational spectroscopy techniques, such as Fourier Transform InfraRed Spectroscopy (FTIRS), Synchrotron InfraRed or Raman Spectroscopy, have been extensively used to study calcified tissues (Ager et al., 2005; Akkus et al., 2003; Boskey et al., 2005; Cazalbou et al., 2004; Farlay et al., 2010b; LeGeros 1981; Miller et al., 2001; Paschalis et al., 1996; Pleshko et al., 1991; Rey et al., 1990, 1991). Spectroscopic techniques allow assessment of physicochemical modifications of mineral induced by mechanical tests (Ager et al., 2005; Akkus et al., 2003; Carden et al., 2003; Morris & Mandair 2011; Tarnowski et al., 2004), agerelated modifications (Ager et al., 2005; Akkus et al., 2003; Miller et al., 2007), and pathologic or treatment-related changes (Boskey et al., 2005; Carden & Morris 2000; Fratzl 2004; Huang et al., 2003; Siris et al., 2004). The application of Fourier Transform InfraRed Microspectroscopy and Imaging (FTIRM, FTIRI) for bone allows in situ analysis of embedded bone samples at the BSU level. These techniques are based on the vibrations of the atoms of a molecule and give complementary informations. The functional groups present in the sample absorb infrared light (or scatter light for Raman) at different wavelengths. In bone, infrared and Raman spectroscopies were used to obtain information on bone mineral and organic matrix, the two major components of bone. The main advantage of these techniques is that they can be used on embedded bone biopsies, on thin sections (2µm-thick MMA sections), keeping the integrity of the bone microarchitecture. By infrared spectroscopy, different vibrations can be analyzed in bone mineral: the phosphate mode vibrations with the 13PO4 corresponding to an antisymmetric stretch, the 4PO4 (bending stretch), the carbonate vibrations 2CO3 and 3CO3. Organic matrix can be analyzed through the Amides I, II and III vibrations. Different parameters can be deduced from these vibrations, as the mineral maturity, crystallinity, carbonate substitution, mineral to matrix ratio, providing informations on bone mineral quality (Boskey et al., 1998, 2005; Miller et al., 2007; Paschalis et al., 1996, 1997a; 1997b; Paschalis & Mendelsohn 2000, Pleshko et al., 1991, Rey et al., 1989, 1995a). By Raman spectroscopy, crystallinity, carbonate type B substitution, and mineral to matrix ratio can be determined (Akkus et al., 2003; Morris & Mandair 2011). Others techniques as vibrational methods have been extensively used to obtain informations on the structural and compound identification, especially on the relative crystallinity (also called crystallinity index).

#### **4.6 Maturity and crystallinity of bone crystals**

As mentioned previously, with maturation, stable apatitic domains grow, whereas hydrated layer decreases. Thus, when bone crystals mature, their crystallinity also increases. These two variables can be assessed separately in infrared spectroscopy (Fig.11). Mineral maturity and mineral crystallinity are two parameters temporally linked and often well correlated in

Bone Mineral Quality 19

was very close to the kinetic of the degree of mineralization, whereas the crystallinity index was biphasic, showing a rapid increase for the six first months, then stabilizing until 18 months, and showing another increase toward highest values after 24 months (Fig.12). In another study performed in women long term-treated by bisphosphonates, an increase in mineral maturity was observed, associated with a decrease in crystallinity, reinforcing the statement that mineral maturity and crystallinity have to be cautiously separately analyzed

Fig. 12. Histograms showing (left) the rapid increase of mineral maturity during the 6 first months of mineralization, followed by a slowdown and stabilization; (right) the biphasic evolution of mineral crystallinity index, with an increase the 6 first months, a stabilization, and then a resumption at 18 months (Reprinted from Bala et al., 2010, with permission from

Finally, in a study on 53 human vertebrae, it was shown that with age of donor, crystallinity was increased and mineral maturity unchanged (Farlay et al., 2010a). This increase in crystallinity was previously shown in osteoporotic bone (Boskey, 2003), suggesting that with aging, an increase in crystal size/perfection occurs, independently of the bone remodeling

In bone mineral, carbonates ions represent about 6-7% of the total mineral ions, thus a non

apatite lattice either to PO43- (major site, type-B carbonate) or to OH- (minor site, type-A carbonate). A third site of CO32- ions corresponds to labile carbonates which decrease with the maturation of the apatite crystal (Rey et al., 1989, 1991). The carbonates decrease the regularity of the atomic arrangement in hydroxyapatite (Blumenthal et al., 1975), thus altering the crystallinity. CO32- determine physical properties of materials, or biological behavior of cells in scaffold or ceramics. The CO32-A/B ratio is very constant among

2- can be incorporated into bone mineral by substitution in the

(Bala et al., 2011).

Elsevier).

(Farlay et al., 2010a).

**4.7 Carbonates in bone mineral** 

negligible proportion. CO3

synthetic apatite. The ratio 1030/1020 cm-1 (apatitic phosphate over non apatitic phosphate, ν3PO4 vibration) was well correlated in synthetic apatites to the crystallinity measured by XRD, and it was established that the ratio 1030/1020 cm-1 was an index of mineral maturity/crystallinity. From that, mineral maturity and crystallinity were associated in a lot of studies (Boskey et al., 2005; Paschalis et al., 1996, 1997a, 1997b). We have defined a new ratio to assess mineral maturity, the 1030/1110 cm-1, (apatitic phosphate over non apatitic phosphate, ν3PO4 vibration) equivalent to the 1030/1020 cm-1 but more sensitive (Farlay et al., 2010b). We agree with the fact that the 1020/1030 cm-1 ratio, which is an index of mineral maturity, evolves simultaneously with crystallinity in synthetic samples or normal bone. However, by definition, those two parameters are different, as mineral maturity corresponds to a stage of maturation, and crystallinity corresponds to the organization of the apatite lattice. We have defined a new mineral crystallinity index, measured by FTIRM on the peak 604 cm-1 (bending vibration of phosphates) (Farlay et al., 2010b). This vibration is often inaccessible for microscopic infrared imaging, due to the cut-off of the detector. A wide band detector allows the access to this vibration and we have shown that the value of the full width at half maximum of the 604 cm-1 peak, was inversely correlated to the crystallinity (Farlay et al., 2010b). This crystallinity index is well correlated, on the same samples, with another crystallinity index measured on the same vibration, the Shemesh ratio (Shemesh 1990) which is itself derived from the splitting factor of Termine & Posner, 1966. In human bone, it has been shown that those two parameters could evolve separately, and thus can independently affect the mineral characteristics. Indeed, mineral maturity can be affected by modification of bone remodeling (and by formative or antiresorptive treatments), whereas crystallinity can be influenced by ionic substitutions. This was verified in bone samples from patients with skeletal fluorosis (Farlay et al., 2010b). Skeletal fluorosis is a pathology caused by an excessive consumption of fluoride, and characterized by ionic substitution of hydroxyl ions by fluoride ions in bone mineral. In these samples, mineral maturity was decreased, due to a stimulation of osteoblasts on bone formation by fluoride, but crystallinity was increased due to the substitution of OH- by F-.

Fig. 11. Infrared spectra of human cortical bone (iliac bone) showing the mineral (13PO4, 4PO4,2CO3) and the organic (amides) vibrations (INSERM UMR 1033).

In the study previously mentioned for determining the chronology of secondary mineralization on ewes (Bala et al., 2010), it has been shown that kinetic of mineral maturity

synthetic apatite. The ratio 1030/1020 cm-1 (apatitic phosphate over non apatitic phosphate, ν3PO4 vibration) was well correlated in synthetic apatites to the crystallinity measured by XRD, and it was established that the ratio 1030/1020 cm-1 was an index of mineral maturity/crystallinity. From that, mineral maturity and crystallinity were associated in a lot of studies (Boskey et al., 2005; Paschalis et al., 1996, 1997a, 1997b). We have defined a new ratio to assess mineral maturity, the 1030/1110 cm-1, (apatitic phosphate over non apatitic phosphate, ν3PO4 vibration) equivalent to the 1030/1020 cm-1 but more sensitive (Farlay et al., 2010b). We agree with the fact that the 1020/1030 cm-1 ratio, which is an index of mineral maturity, evolves simultaneously with crystallinity in synthetic samples or normal bone. However, by definition, those two parameters are different, as mineral maturity corresponds to a stage of maturation, and crystallinity corresponds to the organization of the apatite lattice. We have defined a new mineral crystallinity index, measured by FTIRM on the peak 604 cm-1 (bending vibration of phosphates) (Farlay et al., 2010b). This vibration is often inaccessible for microscopic infrared imaging, due to the cut-off of the detector. A wide band detector allows the access to this vibration and we have shown that the value of the full width at half maximum of the 604 cm-1 peak, was inversely correlated to the crystallinity (Farlay et al., 2010b). This crystallinity index is well correlated, on the same samples, with another crystallinity index measured on the same vibration, the Shemesh ratio (Shemesh 1990) which is itself derived from the splitting factor of Termine & Posner, 1966. In human bone, it has been shown that those two parameters could evolve separately, and thus can independently affect the mineral characteristics. Indeed, mineral maturity can be affected by modification of bone remodeling (and by formative or antiresorptive treatments), whereas crystallinity can be influenced by ionic substitutions. This was verified in bone samples from patients with skeletal fluorosis (Farlay et al., 2010b). Skeletal fluorosis is a pathology caused by an excessive consumption of fluoride, and characterized by ionic substitution of hydroxyl ions by fluoride ions in bone mineral. In these samples, mineral maturity was decreased, due to a stimulation of osteoblasts on bone formation by fluoride,

but crystallinity was increased due to the substitution of OH- by F-.

Fig. 11. Infrared spectra of human cortical bone (iliac bone) showing the mineral (13PO4,

In the study previously mentioned for determining the chronology of secondary mineralization on ewes (Bala et al., 2010), it has been shown that kinetic of mineral maturity

4PO4,2CO3) and the organic (amides) vibrations (INSERM UMR 1033).

was very close to the kinetic of the degree of mineralization, whereas the crystallinity index was biphasic, showing a rapid increase for the six first months, then stabilizing until 18 months, and showing another increase toward highest values after 24 months (Fig.12). In another study performed in women long term-treated by bisphosphonates, an increase in mineral maturity was observed, associated with a decrease in crystallinity, reinforcing the statement that mineral maturity and crystallinity have to be cautiously separately analyzed (Bala et al., 2011).

Fig. 12. Histograms showing (left) the rapid increase of mineral maturity during the 6 first months of mineralization, followed by a slowdown and stabilization; (right) the biphasic evolution of mineral crystallinity index, with an increase the 6 first months, a stabilization, and then a resumption at 18 months (Reprinted from Bala et al., 2010, with permission from Elsevier).

Finally, in a study on 53 human vertebrae, it was shown that with age of donor, crystallinity was increased and mineral maturity unchanged (Farlay et al., 2010a). This increase in crystallinity was previously shown in osteoporotic bone (Boskey, 2003), suggesting that with aging, an increase in crystal size/perfection occurs, independently of the bone remodeling (Farlay et al., 2010a).

#### **4.7 Carbonates in bone mineral**

In bone mineral, carbonates ions represent about 6-7% of the total mineral ions, thus a non negligible proportion. CO32- can be incorporated into bone mineral by substitution in the apatite lattice either to PO43- (major site, type-B carbonate) or to OH- (minor site, type-A carbonate). A third site of CO3 2- ions corresponds to labile carbonates which decrease with the maturation of the apatite crystal (Rey et al., 1989, 1991). The carbonates decrease the regularity of the atomic arrangement in hydroxyapatite (Blumenthal et al., 1975), thus altering the crystallinity. CO32- determine physical properties of materials, or biological behavior of cells in scaffold or ceramics. The CO3 2-A/B ratio is very constant among

Bone Mineral Quality 21

sex, age, ethnicity or skeletal sites in BMDD measurements (22% in w% Ca) (Roschger et al., 2003). A recent study on vertebral bone of 53 donors (age range: 54-95 year-old, 21 men,

Fig. 13. Quantitative microradiography: (top) aluminium step wedge and microradiograph of bone tissue. (bottom) Distribution of the degrees of mineralization in human iliac bone

**5.1.2 Bone mineralization at tissue level in bone of post-menopausal women treated** 

In adult bone, the DMB, i.e., the bone mineral density at the tissue level depends on the rate of remodeling. Thus, agents (parathyroid hormone) or events (menopause, ovariectomy) which provoke an augmentation in the « birthrate » or activation frequency of Basic Multicellular Units (BMUs), induce a decrease of the « lifespan » of BSUs, in other words on the time available for the secondary mineralization. This leads to the fact that new BSUs are resorbed before they have fully completed their secondary mineralization, as proven by the presence of a large amount of uncompletely mineralized BSUs and a low mean DMB (Arlot

(INSERM UMR 1033).

**with anti-osteoporotic treatments** 

27 women) showed no influence of sex and age on the DMB (Follet et al., 2010).

different species, suggesting that CO32- incorporation in bone is a highly regulated process. Carbonates are also important for mineral dissolution (LeGeros 1981). It is well-established in literature that, in synthetic apatites, CO32- content increases with time of maturation (Cazalbou et al., 2004b; LeGeros & LeGeros 1983; LeGeros et al., 1968; Rey et al., 1989, 1995a). However, in bone, there is a controversy with some studies showing an increase of CO3/PO4 with mineral maturation (Petra et al., 2005) or a decrease (Farlay et al., 2006; Ou-Yang et al., 2001; Paschalis et al., 1996). In very young bone, labile carbonates and HPO4 are high, and progressively decreased with time of maturation, this being associated with an increase in mineral crystallinity (Magne et al., 2001). The amount of carbonate ions can be measured by different methods (chemical dosage, thermogravimetric analysis, vibrational spectroscopies…). In infrared spectroscopy, carbonate content is generally determined using the ν2CO3 line (out-of-plane bending vibration) which is free of contribution of amide vibrations, and was generally calculated as CO3/PO4 area ratio. The role of carbonate in bone mineral is not entirely elucidated. The hypothesis of Wopenka and Pasteris is that carbonate could control bone crystal size, and that high concentration of carbonate could play an important role in constraining bone crystals to the nanometer scale (Wopenka & Pasteris 2005). Indeed, dentin, in which the amount of carbonate concentration is the same than in bone, has also a crystal size similar to bone. However, enamel crystals, which are very much larger size, have only about half the carbonate concentration as bone does.

### **5. Modifications of bone mineral characteristics with age or in post-menopausal osteoporosis**

Bone loss occurs in all individuals after middle life. This bone loss can be moderated (osteopenia), or can become more important (osteoporosis). Osteoporosis is a metabolic bone disease, leading to a decrease in the amount of mineralized bone and an increase in the risk of fracture. In post menopausal osteoporosis, the oestrogen deficiency lead to an acceleration of bone remodeling, the balance between resorption and formation is disturbed in favour of resorption. This lead to a decrease in the more mineralized bone, which is only partially replaced by young less mineralized bone, reducing material stiffness.

### **5.1 Modifications of bone mineral at tissue level**

Several studies performed in bone from osteoporotic women have shown not only that bone mass is decreased but that the bone mineral content was decreased, due to the fact the newly formed BSUs have not the time to achieve their mineralization before to be resorbed. Thus the mean age of matrix is decreased, leading to a decrease to the mean degree of mineralization.

#### **5.1.1 Degree of mineralization (DMB) in control humans (Boivin et al., 2008)**

Bone samples from persons of both sexes (sudden died and without known pathology) were studied. This control group was composed of iliac bone samples taken at necropsy form 30 women (aged 48.4 3.7 years; range 20-93 years) and 13 men (aged 66.0 4.4 years; range 43-86 years). The mean DMB expressed in g mineral/cm3 (mean SEM) was 1.082 0.017 in cortical bone and 1.099 0.018 in cancellous bone (Figure 13).

In iliac crests, no significant influence of sex or age on the mean DMB was observed (Boivin et al., 2008). In cancellous bone, another study by qBEI showed the absence of influence of

different species, suggesting that CO32- incorporation in bone is a highly regulated process. Carbonates are also important for mineral dissolution (LeGeros 1981). It is well-established in literature that, in synthetic apatites, CO32- content increases with time of maturation (Cazalbou et al., 2004b; LeGeros & LeGeros 1983; LeGeros et al., 1968; Rey et al., 1989, 1995a). However, in bone, there is a controversy with some studies showing an increase of CO3/PO4 with mineral maturation (Petra et al., 2005) or a decrease (Farlay et al., 2006; Ou-Yang et al., 2001; Paschalis et al., 1996). In very young bone, labile carbonates and HPO4 are high, and progressively decreased with time of maturation, this being associated with an increase in mineral crystallinity (Magne et al., 2001). The amount of carbonate ions can be measured by different methods (chemical dosage, thermogravimetric analysis, vibrational spectroscopies…). In infrared spectroscopy, carbonate content is generally determined using the ν2CO3 line (out-of-plane bending vibration) which is free of contribution of amide vibrations, and was generally calculated as CO3/PO4 area ratio. The role of carbonate in bone mineral is not entirely elucidated. The hypothesis of Wopenka and Pasteris is that carbonate could control bone crystal size, and that high concentration of carbonate could play an important role in constraining bone crystals to the nanometer scale (Wopenka & Pasteris 2005). Indeed, dentin, in which the amount of carbonate concentration is the same than in bone, has also a crystal size similar to bone. However, enamel crystals, which are very much larger size, have only about half the carbonate concentration as bone does.

**5. Modifications of bone mineral characteristics with age or in** 

partially replaced by young less mineralized bone, reducing material stiffness.

**5.1.1 Degree of mineralization (DMB) in control humans (Boivin et al., 2008)** 

Bone loss occurs in all individuals after middle life. This bone loss can be moderated (osteopenia), or can become more important (osteoporosis). Osteoporosis is a metabolic bone disease, leading to a decrease in the amount of mineralized bone and an increase in the risk of fracture. In post menopausal osteoporosis, the oestrogen deficiency lead to an acceleration of bone remodeling, the balance between resorption and formation is disturbed in favour of resorption. This lead to a decrease in the more mineralized bone, which is only

Several studies performed in bone from osteoporotic women have shown not only that bone mass is decreased but that the bone mineral content was decreased, due to the fact the newly formed BSUs have not the time to achieve their mineralization before to be resorbed. Thus the mean age of matrix is decreased, leading to a decrease to the mean degree of

Bone samples from persons of both sexes (sudden died and without known pathology) were studied. This control group was composed of iliac bone samples taken at necropsy form 30 women (aged 48.4 3.7 years; range 20-93 years) and 13 men (aged 66.0 4.4 years; range 43-86 years). The mean DMB expressed in g mineral/cm3 (mean SEM) was 1.082 0.017 in

In iliac crests, no significant influence of sex or age on the mean DMB was observed (Boivin et al., 2008). In cancellous bone, another study by qBEI showed the absence of influence of

**post-menopausal osteoporosis** 

mineralization.

**5.1 Modifications of bone mineral at tissue level** 

cortical bone and 1.099 0.018 in cancellous bone (Figure 13).

sex, age, ethnicity or skeletal sites in BMDD measurements (22% in w% Ca) (Roschger et al., 2003). A recent study on vertebral bone of 53 donors (age range: 54-95 year-old, 21 men, 27 women) showed no influence of sex and age on the DMB (Follet et al., 2010).

Fig. 13. Quantitative microradiography: (top) aluminium step wedge and microradiograph of bone tissue. (bottom) Distribution of the degrees of mineralization in human iliac bone (INSERM UMR 1033).

#### **5.1.2 Bone mineralization at tissue level in bone of post-menopausal women treated with anti-osteoporotic treatments**

In adult bone, the DMB, i.e., the bone mineral density at the tissue level depends on the rate of remodeling. Thus, agents (parathyroid hormone) or events (menopause, ovariectomy) which provoke an augmentation in the « birthrate » or activation frequency of Basic Multicellular Units (BMUs), induce a decrease of the « lifespan » of BSUs, in other words on the time available for the secondary mineralization. This leads to the fact that new BSUs are resorbed before they have fully completed their secondary mineralization, as proven by the presence of a large amount of uncompletely mineralized BSUs and a low mean DMB (Arlot

Bone Mineral Quality 23

physico-chemical composition, some differences appears. However, the techniques were different (FTIR and Raman microspectroscopies), and the bones were not extracted from the

Different factors increase the risk of fracture, independent of bone mineral density. Among these factors, bone mineral features are important in determining some mechanical behavior, especially elastic properties. Besides the decrease in bone mineral volume occurring with age, the alteration of mineral quality plays also a major role in bone fragility. The study of bone mineral quality is a instrumental challenge, due to [1] the dimension and the nature itself of crystal apatite (nanocrystalline), [2] the fact that bone is a composite material (organic matrix intimately linked to bone mineral), and [3] because bone is a

Thus, the using of different complementary techniques (spectroscopic, diffraction, electron microscopy techniques) has been very useful in the comprehension of bone mineral structure the last 50 years. Despite very important findings, there are still lacks in the understanding of the role of certain ions (minor or trace elements) in the biological or

Bone is a fascinating tissue governed by the remodeling activity and the plasma composition. It underlies several characteristics which are interdependent. For example, carbonate ions substitutions into apatite promote the platelet-shape in bone crystals rather than needle-shape found in enamel. But the presence of carbonates, moreover, as mentioned

between different variables is still indispensable to understand the relationship between

From the previous studies performed on bone mineral, it seems that a small crystal size associated with a heterogeneity of crystal size (nanometer size-scale) is important to an optimal mechanical strength. Indeed, the presence of too large crystal is not good for bone strength. Fluoride, as mentioned previously, induces the formation of large crystals, and the formation of a "brittle" bone, despite a high BMD. Moreover, concerning mineralization and its heterogeneity at the tissue level, values comprised between 0.8 and 1.30 g/cm3 of bone could be a great deal to maintain stiffness of the bone. The preservation of mineralization heterogeneity is necessary for bone mechanical strength, allowing, for example, the stopping of microcracks propagation. No modification in degree of mineralization is observed with age or sex, but some differences exist in hip- fracture or osteoporotic cases. Thus a modification in the calcium absorption or an increase in the bone remodeling can lead to a decrease to the degree of mineralization. Differences also exist in studies in degree of mineralization due to the fact that either weight or non-weight-bearing bones are analyzed. We have an ongoing study on these different types of bone (weight or non-weight-bearing bones) in a same donor, in order to analyze the structural and mechanical bone mineral

In conclusion, despite a lot of studies have permitted to understand the characteristics of bone mineral, further studies are needed to clarify the mechanisms of bone fragility at the

in bone apatite. Thus, the study of correlations

complex tissue with different levels of organization (organ, BSU, crystal).

biomechanical properties (such as carbonates, strontium ions etc.).

previously, inhibits the incorporation of OH-

physicochemical and mechanical properties.

same donors.

properties.

different levels of investigation.

**6. Perspectives and conclusion** 

et al., 2005; Boivin & Meunier 2003; Misof et al., 2003). In a study on men and women with idiopathic osteoporosis, a decrease in DMB was observed compared to controls (-7%) (Boivin et al., 2008). However, a decrease in heterogeneity index of mineralization was observed in this study, indicating a homogeneization of the mineralization. This decrease is also accompanied, in men only, by a decrease in microhardness (-10%) (Boivin et al., 2008). However, it is important to mention that, both in control or osteoporotic bone, no variation in DMB or microhardness was correlated with age. Conversely, antiresorptive agents (bisphosphonates, calcitonin, estrogen, SERMs) which cause a marked reduction in the « birthrate » of BMUs, prolong the « lifespan » of the BSUs, allowing a more complete secondary mineralization and an increase of DMB (Bala et al., 2011a, 2011b; Boivin et al., 2000, 2003; Borah et al., 2005, 2006). A dissociating agent (Strontium Ranelate), acting both on resorption and formation, is now used in the treatment of post-menopausal osteoporosis (Meunier et al., 2004; Reginster et al., 2005). After short- and long-term treatment, the DMB was not significantly different from the physiological range (Boivin et al., 2010; Doublier et al., 2011a). Recently, also with a microradiographic method, it has been shown, in hipfracture patients, an increase in DMB heterogeneity in both interstitial and osteons compared to controls (Bousson et al., 2011). Thus, some differences exist, considering osteoporotic or hip-fractures cases, in term of heterogeneity of mineralization.

The mineralization index measured by FTIRM, calculated as the ratio of 13PO4/amide I vibration (Paschalis et al., 1996) is a relative index which has been correlated to ash content (Faibish et al., 2005) and DMB (Farlay et al., unpublished data). The mineralization index (or mineral to matrix ratio) has been shown to be higher in bone of post menopausal woman treated with alendronate (Boskey et al., 2009).

#### **5.2 Modifications of bone crystal characteristics**

Concerning crystal characteristics, it has been shown, by XRD technique that, with age, there was an increase in crystal size. In 117 iliac bone samples (range of age: 0-90 years), there was an increase, between 0-30 years, in crystallinity in c-axis, corresponding to the crystal length, and a decrease in the a axis (Hanschin & Stern 1995). After 30 years, modifications were less evident, only a slight increase in crystallinity within the basal plane was observed.

Modifications of bone crystal size impaired mechanical characteristic of bone. Indeed, it was observed that the bones of older animals or that osteoporotic bone, two cases in which bone fractures easily, contained more large crystals than normal (Boskey 2003). Conversely, young animals, which have mechanically strong bones, have a mixture of small and large crystals. In a recent study performed by Raman microspectroscopy in human femurs (age range: 52-85 years old), an overall reduction in heterogeneity of mineralization, crystallinity, and type-B carbonation was observed (Yerramshetty et al., 2006). In this study, bone became more mineralized and more highly type-B carbonated with age, whereas crystallinity was unchanged. In a study performed by FTIRM on 53 human vertebrae (range of age: 54-93 years), a significant increase in crystallinity was observed (Farlay et al., 2010a). This means that, in vertebrae, either crystal size or crystal perfection is increased. In those vertebrae, the bone volume was also significantly decreased. The reason of the increase in crystallinity is unclear, but it is possible that it corresponds to a mechanism of adaptation of bone, in order to compensate the bone loss due to age. In the same study, a decrease in CO3/PO4 ratio was observed suggesting an alteration of the physico-chemical composition of bone mineral with age. Thus, comparing the results obtained in human femora and vertebrae in term of

et al., 2005; Boivin & Meunier 2003; Misof et al., 2003). In a study on men and women with idiopathic osteoporosis, a decrease in DMB was observed compared to controls (-7%) (Boivin et al., 2008). However, a decrease in heterogeneity index of mineralization was observed in this study, indicating a homogeneization of the mineralization. This decrease is also accompanied, in men only, by a decrease in microhardness (-10%) (Boivin et al., 2008). However, it is important to mention that, both in control or osteoporotic bone, no variation in DMB or microhardness was correlated with age. Conversely, antiresorptive agents (bisphosphonates, calcitonin, estrogen, SERMs) which cause a marked reduction in the « birthrate » of BMUs, prolong the « lifespan » of the BSUs, allowing a more complete secondary mineralization and an increase of DMB (Bala et al., 2011a, 2011b; Boivin et al., 2000, 2003; Borah et al., 2005, 2006). A dissociating agent (Strontium Ranelate), acting both on resorption and formation, is now used in the treatment of post-menopausal osteoporosis (Meunier et al., 2004; Reginster et al., 2005). After short- and long-term treatment, the DMB was not significantly different from the physiological range (Boivin et al., 2010; Doublier et al., 2011a). Recently, also with a microradiographic method, it has been shown, in hipfracture patients, an increase in DMB heterogeneity in both interstitial and osteons compared to controls (Bousson et al., 2011). Thus, some differences exist, considering

osteoporotic or hip-fractures cases, in term of heterogeneity of mineralization.

treated with alendronate (Boskey et al., 2009).

**5.2 Modifications of bone crystal characteristics** 

The mineralization index measured by FTIRM, calculated as the ratio of 13PO4/amide I vibration (Paschalis et al., 1996) is a relative index which has been correlated to ash content (Faibish et al., 2005) and DMB (Farlay et al., unpublished data). The mineralization index (or mineral to matrix ratio) has been shown to be higher in bone of post menopausal woman

Concerning crystal characteristics, it has been shown, by XRD technique that, with age, there was an increase in crystal size. In 117 iliac bone samples (range of age: 0-90 years), there was an increase, between 0-30 years, in crystallinity in c-axis, corresponding to the crystal length, and a decrease in the a axis (Hanschin & Stern 1995). After 30 years, modifications were less

Modifications of bone crystal size impaired mechanical characteristic of bone. Indeed, it was observed that the bones of older animals or that osteoporotic bone, two cases in which bone fractures easily, contained more large crystals than normal (Boskey 2003). Conversely, young animals, which have mechanically strong bones, have a mixture of small and large crystals. In a recent study performed by Raman microspectroscopy in human femurs (age range: 52-85 years old), an overall reduction in heterogeneity of mineralization, crystallinity, and type-B carbonation was observed (Yerramshetty et al., 2006). In this study, bone became more mineralized and more highly type-B carbonated with age, whereas crystallinity was unchanged. In a study performed by FTIRM on 53 human vertebrae (range of age: 54-93 years), a significant increase in crystallinity was observed (Farlay et al., 2010a). This means that, in vertebrae, either crystal size or crystal perfection is increased. In those vertebrae, the bone volume was also significantly decreased. The reason of the increase in crystallinity is unclear, but it is possible that it corresponds to a mechanism of adaptation of bone, in order to compensate the bone loss due to age. In the same study, a decrease in CO3/PO4 ratio was observed suggesting an alteration of the physico-chemical composition of bone mineral with age. Thus, comparing the results obtained in human femora and vertebrae in term of

evident, only a slight increase in crystallinity within the basal plane was observed.

physico-chemical composition, some differences appears. However, the techniques were different (FTIR and Raman microspectroscopies), and the bones were not extracted from the same donors.

### **6. Perspectives and conclusion**

Different factors increase the risk of fracture, independent of bone mineral density. Among these factors, bone mineral features are important in determining some mechanical behavior, especially elastic properties. Besides the decrease in bone mineral volume occurring with age, the alteration of mineral quality plays also a major role in bone fragility. The study of bone mineral quality is a instrumental challenge, due to [1] the dimension and the nature itself of crystal apatite (nanocrystalline), [2] the fact that bone is a composite material (organic matrix intimately linked to bone mineral), and [3] because bone is a complex tissue with different levels of organization (organ, BSU, crystal).

Thus, the using of different complementary techniques (spectroscopic, diffraction, electron microscopy techniques) has been very useful in the comprehension of bone mineral structure the last 50 years. Despite very important findings, there are still lacks in the understanding of the role of certain ions (minor or trace elements) in the biological or biomechanical properties (such as carbonates, strontium ions etc.).

Bone is a fascinating tissue governed by the remodeling activity and the plasma composition. It underlies several characteristics which are interdependent. For example, carbonate ions substitutions into apatite promote the platelet-shape in bone crystals rather than needle-shape found in enamel. But the presence of carbonates, moreover, as mentioned previously, inhibits the incorporation of OH in bone apatite. Thus, the study of correlations between different variables is still indispensable to understand the relationship between physicochemical and mechanical properties.

From the previous studies performed on bone mineral, it seems that a small crystal size associated with a heterogeneity of crystal size (nanometer size-scale) is important to an optimal mechanical strength. Indeed, the presence of too large crystal is not good for bone strength. Fluoride, as mentioned previously, induces the formation of large crystals, and the formation of a "brittle" bone, despite a high BMD. Moreover, concerning mineralization and its heterogeneity at the tissue level, values comprised between 0.8 and 1.30 g/cm3 of bone could be a great deal to maintain stiffness of the bone. The preservation of mineralization heterogeneity is necessary for bone mechanical strength, allowing, for example, the stopping of microcracks propagation. No modification in degree of mineralization is observed with age or sex, but some differences exist in hip- fracture or osteoporotic cases. Thus a modification in the calcium absorption or an increase in the bone remodeling can lead to a decrease to the degree of mineralization. Differences also exist in studies in degree of mineralization due to the fact that either weight or non-weight-bearing bones are analyzed. We have an ongoing study on these different types of bone (weight or non-weight-bearing bones) in a same donor, in order to analyze the structural and mechanical bone mineral properties.

In conclusion, despite a lot of studies have permitted to understand the characteristics of bone mineral, further studies are needed to clarify the mechanisms of bone fragility at the different levels of investigation.

Bone Mineral Quality 25

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### **7. Acknowledgments**

The authors gratefully acknowledge Christian Rey, Yohann Bala, Gérard Panczer, Baptiste Depalle, Audrey Doublier, Hélène Follet, Pascale Chavassieux and Pierre Jean Meunier for their collaboration to the major studies referenced in the present chapter.

#### **8. References**


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**1. Introduction** 

**2** 

*México*

**Genetics and Osteoporosis** 

Margarita Valdés-Flores, Leonora Casas-Avila,

Valeria Ponce de León-Suárez and Edith Falcón-Ramírez *Instituto Nacional de Rehabilitación, Secretaría de Salud, México, D.F.* 

Osteoporosis is a multifactorial disease influenced by multiple factors and characterized by an imbalance in the regulation of bone remodeling that cause microarchitectural deterioration which compromises the bone strength and leads to bone fragility increasing the fracture risk. Since several years ago, the World Health Organization has considered osteoporosis as one of the most important public health issues worldwide, with a great repercussion in patients' life quality and in their familiar, social and work environments. Osteoporosis is an important problem in Latin America, currently its prevalence is similar to that in South Europe and slightly lower than in North Europe and among white population in the USA; World Health Organization estimates that in the forthcoming 50 years, osteoporosis prevalence will increase in Latin America until reach those of the currently observable in Europe and USA (World Health Organization [WHO], 1994; National Institute of Health [NIH], 2001 Consensus Development Panel on Osteoporosis Prevention, Diagnosis and Therapy; Cole ZA et al., 2008). During the last decades, the life expectancy has been increased notoriously and the number of subjects older than 60 years old has been increased. This situation in combination with the adverse environmental conditions and the life style, will cause a notorious increment in the incidence of chronic-degenerative diseases in the next decades, as will occur with osteoporosis. Certainly, primary osteoporosis use to be more frequent in posmenopausal women (Greespan et al., 1993); however occasionally it appears in premenopausal women which present several risk factors and even males may be affected by this disorder. It is important to mention that the actual life style favours the inadequate bone quality of children and young people (Asociación Mexicana de Metabolismo Óseo y Mineral, 2001). There are two forms of osteoporosis; primary OP, named posmenopausal or senile form and secondary OP, which is related to diverse endocrine, renal, rheumatic and genetic diseases, and with the prolonged administration of

some drugs which induce bone loss (Riggs et al., 1986; Elliot-Gibson et al., 2004).

Discussing about osteoporosis it is necessary to mention the term "peak bone mass" (PBM), which refers to the maximum bone mass that an individual reaches in his life and it occurs between 20-30 years old approximately. PBM is the result of the interaction of multiple genetic and environmental factors; upon the PBM is reached, progressive loss of bone mass occurs naturally, depending on the magnitude and speed of subsequent bone loss (Burclar et al., 1989; Kanis et al., 1994; Guéguen et al., 1995.). The annual average bone loss in posmenopausal women is estimated in 1-2%, and 0.2.-0.5 % in males. It is considered that


## **Genetics and Osteoporosis**

Margarita Valdés-Flores, Leonora Casas-Avila, Valeria Ponce de León-Suárez and Edith Falcón-Ramírez *Instituto Nacional de Rehabilitación, Secretaría de Salud, México, D.F. México*

### **1. Introduction**

32 Osteoporosis

Saito, M. & Marumo, K. (2010). Collagen cross-links as a determinant of bone quality: a

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Termine, J. D. & Posner, A. S. (1966). Infra-red determinaion of the percentage of crystallinity in apatitic calcium phosphates. *Nature*, Vol.211, No.5046, pp. 268-270. Traub, W.; Arad, T. & Weiner, S. (1992). Origin of mineral crystal growth in collagen fibrils.

Turner, C. H. & Burr, D. B. (1993). Basic biomechanical measurements of bone: a tutorial. *Bone*, Vol.14, No.4, pp. 595-608. Copyright (2011), with permission from Elsevier. Vedi, S.; Compston, J. E.; Webb, A. & Tighe, J. R. (1983). Histomorphometric analysis of

Viguet-Carrin, S.; Garnero, P. & Delmas, P. D. (2006). The role of collagen in bone strength.

Weaver, J. K. (1966). The microscopic hardness of bone. *J Bone Joint Surg Am*, Vol.48, No.2,

Wopenka, B. & Pasteris, J. D. (2005). A mineralogical perpective on the apatite in bone.

Yerramshetty, J. S.; Lind, C. & Akkus, O. (2006). The compositional and physicochemical

Zhu, P.; Xu, J.; Sahar, N.; Morris, MD., Kohn, DH., Ramamoorthy, A. (2009) Time-resolved

homogeneity of male femoral cortex increases after the sixth decade. *Bone*, Vol.39,

dehydration-induced structural changes in an intact bovine cortical bone revealed by solid-state NMR spectroscopy. *J Am Chem Soc*, Vol.131, No 7, pp 17064-17065.

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strength and fragility. *N Engl J Med*, Vol.354, No.21, pp. 2250-2261.

*Osteoporos Int*, Vol.21, No.2, pp. 195-214.

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possible explanation for bone fragility in aging, osteoporosis, and diabetes mellitus.

& Abbott, T. A. (2004). Predictive value of low BMD for 1-year fracture outcomes is similar for postmenopausal women ages 50-64 and 65 and Older: results from the National Osteoporosis Risk Assessment (NORA). *J Bone Miner Res*, Vol.19, No.8,

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Hydrazine-deproteinated bone mineral. Physical and chemical properties. *Calcif* 

dynamic parameters of trabecular bone formation in the iliac crest of normal British

Osteoporosis is a multifactorial disease influenced by multiple factors and characterized by an imbalance in the regulation of bone remodeling that cause microarchitectural deterioration which compromises the bone strength and leads to bone fragility increasing the fracture risk. Since several years ago, the World Health Organization has considered osteoporosis as one of the most important public health issues worldwide, with a great repercussion in patients' life quality and in their familiar, social and work environments. Osteoporosis is an important problem in Latin America, currently its prevalence is similar to that in South Europe and slightly lower than in North Europe and among white population in the USA; World Health Organization estimates that in the forthcoming 50 years, osteoporosis prevalence will increase in Latin America until reach those of the currently observable in Europe and USA (World Health Organization [WHO], 1994; National Institute of Health [NIH], 2001 Consensus Development Panel on Osteoporosis Prevention, Diagnosis and Therapy; Cole ZA et al., 2008). During the last decades, the life expectancy has been increased notoriously and the number of subjects older than 60 years old has been increased. This situation in combination with the adverse environmental conditions and the life style, will cause a notorious increment in the incidence of chronic-degenerative diseases in the next decades, as will occur with osteoporosis. Certainly, primary osteoporosis use to be more frequent in posmenopausal women (Greespan et al., 1993); however occasionally it appears in premenopausal women which present several risk factors and even males may be affected by this disorder. It is important to mention that the actual life style favours the inadequate bone quality of children and young people (Asociación Mexicana de Metabolismo Óseo y Mineral, 2001). There are two forms of osteoporosis; primary OP, named posmenopausal or senile form and secondary OP, which is related to diverse endocrine, renal, rheumatic and genetic diseases, and with the prolonged administration of some drugs which induce bone loss (Riggs et al., 1986; Elliot-Gibson et al., 2004).

Discussing about osteoporosis it is necessary to mention the term "peak bone mass" (PBM), which refers to the maximum bone mass that an individual reaches in his life and it occurs between 20-30 years old approximately. PBM is the result of the interaction of multiple genetic and environmental factors; upon the PBM is reached, progressive loss of bone mass occurs naturally, depending on the magnitude and speed of subsequent bone loss (Burclar et al., 1989; Kanis et al., 1994; Guéguen et al., 1995.). The annual average bone loss in posmenopausal women is estimated in 1-2%, and 0.2.-0.5 % in males. It is considered that

Genetics and Osteoporosis 35

familial or demographic similarity during the natural history development of the disease or even differences in the pharmacological management response. In accordance to National Osteoporosis Foundation (IOF) 2008 statements, fractures family history represents an important risk factor independently of the bone mineral density and the presence of osteoporosis in first degree relatives has been related to the decrease in peak bone mass. The analysis of genetic susceptibility to osteoporosis has been complicated because it is caused by the effect of multiple genes that exert their effect on the bone phenotype, taking in account that a great number of environmental factors acting on BMD are involved; however, despite all these difficulties, a large amount and variety of worldwide investigations suggest that BMD heritability ranges between 40-70% in spine, between 70-85% in hip and between 50-60% in wrist (Andrew et al., 2005; Michaelsson et al 2005; Deng et al., 2002). Densitometric studies in monozygotic twins (MC) and dicygotic twins (DC) have revealed that spine and femoral neck BMD consistency is higher (6-8:1) in MC twins than in DC twins. Family studies have estimated that fractures heritability ranges between 20-60%, depending on the anatomic region where those occur (Michaelsson et al 2005; MacGregor et al., 2000; Deng et al., 2002). In these cases, classic segregation studies have facilitated

In the other hand, association studies have also been very helpful to associate particular phenotypic characteristics, such as bone mineral density or the occurrence of fractures, with very specific genetic variants (gene polymorphisms, specially single nucleotide variants). Besides, there are other bones characteristics with evident heritable component, among them are: geometry and length of the femoral neck, bone ultrasonic properties (which represent the trabecular interconnectivity degree), growth and speed of bone remodeling, bone dimensions and other conditions that have an impact on bone quality (Slemenda et al., 1996; Arden et al., 1996); for example body mass index and age at which menopause occurs. It is convenient to mention that family history of hip fractures has consistently been shown

The functioning of osteoarticular system is extremely dynamic and complex, it is constantly under remodeling and it have multiple and varied mechanisms to maintain homeostasis; therefore, its genetic regulation mechanisms are also complex to understand and integrate. Genes that have been linked with BMD genetic control are distributed along all the human genome and, they are in practically all chromosomes, each of them fulfills different functions and contributes in a different way to the genetic control of bone phenotype (Stewart et al., 2006; Xiong et al., 2006; Marini et al., 2010). There are some genes with important roles in bone homeostasis because their products are involved in elemental functions related to bone structure and metabolism (formation, growth, differentiation,

Since long time ago, we know that bone metabolism has a great hormonal influence; therefore, genes that encode for its receptors are elemental in bone metabolism genetic regulation, among them we have genes ESR1 and ESR2 which encode for estrogens α and β receptors and are expressed in various bone cells types (osteoblasts, octeocytes and osteoclasts), both receptor types show a different expression pattern in the cortical and trabecular bones. Estrogens represent one of the most important regulators for bone metabolism, they regulate bone growth and maturation, and they also influence the differences between bone maturation and bone consolidation in men and women. These hormones have the capacity to block the osteoclastogenesis process, can interfere with the function of osteoclasts, induce them to

identifying new genes related to the BMD genetic control.

to be a risk factor for osteoporosis(Andrew et al., 2005).

resorption, maintenance, etc.) (Ralston et al., 2002; Williams et al., 2006).

about 30% of women at this phase shows an accelerated bone loss (approximately 5% per year) during the first 5 years after menopause, which represents higher risk to suffer osteoporotic fractures at this moment of their lives (Elliot –Gibson V et al., 2004).

Osteoporosis has been characterized for having a very discrete clinic behavior; it is practically "silent" remaining latent for years or could get worsen without causing significant symptoms. Nevertheless, one of the most frequent clinic manifestations is the back chronic pain, which may be attributed to the presence of vertebral micro-fractures, frequently it can be noted progressive height loss due to vertebral compression and/or slimming; this anomalies can be heterogeneous and cause loss of the spine natural conformation causing abnormal curvatures and scoliosis (Ismail et al., 1999). Fractures are the most frequent and dangerous complication of osteoporosis and may occur practically in all bones, even with a discrete trauma and spontaneously. As has been documented in LAVOS (*Latín American Vertebral Osteoporosis Study*) (Clark et al., 2009). and EVOS (European Vertebral Osteoporosis Study) (Raspe et al., 1998) studies, the spine is the most common site in which fracture occur. Booth studies showed that, the frequency of these fractures are related to gender and age, but also to races geographic distribution. Apparently they are more common in Scandinavian and North American population, whereas they are less frequent in South of Europe. Interestingly, the frequency is higher in urban areas than in rural ones, which outstands the importance of environmental factors in this disease besides of the genetic predisposition. After vertebral fractures, hip fractures occur, followed by forearm. It is estimated that about 25% of individuals showing this kind of fractures die due to complications, and other 25% (even the after the surgery), never recover the life quality they have before the fracture. On the other hand, patients who have suffered one or more fractures (in any place) predispose to have new fractures, independently of their bone mineral density (BMD). The risk for new fractures is higher in individuals who have suffered first fractures at early age and in those who have higher number of previous fractures.

### **2. Genetic susceptibility in osteoporosis**

There are several elements that suggest that bone phenotype is under of an important genetic influence. The first observation is the familial aggregation detected in the clinical practice, in which can be observed the segregation of some phenotypic characteristics, like family history of bad bone quality of osteoporotic fractures (Guéguez et al 1995; Fox et al., 1998; Kannus et al., 1999). On the other hand, description in literature of several diseases of genetic origin with monogenetic inheritance, which phenotype includes the loss or gain of mineral bone density, supports the hypothesis that bone phenotype has an important genetic component. Some of the most studied diseases are the different forms of osteogenesis imperfecta, the diverse varieties of osteopetrosis, pyknodisostosis, sclerostenosis and osteoporosis syndrome accompanied by pseudoglioma (Barros et al., 2007), among others. Besides, there are reports of severe osteoporosis cases in which mutations have been detected in genes which have been previously associated with the genetic control of mineral bone density, as the genes for estrogens receptors 1 and 2 (ESR1, ESR2), androgens receptor (AR) and vitamin D receptor (VDR). Changes in the normal sequence of those genes could cause osteoporosis. However, the primary osteoporosis represents the most common form in all populations (Duncan et al., 2005, 2008, 2010). Primary osteoporosis has a multi-factorial and polygenic origin and the evidences that it shows clearly genetic susceptibility are family history of bad bone quality and fractures,

about 30% of women at this phase shows an accelerated bone loss (approximately 5% per year) during the first 5 years after menopause, which represents higher risk to suffer

Osteoporosis has been characterized for having a very discrete clinic behavior; it is practically "silent" remaining latent for years or could get worsen without causing significant symptoms. Nevertheless, one of the most frequent clinic manifestations is the back chronic pain, which may be attributed to the presence of vertebral micro-fractures, frequently it can be noted progressive height loss due to vertebral compression and/or slimming; this anomalies can be heterogeneous and cause loss of the spine natural conformation causing abnormal curvatures and scoliosis (Ismail et al., 1999). Fractures are the most frequent and dangerous complication of osteoporosis and may occur practically in all bones, even with a discrete trauma and spontaneously. As has been documented in LAVOS (*Latín American Vertebral Osteoporosis Study*) (Clark et al., 2009). and EVOS (European Vertebral Osteoporosis Study) (Raspe et al., 1998) studies, the spine is the most common site in which fracture occur. Booth studies showed that, the frequency of these fractures are related to gender and age, but also to races geographic distribution. Apparently they are more common in Scandinavian and North American population, whereas they are less frequent in South of Europe. Interestingly, the frequency is higher in urban areas than in rural ones, which outstands the importance of environmental factors in this disease besides of the genetic predisposition. After vertebral fractures, hip fractures occur, followed by forearm. It is estimated that about 25% of individuals showing this kind of fractures die due to complications, and other 25% (even the after the surgery), never recover the life quality they have before the fracture. On the other hand, patients who have suffered one or more fractures (in any place) predispose to have new fractures, independently of their bone mineral density (BMD). The risk for new fractures is higher in individuals who have suffered first fractures at early age and in those who have

There are several elements that suggest that bone phenotype is under of an important genetic influence. The first observation is the familial aggregation detected in the clinical practice, in which can be observed the segregation of some phenotypic characteristics, like family history of bad bone quality of osteoporotic fractures (Guéguez et al 1995; Fox et al., 1998; Kannus et al., 1999). On the other hand, description in literature of several diseases of genetic origin with monogenetic inheritance, which phenotype includes the loss or gain of mineral bone density, supports the hypothesis that bone phenotype has an important genetic component. Some of the most studied diseases are the different forms of osteogenesis imperfecta, the diverse varieties of osteopetrosis, pyknodisostosis, sclerostenosis and osteoporosis syndrome accompanied by pseudoglioma (Barros et al., 2007), among others. Besides, there are reports of severe osteoporosis cases in which mutations have been detected in genes which have been previously associated with the genetic control of mineral bone density, as the genes for estrogens receptors 1 and 2 (ESR1, ESR2), androgens receptor (AR) and vitamin D receptor (VDR). Changes in the normal sequence of those genes could cause osteoporosis. However, the primary osteoporosis represents the most common form in all populations (Duncan et al., 2005, 2008, 2010). Primary osteoporosis has a multi-factorial and polygenic origin and the evidences that it shows clearly genetic susceptibility are family history of bad bone quality and fractures,

osteoporotic fractures at this moment of their lives (Elliot –Gibson V et al., 2004).

higher number of previous fractures.

**2. Genetic susceptibility in osteoporosis** 

familial or demographic similarity during the natural history development of the disease or even differences in the pharmacological management response. In accordance to National Osteoporosis Foundation (IOF) 2008 statements, fractures family history represents an important risk factor independently of the bone mineral density and the presence of osteoporosis in first degree relatives has been related to the decrease in peak bone mass.

The analysis of genetic susceptibility to osteoporosis has been complicated because it is caused by the effect of multiple genes that exert their effect on the bone phenotype, taking in account that a great number of environmental factors acting on BMD are involved; however, despite all these difficulties, a large amount and variety of worldwide investigations suggest that BMD heritability ranges between 40-70% in spine, between 70-85% in hip and between 50-60% in wrist (Andrew et al., 2005; Michaelsson et al 2005; Deng et al., 2002). Densitometric studies in monozygotic twins (MC) and dicygotic twins (DC) have revealed that spine and femoral neck BMD consistency is higher (6-8:1) in MC twins than in DC twins. Family studies have estimated that fractures heritability ranges between 20-60%, depending on the anatomic region where those occur (Michaelsson et al 2005; MacGregor et al., 2000; Deng et al., 2002). In these cases, classic segregation studies have facilitated identifying new genes related to the BMD genetic control.

In the other hand, association studies have also been very helpful to associate particular phenotypic characteristics, such as bone mineral density or the occurrence of fractures, with very specific genetic variants (gene polymorphisms, specially single nucleotide variants). Besides, there are other bones characteristics with evident heritable component, among them are: geometry and length of the femoral neck, bone ultrasonic properties (which represent the trabecular interconnectivity degree), growth and speed of bone remodeling, bone dimensions and other conditions that have an impact on bone quality (Slemenda et al., 1996; Arden et al., 1996); for example body mass index and age at which menopause occurs. It is convenient to mention that family history of hip fractures has consistently been shown to be a risk factor for osteoporosis(Andrew et al., 2005).

The functioning of osteoarticular system is extremely dynamic and complex, it is constantly under remodeling and it have multiple and varied mechanisms to maintain homeostasis; therefore, its genetic regulation mechanisms are also complex to understand and integrate. Genes that have been linked with BMD genetic control are distributed along all the human genome and, they are in practically all chromosomes, each of them fulfills different functions and contributes in a different way to the genetic control of bone phenotype (Stewart et al., 2006; Xiong et al., 2006; Marini et al., 2010). There are some genes with important roles in bone homeostasis because their products are involved in elemental functions related to bone structure and metabolism (formation, growth, differentiation, resorption, maintenance, etc.) (Ralston et al., 2002; Williams et al., 2006).

Since long time ago, we know that bone metabolism has a great hormonal influence; therefore, genes that encode for its receptors are elemental in bone metabolism genetic regulation, among them we have genes ESR1 and ESR2 which encode for estrogens α and β receptors and are expressed in various bone cells types (osteoblasts, octeocytes and osteoclasts), both receptor types show a different expression pattern in the cortical and trabecular bones. Estrogens represent one of the most important regulators for bone metabolism, they regulate bone growth and maturation, and they also influence the differences between bone maturation and bone consolidation in men and women. These hormones have the capacity to block the osteoclastogenesis process, can interfere with the function of osteoclasts, induce them to

Genetics and Osteoporosis 37

**Gene Chromosomal location Product** 

ESRα 6q25 Estrogens receptor α ESRβ 14q22 Estrogens receptor β AR Xq11 Androgens receptor VDR 12q12 D vitamin receptor PTH 11p15 Paratohormone

PTHR1 3p22 Paratohormone receptor 1

CASR 3q13 Receptor sensitive to calcium

ALOX12 17p13 Araquinodate 12 lipoxigenase ALOX15 17p13 Araquinodate 15 lipoxigenase BMP2 20p12 Morphogenetic protein of bone 2 BMP4 14q22 Morphogenetic protein of bone 4 BMP7 20q13 Morphogenetic protein of bone 7 IGF-1 12q22 Growth factor similar to insulin LRP5 11q13 Receptor related to lipoprotein

LRP6 12p13 Receptor related to lipoprotein

SOST 17q12 Sclerotin

NOG 17q22 Protein antagonist of

of low density 5

of low density 6

morphogenetic proteins

GR 5q31 Glucocorticoids receptor

CTR 7p21 Calcitonin receptor

CT 11p15 Calcitonin

CYP1A1 15q21 Aromatase

ADPN 3q27 Liponectin

PRL 6p22 Prolactin LEP 7q31 Leptine

INS 11p15 Insulin

LEPR lp31 Leptine receptor

INSR 19p13 Insulin receptor

COL1A1 17p21 Collagen 1A1 COL1A2 7q22 Collagen 1A2 OC 1q25 Osteocalcin OPN 4q21 Osteopontin

*Hormones and their receptors*

*Matrix components* 

*With participation in osteoblastogenic processes* 

apoptosis, and may also modify the expression of genes involved in the bone remodeling process (Slemenda et al., 1996; Kameda et al., 1997; Cummings et al., 1998)). Moreover, hormones contribute to down the expression of the Tumoral Necrosis Factor (TNF), and thereby reducing osteoclasts response to the RANK and RANKL activity (the ligand binding to the activator receptor for the kappa B factor and its ligand) (Hughes et al., 1996).

It is already known that vitamin D, through the interaction with its receptor, plays an important role in calcium homeostasis for the regulation of growth and differentiation of bone cells; that is the reason why the gene that encodes for the vitamin D receptor (VDR) is quite important in bone metabolism. Another important gene is IL6, which codifies for interleukin 6, which is a proinflammatory cytokine that has been related to several biologic processes, as bone resorption, osteoporosis and other diseases as rheumatoid arthritis, diabetes mellitus, cardiovascular diseases, cancer, etc. LRP5 gene, which encodes for protein 5 related to the low density lipoprotein receptor that participates in the development and maintenance of several tissues and represent one of the regulators for the development and proliferation of the osteoblasts (Gong et al., 2001). Other genes relevant for bone metabolism are RANK, RANK-L and OPG which encode for key proteins for bone remodeling process (Capellen et al., 2002). Other genes with higher impact on bone phenotype is the COL1A1 gene, which encodes for one of the most abundant structural proteins in bone (collagen 1A1). A great number of investigations have analyzed the association among osteoporosis and allelic and genotypic variants of these genes (Ralston et al., 2002).

There are some characteristics, for example the body mass index, that could have an impact on bone phenotype. These traits are also under genetic influence so we found genes that are related with more than one phenotype. Since several years ago it is clear that there is an important relation between bone mineral density and body mass, we already know that overweight individuals should support higher weight opposite to individuals with a lower body weight, therefore, bone mineral density is higher in overweight subjects, while thinner subjects, including the ones with alimentary disorders as anorexia or malnutrition, could present low bone quality. Some of the genes with impact on these phenotypes are ESR1, ESR2, VDR, LRP5, IL6 and OPG between others (Deng et al-. 2002; Jie et al., 2009; Frenkel et al., 2010)K. During the last years, the leptin gene and its receptor (LEP and LEPR) have been revealed as an important hormonal factors for the regulation of appetite and energetic metabolism; besides, leptin has an osteogenic effect by stimulating osteoblasts formation and plays a direct osteogenic role on bone marrow stromal cells, which allows its differentiation and maturation to osteoblasts (Esteppman et al., 2000).

Other important genes in both phenotypes are the proinsulin gene (INS), its receptor (INSR), and probably too the gene family of growth factors similar to insulin, since apparently insulin exerts a mitogenic effect on osteoblasts, which could partially explain bone mass increment that is usually noticed in obese individuals.

Table 1 despicts some of the genes related to the bone phenotype and the function that has been attributed to their products. It is evident the genetic influence on different aspects of metabolism and homeostasis of bone tissue (structure, formation, resorption and remodeling) and the number of genes involved is large and their functions are diverse. In the case of bone structure highlights the COL1A1 and COL1A2 genes which code for the type I colagen protein, which represents over 90% of the organic matrix of bone. The osteocalcin and osteopontin are also important, the first one is a calcium binding protein which is secreted by osteoblasts and is encoded by the gene OC, while the phosphoprotein known as osteopontin, encoded by the gene OPN, is essential in the mineralization process.

apoptosis, and may also modify the expression of genes involved in the bone remodeling process (Slemenda et al., 1996; Kameda et al., 1997; Cummings et al., 1998)). Moreover, hormones contribute to down the expression of the Tumoral Necrosis Factor (TNF), and thereby reducing osteoclasts response to the RANK and RANKL activity (the ligand binding

It is already known that vitamin D, through the interaction with its receptor, plays an important role in calcium homeostasis for the regulation of growth and differentiation of bone cells; that is the reason why the gene that encodes for the vitamin D receptor (VDR) is quite important in bone metabolism. Another important gene is IL6, which codifies for interleukin 6, which is a proinflammatory cytokine that has been related to several biologic processes, as bone resorption, osteoporosis and other diseases as rheumatoid arthritis, diabetes mellitus, cardiovascular diseases, cancer, etc. LRP5 gene, which encodes for protein 5 related to the low density lipoprotein receptor that participates in the development and maintenance of several tissues and represent one of the regulators for the development and proliferation of the osteoblasts (Gong et al., 2001). Other genes relevant for bone metabolism are RANK, RANK-L and OPG which encode for key proteins for bone remodeling process (Capellen et al., 2002). Other genes with higher impact on bone phenotype is the COL1A1 gene, which encodes for one of the most abundant structural proteins in bone (collagen 1A1). A great number of investigations have analyzed the association among osteoporosis

There are some characteristics, for example the body mass index, that could have an impact on bone phenotype. These traits are also under genetic influence so we found genes that are related with more than one phenotype. Since several years ago it is clear that there is an important relation between bone mineral density and body mass, we already know that overweight individuals should support higher weight opposite to individuals with a lower body weight, therefore, bone mineral density is higher in overweight subjects, while thinner subjects, including the ones with alimentary disorders as anorexia or malnutrition, could present low bone quality. Some of the genes with impact on these phenotypes are ESR1, ESR2, VDR, LRP5, IL6 and OPG between others (Deng et al-. 2002; Jie et al., 2009; Frenkel et al., 2010)K. During the last years, the leptin gene and its receptor (LEP and LEPR) have been revealed as an important hormonal factors for the regulation of appetite and energetic metabolism; besides, leptin has an osteogenic effect by stimulating osteoblasts formation and plays a direct osteogenic role on bone marrow stromal cells, which allows its

Other important genes in both phenotypes are the proinsulin gene (INS), its receptor (INSR), and probably too the gene family of growth factors similar to insulin, since apparently insulin exerts a mitogenic effect on osteoblasts, which could partially explain bone mass

Table 1 despicts some of the genes related to the bone phenotype and the function that has been attributed to their products. It is evident the genetic influence on different aspects of metabolism and homeostasis of bone tissue (structure, formation, resorption and remodeling) and the number of genes involved is large and their functions are diverse. In the case of bone structure highlights the COL1A1 and COL1A2 genes which code for the type I colagen protein, which represents over 90% of the organic matrix of bone. The osteocalcin and osteopontin are also important, the first one is a calcium binding protein which is secreted by osteoblasts and is encoded by the gene OC, while the phosphoprotein known as osteopontin, encoded by the gene OPN, is essential in the mineralization process.

to the activator receptor for the kappa B factor and its ligand) (Hughes et al., 1996).

and allelic and genotypic variants of these genes (Ralston et al., 2002).

differentiation and maturation to osteoblasts (Esteppman et al., 2000).

increment that is usually noticed in obese individuals.


Genetics and Osteoporosis 39

collagenase with preferential expression in osteoblasts, indubitably play a crucial role in

Different hormones involved in the bone formation and remodeling, including the sex hormones (estrogen, progesterone, androgens), growth hormone, insulin, parathyroid hormone, calcitonin, cortisol and thyroid hormones. These hormones are implicated in different ways in bone metabolism according to the different stages, including intrauterine life, in such a way that the different hormones impact on linear growth of bones, bone maturation, bone homeostasis and the size that will be achieved in adulthood. That's why there are hormonal conditions such as hypothyroidism, hyperthyroidism, postmenopause, andropause and glucocorticoid prolonged intake which are capable to impact on the quality of the bone. Finally we can not ignore that various interleukins, growth factors and their receptors have been identified and the participation in the genetic control of bone mineral density of other proteins are still under study, as in the case of ILα, ILβ, IL6, TNF, TNFR2 among others. On the other hand, it is important to mention that during the last years some investigations have pointed out that some of the genes related with bone phenotype have been related to other disorders as cardiovascular diseases; for example, genes such as osteoprotegerin (OPG), the receptor activator for nuclear factor kappa B ligand (RANKL) and bone morphogenetic protein 2 (BMP) have been associated with osteoporosis and with cardiovascular diseases, particularly atherosclerosis, which suggest that products of these genes take part in the calcification process (Collin-Osdoby et al., 2004; Marini et al., 2010).

**3. Linkage analysis as strategy in the study of osteoporosis** 

and skeletal geometry, have been investigated.

Linkage studies are well validated for identification of responsible genes in monogenic diseases, since the inheritance of marker alleles is related to the inheritance of a bone trait within family members. Combining the use of statistical approaches in quantitative trait loci (QTL) and genome-wide association studies (GWAS), it is possible to establish a strategy to identify chromosomal regions which contain regulating genes of some important traits in complex polygenic diseases with genetically heterogeneous traits as osteoporosis, making possible to evaluate how many of the hundreds of proposed candidate genes are really associated. Most of linkage studies in osteoporosis selected the bone mineral density as the trait of interest; however regions that regulate other relevant phenotypes, such as bone mass

Former studies identified important loci linked to bone mass and geometry. A genome search study in sib pairs recruited from families with a history of osteoporosis, obtained data suggestive of linkage of 1p36, 2p23-24 and 4q32-34 with spine and hip BMD (Devoto et al., 1998; Devoto et al., 2001). Studies with healthy female sib pairs demonstrated linkage of locus 11q12-13 with BMD variation (Koller et al., 1998) and evidence suggestive of linkage of 1q21-23, 5q33-35 and 6p1-12 to femoral neck or lumbar spine BMD was obtained in a genome-wide search study performed in Caucasian and African-American healthy female sib pairs (Koller et al., 2000). Other study identified loci in 5q and 4q that showed linkage to regulation of important aspects of femoral neck geometry (Koller et al., 2001). A QTL not previously described in 22q11 showed suggestive linkage in a study with families from Belgium and France (Kaufman et al., 2008). The presence of genes controlling BMD on 1p36 was suggested too in a multivariate linkage analysis in osteoporosis pedigrees (Zhang et al., 2009). One genome-wide scan for bone loss showed that change in femoral neck BMD in Mexican-American families is significantly linked to 1q23 (Shaffer et al., 2009). Interestingly

bone resorption.


Table 1. Genes related to bone phenotype, their chromosomal location and their products

The osteoclastogenesis and the osteoblastogenesis are fundamental processes for the homeostasis of bone tissue as the speed and intensity of bone formation and bone resorption depending on several conditions. Both mechanisms show a significant genetic influence, so the amount of genes and therefore of proteins with participation in both processes is very significant. Among them are genes that encode for the family of bone morphogenetic proteins (BMP´s), the LRP5 and LRP6 genes that code for receptors for low density lipoproteins, which are involved in the osteoblastogenesis most likely to regulate the level of bone mineralization. The osteoclastogenesis is determined by the differential expression of genes of the RANK/RANK-L/OPG route. The P53 oncogene which product is very important for multiple biological processes and the cathepsin K gen (CPK) wich codes for a

**Gene Chromosomal location Product** P53 17p13 Tumor suppressor P53 protein

RANK 18q22 Receptor activator of NF-

CLC7 16p13 Chlorine channel 7

TNF 6p21 Tumoral necrosis factor

APOE1 19q13 Apolipoprotein E MMP-1 11q22 Metalloproteinase MMP-2 16q13 Metalloproteinase MMP-9 20q11 Collagenase PON-1 7q21 Esterase

SHH 7q36

TNFR2 1p36 Tumoral necrosis factor receptor

MTHFR 1p36 5,10-Methylenetetrahydrofolate

Table 1. Genes related to bone phenotype, their chromosomal location and their products The osteoclastogenesis and the osteoblastogenesis are fundamental processes for the homeostasis of bone tissue as the speed and intensity of bone formation and bone resorption depending on several conditions. Both mechanisms show a significant genetic influence, so the amount of genes and therefore of proteins with participation in both processes is very significant. Among them are genes that encode for the family of bone morphogenetic proteins (BMP´s), the LRP5 and LRP6 genes that code for receptors for low density lipoproteins, which are involved in the osteoblastogenesis most likely to regulate the level of bone mineralization. The osteoclastogenesis is determined by the differential expression of genes of the RANK/RANK-L/OPG route. The P53 oncogene which product is very important for multiple biological processes and the cathepsin K gen (CPK) wich codes for a

IL1α 2q14 Interleukin 1A IL1β 2q14 Interleukin 1B IL6 7p21 Interleukin 6

RANK-L 13q14 Ligand of the receptor activator

KAPPA-B

2

reductase

Hedgehog protein (it participates in skeleton

embryogenesis)

of NF-KAPPA-B

CPK 1q21 Catepsine K OC 1q25 Osteocalcin OPN 4q21 Osteopontin OPG 8q24 Osteoprogeterin

*With participation in* 

*Cytokines and their receptors*

*Others functions* 

*osteoclastogenesis processes*

collagenase with preferential expression in osteoblasts, indubitably play a crucial role in bone resorption.

Different hormones involved in the bone formation and remodeling, including the sex hormones (estrogen, progesterone, androgens), growth hormone, insulin, parathyroid hormone, calcitonin, cortisol and thyroid hormones. These hormones are implicated in different ways in bone metabolism according to the different stages, including intrauterine life, in such a way that the different hormones impact on linear growth of bones, bone maturation, bone homeostasis and the size that will be achieved in adulthood. That's why there are hormonal conditions such as hypothyroidism, hyperthyroidism, postmenopause, andropause and glucocorticoid prolonged intake which are capable to impact on the quality of the bone. Finally we can not ignore that various interleukins, growth factors and their receptors have been identified and the participation in the genetic control of bone mineral density of other proteins are still under study, as in the case of ILα, ILβ, IL6, TNF, TNFR2 among others.

On the other hand, it is important to mention that during the last years some investigations have pointed out that some of the genes related with bone phenotype have been related to other disorders as cardiovascular diseases; for example, genes such as osteoprotegerin (OPG), the receptor activator for nuclear factor kappa B ligand (RANKL) and bone morphogenetic protein 2 (BMP) have been associated with osteoporosis and with cardiovascular diseases, particularly atherosclerosis, which suggest that products of these genes take part in the calcification process (Collin-Osdoby et al., 2004; Marini et al., 2010).

### **3. Linkage analysis as strategy in the study of osteoporosis**

Linkage studies are well validated for identification of responsible genes in monogenic diseases, since the inheritance of marker alleles is related to the inheritance of a bone trait within family members. Combining the use of statistical approaches in quantitative trait loci (QTL) and genome-wide association studies (GWAS), it is possible to establish a strategy to identify chromosomal regions which contain regulating genes of some important traits in complex polygenic diseases with genetically heterogeneous traits as osteoporosis, making possible to evaluate how many of the hundreds of proposed candidate genes are really associated. Most of linkage studies in osteoporosis selected the bone mineral density as the trait of interest; however regions that regulate other relevant phenotypes, such as bone mass and skeletal geometry, have been investigated.

Former studies identified important loci linked to bone mass and geometry. A genome search study in sib pairs recruited from families with a history of osteoporosis, obtained data suggestive of linkage of 1p36, 2p23-24 and 4q32-34 with spine and hip BMD (Devoto et al., 1998; Devoto et al., 2001). Studies with healthy female sib pairs demonstrated linkage of locus 11q12-13 with BMD variation (Koller et al., 1998) and evidence suggestive of linkage of 1q21-23, 5q33-35 and 6p1-12 to femoral neck or lumbar spine BMD was obtained in a genome-wide search study performed in Caucasian and African-American healthy female sib pairs (Koller et al., 2000). Other study identified loci in 5q and 4q that showed linkage to regulation of important aspects of femoral neck geometry (Koller et al., 2001). A QTL not previously described in 22q11 showed suggestive linkage in a study with families from Belgium and France (Kaufman et al., 2008). The presence of genes controlling BMD on 1p36 was suggested too in a multivariate linkage analysis in osteoporosis pedigrees (Zhang et al., 2009). One genome-wide scan for bone loss showed that change in femoral neck BMD in Mexican-American families is significantly linked to 1q23 (Shaffer et al., 2009). Interestingly

Genetics and Osteoporosis 41

populations. The results in many cases have been controversial, for example the SNP G/A in ERα gene exon 8, have been associated with osteoporosis in Thailander (Ongphiphadhanakul et al., 2001) and in Mexican women (Gómez et al., 2007), but association was denied when it was studied in Spanish women (Riancho et al., 2006), in spite all three investigations were performed with posmenopausal women. The T/C SNP of ERα gene was associated with low BMD in Japanese women, but not in Afro-American, Caucasian or Chineese women and the A/G SNP of the same gene, was associated with low BMD only in Afro-American Women, but not in Caucasian, Chinese nor in Japanese women (Greendale et al., 2006). The differences between studies results might be due to the genetic background of studied populations, which emphasize the importance of performing studies to explore the polymorphisms in specific groups with the same characteristics to avoid the incorrect use of genetic markers. Differences between races were evident too in studies with the IL6 G572C polymorphism in which the results in Korean (Chung et al., 2003) and Japanese (Ota et al., 2001) populations were consistent associating the G allele with low BMD, meanwhile in the study performed with Caucasian US women (Ferrari et al., 2003),

Discordances can certainly be seen due to the frequencies of some alleles in different populations. It is important to determine the frequency of the polymorphism in a general population study before to perform a case-control study, since some genetic sites could be not polymorphic in some populations or the variant might be present in very low frequencies and their analysis could give spurious or no association results. An example of a SNPs which could not be used as osteoporosis genetic markers in Korean population are the G174C and G/A polymorphisms in the promoter of the IL6 gene because they show a very low frequency of this polymorphisms which difficult to found associations (Chung et al., 2003). However, the same G174C SNP was analyzed in Caucasian American healthy women (Ferrari et al., 2003) and in Mexican osteoporotic and non osteoporotic women as well as in general population (Magaña, et al., 2008), obtaining that the C allele is a protective factor from bone resorption and from osteoporosis respectively. However, most of the VDR gene SNPs showed in table 2, were consistently associated with low BMD or with osteoporosis in a great variety of populations. SNPs in intron 10, exon 2 and promoter of the gene, have resulted associated in European (Bustamante et al., 2007b; Utterlinden et al., 2001) American (Kiel et al., 2007; Pérez et al., 2008; Moffet et al., 2007) and Asiatic (Mencej et al., 2009) populations and even in large scale studies with world´s population (Morrison, 2004). The colagen IA1 is one of the most studied genes involved in osteoporosis. Many SNPs have been consistently associated with BMD and osteoporosis in several populations in this gene. The G/T change has been associated with osteoporosis in almost all studied populations, for example in Mexican (Falcón-Ramírez et al., 20011) and in British (Stewart et al., 2006). Not all the polymorphisms have a functional effect on bone traits, but the presence of the polymorphism G/T in Sp1 site, alters the recognition of the Sp1 factor having effects on

the G allele appears as a protective factor from bone resorption.

transcription, protein production and mechanical strength of bone.

The appropriate expression of the genes of the route of signaling RANK/RANK-L/OPG is essential in osteoclastogenesis process, and makes them some of the most investigated genes performing studies with specific allelic, genotypic and haplotypic variants in this genes searching for associations with bone mineral density. In this case, variations of a single nucleotide in the intron 1, 9, and others located in the 3´del region gene RANK have consistently shown their association with low bone mineral density in spine and hip in European populations (Paternoster et al., 2010; Styrkarsdottir et al., 2009, Xiong et al., 2006).

a study with pairs of brothers suggested that QTL on 7q34, 14q32 and 21q21 were malespecific (Peacock et al., 2009) and other report provides evidence of gender specific QTL on 10q21 and 18p11 (Ralston et al., 2005). Suggestive evidence of linkage of novel regions related with BMD and hip geometry on chromosomes 4, 5, 11, 16 and 20 was obtained in a sample of Caucasian Europeans (Karasik et al., 2010).

Two important large scale studies with a cohort of more than 19,000 european subjects, identified SNPs in previously proposed osteoporosis candidate genes and in regions not previously associated with femoral neck and lumbar spine BMD. SNPs from ESR1, LRP4, ITGA1, LRP5, SOST, SPP1, TNFRSF11A, TNFRSF11B AND TNFSN11 associated with either femoral neck or lumbar spine BMD in a cohort of more than 19,000 subjects. In the same study, SNPs from LRP5, SOST, SPP1 and TNFSF11A, were associated with fracture risk (Richards et al., 2009). The other study, confirmed the significant association of previously known BMD loci: ESR1, TNFRSF11B, LRP5, SP7, ZBTB40, TNFSF11 and TNFRSF11A, but interestingly they identified several loci in regions not previously associated with BMD (Rivadeneira et al., 2009). Recently, variants in CATSPERB (Koller et al., 2010), MATN3, IGF1 (Li et al., 2011), SOD2 (Deng et al., 2011) and FONG (Kou et al., 2011) genes between many others, have been involved in BMD regulation and in the pathogenesis of osteoporosis. Evidences for genes or loci association with BMD are controversial in many cases (Ralston & Uterlinden, 2010). Further large scale studies will be necessary to address the role of gene variants on BMD and osteoporosis, but the importance of this studies lies in the potential uses and clinical implications since, besides of differences in the effect of variants, the identified genes might be important for drugs design to prevention and treatment of osteoporosis.

### **4. Association studies**

During the last years, association studies among natural variations of our genome (gene polymorphisms) and particular phenotypic characteristics such as OP, have shown that the mechanisms that condition this heritable susceptibility are defined by the presence of mutations or polymorphisms in one or several genes that influence bone phenotype. In this case, it is important clarifying that the term polymorphism refers to the presence of two or more gene variants in the same allele, in such a way that the less common variant must have a frequency equal or higher on 1% of the population, otherwise, the variation is considered as a mutation. These changes in the normal sequence may involve several bases, as in case minisatellites or VNTR (*variable number of tandem repeat*), where the size of repeated fragments range from 15 to 70 pairs of bases in tandem. Other kind of polymorphisms are of microsatellite also known as STR (*short tandem repeats*), which characterize for showing variations in the nucleotide number (2-6 base pairs). Recently, single nucleotide variations also known as SNPs (*single nucleotide polymorphisms*) have been analyzed; in this case, the analysis of these variations represents a very commonly used tool in studies that intend associating certain allelic variants with phenotypic characteristics, specially the ones attributed to polygenic diseases (multi-factorial and complex). Table 2 shows several single nucleotide polymorphisms studied in relation to osteoporosis and bone mineral density. It can be observed that some polymorphisms have been consistently studied with respect to particular bone traits, such as BMD in specific anatomic regions and in some cases with fracture risk.

Polymorphisms in genes as ER α and β, IL6, VDR, Aromatase (CYP19), COL IA1, RANK and RANKL are between the most studied. There are several polymorphic sites which association with BMD or with osteoporosis has been demonstrated in many different

a study with pairs of brothers suggested that QTL on 7q34, 14q32 and 21q21 were malespecific (Peacock et al., 2009) and other report provides evidence of gender specific QTL on 10q21 and 18p11 (Ralston et al., 2005). Suggestive evidence of linkage of novel regions related with BMD and hip geometry on chromosomes 4, 5, 11, 16 and 20 was obtained in a

Two important large scale studies with a cohort of more than 19,000 european subjects, identified SNPs in previously proposed osteoporosis candidate genes and in regions not previously associated with femoral neck and lumbar spine BMD. SNPs from ESR1, LRP4, ITGA1, LRP5, SOST, SPP1, TNFRSF11A, TNFRSF11B AND TNFSN11 associated with either femoral neck or lumbar spine BMD in a cohort of more than 19,000 subjects. In the same study, SNPs from LRP5, SOST, SPP1 and TNFSF11A, were associated with fracture risk (Richards et al., 2009). The other study, confirmed the significant association of previously known BMD loci: ESR1, TNFRSF11B, LRP5, SP7, ZBTB40, TNFSF11 and TNFRSF11A, but interestingly they identified several loci in regions not previously associated with BMD (Rivadeneira et al., 2009). Recently, variants in CATSPERB (Koller et al., 2010), MATN3, IGF1 (Li et al., 2011), SOD2 (Deng et al., 2011) and FONG (Kou et al., 2011) genes between many others, have been involved in BMD regulation and in the pathogenesis of osteoporosis. Evidences for genes or loci association with BMD are controversial in many cases (Ralston & Uterlinden, 2010). Further large scale studies will be necessary to address the role of gene variants on BMD and osteoporosis, but the importance of this studies lies in the potential uses and clinical implications since, besides of differences in the effect of variants, the identified genes might be

During the last years, association studies among natural variations of our genome (gene polymorphisms) and particular phenotypic characteristics such as OP, have shown that the mechanisms that condition this heritable susceptibility are defined by the presence of mutations or polymorphisms in one or several genes that influence bone phenotype. In this case, it is important clarifying that the term polymorphism refers to the presence of two or more gene variants in the same allele, in such a way that the less common variant must have a frequency equal or higher on 1% of the population, otherwise, the variation is considered as a mutation. These changes in the normal sequence may involve several bases, as in case minisatellites or VNTR (*variable number of tandem repeat*), where the size of repeated fragments range from 15 to 70 pairs of bases in tandem. Other kind of polymorphisms are of microsatellite also known as STR (*short tandem repeats*), which characterize for showing variations in the nucleotide number (2-6 base pairs). Recently, single nucleotide variations also known as SNPs (*single nucleotide polymorphisms*) have been analyzed; in this case, the analysis of these variations represents a very commonly used tool in studies that intend associating certain allelic variants with phenotypic characteristics, specially the ones attributed to polygenic diseases (multi-factorial and complex). Table 2 shows several single nucleotide polymorphisms studied in relation to osteoporosis and bone mineral density. It can be observed that some polymorphisms have been consistently studied with respect to particular bone traits, such as BMD in specific anatomic regions and in some cases with fracture risk. Polymorphisms in genes as ER α and β, IL6, VDR, Aromatase (CYP19), COL IA1, RANK and RANKL are between the most studied. There are several polymorphic sites which association with BMD or with osteoporosis has been demonstrated in many different

sample of Caucasian Europeans (Karasik et al., 2010).

important for drugs design to prevention and treatment of osteoporosis.

**4. Association studies** 

populations. The results in many cases have been controversial, for example the SNP G/A in ERα gene exon 8, have been associated with osteoporosis in Thailander (Ongphiphadhanakul et al., 2001) and in Mexican women (Gómez et al., 2007), but association was denied when it was studied in Spanish women (Riancho et al., 2006), in spite all three investigations were performed with posmenopausal women. The T/C SNP of ERα gene was associated with low BMD in Japanese women, but not in Afro-American, Caucasian or Chineese women and the A/G SNP of the same gene, was associated with low BMD only in Afro-American Women, but not in Caucasian, Chinese nor in Japanese women (Greendale et al., 2006). The differences between studies results might be due to the genetic background of studied populations, which emphasize the importance of performing studies to explore the polymorphisms in specific groups with the same characteristics to avoid the incorrect use of genetic markers. Differences between races were evident too in studies with the IL6 G572C polymorphism in which the results in Korean (Chung et al., 2003) and Japanese (Ota et al., 2001) populations were consistent associating the G allele with low BMD, meanwhile in the study performed with Caucasian US women (Ferrari et al., 2003), the G allele appears as a protective factor from bone resorption.

Discordances can certainly be seen due to the frequencies of some alleles in different populations. It is important to determine the frequency of the polymorphism in a general population study before to perform a case-control study, since some genetic sites could be not polymorphic in some populations or the variant might be present in very low frequencies and their analysis could give spurious or no association results. An example of a SNPs which could not be used as osteoporosis genetic markers in Korean population are the G174C and G/A polymorphisms in the promoter of the IL6 gene because they show a very low frequency of this polymorphisms which difficult to found associations (Chung et al., 2003). However, the same G174C SNP was analyzed in Caucasian American healthy women (Ferrari et al., 2003) and in Mexican osteoporotic and non osteoporotic women as well as in general population (Magaña, et al., 2008), obtaining that the C allele is a protective factor from bone resorption and from osteoporosis respectively. However, most of the VDR gene SNPs showed in table 2, were consistently associated with low BMD or with osteoporosis in a great variety of populations. SNPs in intron 10, exon 2 and promoter of the gene, have resulted associated in European (Bustamante et al., 2007b; Utterlinden et al., 2001) American (Kiel et al., 2007; Pérez et al., 2008; Moffet et al., 2007) and Asiatic (Mencej et al., 2009) populations and even in large scale studies with world´s population (Morrison, 2004). The colagen IA1 is one of the most studied genes involved in osteoporosis. Many SNPs have been consistently associated with BMD and osteoporosis in several populations in this gene. The G/T change has been associated with osteoporosis in almost all studied populations, for example in Mexican (Falcón-Ramírez et al., 20011) and in British (Stewart et al., 2006). Not all the polymorphisms have a functional effect on bone traits, but the presence of the polymorphism G/T in Sp1 site, alters the recognition of the Sp1 factor having effects on transcription, protein production and mechanical strength of bone.

The appropriate expression of the genes of the route of signaling RANK/RANK-L/OPG is essential in osteoclastogenesis process, and makes them some of the most investigated genes performing studies with specific allelic, genotypic and haplotypic variants in this genes searching for associations with bone mineral density. In this case, variations of a single nucleotide in the intron 1, 9, and others located in the 3´del region gene RANK have consistently shown their association with low bone mineral density in spine and hip in European populations (Paternoster et al., 2010; Styrkarsdottir et al., 2009, Xiong et al., 2006).


**GEN**

**IL-6**

G/C (G174C)

Promoter

**POLYMORPHISM**

**LOCATION**

**REFERENCES**

Chung et al., 2003.

Ferrari et al., 2003.

Magaña et al., 2008.

Chung et al., 2003.

G/A

**IL6R** **VDR**

C/T A/C

A/C

A/G

 Intron 10

 Intron 10

Bustamante et al., 2007b.

Uitterlinden et al., 2001.

Kiel et al., 2007.

Bustamante et al., 2007b.

Morrison, 2004.

Pérez et al., 2008.

C/T A/C/G/T

 Exon 2

Exon 2

Bustamante et al., 2007b.

Pérez et al., 2008.

Kiel et al., 2007.

Morrison, 2004.

Moffett et al., 2007.

Associated with osteoporosis. World population.

Low BMD in spine and/or femoral neck in postmenopausal-

menopausal Argentinean women.

Associated with BMD; not clearly to osteoporosis in Spanish women.

Low BMD in spine and/or femoral neck in postmenopausal-

menopausal Argentinean women.

Associated with osteoporosis and BMD of femoral neck and spine in US population (Framingham). Associated with osteoporosis. World population.

C/C genotype Associated with low BMD in wrist and fracture risk in Caucasian postmenopausal US women.

 Intron 10

3' UTR

Kiel et al., 2007.

Bustamante et al., 2007b.

Kiel et al., 2007.

Morrison, 2004.

C/T G/A A/C

Promoter Promoter Exon 9

Bustamante et al., 2007a.

 Promoter

**OUTCOME** No association with BMD of Korean premenopausal women, due to its low frequency among Korean population. The C allele as a protective factor from bone resorption in healthy Caucasian US women older than 65 years.

The C allele is associated as a protective factor in Mexican women.

Korean premenopausal women. Not associated with BMD due to its low frequency among Korean population.

C/T and G/A polymorphisms associated with femoral BMD and body mass ratio; A/C associated with lumbar spine BMD. Spanish postmenopausal women.

Associated with low BMD of femoral neck and spine in US population (Framingham). Associated with low BMD in Spanish postmenopausal women.

Associated with low BMD femoral neck and spine in US population (Framingham).

Associated with low BMD. World population.

Associated with low BMD in Spanish postmenopausal women.

Not associated with BMD or fracture. Meta-analysis with world population.

Associated with osteoporosis and BMD of femoral neck and spine in US population (Framingham). Associated with low BMD in Spanish postmenopausal women.

Genetics and Osteoporosis 43


#### Genetics and Osteoporosis 43

42 Osteoporosis

**GEN**

**CALCR** C/T

**ER α**

G/A C/T

Intron 1

C/T rs2234693

C/G C/T C/T C/T T/C A/G C/A C/G

> **ER β**

G/C G/A

 C/A T/C C/T C/T  A/G

> **IL-6**

G/C (G572C)

Promoter

Chung et al., 2003.

Ota et al., 2001.

Ferrari et al., 2003

Magaña et al., 2008.

Intron 7

Promoter Promoter

Shearman et al., 2004.

Ichikawa et al., 2005.

 Low hip BMD in US population (Framingham study) T/C

Associated with spine BMD normal variations in US Caucasian men and women.

C allele and increased BMD in premenopausal Korean women

G allele associated with low BMD in Japanese postmenopausal women

G allele as protective factor from bone resorption in healthy Caucasian US women older than 65 years.

Intron 3 Intron 8

Rivadeneira et al., 2006.

 Intron 8 Intron 2 Intron 8

Intron 3

Wang et al., 2008.

Massart et al., 2009.

Greendale et al., 2006.

 AA and AC genotypes associated with hip fracture in Italian C/T

Associated with low spine BMD in Caucasian and high hip BMD in Chinese women.

Vertebral fracture risk in carriers of haplotype 1 (CC) in Dutch population.

Associated with hip fracture in Chinese population.

rs726282 rs1801132

Limer et al., 2009.

Low BMD in European males.

3' UTR rs728524

Greendale et al., 2006.

Low BMD in hip and/or spine in Japanese and Afro-American women, respectively.

rs3020314 rs1884051

Wang et al., 2008.

Associated with hip fracture in Chinese population.

rs1884052 rs3778099

Greendale et al., 2006. Bustamante et al, 2007b

Kiel et al., 2007.

Ongphiphadhanakul t al., 1998.

Wang et al., 2008.

Exon 8

Ongphiphadhanakul et al., 2001. Riancho et al., 2006

Gómez et al., 2007.

 C/T

**POLYMORPHISM**

**LOCATION**

Exon 13 Intron 12

**REFERENCES**

Xiong et al., 2006.

**OUTCOME** Associated with spine osteoporosis in European families

Associated with osteoporosis in postmenopausal Thailander women.

Not associated with BMD in postmenpausal Spanish women.

Associated with spine osteoporosis in Mexican women.

Associated with high BMD of spine and radius in Thailander males.

No association in Chinese of both genders.

Low BMD in spine in Afro-American and Japanese women.

Low BMD in femoral neck in Spanish women.

Associated with hip/spine osteoporosis, with bone mass and geometry in US families of European origin (Framingham study).


**GEN**

**CYP19** **PTHR1**

A/T C/T A/G T/C

Vilariño-Güell et al., 2007.

As haplotype, they are associated with total BMD, bone mass peak, and/or loss of BMD in spine and/or hip in European families (FAMOS), in Caucasian and British women (ALSPAC) .

Intron 1 Intron 2 Intron 8 Intron 10

3' UTR

Richards et al., 2008.

Paternoster et al., 2010.

Associated with low BMD in spine in European population (Rotterdam study).

Associated with low BMD cortical. Analyzed in women of the United Kingdom (ALSPAC) and Swedish men (GOOD).

High BMD with CC genotype in Spanish women.

High BMD with CC genotype; dose effect of C allele in Korean women.

Low spine BMD in European and Asiatic population.

BMD high con AA genotype in Chinese women.

**OPG** G/A

G/C A/G

**ITGA1** **COLIA1**

G/T

Intron 1

Stewart et al., 2006.

Jin et al., 2009.

Falcón-Ramírez et al., 2011.

Stewart et al., 2006.

Stewart et al., 2006.

Jin et al., 2009.

G/T

Ins/del T

C/A

 Intron 11

Kiel et al., 2007.

 Promoter

 Promoter

Low BMD with haplotype -1997G/-1663 of lT/+1245T in hip and spine in British women.

Low BMD and increment of fracture risk in hip, in British men and women.

Associated with spine osteoporosis in Mexican women.

Low BMD in hip and spine of British women. BMD increment as haplotype with other SNPs of the gene, only in spine.

Low BMD in hip and spine in British women.

Low BMD and increment of hip fracture risk, in British men and women.

Associated with the width of the femoral neck in US women.

C/T T/G A/C

Exon 3 Intron 5 Intron 28

Lee et al., 2007.

Associated as alleles and also as haplotypes with hip osteoporosis in Korean women.

5' proximal region

Geng et al., 2007.

Exon 1 García-Unzueta et al., 2008. Kim et al., 2008.

Lee et al., 2010.

T/C G/A C/T

Intron 3 Intron 4 Intron 5

**POLYMORPHISM**

**LOCATION**

**REFERENCES**

Hong et al., 2007.

**OUTCOME** Associated with low (T/C) and high (G/A and C/T) BMD in Chinese men.

Genetics and Osteoporosis 45


**GEN**

**VDR**

C/T A/G

 Promoter

Kiel et al., 2007.

Morrison, 2004.

Mencej et al., 2009.

Uitterlinden et al., 2001.

A/G

A/C

**CYP19**

ins/del TTC

 Intron 4

rs2189480 Kiel et al., 2007.

Bustamante et al., 2007b.

Limer et al., 2009.

Riancho et al., 2005.

Mendoza et al., 2006.

Riancho et al., 2007.

Riancho et al., 2009.

Associated with low hip and spine BMD with TT genotype in Spanish women. C/T

Low heel BMD in males of many European countries.

Low hip and spine BMD in Spanish women.

Associated with vertebral fractures risk in Spanish women.

Higher hip BMD with GG genotype in Spanish women.

Associated with high hip BMD with TT genotype in Spanish women.

Associated with vertebral fracture risk in Spanish women.

T/C

C/G C/T A/G

C/G G/A C/T T/C C/T

3' UTR Intron 8 Intron 2

Xiong et al., 2006.

Xiong et al., 2006.

Associated with hip/spine osteoporosis in US families.

Associated with hip/spine osteoporosis. US/European origin families.

3' UTR Intron 2

Kiel et al., 2007.

Associated with osteoporosis and femoral neck BMD in US families of European origin (Framingham).

 Between exons I.2 y I.6

Exon I.6

5' UTR

3' UTR

Limer et al., 2009.

Mendoza et al., 2006.

Riancho et al., 2007.

Riancho et al., 2009.

Riancho et al., 2009.

Riancho et al., 2007.

Exon 3

Low heel BMD with 1 or 2 copies of TTC in males of European countries.

Low hip and spine BMD with TTC in Spanish women.

Low hip and spine BMD with TTC/G (rs10046) in Spanish women.

Associated with higher vertebral fracture risk in Spanish women.

 Promoter region

Exon 2

Uitterlinden et al., 2001.

**POLYMORPHISM**

**LOCATION**

**REFERENCES**

**OUTCOME** Associated with a larger number of fractures; it does not show significant differences as risk factor for osteoporosis in Dutch women.

Associated with osteoporosis and BMD of femoral neck and spine in US population (Framingham). Associated with osteoporosis. World population.

Associated with osteoporosis in Slovenia women.

Associated with fractures but not with osteoporosis in Dutch women.

Associated with low BMD of femoral neck and spine in US population (Framingham). Associated with low BMD of femoral neck and spine in postmenopausal Spanish women.


Genetics and Osteoporosis 47

**GEN**

**RANKL**

C/T

Intron 1

**POLYMORPHISM**

**LOCATION**

**REFERENCES**

Xiong et al., 2006.

Mencej et al., 2006.

Mencej et al., 2008.

Mencej et al., 2009

C/T C/T C/G

C/T C/T **HDC** C/T

**ADCY10**

G/A C/T

**TWIST1**

A/G

 3' region

Hwang et al., 2010.

Associated with osteoporosis in postmenopausal Korean women. Table 2. Gene polymorphisms associated with osteoporosis and bone mineral density.

 Intron 14

Ichikawa et al., 2009.

Exon 7

Ichikawa et al., 2009.

Positive association to spine BMD; modest effect on the BMD peak in spine of US population (sisters study). US men presented association to hip BMD and trend to association to spine BMD.

 A/C A/C C/T

3' region 3' region 5' region 5' region

Xiong et al., 2006.

rs9594738 rs9594759

Styrkarsdottir et al., 2008.

 Intron 1

Intron 1

Mencej et al., 2006.

Mencej et al., 2008.

Mencej et al., 2008.

Associated with a BMD decrease in spine in postmenopausal Slovenia women.

Associated with low spine BMD and moderately associated with fractures, in Australian, Danish and Icelandic subjects.

Polymorphisms associated with hip osteoporosis in European origin families.

CC genotype associated with low hip and spine BMD of postmenopausal Slovenia women.

 Association to low spine BMD of postmenopausal Slovenia women.

Intron 2

Xiong et al., 2006.

**OUTCOME** Associated with hip BMD decrease in European origin families.

CC genotype Associated with a low BMD in postmenopausal Slovenia women.

Associated with low spine BMD in osteoporotic Slovenia women.

Associated with spine BMD decrease in postmenopausal Slovenia women.

Associated with hip BMD decrease in European origin families.


**GEN**

**RUNX2** A/T

 A/T T/C C/T

A/G

> **Unknown gene**

**RANK**

rs3018362

Paternoster et al., 2010.

Styrkarsdottir et al., 2009.

Xiong et al., 2006.

A/G A/G A/C C/G A/G A/G A/T G/T A/T C/T A/G G/T G/T C/T C/G G/T C/T

A/G

 Intron 6

Koh et al., 2007.

Polymorphism associated with low BMD in ward´s triangle, trocanter and femur, in Korean population.

Intron 1 Intron 1 Intron 1 Intron 1 Intron 1 Intron 2 Intron 3 Intron 3 Intron 4 Intron 7 Intron 7 Intron 9 Intron 9 Intron 9 Intron 9 3' region 3' region

G/T A/G

rs6696981 rs7524102

Styrkarsdottir et al., 2008.

Exon 2

Vaughan et al., 2002.

Promoter 2

Lee et al., 2009.

CC genotype shows a low BMD in postmenopausal Korean women in spine and hip.

The A allele was associated with an increment of BMD in Australian women.

Associated with hip and spine fractures in Australian, Icelandic and Danish women.

Associated with low cortical BMD. Analyzed in women of the United Kingdom (ALSPAC) and Swedish men (GOOD).

Associated with low BMD in hip of Icelandic and European subjects.

Analyzed as haplotypes, these 17 polymorphisms showed association with osteoporosis and decrease of BMD in hip and spine in European families.

Intron 3 Intron 4 Intron 4

**POLYMORPHISM**

**LOCATION**

**REFERENCES**

Ermakov et al., 2006.

**OUTCOME** Associated with anthropometric femoral length in a study conducted in Israel.

Genetics and Osteoporosis 49

development are characteristics of modern civilizations. This fact generates without a doubt a glaring increment in the incidence of several chronic degenerative diseases which may become crippling as occurs with osteoporosis, where the complications directly or indirectly cause great social and economic costs; thereby, they represent a social and health services challenge. Considering environment effects on the bone phenotype and the modifications in life style of populations in present time, osteoporosis could be in the future a disorder that occurs in younger population, rather than preferentially in elder people. This situation could overpass the medical services answer capacity and the governmental budget assigned to the medical care and rehabilitation of these patients; so it is important to intensify the investigations leading to elucidate the physiopathology of this disorder and the most

Genetic association studies enable identification of new genes related to bone metabolism. Knowledge of the function of its products will allow us attaining a better understanding of some aspects of bone metabolism not entirely explored yet and will open new opportunities for therapeutic development in osteoporosis. On the other hand, clinical research from which results association studies, makes possible to identify and associate genotypic profiles (haplotypes) of risk in families and populations and even in ethnic groups. There is no doubt that progress in this scientific knowledge field, technological progress and especially the various preventative strategies at different stages of life, including prenatal stage through the integral care of maternal health, will surely contribute to achieve a better

Andrew T, Antioniades L, Scurrah KJ, Macgregor AJ & Spector TD. (2005). Risk of wrist

Arden NK, Baker J, Hogg C, Baan K & Spector TD. (1996). The heritability of bone mineral

Barros ER, Dias da Silva MR, Kunii IS, Hauache OM & Lazaretti-Castro M. (2007). A novel

Bustamante M, Nogués X, Enjuanes A, Elosua R, García-Giralt N, Pérez-Edo L, Cáceres E,

Bustamante M, Nogués X, Mellibovsky L, Agueda L, Jurado S, Cáceres E, Blanch J, Carreras

syndrome. *Osteoporos Int*, Vol. 18, No. 7, (July 2007), pp. (1017-1018). Burckardt P. (1989). The peak bone mass concept. *Clin Rehumatol*, Vol. 8, No. S2, (June 1989),

fracture in women is heritable and is influenced by genes that are largely independent of those influencing BMD. *J Bone Miner Res*, Vol. 20, No. 1, (January

density, ultrasound of the calcaneus and hip axis length: a study of postmenopausal twins. *J Bone Miner Res*, Vol. 11, No. 4, (April 1996), pp. (530-534). Asociación Mexicana de Metabolismo Óseo y Mineral. (2001). Consenso Mexicano de

Osteoporosis. *Rev Invest Clin,* Vol. 5, No. 53, (September-October 2001), pp. (469-495).

mutation in the LRP5 gene, is associated with osteoporosis-pseudoglioma

Carreras R, Mellibovsky L, Balcells S, Díez-Pérez A & Grinberg D. (2007). COL1A1, ESR1, VDR and TGFB1 polymorphisms and haplotypes in relation to BMD in Spanish postmenopausal women. *Osteoporos Int*, Vol.18, No.2, (February 2007), pp.

R, Díez-Pérez A, Grinberg D & Balcells S. (2007). Polymorphisms in the interleukin-6 receptor gene are associated with bone mineral density and body mass index in

understanding of the disease, a better care and especially a better prevention.

relevant processes in bone metabolism.

2005), pp. (67–74).

pp. (16-21).

(235-243).

**7. References** 

Other variations of a single nucleotide in intron 1 of the RANK-L gene have repeatedly been associated with low BMD of hip and spine in European, Asiatic and European populations (Xiong et al., 2006; Mencej et al., 2006; Mencej et al., 2008; Styrkarsdottir et al., 2008). The presence of these polymorphisms on human genome, are relatively easy to identify since birth or even in prenatal stage. These polymorphisms show a well defined inheritance pattern and their distribution may show differences not only among family groups but among populations and ethnic groups. However, in this kind of studies, we must be extremely careful and constantly consider the potentially confusing effect of some variables, such as: heterogeneity of populations, caused by genetic admixture, specially product of population's migration, the number of individuals included in studies is very important as well as the proper selection of cases and controls, and finally, the method to analyze data (Spencer et al., 2009; Duncan et al., 2002; Macarty et al., 2008). Not considering these elements in association studies would easily led us to establish spurious associations (Koller et al., 2004). Defining the genetic basis of primary osteoporosis in any population is not a simple task, we face a multi-factorial and polygenic entity present in populations that may have a great genetic heterogeneity; however the exploration of bone structure and metabolism genetic control, would allow to know the molecular basis of diseases such as osteoporosis, which represents a new window to explore therapeutic opportunities that would facilitate management of bone disorders.

### **5. Epigenetics and osteoporosis**

During the last years attempts have been made to analyze the relation between environmental and genetic factors in the so called "complex diseases" using epigenetic studies. Epigenetics studies causal interactions among "genes" and their "products" which give place to the "phenotype", which represents the body manifestation of a specific genetic profile. Epigenetics analyzes hereditary changes in the gene expression without changes in the DNA sequence, thus representing an important nexus between genotype, environment and the presence of a disease (Dupont et al., 2009). In osteoporosis, as a polygenic entity in which environmental component plays a determinant role, several risk conditions of maternal origin as bad nutrition of the mother, particularly the lack of vitamin D, habits as smoking and exposition to chemical agents (possibly including some drugs that impact bone quality), have the capacity to induce hereditary changes on future generations, which may occur in very early stages of the embrionary development, even during the neonatal period and they can generate an "imprinting" in the pattern of gene expression; this pattern is hereditary and "semi-permanent" because epigenetic modifications are reversible (Jiang et al., 2004; Dupont et al., 2009). On the other hand, apparently there is a relationship between low weight and size at time of birth and a higher risk of osteoporotic fractures during adult stage. Then we should understand that besides genetic and environmental factors, "epigenetic" can influence genome expression, so the prevention of some maternal conditions represents a valuable opportunity to develop preventative strategies aimed to improve bone quality in future generations.

#### **6. Conclusion**

Increment in life expectancy in some populations, ageing, changes in life style, especially the ones related to nutrition quality and physical activity, plus the vertiginous technological

Other variations of a single nucleotide in intron 1 of the RANK-L gene have repeatedly been associated with low BMD of hip and spine in European, Asiatic and European populations (Xiong et al., 2006; Mencej et al., 2006; Mencej et al., 2008; Styrkarsdottir et al., 2008). The presence of these polymorphisms on human genome, are relatively easy to identify since birth or even in prenatal stage. These polymorphisms show a well defined inheritance pattern and their distribution may show differences not only among family groups but among populations and ethnic groups. However, in this kind of studies, we must be extremely careful and constantly consider the potentially confusing effect of some variables, such as: heterogeneity of populations, caused by genetic admixture, specially product of population's migration, the number of individuals included in studies is very important as well as the proper selection of cases and controls, and finally, the method to analyze data (Spencer et al., 2009; Duncan et al., 2002; Macarty et al., 2008). Not considering these elements in association studies would easily led us to establish spurious associations (Koller et al., 2004). Defining the genetic basis of primary osteoporosis in any population is not a simple task, we face a multi-factorial and polygenic entity present in populations that may have a great genetic heterogeneity; however the exploration of bone structure and metabolism genetic control, would allow to know the molecular basis of diseases such as osteoporosis, which represents a new window to explore therapeutic opportunities that

During the last years attempts have been made to analyze the relation between environmental and genetic factors in the so called "complex diseases" using epigenetic studies. Epigenetics studies causal interactions among "genes" and their "products" which give place to the "phenotype", which represents the body manifestation of a specific genetic profile. Epigenetics analyzes hereditary changes in the gene expression without changes in the DNA sequence, thus representing an important nexus between genotype, environment and the presence of a disease (Dupont et al., 2009). In osteoporosis, as a polygenic entity in which environmental component plays a determinant role, several risk conditions of maternal origin as bad nutrition of the mother, particularly the lack of vitamin D, habits as smoking and exposition to chemical agents (possibly including some drugs that impact bone quality), have the capacity to induce hereditary changes on future generations, which may occur in very early stages of the embrionary development, even during the neonatal period and they can generate an "imprinting" in the pattern of gene expression; this pattern is hereditary and "semi-permanent" because epigenetic modifications are reversible (Jiang et al., 2004; Dupont et al., 2009). On the other hand, apparently there is a relationship between low weight and size at time of birth and a higher risk of osteoporotic fractures during adult stage. Then we should understand that besides genetic and environmental factors, "epigenetic" can influence genome expression, so the prevention of some maternal conditions represents a valuable opportunity to develop preventative strategies aimed to

Increment in life expectancy in some populations, ageing, changes in life style, especially the ones related to nutrition quality and physical activity, plus the vertiginous technological

would facilitate management of bone disorders.

**5. Epigenetics and osteoporosis** 

improve bone quality in future generations.

**6. Conclusion** 

development are characteristics of modern civilizations. This fact generates without a doubt a glaring increment in the incidence of several chronic degenerative diseases which may become crippling as occurs with osteoporosis, where the complications directly or indirectly cause great social and economic costs; thereby, they represent a social and health services challenge. Considering environment effects on the bone phenotype and the modifications in life style of populations in present time, osteoporosis could be in the future a disorder that occurs in younger population, rather than preferentially in elder people. This situation could overpass the medical services answer capacity and the governmental budget assigned to the medical care and rehabilitation of these patients; so it is important to intensify the investigations leading to elucidate the physiopathology of this disorder and the most relevant processes in bone metabolism.

Genetic association studies enable identification of new genes related to bone metabolism. Knowledge of the function of its products will allow us attaining a better understanding of some aspects of bone metabolism not entirely explored yet and will open new opportunities for therapeutic development in osteoporosis. On the other hand, clinical research from which results association studies, makes possible to identify and associate genotypic profiles (haplotypes) of risk in families and populations and even in ethnic groups. There is no doubt that progress in this scientific knowledge field, technological progress and especially the various preventative strategies at different stages of life, including prenatal stage through the integral care of maternal health, will surely contribute to achieve a better understanding of the disease, a better care and especially a better prevention.

### **7. References**


Genetics and Osteoporosis 51

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**3** 

*1Iran 2Canada* 

**Biomechanics of Osteoporosis:** 

**and Remodeling Processes** 

Gholamreza Rouhi

*1Faculty of Biomedical Engineering,* 

*University of Ottawa, Ontario,* 

*Amirkabir University of Technology, Tehran,* 

**The Importance of Bone Resorption** 

*2Department of Mechanical Engineering & School of Human Kinetics,* 

Bone is a vital, dynamic connective tissue that gives form to the body, supporting its weight, protecting vital organs, and facilitating locomotion by providing attachments for muscles to act as levers. It also acts as a reservoir for ions, especially for calcium and phosphate, the homeostasis of which is essential to life. These functions place serious requirements on the mechanical properties of bone, which should be stiff enough to support the body's weight and tough enough to prevent easy fracturing, as well as it should be able to be resorbed and/or formed depending on the mechanical and biological requirements of the body. Under normal physiological conditions, the structure/function relationships observed in bone, coupled with its role in maintaining mineral homeostasis, strongly suggest that it is an organ of optimum structural design. To fulfill these structure/function relationships adequately, bone is constantly being broken down and rebuilt in a process called remodeling. Bone has the potential to adapt its architecture, shape, and mechanical properties via a continuous process termed adaptation in response to altered loading conditions (Burr et al., 2002; Forwood & Turner, 1995; Hsieh & Turner, 2001). Under normal states of bone homeostasis, the remodeling activities in bone serve to remove bone mass where the mechanical demands of the skeleton are low, and form bone at those sites where mechanical loads are transmitted sufficiently and repeatedly. An early hypothesis about the dependence of the structure and form of bones, and the mechanical loads they carry, was proposed by Galileo in 1638 (Ascenzi, 1993), and was first described in a semiquantitative manner by Wolff (Wolff, 1892). The adaptive response of bone has been a subject of research for more than a century and many researchers have attempted to develop mathematical

In this chapter, a brief explanation about the bone structure and mechanics will be provided first. Then, the bone remodeling process and its relation with osteoporosis will be discussed. The important issue of bone quality makes another section of this chapter.

**1. Introduction** 

models for functional adaptation of bone.


## **Biomechanics of Osteoporosis: The Importance of Bone Resorption and Remodeling Processes**

### Gholamreza Rouhi

*1Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran, 2Department of Mechanical Engineering & School of Human Kinetics, University of Ottawa, Ontario, 1Iran 2Canada* 

### **1. Introduction**

58 Osteoporosis

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resolution linkage and linkage disequilibrium analyses of chromosome 1p36 SNPs identify new positional candidate genes for low bone mineral density. *Osteoporos*  Bone is a vital, dynamic connective tissue that gives form to the body, supporting its weight, protecting vital organs, and facilitating locomotion by providing attachments for muscles to act as levers. It also acts as a reservoir for ions, especially for calcium and phosphate, the homeostasis of which is essential to life. These functions place serious requirements on the mechanical properties of bone, which should be stiff enough to support the body's weight and tough enough to prevent easy fracturing, as well as it should be able to be resorbed and/or formed depending on the mechanical and biological requirements of the body. Under normal physiological conditions, the structure/function relationships observed in bone, coupled with its role in maintaining mineral homeostasis, strongly suggest that it is an organ of optimum structural design. To fulfill these structure/function relationships adequately, bone is constantly being broken down and rebuilt in a process called remodeling. Bone has the potential to adapt its architecture, shape, and mechanical properties via a continuous process termed adaptation in response to altered loading conditions (Burr et al., 2002; Forwood & Turner, 1995; Hsieh & Turner, 2001). Under normal states of bone homeostasis, the remodeling activities in bone serve to remove bone mass where the mechanical demands of the skeleton are low, and form bone at those sites where mechanical loads are transmitted sufficiently and repeatedly. An early hypothesis about the dependence of the structure and form of bones, and the mechanical loads they carry, was proposed by Galileo in 1638 (Ascenzi, 1993), and was first described in a semiquantitative manner by Wolff (Wolff, 1892). The adaptive response of bone has been a subject of research for more than a century and many researchers have attempted to develop mathematical models for functional adaptation of bone.

In this chapter, a brief explanation about the bone structure and mechanics will be provided first. Then, the bone remodeling process and its relation with osteoporosis will be discussed. The important issue of bone quality makes another section of this chapter.

Biomechanics of Osteoporosis: The Importance of Bone Resorption and Remodeling Processes 61

phases of bone?; How do the organic and the inorganic phases of bone interact to offer the superior mechanical properties?; How do the cross-links within and between collagen fibrils contribute to the mechanical properties of the collagen?; and How is load and stress distributed between the collagen and mineral?. It is known that improperly mineralized tissues are often resulted when there is flaw in organic phase of bone for mineral deposition (Lucchinetti, 2001). At the nanoscale, bone is a composite of a collagen-rich organic matrix and mineral nanoparticles made from carbonated hydroxyapatite. The basic building block of the bone material is a mineralized collagen fibril of between 50 and 200 nm diameter. The collagen fibrils are filled and coated by mineral crystallites (Rubin et al., 2004); the latter are mainly flat plates that are mostly distributed parallel to each other in a fibril, and parallel to the long axis of the collagen fibrils (Landis, 1996). These well organized structural features have been associated with various unique structural properties of bone. For instance, the stiffness of bone is related to the composite structure of mineral micro-crystals and collagen fibers (Lakes & Saha, 1979); and the cement lines as weak interfaces convey a degree of

Bone is a porous structure with different values of porosity depending on its macrostructure. At the macroscopic level, there are basically two types of bone structures: cortical (compact or Haversian) and cancellous (spongy, or trabecular) bone. Cortical bone is a dense, solid mass with only microscopic channels, and with a maximal density of about 1.8 gr/cm3. Approximately 80% of the skeletal mass in the adult human is cortical bone, which forms the outer wall of all bones and is largely responsible for the supportive and protective function of the skeleton. The main structural unit of the cortical bone is called osteon, or a Haversian system (Rouhi, 2006a). A typical osteon is a hollow cylinder with the outer and inner diameters of about 200 (or 250) and 50 μm, respectively. An osteon is made up of 20 to 30 concentric lamellae, and surrounding the outer border of each osteon there is a cement line, a 1-2 μm thick layer of mineralized matrix deficient in collagen fibers, which it is believed they act as crack stoppers when cracks are present. On the other hand, cancellous (spongy or trabecular) bone is a lattice of narrow rods and plates (70 to 200 μm in thickness) of calcified bone tissue called trabeculae, with an average thickness of 100-150 μm (Van der Meulen & Prendergast, 2000). The trabeculae are surrounded by bone marrow that is vascular and provides nutrients and waste disposal for the bone cells. The symmetry of structure in cancellous bone depends upon the direction of applied loads. If the stress pattern in spongy bone is complex, then the structure of the network of trabeculae is also complex and highly asymmetric. Comparison of micrograph structures with the density maps show that low density, open cell, rod like structure develops in regions of low stress while greater density, closed cell, plate like structures appear in regions of higher density in cancellous bone (Gibson, 1985). There are no blood vessels within the trabeculae, but there are vessels immediately adjacent to the tissue. Trabecualr bone is less mineralized than cortical bone, and experimental evidence and data suggest that spongy bone is much more active in remodeling than that of cortical bone (Guo & Goldstein, 1997). With ageing there are changes in the microarchitecture of bone. There is thinning of the cortex and of trabeculae, and a loss of connectivity, in particular of the horizontal trabeculae. The major cellular elements of bone include osteoclasts (bone resorbing cells), osteoblasts (bone making cells), osteocytes (bone sensor cells) and bone lining cells (inactive cells on the resting surfaces of bone) (Burger & Klein-Nulend, 1999). While osteoblasts and osteoclasts have

toughness to bone (Piekarski, 1970).

Two mixture models of bone resorption, a bi- and a tri-phasic model of bone resorption will be reviewed, followed by a 2D model investigating the effects of osteocytes number and mechanosensitivity on bone loss. Discussion and conclusions make the last section of this chapter.

### **2. Bone structure and mechanics**

Bone is the main constituent of the skeletal system and differs from the connective tissues in rigidity and hardness. The rigidity and hardness of bone enable the skeleton to maintain the shape of the body; to protect the vital organs; to supply the framework for the bone marrow; and also to transmit the force of muscular contraction from one part to another during movement. It is made basically of the fibrous protein collagen, impregnated with a mineral closely resembling calcium phosphate (Currey, 2002). The mineral content of bone acts as a reservoir for ions, particularly calcium (almost 99% of the calcium of our body is stored in bone), and it also contributes to the regulation of extracellular fluid composition. It also contains water, which is very important mechanically, some not well understood proteins and polysaccharides, living cells and blood vessels. The organic matrix of bone consists of 90% collagen, the most abundant protein in the body, and about 10% of various noncollagenous proteins (Behari, 1991). The protein part, mainly collagen type I, forms a model for the subsequent deposition of hydroxyapatite, the mineral phase of bone which provides rigidity to the structure. From mechanical point of view, bone is a nonhomogeneous and anisotropic material. Spongy and cortical bones can be considered as orthotropic and transversely isotropic materials, respectively. In the physiological range of loading, bone can be assumed as a linear elastic material, with negligible viscoelastic effects (Rouhi, 2006a). Bone is stronger in compression than in tension, and much greater young's moduli of elasticity than shear modulus (Bartel et al., 2006).

Outstanding mechanical properties of bone can be achieved by a very complex hierarchical structure of bone tissue, which has been explained in a number of reviews (Weiner and Wagner, 1998; Fratzl et al., 2004; Fratzl and Weinkamer, 2007). The mechanical performance of bone tissue depends on all levels of hierarchy. The term composite is usually employed for those materials in which two or more distinct phases are separated on a scale larger than the atomic, and in which their material properties such as stiffness and strength are altered compared with those of a homogeneous material. On the basis of the definition of a composite and also by considering bone structure, it is clear that bone is a composite material. Bone, as a biocomposite, shows hierarchical structures at different scales (Lakes, 1993). For example, in cortical bone, on the microstructural level, there are osteons or Haversian systems, which are large hollow fibers (200 to 250 μm outer diameter) composed of concentric lamellae and of pores. The lamellae are made up of fibers, and the fibers contain fibrils.

At the molecular level, the sophisticated structural interaction between the organic and inorganic phases is one of the fundamental determinants of the astonishing mechanical properties of bone. The underlying assumption is that a strong bonding between mineral and collagen allows the former to stiffen the collagen matrix through shear stress transfer. There are some important questions related to the composite nature of bone, which need to be addressed in order to make one able to understand the mechanics of bone as a composite at different hierarchical levels, such as: What are the properties of organic and mineral

Two mixture models of bone resorption, a bi- and a tri-phasic model of bone resorption will be reviewed, followed by a 2D model investigating the effects of osteocytes number and mechanosensitivity on bone loss. Discussion and conclusions make the last section of

Bone is the main constituent of the skeletal system and differs from the connective tissues in rigidity and hardness. The rigidity and hardness of bone enable the skeleton to maintain the shape of the body; to protect the vital organs; to supply the framework for the bone marrow; and also to transmit the force of muscular contraction from one part to another during movement. It is made basically of the fibrous protein collagen, impregnated with a mineral closely resembling calcium phosphate (Currey, 2002). The mineral content of bone acts as a reservoir for ions, particularly calcium (almost 99% of the calcium of our body is stored in bone), and it also contributes to the regulation of extracellular fluid composition. It also contains water, which is very important mechanically, some not well understood proteins and polysaccharides, living cells and blood vessels. The organic matrix of bone consists of 90% collagen, the most abundant protein in the body, and about 10% of various noncollagenous proteins (Behari, 1991). The protein part, mainly collagen type I, forms a model for the subsequent deposition of hydroxyapatite, the mineral phase of bone which provides rigidity to the structure. From mechanical point of view, bone is a nonhomogeneous and anisotropic material. Spongy and cortical bones can be considered as orthotropic and transversely isotropic materials, respectively. In the physiological range of loading, bone can be assumed as a linear elastic material, with negligible viscoelastic effects (Rouhi, 2006a). Bone is stronger in compression than in tension, and much greater young's

Outstanding mechanical properties of bone can be achieved by a very complex hierarchical structure of bone tissue, which has been explained in a number of reviews (Weiner and Wagner, 1998; Fratzl et al., 2004; Fratzl and Weinkamer, 2007). The mechanical performance of bone tissue depends on all levels of hierarchy. The term composite is usually employed for those materials in which two or more distinct phases are separated on a scale larger than the atomic, and in which their material properties such as stiffness and strength are altered compared with those of a homogeneous material. On the basis of the definition of a composite and also by considering bone structure, it is clear that bone is a composite material. Bone, as a biocomposite, shows hierarchical structures at different scales (Lakes, 1993). For example, in cortical bone, on the microstructural level, there are osteons or Haversian systems, which are large hollow fibers (200 to 250 μm outer diameter) composed of concentric lamellae and of pores. The lamellae are made up of fibers, and the fibers

At the molecular level, the sophisticated structural interaction between the organic and inorganic phases is one of the fundamental determinants of the astonishing mechanical properties of bone. The underlying assumption is that a strong bonding between mineral and collagen allows the former to stiffen the collagen matrix through shear stress transfer. There are some important questions related to the composite nature of bone, which need to be addressed in order to make one able to understand the mechanics of bone as a composite at different hierarchical levels, such as: What are the properties of organic and mineral

this chapter.

contain fibrils.

**2. Bone structure and mechanics** 

moduli of elasticity than shear modulus (Bartel et al., 2006).

phases of bone?; How do the organic and the inorganic phases of bone interact to offer the superior mechanical properties?; How do the cross-links within and between collagen fibrils contribute to the mechanical properties of the collagen?; and How is load and stress distributed between the collagen and mineral?. It is known that improperly mineralized tissues are often resulted when there is flaw in organic phase of bone for mineral deposition (Lucchinetti, 2001). At the nanoscale, bone is a composite of a collagen-rich organic matrix and mineral nanoparticles made from carbonated hydroxyapatite. The basic building block of the bone material is a mineralized collagen fibril of between 50 and 200 nm diameter. The collagen fibrils are filled and coated by mineral crystallites (Rubin et al., 2004); the latter are mainly flat plates that are mostly distributed parallel to each other in a fibril, and parallel to the long axis of the collagen fibrils (Landis, 1996). These well organized structural features have been associated with various unique structural properties of bone. For instance, the stiffness of bone is related to the composite structure of mineral micro-crystals and collagen fibers (Lakes & Saha, 1979); and the cement lines as weak interfaces convey a degree of toughness to bone (Piekarski, 1970).

Bone is a porous structure with different values of porosity depending on its macrostructure. At the macroscopic level, there are basically two types of bone structures: cortical (compact or Haversian) and cancellous (spongy, or trabecular) bone. Cortical bone is a dense, solid mass with only microscopic channels, and with a maximal density of about 1.8 gr/cm3. Approximately 80% of the skeletal mass in the adult human is cortical bone, which forms the outer wall of all bones and is largely responsible for the supportive and protective function of the skeleton. The main structural unit of the cortical bone is called osteon, or a Haversian system (Rouhi, 2006a). A typical osteon is a hollow cylinder with the outer and inner diameters of about 200 (or 250) and 50 μm, respectively. An osteon is made up of 20 to 30 concentric lamellae, and surrounding the outer border of each osteon there is a cement line, a 1-2 μm thick layer of mineralized matrix deficient in collagen fibers, which it is believed they act as crack stoppers when cracks are present. On the other hand, cancellous (spongy or trabecular) bone is a lattice of narrow rods and plates (70 to 200 μm in thickness) of calcified bone tissue called trabeculae, with an average thickness of 100-150 μm (Van der Meulen & Prendergast, 2000). The trabeculae are surrounded by bone marrow that is vascular and provides nutrients and waste disposal for the bone cells. The symmetry of structure in cancellous bone depends upon the direction of applied loads. If the stress pattern in spongy bone is complex, then the structure of the network of trabeculae is also complex and highly asymmetric. Comparison of micrograph structures with the density maps show that low density, open cell, rod like structure develops in regions of low stress while greater density, closed cell, plate like structures appear in regions of higher density in cancellous bone (Gibson, 1985). There are no blood vessels within the trabeculae, but there are vessels immediately adjacent to the tissue. Trabecualr bone is less mineralized than cortical bone, and experimental evidence and data suggest that spongy bone is much more active in remodeling than that of cortical bone (Guo & Goldstein, 1997). With ageing there are changes in the microarchitecture of bone. There is thinning of the cortex and of trabeculae, and a loss of connectivity, in particular of the horizontal trabeculae. The major cellular elements of bone include osteoclasts (bone resorbing cells), osteoblasts (bone making cells), osteocytes (bone sensor cells) and bone lining cells (inactive cells on the resting surfaces of bone) (Burger & Klein-Nulend, 1999). While osteoblasts and osteoclasts have

Biomechanics of Osteoporosis: The Importance of Bone Resorption and Remodeling Processes 63

to osteoclasts and so bone resorption starts (Martin, 2000). Gradual and diffusive osteocyte death has been reported with aging that can lead to enhanced bone remodeling and bone loss. Moreover, osteocyte death can make bones more brittle and vulnerable to fatigue damage, and bone remodeling bone loss (Jee, 2001). There are several reasons for the necessity of remodeling process, for examples: immature bone formed at the metaphyses is structurally inferior to mature bone; or the quality of adult bone deteriorates with time; or microcracks produced in bone by daily activity should be removed to attain a desired strength in bone; and/or ions concentration (e.g. calcium) should be adjusted to lie in an acceptable range; and, most likely, other factors that will be known in the future (Rouhi, 2006a). Assuming normal rates of adult bone remodeling, cortical bone has a mean age of 20 years and cancellous bone 1 to 4 years (Parfitt, 1983). Numerous theories related to the bone remodeling process have been proposed so far (see for instance, (Cowin & Hegedus, 1976; Hegedus & Cowin, 1976; Beaupre et al., 1990; Mullender et al., 1994; Jacobs et al., 1997;

Many diseases are related to global shift in the bone remodeling balance, for example: Osteoporosis, which is caused by increased osteoclast activity; Osteopetrosis, which is an abnormal increase in bone density by reduced osteoclast activity, Osteopenia, which is the bone loss by decreased osteoblast activity. The balance between bone resorption and bone formation is maintained through a complex regulatory system of systemic local factors acting on bone cells, such as calcium regulating factors, sex hormones, growth factors, and cytokine. The signal responsible for termination of bone resorption and initiation of bone formation are not well understood; however, evidence suggests that liberation of matrix embedded insulin- like growth factor system components may induce the shift. During bone turnover, surplus products synthesized by the osteoblasts during bone formation or fragments released during bone resorption are found in blood and urine. Too much bone resorption at the expense of formation results in osteoporosis, a loss of bone strength and integrity, resulting in fractures after minimal trauma. This leads to a disturbance in the bone's microarchitecture, which increases the probability of fractures. Osteoporosis is often called a "silent disease" because there are no symptoms until a bone breaks. Osteoporosis is a condition characterized by low bone mineral density and microstructural deterioration of bone tissue, leading to enhanced bone fragility and structural failure of the skeleton under low loads. Osteoporosis is a disease of enormous socioeconomic impact that is characterized by increase bone fragility (Seeman and Delmas, 2006). Such fragility is generally associated with an abnormal loss in bone volume, deterioration in the quality of the bone microarchitecture, an increased bone turnover rate, and also a shift of bone mineral density towards a lower mineralization density. Bone fragility can be defined from the pathophysiological point of view as "...the consequence of a stochastic process, that is, multiple genetic, physical, hormonal and nutritional factors acting alone or in concert to diminish skeletal integrity (Marcus, 1996)". The treatment of the bone diseases is based on drugs that intend to restore the remodeling equilibrium. Most of the work on osteoporosis, probably the most important of these diseases, seems to be currently in the osteoclast inhibition side (Rodan & Martin, 2000;

Peak bone mass (PBM) corresponds to the amount of bony tissue present at the end of skeletal maturation. It is a major determinant of the risk of fracture later in life, because there is an inverse relationship between fracture risk and areal bone mineral density, in

Rouhi et al., 2004 & 2006b))

Teitelbaum, 2000; Rouhi et al., 2007).

opposite functions and have different developmental origins, they exhibit several parallel features, particularly with respect to their life cycles. Osteoblasts and osteoclasts are both temporary cells with relatively short life spans (Parfitt, 1995).

#### **3. Bone remodeling process and osteoporosis**

During growth, bone is formed in the necessary places and resorbed as needed to attain the final shape, in a process called modeling. Modeling involves resorption drifts and formation drifts that remove or add bone over wide regions of bone surfaces. Thus, in modeling, bone resorbing and making cells act independently and at different spots. Modeling controls the growth, shape, size, strength, and anatomy of bones and joints. Collectively, modeling leads to increasing the outside cortex and marrow cavity diameters, shaping the ends of long bones. Modeling allows not only the development of normal architecture during growth, but also the modulation of this architecture and mass when the mechanical condition changes. When bone strains exceed a modeling threshold window, the minimum effective strain, modeling in the formation mode is turned on to increase bone mass and strength, and lower its strains toward the bottom of the window. When strains remain below the modeling threshold, mechanically controlled formation drifts stay inactive. As the forces on bone increases 20 times in size between birth and maturity, modeling in the formation mode keeps making bones strong enough to keep their strains from exceeding the modeling threshold, and therefore from reaching the microdamage threshold (Jee, 2001). In the adult age, the localized and independent activities of cells in modeling, are replaced by a distributed and coordinated work of the cells, resulting in a dynamic state called remodeling process. The actual remodeling occurs in two steps: the osteoclasts attach to the bone surface, dissolve the mineral, and later the organic phase of the bone, opening a hole that is subsequently filled by a number of osteoblasts, which produce the collagen matrix and secrete a protein which stimulates the calcium phosphate deposition. In the bone remdoeling process, resorption of extra-cellular matrices by osteoclasts (Teitelbaum & Ross 2003) is followed by osteoblastic invasion of the cavity, and subsequent secretion of extracellular matrix that is then mineralized (Ducy et al. 2000). These two processes, which together are called bone remodeling, occur continuously from birth to death and are in balance in a healthy bone (Riggs et al. 2002). This state can be shifted in favour of bone formation or resorption by mechanical stimulation, hormonal effects, nutrition, or diseases among other factors (Rouhi, 2006). Optimal remodeling is responsible for bone health and strength throughout life. An imbalance in bone remodeling may cause diseases such as osteoporosis. Bone remodeling occurs throughout life in thousands of sites within the human skeleton. The cellular link between bone resorbing cells or osteoclasts, and bone forming cells or osteoblasts, is known as coupling. How bone resorption and bone formation are linked is not entirely understood, but the consequences of accentuating one or the other preferentially leads to disease.

It was postulated that bone remodeling occurs to repair microdamage in bone (Frost, 1985; Mori & Burr, 1993). It was suggested that disruption of the canlicular connections occur when microcracks cut across them and can provide the stimulus to launch remodeling. It is well accepted that an unharmed gap junction intercellular communication or osteocytecanalicular system inhibits the activation of osteoclast resorption and that interruption of the connection, for instance osteocyte apoptosis or microdamage, prevent the inhibition signals

opposite functions and have different developmental origins, they exhibit several parallel features, particularly with respect to their life cycles. Osteoblasts and osteoclasts are both

During growth, bone is formed in the necessary places and resorbed as needed to attain the final shape, in a process called modeling. Modeling involves resorption drifts and formation drifts that remove or add bone over wide regions of bone surfaces. Thus, in modeling, bone resorbing and making cells act independently and at different spots. Modeling controls the growth, shape, size, strength, and anatomy of bones and joints. Collectively, modeling leads to increasing the outside cortex and marrow cavity diameters, shaping the ends of long bones. Modeling allows not only the development of normal architecture during growth, but also the modulation of this architecture and mass when the mechanical condition changes. When bone strains exceed a modeling threshold window, the minimum effective strain, modeling in the formation mode is turned on to increase bone mass and strength, and lower its strains toward the bottom of the window. When strains remain below the modeling threshold, mechanically controlled formation drifts stay inactive. As the forces on bone increases 20 times in size between birth and maturity, modeling in the formation mode keeps making bones strong enough to keep their strains from exceeding the modeling threshold, and therefore from reaching the microdamage threshold (Jee, 2001). In the adult age, the localized and independent activities of cells in modeling, are replaced by a distributed and coordinated work of the cells, resulting in a dynamic state called remodeling process. The actual remodeling occurs in two steps: the osteoclasts attach to the bone surface, dissolve the mineral, and later the organic phase of the bone, opening a hole that is subsequently filled by a number of osteoblasts, which produce the collagen matrix and secrete a protein which stimulates the calcium phosphate deposition. In the bone remdoeling process, resorption of extra-cellular matrices by osteoclasts (Teitelbaum & Ross 2003) is followed by osteoblastic invasion of the cavity, and subsequent secretion of extracellular matrix that is then mineralized (Ducy et al. 2000). These two processes, which together are called bone remodeling, occur continuously from birth to death and are in balance in a healthy bone (Riggs et al. 2002). This state can be shifted in favour of bone formation or resorption by mechanical stimulation, hormonal effects, nutrition, or diseases among other factors (Rouhi, 2006). Optimal remodeling is responsible for bone health and strength throughout life. An imbalance in bone remodeling may cause diseases such as osteoporosis. Bone remodeling occurs throughout life in thousands of sites within the human skeleton. The cellular link between bone resorbing cells or osteoclasts, and bone forming cells or osteoblasts, is known as coupling. How bone resorption and bone formation are linked is not entirely understood, but the consequences of accentuating one or the other

It was postulated that bone remodeling occurs to repair microdamage in bone (Frost, 1985; Mori & Burr, 1993). It was suggested that disruption of the canlicular connections occur when microcracks cut across them and can provide the stimulus to launch remodeling. It is well accepted that an unharmed gap junction intercellular communication or osteocytecanalicular system inhibits the activation of osteoclast resorption and that interruption of the connection, for instance osteocyte apoptosis or microdamage, prevent the inhibition signals

temporary cells with relatively short life spans (Parfitt, 1995).

**3. Bone remodeling process and osteoporosis** 

preferentially leads to disease.

to osteoclasts and so bone resorption starts (Martin, 2000). Gradual and diffusive osteocyte death has been reported with aging that can lead to enhanced bone remodeling and bone loss. Moreover, osteocyte death can make bones more brittle and vulnerable to fatigue damage, and bone remodeling bone loss (Jee, 2001). There are several reasons for the necessity of remodeling process, for examples: immature bone formed at the metaphyses is structurally inferior to mature bone; or the quality of adult bone deteriorates with time; or microcracks produced in bone by daily activity should be removed to attain a desired strength in bone; and/or ions concentration (e.g. calcium) should be adjusted to lie in an acceptable range; and, most likely, other factors that will be known in the future (Rouhi, 2006a). Assuming normal rates of adult bone remodeling, cortical bone has a mean age of 20 years and cancellous bone 1 to 4 years (Parfitt, 1983). Numerous theories related to the bone remodeling process have been proposed so far (see for instance, (Cowin & Hegedus, 1976; Hegedus & Cowin, 1976; Beaupre et al., 1990; Mullender et al., 1994; Jacobs et al., 1997; Rouhi et al., 2004 & 2006b))

Many diseases are related to global shift in the bone remodeling balance, for example: Osteoporosis, which is caused by increased osteoclast activity; Osteopetrosis, which is an abnormal increase in bone density by reduced osteoclast activity, Osteopenia, which is the bone loss by decreased osteoblast activity. The balance between bone resorption and bone formation is maintained through a complex regulatory system of systemic local factors acting on bone cells, such as calcium regulating factors, sex hormones, growth factors, and cytokine. The signal responsible for termination of bone resorption and initiation of bone formation are not well understood; however, evidence suggests that liberation of matrix embedded insulin- like growth factor system components may induce the shift. During bone turnover, surplus products synthesized by the osteoblasts during bone formation or fragments released during bone resorption are found in blood and urine. Too much bone resorption at the expense of formation results in osteoporosis, a loss of bone strength and integrity, resulting in fractures after minimal trauma. This leads to a disturbance in the bone's microarchitecture, which increases the probability of fractures. Osteoporosis is often called a "silent disease" because there are no symptoms until a bone breaks. Osteoporosis is a condition characterized by low bone mineral density and microstructural deterioration of bone tissue, leading to enhanced bone fragility and structural failure of the skeleton under low loads. Osteoporosis is a disease of enormous socioeconomic impact that is characterized by increase bone fragility (Seeman and Delmas, 2006). Such fragility is generally associated with an abnormal loss in bone volume, deterioration in the quality of the bone microarchitecture, an increased bone turnover rate, and also a shift of bone mineral density towards a lower mineralization density. Bone fragility can be defined from the pathophysiological point of view as "...the consequence of a stochastic process, that is, multiple genetic, physical, hormonal and nutritional factors acting alone or in concert to diminish skeletal integrity (Marcus, 1996)". The treatment of the bone diseases is based on drugs that intend to restore the remodeling equilibrium. Most of the work on osteoporosis, probably the most important of these diseases, seems to be currently in the osteoclast inhibition side (Rodan & Martin, 2000; Teitelbaum, 2000; Rouhi et al., 2007).

Peak bone mass (PBM) corresponds to the amount of bony tissue present at the end of skeletal maturation. It is a major determinant of the risk of fracture later in life, because there is an inverse relationship between fracture risk and areal bone mineral density, in

Biomechanics of Osteoporosis: The Importance of Bone Resorption and Remodeling Processes 65

properties in compact bone is the anisotropy of the fracture toughness, which differs by almost two orders of magnitude between a crack that propagates parallel or perpendicular to the fibril direction. This dependence of fracture properties on collagen orientation underlines the general importance of the organic matrix and its organization for bone toughness (Seeman & Delmas, 2006). Mechanical properties of bone are determined by a number of structural features, including: the mineral concentration inside the organic matrix; the size and mechanical properties of mineral particles; the quality of the collagen, in term of its amino-acid sequence, crosslinks and hydration; the quality and composition of the extrafibrillar organic matrix between the collagen fibrils; and the orientation distribution of the mineralized collagen fibrils. The mineral concentration inside the organic bone matrix is a major determinant of bone stiffness and strength (Seeman & Delmas, 2006; Currey, 2001; Currey, 2002). However, the mineral content within both the trabecular and the cortical bone is far from homogeneous. At least two processes that occur in bones over the whole lifetime of an adult individual are responsible for this situation: bone remodeling and kinetics of matrix mineralization. The newly formed bone matrix is initially unmineralized (osteoid), but after an initial maturation time of about 2 weeks, the bone goes through a stage of rapid mineralization, where 70% of the full matrix mineral content is achieved in a few days (primary mineralization). Then, the mineral content increases very slowly to reach full

mineralization within years (secondary mineralization) (Boivin & Meunier, 2003).

Fracture risk increases with age, partly as a function of changes in bone mineral density. Aging is associated with a reduction in collagen content. In osteoporosis, there is an increase in both synthesis and degradation of collagen, and an increase in the number of immature cross-links. Osteoporotic bone may be more fragile due to fewer collagen fibers and weaker cross-linking. Questions such as: How do therapeutic treatments for osteoporosis alter collagen quality (contents, cross linking, turnover rate)?; and How does increased bone turnover affect collagen quality?, are still open and need to be addressed in the future. Although changes in bone mineral content are widely recognized to occur in aging and osteoporosis, the physicochemical properties of the mineral crystal may also be changed. Mineral crystallinity increases with age, and this in itself may make the tissue more brittle. Anti-resorptive therapies increase tissue mineralization by increasing the mean tissue age. Whether this is beneficial or deleterious is not clear yet. However, the increase in mineralization never achieves the level of mineral in normal non-osteoporotic age-matched controls, so it is likely to be a positive change. However, anti-resorptive therapies also have a tendency to make the tissue mineralization more uniform, from a fracture mechanics point of view, and this would make it more likely for cracks that are introduced into the matrix to grow. There are still many questions in regard to the mineral phase of bone, such as: How is bone crystallinity affected by long-term antiresorptive therapies?; What role do osteocytes play in matrix mineralization?; What is the relationship between mineral crystallinity and brittleness?; What is the mechanical effect of reduced variability in bone mineral distribution (i.e. increasing homogeneity of tissue properties)?, which need to be addressed in the future. Structural changes, some of which are independent of bone mass, also occur in osteoporosis. In osteoporosis, there is a tendency to convert to a more rod-like and more anisotropic structure, whereas bisphosphonate treatments tend to make the bone more plate-like and more isotropic.

women, as well as in men. Interaction between genetic and non-genetic factors on bone mineral mass and structure changes during puberty. Genetic factors are either acting directly on bone or indirectly by modulating the sensitivity to environmental factors. Similarly, environmental factors are acting either directly on bone or indirectly by modulating the genetic potential. Human bone mass increases during growth, levels off in young adult life, and after about 30 years it starts to decrease. The most common sites of bone fracture are spine, hip, and wrist. The main cause of osteoporosis is the continuous loss of bone during life, which is intensified in female after menopause and male with andropause. At age 70 years, 70% of the young adult mass can remain (Wanich, 1999). It is known that with ageing, bone is lost from all parts of the skeleton, but not in equal amounts. Another factor is a lesser bone production during maturation, which cause a reduction in peak bone mass. Both cortical and cancellous bones are primarily thinned by the removal of bone at the endosteal surfaces adjacent to bone marrow. Cortical bone loss occurs mostly at the cortical endosteal surface and to a small degree from the increase in the radius of the Haversian canals. A small net gain of bone partly offsets this lost at the periosteal surface (Martin and Burr, 1989; Frost, 1999a). Age-related cancellous bone loss is because of the imbalance in bone remodeling with excessive bone resorption relative to bone formation. The sequence of Activation-Resorption-Formation is often uncoupled because of reducing the available trabecular rods/plates surfaces for bone formation. In elderly people, the most common cause of increased bone resorption is calcium and vitamin D deficiency, which will result in secondary hyperparathyroidism. Muscle mass and strength increases during growth and plateaus in young adults and then declines. Interesting to know that muscles apply the largest loads on bone, and bones normally adapt their mass and strength to the largest load. Thus, age-related reduction in muscle mass and strength can be deemed as a major factor for the age-related reduction in bone apparent density and strength (Bucwalter, et al., 1993; Frost, 1999 a&b; Burr, 1997). Needless to emphasize that loss of muscle mass and strength will increase the tendency to fall, and thus will increase the fracture risk.

### **4. Factors determining bone quality**

The quality of bone tissue relates to its composition and microstructure, whereas its quality as an organ depends also on its macrostructure. The strength of a bone and its ability to perform these physical functions depend on its structure and the intrinsic properties of the materials of which it is composed. The amount of bone, its spatial arrangement, its composition, and its turnover are all determinants of its ability to perform mechanical functions and to resist fracture. Bone quality is determined by at least four factors as follows: Properties of the organic and mineral phases of bone, also the collagen-HAp composite structure; Microdamage accumulation; Architecture and geometry of cancellous and cortical bone; and finally Rate of bone turnover and remodeling. Organic and mineral phases, i.e. collagen and hydroxyapatite, and architecture changes with age, bone diseases, such as osteoporosis, and therapeutic treatment. The risk of fracture in a 75-year-old woman can be 4-7 times that of a 45-yr-old woman with identical bone mass, demonstrating a bone quality component of fragility that is independent of bone mass.

The fracture resistance of bone results from the ability of its microstructure to dissipate deformation energy, without the propagation of large cracks leading to eventual material failure (Currey, 1999; Currey, 2003; Taylor et al., 2007). One striking feature of the fracture

women, as well as in men. Interaction between genetic and non-genetic factors on bone mineral mass and structure changes during puberty. Genetic factors are either acting directly on bone or indirectly by modulating the sensitivity to environmental factors. Similarly, environmental factors are acting either directly on bone or indirectly by modulating the genetic potential. Human bone mass increases during growth, levels off in young adult life, and after about 30 years it starts to decrease. The most common sites of bone fracture are spine, hip, and wrist. The main cause of osteoporosis is the continuous loss of bone during life, which is intensified in female after menopause and male with andropause. At age 70 years, 70% of the young adult mass can remain (Wanich, 1999). It is known that with ageing, bone is lost from all parts of the skeleton, but not in equal amounts. Another factor is a lesser bone production during maturation, which cause a reduction in peak bone mass. Both cortical and cancellous bones are primarily thinned by the removal of bone at the endosteal surfaces adjacent to bone marrow. Cortical bone loss occurs mostly at the cortical endosteal surface and to a small degree from the increase in the radius of the Haversian canals. A small net gain of bone partly offsets this lost at the periosteal surface (Martin and Burr, 1989; Frost, 1999a). Age-related cancellous bone loss is because of the imbalance in bone remodeling with excessive bone resorption relative to bone formation. The sequence of Activation-Resorption-Formation is often uncoupled because of reducing the available trabecular rods/plates surfaces for bone formation. In elderly people, the most common cause of increased bone resorption is calcium and vitamin D deficiency, which will result in secondary hyperparathyroidism. Muscle mass and strength increases during growth and plateaus in young adults and then declines. Interesting to know that muscles apply the largest loads on bone, and bones normally adapt their mass and strength to the largest load. Thus, age-related reduction in muscle mass and strength can be deemed as a major factor for the age-related reduction in bone apparent density and strength (Bucwalter, et al., 1993; Frost, 1999 a&b; Burr, 1997). Needless to emphasize that loss of muscle mass and

strength will increase the tendency to fall, and thus will increase the fracture risk.

The quality of bone tissue relates to its composition and microstructure, whereas its quality as an organ depends also on its macrostructure. The strength of a bone and its ability to perform these physical functions depend on its structure and the intrinsic properties of the materials of which it is composed. The amount of bone, its spatial arrangement, its composition, and its turnover are all determinants of its ability to perform mechanical functions and to resist fracture. Bone quality is determined by at least four factors as follows: Properties of the organic and mineral phases of bone, also the collagen-HAp composite structure; Microdamage accumulation; Architecture and geometry of cancellous and cortical bone; and finally Rate of bone turnover and remodeling. Organic and mineral phases, i.e. collagen and hydroxyapatite, and architecture changes with age, bone diseases, such as osteoporosis, and therapeutic treatment. The risk of fracture in a 75-year-old woman can be 4-7 times that of a 45-yr-old woman with identical bone mass, demonstrating a bone quality

The fracture resistance of bone results from the ability of its microstructure to dissipate deformation energy, without the propagation of large cracks leading to eventual material failure (Currey, 1999; Currey, 2003; Taylor et al., 2007). One striking feature of the fracture

**4. Factors determining bone quality** 

component of fragility that is independent of bone mass.

properties in compact bone is the anisotropy of the fracture toughness, which differs by almost two orders of magnitude between a crack that propagates parallel or perpendicular to the fibril direction. This dependence of fracture properties on collagen orientation underlines the general importance of the organic matrix and its organization for bone toughness (Seeman & Delmas, 2006). Mechanical properties of bone are determined by a number of structural features, including: the mineral concentration inside the organic matrix; the size and mechanical properties of mineral particles; the quality of the collagen, in term of its amino-acid sequence, crosslinks and hydration; the quality and composition of the extrafibrillar organic matrix between the collagen fibrils; and the orientation distribution of the mineralized collagen fibrils. The mineral concentration inside the organic bone matrix is a major determinant of bone stiffness and strength (Seeman & Delmas, 2006; Currey, 2001; Currey, 2002). However, the mineral content within both the trabecular and the cortical bone is far from homogeneous. At least two processes that occur in bones over the whole lifetime of an adult individual are responsible for this situation: bone remodeling and kinetics of matrix mineralization. The newly formed bone matrix is initially unmineralized (osteoid), but after an initial maturation time of about 2 weeks, the bone goes through a stage of rapid mineralization, where 70% of the full matrix mineral content is achieved in a few days (primary mineralization). Then, the mineral content increases very slowly to reach full mineralization within years (secondary mineralization) (Boivin & Meunier, 2003).

Fracture risk increases with age, partly as a function of changes in bone mineral density. Aging is associated with a reduction in collagen content. In osteoporosis, there is an increase in both synthesis and degradation of collagen, and an increase in the number of immature cross-links. Osteoporotic bone may be more fragile due to fewer collagen fibers and weaker cross-linking. Questions such as: How do therapeutic treatments for osteoporosis alter collagen quality (contents, cross linking, turnover rate)?; and How does increased bone turnover affect collagen quality?, are still open and need to be addressed in the future. Although changes in bone mineral content are widely recognized to occur in aging and osteoporosis, the physicochemical properties of the mineral crystal may also be changed. Mineral crystallinity increases with age, and this in itself may make the tissue more brittle. Anti-resorptive therapies increase tissue mineralization by increasing the mean tissue age. Whether this is beneficial or deleterious is not clear yet. However, the increase in mineralization never achieves the level of mineral in normal non-osteoporotic age-matched controls, so it is likely to be a positive change. However, anti-resorptive therapies also have a tendency to make the tissue mineralization more uniform, from a fracture mechanics point of view, and this would make it more likely for cracks that are introduced into the matrix to grow. There are still many questions in regard to the mineral phase of bone, such as: How is bone crystallinity affected by long-term antiresorptive therapies?; What role do osteocytes play in matrix mineralization?; What is the relationship between mineral crystallinity and brittleness?; What is the mechanical effect of reduced variability in bone mineral distribution (i.e. increasing homogeneity of tissue properties)?, which need to be addressed in the future. Structural changes, some of which are independent of bone mass, also occur in osteoporosis. In osteoporosis, there is a tendency to convert to a more rod-like and more anisotropic structure, whereas bisphosphonate treatments tend to make the bone more plate-like and more isotropic.

Biomechanics of Osteoporosis: The Importance of Bone Resorption and Remodeling Processes 67

In the conservation of mass equations, the rate of mass transferred to different constituents is assumed to be given by an empirical relation arising from the dissolution kinetics of the solid phase. In the constitutive equations, it is assumed that dependent variables, such as free energy, are a function of temperature, deformation gradient, rate of deformation

It should be noted that bone mineral (hydroxyapatite) and organic (collagen I) matrix are degraded independently. Thus, a bone resorption model needs two separate expressions, one because of the each phase. Because of the lack of information about the dissolution of the organic phase, we only considered the mineral phase dissolution and assumed that it is equivalent to the dissolution of the bone matrix. Microscopic observations suggest that degradation of collagen closely follows mineral degradation (Chambers et al. 1984), so our assumption may be justified. Dissolution of minerals occurs at the bone surface. A major source of uncertainty is the surface reactivity, which depends on chemical composition, atomic structure, and surface topography. The free energy of surface sites changes as a function of the aforementioned factors. Thus, no universal expression for the dissolution kinetics exists and experimental studies are needed to derive a dissolution kinetics relation for each case. The dissolution kinetics of hydroxyapatite has been the subject of numerous studies so far (Christoffersen et al. 1996; Dorozhkin 1997a; 1997b; 1997c; Thomann et al. 1989; 1990; 1991; Margolis and Moreno 1992; Hankermeyer et al. 2002; Fulmer et al. 2002; Chow et al. 2003). Because of the small dimensions of the resorption microenvironment between the osteoclasts and the bone matrix assuming that dissolution is governed by the

In order to develop a general framework for the description of bio-chemo-mechanically driven bone resorption, some basic assumptions should be made as follows: Bone is a biphasic mixture of a solid phase and a fluid phase; The transfer of mass, energy and entropy between the solid and the fluid phases are a result of biochemical reactions that occur between the osteoclasts and the matrix; The characteristic time of chemical reactions is several orders of magnitude greater than the characteristic time associated with a complete perfusion of the blood plasma in bone, so the resorption process can be considered isothermal; The bone matrix is isotropic and linearly elastic; Mechanical, chemical, and biological factors affect the rate of bone resorption, thus they all appear in the bio-chemomechanical affinity as the driving forces of the chemical reactions; and finally Dissolution of the matrix is the same as resorption of the mineral phase. Furthermore, it is assumed that the degree of saturation is a function of the bio-chemo-mechanical affinity, but not just of the

Bone resorption can be simplified to (see (Blair 1998; Dorozhkin 1997a; 1997b; 1997c)):

 Ca 10(PO4)6(OH)2 +2H+ 10Ca2+ +6PO4− 3 +2H2O (1) The chemical driving force for bone resorption, i.e., the chemical reaction shown in Equation (1), can be expressed by the Gibbs free energy variation per mole. In 1992, Margolis and Moreno (Margolis & Moreno, 1992) performed dissolution experiments with hydroxyapatite crystals, in which they measured pH, calcium and phosphate concentrations at a constant temperature. They proposed the following equation for the rate of dissolution

J = k(1− DS)m [H+]n (2)

gradient, and the extent of chemical reactions (Rouhi et al., 2007).

reaction kinetics seems logical and acceptable.

of the mineral phase of the bone matrix:

Gibbs free energy.

Complete trabecular perforations increase as the remodeling rate increase. These may weaken the structure more than expected based on the loss of bone mass alone. Regarding the effects of bone architecture on its quality and mechanical properties, there are some questions such as: Does maintenance of anisotropy reduce bone fracture risk?; To what extent do resorption bays in trabeculae waken bone?; and What is the relative role of trabecular and cortical bone in vertebral and hip fracture risk?, which need to be answered.

A reduction in fracture toughness of bone with age was reported in the literature, which was caused either because of an increase in mineralization (Currey et al., 1996; Zioupos et al., 1998) or alterations in the collagen matrix (Zioupos et al., 1999). In an animal model of disuse osteoporosis, a reduction in collagen cross-links can be seen (Yamauchi et al., 1988). Other experimental evidence supports the idea that the concentration of collagen cross-links is considerably lower in osteoporotic individuals compared to age-matched controls (Oxlund et al., 1996). It should be noted that the initial cross-links between collagen molecules are unstable, but as bone matures, the cross-links also mature into more stable nonreducible forms. So, there is an increase in collagen matrix's density, stiffness, and strength during maturation (Bailey & Paul, 1999). It should also be noted that the content of mature cross-links is lower in cancellous bone as compared to cortical bone, due to the greater rate of the cancellous bone remodeling (Eyre et al., 1988). The bone collagen crosslinks are usually modified in the mineralization process.

### **5. A bi-phasic mixture model of bone resorption process**

Osteoporosis, regardless of etiology, always represents enhanced bone resorption relative to formation. Thus, insights into the pathogenesis of this disease, and progress in its prevention and/or cure, depend on understanding the mechanisms by which bone is degraded. The osteoclast is the principal resorptive cell of bone, and the most successful treatments of osteoporosis, to date, target osteoclastic bone resorption. The osteoclast is a multinucleated cell whose capacity to degrade hard tissues, among other factors, depends on cell/matrix contact. All forms of adult osteoporosis reflect enhanced bone resorption relative to formation, and should be viewed in the context of the remodeling cycle. The reason for using this way of treatment is the lack of information about all various factors affecting osteoclasts' activity. Biological tissues, including bones, are all composed of multiphase constituents, and there are chemical reactions and/or diffusions between different components of them. Cells, as live organs in the biological tissues, can dictate rate of growth and adaptation, and their activities are affected by different, including mechanical, chemical, and biological factors.

Here a brief explanation about a recently proposed biphasic mixture model of bone resorption is presented (Rouhi et al., 2007). This model aims at shedding some light on the bone resorption process using a multi-constituents continuum mechanics model. In this model, bone is treated as a biphasic mixture of matrix and fluid, and bone resorption is considered as an exchange of mass between the solid and fluid phases. This exchange is caused by the secretion of H+ and Cl− from osteoclasts, which creates an acidic environment in a sealed microenvironment between the osteoclasts and the bone matrix (Blair 1998; Rousselle and Heymann 2002). The governing equations for bone resorption can be derived using the conservation laws, entropy inequality, and the appropriate constitutive equations.

Complete trabecular perforations increase as the remodeling rate increase. These may weaken the structure more than expected based on the loss of bone mass alone. Regarding the effects of bone architecture on its quality and mechanical properties, there are some questions such as: Does maintenance of anisotropy reduce bone fracture risk?; To what extent do resorption bays in trabeculae waken bone?; and What is the relative role of trabecular and cortical bone in vertebral and hip fracture risk?, which need to be

A reduction in fracture toughness of bone with age was reported in the literature, which was caused either because of an increase in mineralization (Currey et al., 1996; Zioupos et al., 1998) or alterations in the collagen matrix (Zioupos et al., 1999). In an animal model of disuse osteoporosis, a reduction in collagen cross-links can be seen (Yamauchi et al., 1988). Other experimental evidence supports the idea that the concentration of collagen cross-links is considerably lower in osteoporotic individuals compared to age-matched controls (Oxlund et al., 1996). It should be noted that the initial cross-links between collagen molecules are unstable, but as bone matures, the cross-links also mature into more stable nonreducible forms. So, there is an increase in collagen matrix's density, stiffness, and strength during maturation (Bailey & Paul, 1999). It should also be noted that the content of mature cross-links is lower in cancellous bone as compared to cortical bone, due to the greater rate of the cancellous bone remodeling (Eyre et al., 1988). The bone collagen cross-

Osteoporosis, regardless of etiology, always represents enhanced bone resorption relative to formation. Thus, insights into the pathogenesis of this disease, and progress in its prevention and/or cure, depend on understanding the mechanisms by which bone is degraded. The osteoclast is the principal resorptive cell of bone, and the most successful treatments of osteoporosis, to date, target osteoclastic bone resorption. The osteoclast is a multinucleated cell whose capacity to degrade hard tissues, among other factors, depends on cell/matrix contact. All forms of adult osteoporosis reflect enhanced bone resorption relative to formation, and should be viewed in the context of the remodeling cycle. The reason for using this way of treatment is the lack of information about all various factors affecting osteoclasts' activity. Biological tissues, including bones, are all composed of multiphase constituents, and there are chemical reactions and/or diffusions between different components of them. Cells, as live organs in the biological tissues, can dictate rate of growth and adaptation, and their activities are affected by different, including

Here a brief explanation about a recently proposed biphasic mixture model of bone resorption is presented (Rouhi et al., 2007). This model aims at shedding some light on the bone resorption process using a multi-constituents continuum mechanics model. In this model, bone is treated as a biphasic mixture of matrix and fluid, and bone resorption is considered as an exchange of mass between the solid and fluid phases. This exchange is caused by the secretion of H+ and Cl− from osteoclasts, which creates an acidic environment in a sealed microenvironment between the osteoclasts and the bone matrix (Blair 1998; Rousselle and Heymann 2002). The governing equations for bone resorption can be derived using the conservation laws, entropy inequality, and the appropriate constitutive equations.

links are usually modified in the mineralization process.

mechanical, chemical, and biological factors.

**5. A bi-phasic mixture model of bone resorption process** 

answered.

In the conservation of mass equations, the rate of mass transferred to different constituents is assumed to be given by an empirical relation arising from the dissolution kinetics of the solid phase. In the constitutive equations, it is assumed that dependent variables, such as free energy, are a function of temperature, deformation gradient, rate of deformation gradient, and the extent of chemical reactions (Rouhi et al., 2007).

It should be noted that bone mineral (hydroxyapatite) and organic (collagen I) matrix are degraded independently. Thus, a bone resorption model needs two separate expressions, one because of the each phase. Because of the lack of information about the dissolution of the organic phase, we only considered the mineral phase dissolution and assumed that it is equivalent to the dissolution of the bone matrix. Microscopic observations suggest that degradation of collagen closely follows mineral degradation (Chambers et al. 1984), so our assumption may be justified. Dissolution of minerals occurs at the bone surface. A major source of uncertainty is the surface reactivity, which depends on chemical composition, atomic structure, and surface topography. The free energy of surface sites changes as a function of the aforementioned factors. Thus, no universal expression for the dissolution kinetics exists and experimental studies are needed to derive a dissolution kinetics relation for each case. The dissolution kinetics of hydroxyapatite has been the subject of numerous studies so far (Christoffersen et al. 1996; Dorozhkin 1997a; 1997b; 1997c; Thomann et al. 1989; 1990; 1991; Margolis and Moreno 1992; Hankermeyer et al. 2002; Fulmer et al. 2002; Chow et al. 2003). Because of the small dimensions of the resorption microenvironment between the osteoclasts and the bone matrix assuming that dissolution is governed by the reaction kinetics seems logical and acceptable.

In order to develop a general framework for the description of bio-chemo-mechanically driven bone resorption, some basic assumptions should be made as follows: Bone is a biphasic mixture of a solid phase and a fluid phase; The transfer of mass, energy and entropy between the solid and the fluid phases are a result of biochemical reactions that occur between the osteoclasts and the matrix; The characteristic time of chemical reactions is several orders of magnitude greater than the characteristic time associated with a complete perfusion of the blood plasma in bone, so the resorption process can be considered isothermal; The bone matrix is isotropic and linearly elastic; Mechanical, chemical, and biological factors affect the rate of bone resorption, thus they all appear in the bio-chemomechanical affinity as the driving forces of the chemical reactions; and finally Dissolution of the matrix is the same as resorption of the mineral phase. Furthermore, it is assumed that the degree of saturation is a function of the bio-chemo-mechanical affinity, but not just of the Gibbs free energy.

Bone resorption can be simplified to (see (Blair 1998; Dorozhkin 1997a; 1997b; 1997c)):

$$\text{Ca}\_{10}\text{(PO}\_{6}\text{)}\_{6}\text{(OH)}\_{2} + 2\text{H}^{+} \longrightarrow \text{10Ca}^{2+} + 6\text{PO}\_{4}\text{-}^{-3} + 2\text{H}\_{2}\text{O}\tag{1}$$

The chemical driving force for bone resorption, i.e., the chemical reaction shown in Equation (1), can be expressed by the Gibbs free energy variation per mole. In 1992, Margolis and Moreno (Margolis & Moreno, 1992) performed dissolution experiments with hydroxyapatite crystals, in which they measured pH, calcium and phosphate concentrations at a constant temperature. They proposed the following equation for the rate of dissolution of the mineral phase of the bone matrix:

$$\mathbf{J} = \mathbf{k} (\mathbf{1} - \mathbf{D} \mathbf{S})^{\mathbf{m}} [\mathbf{H}^{+}]^{\mathbf{n}} \tag{2}$$

Biomechanics of Osteoporosis: The Importance of Bone Resorption and Remodeling Processes 69

Thermodynamics, it was also shown that the maximum rate of bone resorption in cortical bone is greater than that of cancellous bone. This behaviour of cortical and trabecular bone, which is well accepted experimentally (Martin & Burr, 1989), can also be predicted

For more detailed information about the basic assumptions, also governing equations of the bi-phasic model of bone resorption, interested readers are encouraged to consult the

Recently, a tri-phasic model of bone resorption using mixture theory with chemical reactions was proposed (Rouhi, 2011). In this model, three different constituents (matrix, fluid, and cells) have been considered. Bone resorption is considered as a chemical reaction caused by the secretion of *H+* and *Cl-* from osteoclasts which creates an acidic environment in a sealed zone between osteoclasts and bone matrix. It is assumed that the solid phase obeys small deformation theory and is isotropic and linearly elastic. The velocity of the matrix and cells is assumed to be zero. The fluid phase is assumed to be viscous, and inertial effects are neglected because of the slow velocities that are at play. A non-rotational fluid is assumed for deriving the final form of the entropy inequality for the mixture as a whole. In the constitutive equations, similar to our bi-phasic model (Rouhi et al., 2007), it is assumed that the free energy, enthalpy, specific entropy, heat flux, and stress tensor are functions of temperature, deformation gradient, and the extent of chemical reactions. Bone resorption was considered as an isothermal and a quasi-static process. For the sake of simplicity, presence of ostocytes in the bone matrix was discarded in this model, despite the fact that fluid flow in the bone matrix (e.g. in the lacuno-canalicular network) has a definite effect on the osteocytes, and, most likely, on the osteoclasts and thus on the rate of bone resorption. Using these assumptions, the governing equations for bone resorption were derived using the conservation laws (mass, momentum, and energy), as well as entropy inequality and the

By using mixture theory with chemical reactions, first, contribution of different phases present in the mixture can be observed. Secondly, using consistency requirement for energy balance, it was found that rate of bone resorption is a function of different factors including apparent density of bone matrix and bone fluid; fluid velocity; momentum supply to the fluid or solid phase; and internal energy densities of different constituents. Thirdly, using the relation between momentum supply to the solid and fluid phase, one can conclude that rate of bone resorption is inversely proportional to the bone fluid velocity. Also, it was found that in spongy bone, by increasing the porosity, rate of resorption will decrease and vice versa. Based on our results, it is speculated that bone resorption in cortical and cancellous bones might be affected by a control system which is resulted from the relation between the specific surface of bone and its apparent density and volume fraction. As it is known, one reason of osteoporosis is the lack of calcium ions in our body, so in the case of need of calcium, where is better than a rich reservoir, i.e. bones, to take away calcium ions via bone resorption process and giving back in the bone formation process. It seems necessary and feasible, as a future task, to investigate the relation between calcium concentration in the bone fluid and the rate of bone resotrpiton

using the axiom of mass balance in this bi-phasic model.

**6. A tri-phasic mixture model of bone resorption process** 

following reference (Rouhi et al., 2007).

appropriate constitutive equations.

using mixture theory.

where *J* is the mineral flux across the real surface of the mineral phase, *DS* is the degree of saturation, [*H+*] is the concentration of hydrogen ion, and *k*, *m*, and *n* are empirical constants. As stated earlier, it is assumed that the mineral flux, *J,* is almost the same as the dissolution rate of the solid phase, i.e. hydroxyapatite + collagen fibers.

The degree of saturation (*DS*) is expressed as:

$$\text{DS} = \left| \left( [\text{Ca}^{2+}]^5 \left[ \text{PO}\_4^{-,3} \right]^3 [\text{OH}^-] \right) / \text{Kso} \right| ^{1/9} \tag{3}$$

where [X] is the concentration of ion X, and Kso is the solubility product of hydroxyapatite (Margolis and Moreno 1992).

Since biological, chemical, and mechanical factors have a definite effect on the rate of dissolution, it is hypothesized that a bio-chemo-mechanical driving force should be considered in the dissolution relation, instead of just a chemical driving force (i.e. just changes in the Gibbs free energy). Dissipation law can be used to find the bio-chemomechanical affinity, and it is defined as the difference between the external work rate and the rate of change in free energy. According to the Second Law of Thermodynamics, this quantity should be nonnegative. Using the dissipation law and after some manipulations, the driving force for the dissolution process of bone can take the following form:

$$\mathbf{A} = \mathbf{u}\mu\_{\text{mech}} + \mathbf{P} + \mathbf{C}\_s \left(\mu\_s - \mu\_{\text{ext}}\right) \tag{4}$$

where *ψmech.* is the mechanical part of the free energy, *P* is the hydrostatic pressure, Cs is defined as ߩȀܯ, where ߩ and *M* are the density and the molar mass of the matrix, respectively, and *μs* & *μext* are the chemical potential of the solid phase in the unstressed condition and the external potential energy, respectively.

Our bi-phasci mixture model of bone resorption shows that the activity of osteoclasts and, thus, the rate of bone resorption are not only dictated by biological factors (e.g., hormone levels), but also by engineering quantities, i.e. hydrostatic pressure, strain energy density, and concentration of different ions before and after the resorption process. Interesting to note that the exact stimulus for the initiation of the remodeling process of bone is not known yet and is a place of debate (Rouhi, 2006a). In 1990, Brown and co-workers have shown experimentally that strain energy density can be a likely stimulus for bone remodeling (Brown et al. 1990), and it was used extensively in many theoretical modeling of bone adaptation; for instance (Jacobs et al. 1997; Huiskes et al. 2000; Doblar´e and Garc´a 2001; Garcia et al. 2002; Ruimerman et al. 2005). As can be seen in Eq. 4, in this biphasic model, strain energy density is appeared as an effective mechanical stimulus for the bone resorption. Moreover, using our bi-phasic model, hydrostatic pressure was introduced as another mechanical stimulus for the bone resorption process (see Eq. (4)). Using this model, it was also shown that increasing either strain energy density or hydrostatic pressure will increase rate of bone resorption. The former point can be used as a theoretical justification for many experimental observations (e.g., (Burr et al. 1985; Burr and Martin 1993; Mori and Burr 1993; Schaffler and Jepsen 2000; Li et al. 2001; Martin 2003; Van Der Vis et al. 1998; Skripitz and Aspenberg 2000; Astrand et al. 2003). This model also shows that an increase in the concentration of H+, or a decrease in the concentrations of *PO4− 3* and Ca2+ can cause a reduction in the rate of bone resorption. Experimental data can be found in support of this model's predictions of the effect of Ca2+ concentration on the rate of bone resorption (Lorget et al. 2000). Using the Second Law of Thermodynamics, it was also shown that the maximum rate of bone resorption in cortical bone is greater than that of cancellous bone. This behaviour of cortical and trabecular bone, which is well accepted experimentally (Martin & Burr, 1989), can also be predicted using the axiom of mass balance in this bi-phasic model.

For more detailed information about the basic assumptions, also governing equations of the bi-phasic model of bone resorption, interested readers are encouraged to consult the following reference (Rouhi et al., 2007).

### **6. A tri-phasic mixture model of bone resorption process**

68 Osteoporosis

where *J* is the mineral flux across the real surface of the mineral phase, *DS* is the degree of saturation, [*H+*] is the concentration of hydrogen ion, and *k*, *m*, and *n* are empirical constants. As stated earlier, it is assumed that the mineral flux, *J,* is almost the same as the

where [X] is the concentration of ion X, and Kso is the solubility product of hydroxyapatite

Since biological, chemical, and mechanical factors have a definite effect on the rate of dissolution, it is hypothesized that a bio-chemo-mechanical driving force should be considered in the dissolution relation, instead of just a chemical driving force (i.e. just changes in the Gibbs free energy). Dissipation law can be used to find the bio-chemomechanical affinity, and it is defined as the difference between the external work rate and the rate of change in free energy. According to the Second Law of Thermodynamics, this quantity should be nonnegative. Using the dissipation law and after some manipulations,

 A = ψmech. + P + Cs (μs –μext) (4) where *ψmech.* is the mechanical part of the free energy, *P* is the hydrostatic pressure, Cs is defined as ߩȀܯ, where ߩ and *M* are the density and the molar mass of the matrix, respectively, and *μs* & *μext* are the chemical potential of the solid phase in the unstressed

Our bi-phasci mixture model of bone resorption shows that the activity of osteoclasts and, thus, the rate of bone resorption are not only dictated by biological factors (e.g., hormone levels), but also by engineering quantities, i.e. hydrostatic pressure, strain energy density, and concentration of different ions before and after the resorption process. Interesting to note that the exact stimulus for the initiation of the remodeling process of bone is not known yet and is a place of debate (Rouhi, 2006a). In 1990, Brown and co-workers have shown experimentally that strain energy density can be a likely stimulus for bone remodeling (Brown et al. 1990), and it was used extensively in many theoretical modeling of bone adaptation; for instance (Jacobs et al. 1997; Huiskes et al. 2000; Doblar´e and Garc´a 2001; Garcia et al. 2002; Ruimerman et al. 2005). As can be seen in Eq. 4, in this biphasic model, strain energy density is appeared as an effective mechanical stimulus for the bone resorption. Moreover, using our bi-phasic model, hydrostatic pressure was introduced as another mechanical stimulus for the bone resorption process (see Eq. (4)). Using this model, it was also shown that increasing either strain energy density or hydrostatic pressure will increase rate of bone resorption. The former point can be used as a theoretical justification for many experimental observations (e.g., (Burr et al. 1985; Burr and Martin 1993; Mori and Burr 1993; Schaffler and Jepsen 2000; Li et al. 2001; Martin 2003; Van Der Vis et al. 1998; Skripitz and Aspenberg 2000; Astrand et al. 2003). This model also shows that an increase in the concentration of H+, or a decrease in the concentrations of *PO4− 3* and Ca2+ can cause a reduction in the rate of bone resorption. Experimental data can be found in support of this model's predictions of the effect of Ca2+ concentration on the rate of bone resorption (Lorget et al. 2000). Using the Second Law of

the driving force for the dissolution process of bone can take the following form:

condition and the external potential energy, respectively.

DS = {([Ca2+]5 [PO4 <sup>−</sup> 3 ]3 [OH−])/Kso}1/9 (3)

dissolution rate of the solid phase, i.e. hydroxyapatite + collagen fibers.

The degree of saturation (*DS*) is expressed as:

(Margolis and Moreno 1992).

Recently, a tri-phasic model of bone resorption using mixture theory with chemical reactions was proposed (Rouhi, 2011). In this model, three different constituents (matrix, fluid, and cells) have been considered. Bone resorption is considered as a chemical reaction caused by the secretion of *H+* and *Cl-* from osteoclasts which creates an acidic environment in a sealed zone between osteoclasts and bone matrix. It is assumed that the solid phase obeys small deformation theory and is isotropic and linearly elastic. The velocity of the matrix and cells is assumed to be zero. The fluid phase is assumed to be viscous, and inertial effects are neglected because of the slow velocities that are at play. A non-rotational fluid is assumed for deriving the final form of the entropy inequality for the mixture as a whole. In the constitutive equations, similar to our bi-phasic model (Rouhi et al., 2007), it is assumed that the free energy, enthalpy, specific entropy, heat flux, and stress tensor are functions of temperature, deformation gradient, and the extent of chemical reactions. Bone resorption was considered as an isothermal and a quasi-static process. For the sake of simplicity, presence of ostocytes in the bone matrix was discarded in this model, despite the fact that fluid flow in the bone matrix (e.g. in the lacuno-canalicular network) has a definite effect on the osteocytes, and, most likely, on the osteoclasts and thus on the rate of bone resorption. Using these assumptions, the governing equations for bone resorption were derived using the conservation laws (mass, momentum, and energy), as well as entropy inequality and the appropriate constitutive equations.

By using mixture theory with chemical reactions, first, contribution of different phases present in the mixture can be observed. Secondly, using consistency requirement for energy balance, it was found that rate of bone resorption is a function of different factors including apparent density of bone matrix and bone fluid; fluid velocity; momentum supply to the fluid or solid phase; and internal energy densities of different constituents. Thirdly, using the relation between momentum supply to the solid and fluid phase, one can conclude that rate of bone resorption is inversely proportional to the bone fluid velocity. Also, it was found that in spongy bone, by increasing the porosity, rate of resorption will decrease and vice versa. Based on our results, it is speculated that bone resorption in cortical and cancellous bones might be affected by a control system which is resulted from the relation between the specific surface of bone and its apparent density and volume fraction. As it is known, one reason of osteoporosis is the lack of calcium ions in our body, so in the case of need of calcium, where is better than a rich reservoir, i.e. bones, to take away calcium ions via bone resorption process and giving back in the bone formation process. It seems necessary and feasible, as a future task, to investigate the relation between calcium concentration in the bone fluid and the rate of bone resotrpiton using mixture theory.

Biomechanics of Osteoporosis: The Importance of Bone Resorption and Remodeling Processes 71

an osteoporotic bone, bone apparent density will also decrease even by increasing the

Some of the possible explanations for the abnormal bone loss in an osteoporotic bone suggested by different researchers are as follows: (1) a higher percentage of the bone forming cells is embedded in bone matrix as osteocytes (Mullender et al., 1996), so a reduction in the number of bone forming cells can be seen; (2) the bone forming activity of osteoblasts is reduced (Mullender et al., 1996; Ruimerman et al., 200?), thus less bone apposition will occur; (3) the average life-span of osteoblasts is reduced (Mullender et al., 1996; Eriksen and Kassem, 1992); (4) a reduction in bone sensor cells mechanosensitivity (Sterck et al., 1998), thus they cannot make a true picture of the mechanical environment of the bone and so there will be a reduction in the smartness of bone structure. It seems reasonable to assume that bone loss in the case of osteoporosis is the result of a combination

For more detailed information about this work, interested readers are encouraged to consult

*i=*0.95

*i=*0.8

> *i=*0.2

*i=*0.9

*i=*0.7

*i=*0.1

*i=*0.4 *i=*0.5

Fig. 1. Results of simulation of the spongy bone remodeling for different levels of osteocyte mechanosensitivity (*μi*), representing the level of activity of bone sensor cells (Li, 2011).

of all the above mentioned, and likely some other, factors.

*i=*1

*i=*0.85

> *i=*0.6

*i=0.3*

the following references (Li & Rouhi, 2011; Li, 2011).

number of osteocytes".

For more detailed information about the basic assumptions, also governing equations of the tri-phasic model of bone resorption, interested readers are encouraged to consult the following reference (Rouhi, 2011).

### **7. The effects of osteocytes number and mechanosensitivity on bone loss**

Based on the experimental data and evidence, it is known that osteocyte density (the number of osteocytes per unit surface of bone) changes with aging and also in osteoporotic bones (Gong et al., 2008; Mullender et al., 1996). Moreover, they interestingly found that the osteocyte density increased in osteoporotic patients compared to that of healthy adults, although excessive bone loss and reduced spongy bone wall thickness have been described as characteristic for osteoporotic bones. Experimental evidence for altered mechanosensitivity of osteocytes derived from osteoporotic patients has also been reported (Sterck et al., 1998). According to the semi-mechanistic bone remodeling theory (Huiskes et al., 2000; Ruimerman et al., 2005), and based on the fact that the number of osteocytes per unit surface of bone decreases with aging, we hypothesized that bone loss with the age is correlated with the reduction of either the number of osteocytes, or the strength of the recruitment signal sent by osteocytes to osteoblasts (Li, 2011; Li & Rouhi, 2011).

In the semimechanistic model of Huiskes and co-workers, bone remodeling is considered as a coupling process of bone resorption and bone formation on the bone free surfaces. Osteoclasts are assumed to resorb bone stochastically. Osteocytes are suggested to act as strain energy density (SED) rate sensing cells, and to play a role in the regulation of bone remodeling. It is assumed that osteocytes locally sense the SED rate perturbation generated by either the external load or by cavities made by osteoclasts (bone resorbing cells), and then recruit osteoblasts to form bone tissue to fill the resorption cavities. Osteoclasts are assumed to resorb a constant amount of bone per day. The probability of osteoclast activities may be regulated by the presence of either micro-cracks or in the case of disuse. Since the changes in bone structure because of osteoporosis are similar to changes resulting from disuse (Frost, 1988; Rodan, 1991), it was assumed that one of the causes for bone loss in osteoporotic bones can be the reduction in osteocyte mechanosensitivity.

In our study, we developed a two dimensional finite element model of spongy bone using a semi-mechanistic bone remodeling theory (Huiskes et al., 2000) to simulate spongy bone remodeling and investigate the validity of our hypotheses (Li, 2011; Li & Rouhi, 2011). Results of our study showed that the osteocyte density has a significant role in the final geometry of spongy bone in the bone remodeling process. It was also shown that by decreasing the osteocyte density (knowing that the osteocyte density decrease as a healthy adult ages), bone loss will occur and there will be a decrease in bone apparent density. Moreover, it was shown that when osteocyte mechanosensitivity is less than a certain level, osteoporotic patients lose more spongy bone than healthy old adults even though osteoporotic patients have greater osteocyte number than in healthy old adults. Figure 1 shows the final simulation results of spongy bone with different mechanosensitivities of osteocytes, but the same osteocytes' number and the same form of osteocyte distribution. As can be seen, by decreasing the mechanosensitivity of osteocytes, there will be a reduction in spongy bone apparent density. Results of this study were in favour of our hypothesis stating that "by decreasing the osteocyte mechanosensitivity, as is the case in

For more detailed information about the basic assumptions, also governing equations of the tri-phasic model of bone resorption, interested readers are encouraged to consult the

**7. The effects of osteocytes number and mechanosensitivity on bone loss** 

Based on the experimental data and evidence, it is known that osteocyte density (the number of osteocytes per unit surface of bone) changes with aging and also in osteoporotic bones (Gong et al., 2008; Mullender et al., 1996). Moreover, they interestingly found that the osteocyte density increased in osteoporotic patients compared to that of healthy adults, although excessive bone loss and reduced spongy bone wall thickness have been described as characteristic for osteoporotic bones. Experimental evidence for altered mechanosensitivity of osteocytes derived from osteoporotic patients has also been reported (Sterck et al., 1998). According to the semi-mechanistic bone remodeling theory (Huiskes et al., 2000; Ruimerman et al., 2005), and based on the fact that the number of osteocytes per unit surface of bone decreases with aging, we hypothesized that bone loss with the age is correlated with the reduction of either the number of osteocytes, or the strength of the recruitment signal sent by osteocytes to osteoblasts (Li, 2011; Li & Rouhi,

In the semimechanistic model of Huiskes and co-workers, bone remodeling is considered as a coupling process of bone resorption and bone formation on the bone free surfaces. Osteoclasts are assumed to resorb bone stochastically. Osteocytes are suggested to act as strain energy density (SED) rate sensing cells, and to play a role in the regulation of bone remodeling. It is assumed that osteocytes locally sense the SED rate perturbation generated by either the external load or by cavities made by osteoclasts (bone resorbing cells), and then recruit osteoblasts to form bone tissue to fill the resorption cavities. Osteoclasts are assumed to resorb a constant amount of bone per day. The probability of osteoclast activities may be regulated by the presence of either micro-cracks or in the case of disuse. Since the changes in bone structure because of osteoporosis are similar to changes resulting from disuse (Frost, 1988; Rodan, 1991), it was assumed that one of the causes for bone loss in osteoporotic bones

In our study, we developed a two dimensional finite element model of spongy bone using a semi-mechanistic bone remodeling theory (Huiskes et al., 2000) to simulate spongy bone remodeling and investigate the validity of our hypotheses (Li, 2011; Li & Rouhi, 2011). Results of our study showed that the osteocyte density has a significant role in the final geometry of spongy bone in the bone remodeling process. It was also shown that by decreasing the osteocyte density (knowing that the osteocyte density decrease as a healthy adult ages), bone loss will occur and there will be a decrease in bone apparent density. Moreover, it was shown that when osteocyte mechanosensitivity is less than a certain level, osteoporotic patients lose more spongy bone than healthy old adults even though osteoporotic patients have greater osteocyte number than in healthy old adults. Figure 1 shows the final simulation results of spongy bone with different mechanosensitivities of osteocytes, but the same osteocytes' number and the same form of osteocyte distribution. As can be seen, by decreasing the mechanosensitivity of osteocytes, there will be a reduction in spongy bone apparent density. Results of this study were in favour of our hypothesis stating that "by decreasing the osteocyte mechanosensitivity, as is the case in

following reference (Rouhi, 2011).

can be the reduction in osteocyte mechanosensitivity.

2011).

an osteoporotic bone, bone apparent density will also decrease even by increasing the number of osteocytes".

Some of the possible explanations for the abnormal bone loss in an osteoporotic bone suggested by different researchers are as follows: (1) a higher percentage of the bone forming cells is embedded in bone matrix as osteocytes (Mullender et al., 1996), so a reduction in the number of bone forming cells can be seen; (2) the bone forming activity of osteoblasts is reduced (Mullender et al., 1996; Ruimerman et al., 200?), thus less bone apposition will occur; (3) the average life-span of osteoblasts is reduced (Mullender et al., 1996; Eriksen and Kassem, 1992); (4) a reduction in bone sensor cells mechanosensitivity (Sterck et al., 1998), thus they cannot make a true picture of the mechanical environment of the bone and so there will be a reduction in the smartness of bone structure. It seems reasonable to assume that bone loss in the case of osteoporosis is the result of a combination of all the above mentioned, and likely some other, factors.

For more detailed information about this work, interested readers are encouraged to consult the following references (Li & Rouhi, 2011; Li, 2011).

Fig. 1. Results of simulation of the spongy bone remodeling for different levels of osteocyte mechanosensitivity (*μi*), representing the level of activity of bone sensor cells (Li, 2011).

Biomechanics of Osteoporosis: The Importance of Bone Resorption and Remodeling Processes 73

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### **8. Discussion and conclusions**

Unlike Engineering materials and structures, biological materials including bone, are sensitive to the mechanical stimuli placed on them. Moreover, their mechanical properties are changing continuously as a function of time, mechanical load, and biological factors (e.g. various hormones levels and nutrition). Osteoporosis is caused when there is an imbalance in the bone remodeling process. So, in order to be able to find a solid cure for this disease, a clear and comprehensive understanding of the bone remodeling process at different level of considerations, i.e. molecular; cellular; and tissue level, is needed. A wealth of evidence has been accumulated during the past few years supporting the concept that the study of bone micro- and nano-structures will not only improve our understanding of the mechanisms that underlie bone fragility, but also help to discover the effects of treatments. For instance, nanomedicine and its application to bone research can undoubtedly broaden our knowledge of patho-physiology and improve the diagnostic, prevention and treatment of bone diseases including osteoporosis. Considering the complexity and multifactorial aspect of the remodeling process, the best way to tackle this problem seems to be working in a multidisciplinary group including researchers from various disciplines of medicine and bioengineering.

Based on the fact that skeletal integrity is determined by the outstanding and variant mechanical properties of bone at different hierarchical levels of its structure, it becomes clear that a simple diagnostic parameter such as hip bone mineral density (BMD) does not have enough diagnostic strength to determine the complex patho-physiological mechanisms that determine bone fragility. Thus, new diagnostic tools developed by bioengineering scientists, coupled with a possible combinatorial approach using different methods to define the material qualities of bone at different hierarchical levels of bone's structure, are needed in identifying the initiation and also the progression of the silent and dangerous disease, socalled osteoporosis.

The responsiveness to either an increase or a decrease in mechanical stimulus is very likely greater in growing than adult bones. So, the concept of public health programs aimed at increasing physical activity among healthy children and adolescents in order to maximize peak bone mass, and thus to minimize the probability of bone fracture due t low strength, seems reasonable and should be considered seriously.

### **9. Acknowledgement**

Amirkabir University of Technology, Iran & University of Ottawa, Canada, as well as Dr. M. Esptein, Dr. W. Herzog, Dr. L. Sudak from the University of Calgary, and Mr. X. Li.

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Unlike Engineering materials and structures, biological materials including bone, are sensitive to the mechanical stimuli placed on them. Moreover, their mechanical properties are changing continuously as a function of time, mechanical load, and biological factors (e.g. various hormones levels and nutrition). Osteoporosis is caused when there is an imbalance in the bone remodeling process. So, in order to be able to find a solid cure for this disease, a clear and comprehensive understanding of the bone remodeling process at different level of considerations, i.e. molecular; cellular; and tissue level, is needed. A wealth of evidence has been accumulated during the past few years supporting the concept that the study of bone micro- and nano-structures will not only improve our understanding of the mechanisms that underlie bone fragility, but also help to discover the effects of treatments. For instance, nanomedicine and its application to bone research can undoubtedly broaden our knowledge of patho-physiology and improve the diagnostic, prevention and treatment of bone diseases including osteoporosis. Considering the complexity and multifactorial aspect of the remodeling process, the best way to tackle this problem seems to be working in a multidisciplinary group including researchers from various disciplines of medicine and

Based on the fact that skeletal integrity is determined by the outstanding and variant mechanical properties of bone at different hierarchical levels of its structure, it becomes clear that a simple diagnostic parameter such as hip bone mineral density (BMD) does not have enough diagnostic strength to determine the complex patho-physiological mechanisms that determine bone fragility. Thus, new diagnostic tools developed by bioengineering scientists, coupled with a possible combinatorial approach using different methods to define the material qualities of bone at different hierarchical levels of bone's structure, are needed in identifying the initiation and also the progression of the silent and dangerous disease, so-

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