**Stem Cell-Mediated Intervertebral Disc Regeneration**

Namath S. Hussain, Vickram Tejwani and Mick Perez-Cruet *Oakland University William Beaumont School of Medicine, Royal Oak, Michigan USA* 

#### **1. Introduction**

282 Tissue Regeneration – From Basic Biology to Clinical Application

Zscharnack M; Hepp P; Richter R et al. (2010). Repair of chronic osteochondral defects using

38, No. 9, pp. (1857-1869), ISSN 1552-3365

predifferentiated mesenchymal stem cells in an ovine model. *Am J Sports Med*, Vol.

Currently, degenerative disk disease (DDD) and the subsequent chronic lower back pain that results from it represent a significant source of morbidity and mortality worldwide. The available treatment modalities such as pain therapy and surgical interventions aim to provide symptomatic relief; however, they do not address the underlying pathophysiology of DDD. The disease also has high societal health care costs (Chan et al., 2006; Cassinelli et al, 2001). Many modalities exist for symptomatic treatment of this condition, including bed rest, massage, stretching, strengthening exercises, physical therapy, epidural injections and other pain management therapies, and spinal surgery. Most conservative therapies are

Fig. 1. Anatomy of the spine with the compartmentalization of the IVD.

Stem Cell-Mediated Intervertebral Disc Regeneration 285

Fig. 4. Postmortem specimen and hematoxylin and eosin staining showing multilevel

attempted before surgery with the intent to spare patients the possible complications associated with surgical intervention. However, these conservative measures and even surgery itself with its associated risks only address the symptoms with no impact on the disease process in the disc itself. Recent research has given further insight into the pathogenesis of DDD, which has borne out a renewed interest in biologic therapies centered on the nucleus pulposus (NP) and the annulus fibrosus and the potential of stem cells to reverse the disease process at a histological and cellular level. In this chapter, we will systemically review the current literature and the most salient studies regarding biologic therapies in the regeneration of the intervertebral disc (IVD). We go on to describe the direction this field is heading in and

Before examining the utility of stem cells in human and animal models, it is important to review several of the basic science benchtop laboratory studies that have provided the rationale for in-vivo testable treatments and hypotheses. These studies examined factors influencing both mesenchymal and embryonic stem cell proliferation and differentiation towards a NP-like phenotype. We will examine how these studies have provided valuable information regarding multiple factors that can stimulate embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) towards a chondrocytic lineage, as well as factors that can

DDD is a condition that rises from a combination of a genetic predisposition (Chan et al., 2006) along with environmental modifiers (Stokes & Iatridis, 2004). Several causes of age-

degeneration of the IVDs with fissuring of the actual disc structure

the future potential of the therapies being developed using ESCs.

**2. Basic science laboratory studies** 

inhibit this differentiation in basic in-vitro models.

**2.1 Genetic studies** 

Fig. 2. Axial slice model of the intervertebral disc with an image of a disc herniation

Fig. 3. Sagittal T2-weighted MRI showing degeneration and loss of T2 signal in the L5-S1 IVD

Fig. 2. Axial slice model of the intervertebral disc with an image of a disc herniation

Fig. 3. Sagittal T2-weighted MRI showing degeneration and loss of T2 signal in the L5-S1

IVD

Fig. 4. Postmortem specimen and hematoxylin and eosin staining showing multilevel degeneration of the IVDs with fissuring of the actual disc structure

attempted before surgery with the intent to spare patients the possible complications associated with surgical intervention. However, these conservative measures and even surgery itself with its associated risks only address the symptoms with no impact on the disease process in the disc itself. Recent research has given further insight into the pathogenesis of DDD, which has borne out a renewed interest in biologic therapies centered on the nucleus pulposus (NP) and the annulus fibrosus and the potential of stem cells to reverse the disease process at a histological and cellular level. In this chapter, we will systemically review the current literature and the most salient studies regarding biologic therapies in the regeneration of the intervertebral disc (IVD). We go on to describe the direction this field is heading in and the future potential of the therapies being developed using ESCs.

#### **2. Basic science laboratory studies**

Before examining the utility of stem cells in human and animal models, it is important to review several of the basic science benchtop laboratory studies that have provided the rationale for in-vivo testable treatments and hypotheses. These studies examined factors influencing both mesenchymal and embryonic stem cell proliferation and differentiation towards a NP-like phenotype. We will examine how these studies have provided valuable information regarding multiple factors that can stimulate embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs) towards a chondrocytic lineage, as well as factors that can inhibit this differentiation in basic in-vitro models.

#### **2.1 Genetic studies**

DDD is a condition that rises from a combination of a genetic predisposition (Chan et al., 2006) along with environmental modifiers (Stokes & Iatridis, 2004). Several causes of age-

Stem Cell-Mediated Intervertebral Disc Regeneration 287

be understated with regard to stem cell development and has important implications pertaining to harvesting and large-scale production of these cells for future potential

Several groups have conducted well-designed in-vitro studies that have gone one step beyond identifying environmental factors that affect differentiation of stem cells into NPlike cells, and have actually studied how these factors may correlate to the current in-vivo microenvironment of the IVD. This was done in order to obtain a clear picture of what would happen if these stem cells were implanted into these native biological conditions. Culturing under IVD-like glucose conditions (1.0 mg/mL glucose) stimulated aggrecan and collagen I expression and deposition. IVD-like osmolarity (485 mOsm) and pH (pH = 6.8) conditions, on the other hand, strongly decreased proliferation and expression of matrix proteins. Combining these conditions resulted in decreased proliferation and gene expression of matrix proteins, demonstrating that, in this case, osmolarity and pH play a larger impact in inhibiting differentiation than glucose does in stimulating it (Wuertz et al.,

Another study by the same group showed that acidity caused an inhibition of aggrecan and collagen I expression, as well as a decrease in proliferation and cell viability. This demonstrates that pH may be the major limitation for stem cell-based IVD repair (Wuertz, 2009). This also illustrates the importance of early intervention and the role of predifferentiation when planning to use stem cells for reparative treatments. However, some studies have shown that implantation of stem cells at a later stage in the DDD process may result in a greater increase in disc height when compared to implantation at an earlier stage (Ho et al., 2008). This finding highlights the importance of studies involving stem cellbased intervertebral disc regeneration being carefully controlled in the context of stage of disc degeneration. Again, this point highlights the importance of temporal sequence when examining therapeutics with stem cells. Additionally, inflammatory processes have been shown to inhibit the chondrogenic differentiation of stem cells, whereas hypoxic conditions exert beneficial effects on chondrogenesis and phenotype stability of transplanted stem cells

There is currently an avid interest in using our accumulated data and knowledge of the factors influencing stem cell proliferation and the exact conditions in the native IVD

Multiple studies have investigated culturing MSCs with NP cells in a co-culture system, allowing for cell-to-cell contact (Yang et al., 2009; Le Maitre et al., 2009; Vadalà et al., 2009; Richardson et al., 2006; Richardson et al., 2008). This contact has been shown to stimulate these MSCs to differentiate toward a chondrocytic lineage, therefore removing the need for pre-differentiation in-vitro (Watanabe et al., 2010; Svanvik et al., 2010; Niu et al., 2009; Wei et al., 2009; Tao et al., 2008; Le Visage et al., 2006; Richardson et al., 2006). This was evidenced by mRNA expression levels of Type II collagen and aggrecan being elevated in co-cultured cells and cells undergoing morphological changes to form three-dimensional micromasses

**2.3 Stem cell growth in the native IVD microenvironment** 

therapeutic uses.

2008).

(Felka et al., 2009).

**2.4 Optimizing conditions to promote proliferation** 

microenvironment to optimize the chances for stem cell proliferation.

related degeneration of the IVD include loss of biomechanical support by surrounding muscular and ligamentous structures, uneven force loading as the aging spine deforms while trying to compensate for these changes, cell senescence, loss of viable progenitor cells, accumulation of degraded matrix molecules, and fatigue failure of both the disc matrix and surrounding annulus fibrosus. Correlations have been made between DDD and collagen, aggrecan, and matrix metalloproteinase polymorphisms coding for structural proteins (Ala-Kokko, 2002).

#### **2.2 Factors influencing stem cell proliferation**

In order to further study how these cells would interact in various factor environments, it became crucial to more fully characterize these cells. This point is very important with regard to stem cell research because it is essential to characterize and identify what factors provide the best type of environment to stimulate ESCs and MSCs to differentiate toward a chondrocytic-type cell lineage.

#### **2.2.1 Mesenchymal Stem Cells**

Transforming growth factor-β3 (TGF-β3) is one factor that has been shown in multiple studies (Steck et al., 2005; Risbud et al., 2004; Shen, 2009) to stimulate cells to differentiate into chondrocytes. Several studies have shown that after TGF-β3 stimulation, MSCs turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, such as aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein (Steck et al., 2005; Risbud et al., 2004). Shen et al. have shown that bone morphogenic protein-2 (BMP-2) can help to enhance TGF-β3-mediated chondrogenesis in MSCs (Shen, 2009). The combination of BMP-2 and TGF-β3 in alginate culture was found to be superior to the standard differentiation method using TGF-β3 alone as evinced by increased mRNA expression of aggrecan, type II collagen, Sox-9, BMP-2, and BMP-7, all of which are chondrocyte markers. This effect was even more pronounced when TGF-β3 and rhBMP-2 were both added (Kuh et al., 2008). This synergistic effect was consistently found in the study, providing further support as to an as yet unknown pathway towards chondrocytic differentiation.

#### **2.2.2 Embryonic Stem Cells**

Hoben et al performed a similar characterization study using human ESCs (Hoben et al., 2009). Growth factors were studied with a coculture method for 3 weeks and evaluated for collagen and glycosaminoglycan (GAG) synthesis. The growth factors studied were TGF-β3, BMP-2, BMP-4, BMP-6, and sonic hedgehog protein. The investigators found that the combination of BMP-4 and TGF-β3 within the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone. Koay et al had similar results with BMP-2 and TGF-β3 leading human ESCs down a differentiation path that produced an end product with high type I collagen content (Koay et al., 2007). However, they also found that human ESCs treated with TGF-β3 followed by TGF-β1 and IGF-1 produced constructs with no collagen I, showing that different growth factor application in different temporal sequences can have a marked impact on end-product composition and biomechanical properties. The importance of temporal sequences cannot

related degeneration of the IVD include loss of biomechanical support by surrounding muscular and ligamentous structures, uneven force loading as the aging spine deforms while trying to compensate for these changes, cell senescence, loss of viable progenitor cells, accumulation of degraded matrix molecules, and fatigue failure of both the disc matrix and surrounding annulus fibrosus. Correlations have been made between DDD and collagen, aggrecan, and matrix metalloproteinase polymorphisms coding for structural proteins (Ala-

In order to further study how these cells would interact in various factor environments, it became crucial to more fully characterize these cells. This point is very important with regard to stem cell research because it is essential to characterize and identify what factors provide the best type of environment to stimulate ESCs and MSCs to differentiate toward a

Transforming growth factor-β3 (TGF-β3) is one factor that has been shown in multiple studies (Steck et al., 2005; Risbud et al., 2004; Shen, 2009) to stimulate cells to differentiate into chondrocytes. Several studies have shown that after TGF-β3 stimulation, MSCs turned positive for collagen type II protein and expressed a large panel of genes characteristic for chondrocytes, such as aggrecan, decorin, fibromodulin, and cartilage oligomeric matrix protein (Steck et al., 2005; Risbud et al., 2004). Shen et al. have shown that bone morphogenic protein-2 (BMP-2) can help to enhance TGF-β3-mediated chondrogenesis in MSCs (Shen, 2009). The combination of BMP-2 and TGF-β3 in alginate culture was found to be superior to the standard differentiation method using TGF-β3 alone as evinced by increased mRNA expression of aggrecan, type II collagen, Sox-9, BMP-2, and BMP-7, all of which are chondrocyte markers. This effect was even more pronounced when TGF-β3 and rhBMP-2 were both added (Kuh et al., 2008). This synergistic effect was consistently found in the study, providing further support as to an as yet unknown pathway towards

Hoben et al performed a similar characterization study using human ESCs (Hoben et al., 2009). Growth factors were studied with a coculture method for 3 weeks and evaluated for collagen and glycosaminoglycan (GAG) synthesis. The growth factors studied were TGF-β3, BMP-2, BMP-4, BMP-6, and sonic hedgehog protein. The investigators found that the combination of BMP-4 and TGF-β3 within the fibrochondrocyte coculture led to an increase in cell proliferation and GAG production compared to either treatment alone. Koay et al had similar results with BMP-2 and TGF-β3 leading human ESCs down a differentiation path that produced an end product with high type I collagen content (Koay et al., 2007). However, they also found that human ESCs treated with TGF-β3 followed by TGF-β1 and IGF-1 produced constructs with no collagen I, showing that different growth factor application in different temporal sequences can have a marked impact on end-product composition and biomechanical properties. The importance of temporal sequences cannot

Kokko, 2002).

**2.2 Factors influencing stem cell proliferation** 

chondrocytic-type cell lineage.

**2.2.1 Mesenchymal Stem Cells** 

chondrocytic differentiation.

**2.2.2 Embryonic Stem Cells** 

be understated with regard to stem cell development and has important implications pertaining to harvesting and large-scale production of these cells for future potential therapeutic uses.

#### **2.3 Stem cell growth in the native IVD microenvironment**

Several groups have conducted well-designed in-vitro studies that have gone one step beyond identifying environmental factors that affect differentiation of stem cells into NPlike cells, and have actually studied how these factors may correlate to the current in-vivo microenvironment of the IVD. This was done in order to obtain a clear picture of what would happen if these stem cells were implanted into these native biological conditions. Culturing under IVD-like glucose conditions (1.0 mg/mL glucose) stimulated aggrecan and collagen I expression and deposition. IVD-like osmolarity (485 mOsm) and pH (pH = 6.8) conditions, on the other hand, strongly decreased proliferation and expression of matrix proteins. Combining these conditions resulted in decreased proliferation and gene expression of matrix proteins, demonstrating that, in this case, osmolarity and pH play a larger impact in inhibiting differentiation than glucose does in stimulating it (Wuertz et al., 2008).

Another study by the same group showed that acidity caused an inhibition of aggrecan and collagen I expression, as well as a decrease in proliferation and cell viability. This demonstrates that pH may be the major limitation for stem cell-based IVD repair (Wuertz, 2009). This also illustrates the importance of early intervention and the role of predifferentiation when planning to use stem cells for reparative treatments. However, some studies have shown that implantation of stem cells at a later stage in the DDD process may result in a greater increase in disc height when compared to implantation at an earlier stage (Ho et al., 2008). This finding highlights the importance of studies involving stem cellbased intervertebral disc regeneration being carefully controlled in the context of stage of disc degeneration. Again, this point highlights the importance of temporal sequence when examining therapeutics with stem cells. Additionally, inflammatory processes have been shown to inhibit the chondrogenic differentiation of stem cells, whereas hypoxic conditions exert beneficial effects on chondrogenesis and phenotype stability of transplanted stem cells (Felka et al., 2009).

#### **2.4 Optimizing conditions to promote proliferation**

There is currently an avid interest in using our accumulated data and knowledge of the factors influencing stem cell proliferation and the exact conditions in the native IVD microenvironment to optimize the chances for stem cell proliferation.

Multiple studies have investigated culturing MSCs with NP cells in a co-culture system, allowing for cell-to-cell contact (Yang et al., 2009; Le Maitre et al., 2009; Vadalà et al., 2009; Richardson et al., 2006; Richardson et al., 2008). This contact has been shown to stimulate these MSCs to differentiate toward a chondrocytic lineage, therefore removing the need for pre-differentiation in-vitro (Watanabe et al., 2010; Svanvik et al., 2010; Niu et al., 2009; Wei et al., 2009; Tao et al., 2008; Le Visage et al., 2006; Richardson et al., 2006). This was evidenced by mRNA expression levels of Type II collagen and aggrecan being elevated in co-cultured cells and cells undergoing morphological changes to form three-dimensional micromasses

Stem Cell-Mediated Intervertebral Disc Regeneration 289

markers (Sakai et al., 2005). The injected discs had a central NP-like region which had a close similarity to the normal biconvex structure of the IVD and contained viable chondrocytes forming a matrix like that of the normal disc (Sakai et al., 2003; Revell et al., 2007). Omlor et al. studied the practical phenomenon of transplanted stem cell loss through the actual annular puncture which was used to not only simulate disc damage and herniation but also to inject the stem cells themselves. They made a logical conclusion that IVD regeneration strategies should increasingly focus on annulus reconstruction in order to reduce implant loss due to annular failure (Omlor et al., 2010). Most studies

Fig. 5. Hematoxylin and eosin staining of the rabbit IVD, showing healthy notochordal cell

Several xenotransplant studies involving ESCs have been conducted with promising results. Jeong et al have shown that rats receiving human ESCs showed relative restoration of the inner annulus structure compared to a control group (Jeong et al., 2010). This finding may help to address the concern of loss of implanted material through the

Many of the stem cells in these studies were xenografted from other species and the recipient animals were not treated with immunosuppressive agents. In spite of this, there was a lack of immune response suggesting an unrecognized immune-privileged site within the intervertebral disc space (Wei et al., 2009; Sheikh et al., 2009). On top of this, there has been some study with MSC showing that transplantation contributes to this immunosuppressive phenomenon by the differentiation of these cells into cells expressing FasL, which has been shown to be an immunosuppressive factor (Hiyama et al., 2008).

focusing on this point are still ongoing.

rests

needle puncture.

expressing collagen-2, aggrecan, and Sox-9 at RNA and protein levels after 14 days of coculture. These changes were unique and not detected in the samples of stem cells cultured alone (Svanvik et al., 2010; Niu et al., 2009; Wei et al., 2009). Furthermore, MSCs from older individuals differentiate spontaneously into chondrocyte-like NP cells upon insertion into NP tissue in-vitro, and thus may not require additional stimulation to induce differentiation. This is a key finding, as such a strategy would minimize the level of external manipulation required prior to insertion of these cells into the patient, thus simplifying the treatment strategy and reducing costs (Le Maitre et al., 2009).

Adipose-Derived Stem Cells (ADSCs) have also been shown to be able to differentiate into NP cells in multiple in-vitro studies (Xie et al., 2009; Tapp et al., 2008; Lu et al., 2007; Lu et al., 2008; Li et al., 2005). Soluble factors released by NP cells direct chondrogenic differentiation of ADSCs in collagen hydrogels, and combination with a nucleus-mimicking collagen type II microenvironment enhances differentiation towards a more pronounced cartilaginous lineage (Lu et al., 2007; Lu et al., 2008).

Studies using annulus fibrosus cells isolated from nondegenerated intervertebral discs have shown that these cells have the capability of differentiating into adipocytes, osteoblasts, chondrocytes, neurons, and endothelial cells in-vitro. These cells may also be induced to become more plastic, allowing them to differentiate along more mesenchymal lineages (Li et al., 2005; Feng et al., 2010; Saraiya et al., 2010). However, when annulus cells are differentiated into a chondrocyte micromass, it was not as rounded or compact as that which occurs with stem cells induced into chondrocyte differentiation (Saraiya et al., 2010). TGF-β stimulation of fetal cells cultured in high cell density led to the production of aggrecan, type I and II collagens and variable levels of type X collagen, although fetal cells had lower adipogenic and osteogenic differentiation capacity than MSCs and variability in matrix synthesis was observed between specific donors (Quintin et al., 2009; Quintin et al., 2010).

### **3. Animal studies**

Many studies using stem cells for disc regeneration have been performed in a wide array of animal models with promising results. Two recent studies were conducted utilizing ADSCs in a murine (Jeong et al., 2010) and a canine model (Ganey et al., 2009). Staining in both studies demonstrated increased Type II collagen and aggrecan in the transplantation group. Additionally, at 6 weeks after transplantation, discs exhibited a restoration of disc hydration and MRI T2 signal intensity and more closely resembled the healthy controls as evidenced by matrix translucency, compartmentalization of the annulus, and increased cell density within the nucleus pulposus. Discs also showed a significantly smaller reduction in disc height when compared with controls.

Multiple studies have shown that MSCs are able to proliferate and survive inside the IVD, with assessments being made as far out as six months post-transplant (Tan et al., 2009; Jeong et al., 2009; Henriksson et al., 2009; Sobajima et al., 2008; Zhang et al., 2005; Crevensten et al., 2004). Additionally, these cells have been proven to differentiate into cells expressing chondrocytic phenotypes, as evidenced by positive immunostaining of collagen type II, aggrecan, and other markers (Henriksson et al., 2009; Yang et al., 2010; Wei et al., 2009; Sakai et al., 2005). Cells were also shown to exhibit NP phenotypic

expressing collagen-2, aggrecan, and Sox-9 at RNA and protein levels after 14 days of coculture. These changes were unique and not detected in the samples of stem cells cultured alone (Svanvik et al., 2010; Niu et al., 2009; Wei et al., 2009). Furthermore, MSCs from older individuals differentiate spontaneously into chondrocyte-like NP cells upon insertion into NP tissue in-vitro, and thus may not require additional stimulation to induce differentiation. This is a key finding, as such a strategy would minimize the level of external manipulation required prior to insertion of these cells into the patient, thus simplifying the treatment

Adipose-Derived Stem Cells (ADSCs) have also been shown to be able to differentiate into NP cells in multiple in-vitro studies (Xie et al., 2009; Tapp et al., 2008; Lu et al., 2007; Lu et al., 2008; Li et al., 2005). Soluble factors released by NP cells direct chondrogenic differentiation of ADSCs in collagen hydrogels, and combination with a nucleus-mimicking collagen type II microenvironment enhances differentiation towards a more pronounced

Studies using annulus fibrosus cells isolated from nondegenerated intervertebral discs have shown that these cells have the capability of differentiating into adipocytes, osteoblasts, chondrocytes, neurons, and endothelial cells in-vitro. These cells may also be induced to become more plastic, allowing them to differentiate along more mesenchymal lineages (Li et al., 2005; Feng et al., 2010; Saraiya et al., 2010). However, when annulus cells are differentiated into a chondrocyte micromass, it was not as rounded or compact as that which occurs with stem cells induced into chondrocyte differentiation (Saraiya et al., 2010). TGF-β stimulation of fetal cells cultured in high cell density led to the production of aggrecan, type I and II collagens and variable levels of type X collagen, although fetal cells had lower adipogenic and osteogenic differentiation capacity than MSCs and variability in matrix synthesis was observed between specific donors (Quintin et al., 2009; Quintin et al.,

Many studies using stem cells for disc regeneration have been performed in a wide array of animal models with promising results. Two recent studies were conducted utilizing ADSCs in a murine (Jeong et al., 2010) and a canine model (Ganey et al., 2009). Staining in both studies demonstrated increased Type II collagen and aggrecan in the transplantation group. Additionally, at 6 weeks after transplantation, discs exhibited a restoration of disc hydration and MRI T2 signal intensity and more closely resembled the healthy controls as evidenced by matrix translucency, compartmentalization of the annulus, and increased cell density within the nucleus pulposus. Discs also showed a significantly smaller reduction in disc

Multiple studies have shown that MSCs are able to proliferate and survive inside the IVD, with assessments being made as far out as six months post-transplant (Tan et al., 2009; Jeong et al., 2009; Henriksson et al., 2009; Sobajima et al., 2008; Zhang et al., 2005; Crevensten et al., 2004). Additionally, these cells have been proven to differentiate into cells expressing chondrocytic phenotypes, as evidenced by positive immunostaining of collagen type II, aggrecan, and other markers (Henriksson et al., 2009; Yang et al., 2010; Wei et al., 2009; Sakai et al., 2005). Cells were also shown to exhibit NP phenotypic

strategy and reducing costs (Le Maitre et al., 2009).

cartilaginous lineage (Lu et al., 2007; Lu et al., 2008).

2010).

**3. Animal studies** 

height when compared with controls.

markers (Sakai et al., 2005). The injected discs had a central NP-like region which had a close similarity to the normal biconvex structure of the IVD and contained viable chondrocytes forming a matrix like that of the normal disc (Sakai et al., 2003; Revell et al., 2007). Omlor et al. studied the practical phenomenon of transplanted stem cell loss through the actual annular puncture which was used to not only simulate disc damage and herniation but also to inject the stem cells themselves. They made a logical conclusion that IVD regeneration strategies should increasingly focus on annulus reconstruction in order to reduce implant loss due to annular failure (Omlor et al., 2010). Most studies focusing on this point are still ongoing.

Fig. 5. Hematoxylin and eosin staining of the rabbit IVD, showing healthy notochordal cell rests

Several xenotransplant studies involving ESCs have been conducted with promising results. Jeong et al have shown that rats receiving human ESCs showed relative restoration of the inner annulus structure compared to a control group (Jeong et al., 2010). This finding may help to address the concern of loss of implanted material through the needle puncture.

Many of the stem cells in these studies were xenografted from other species and the recipient animals were not treated with immunosuppressive agents. In spite of this, there was a lack of immune response suggesting an unrecognized immune-privileged site within the intervertebral disc space (Wei et al., 2009; Sheikh et al., 2009). On top of this, there has been some study with MSC showing that transplantation contributes to this immunosuppressive phenomenon by the differentiation of these cells into cells expressing FasL, which has been shown to be an immunosuppressive factor (Hiyama et al., 2008).

Stem Cell-Mediated Intervertebral Disc Regeneration 291

was done to assess cell survival and localization.

of 9 were damaged with needle puncture. 2 weeks later human MSCs were injected The animals were sacrificed after 1, 3, or 6 months. Disc appearance was visualized by MRI. Immunohistochemistry was used to detect human MSCs.

rabbits underwent needle puncture of the disc with MRIs before and after injection with ESCs

expressing green fluorescent protein. At 8 weeks post-ESC implantation, the animals were killed and the intervertebral discs were harvested and analyzed using H & E staining and immunohistochemical

green fluorescent protein, were transplanted into mature rabbits. Consecutive counts of transplanted cells in the nucleus area were performed for 48 weeks with immunohistochemical and proteoglycan content analyses along with PCR detection of mRNA expression of Type I and II collagen, aggrecan and

analysis.

versican.

All injured discs

by real-time PCR.

Cells that were positive for green fluorescent protein were observed in the nucleus pulposus of celltransplanted rabbit discs 2 weeks after transplantation. GFP-positive cells were positive for Type II collagen, keratan sulfate, chondroitin sulfate, and aggrecan.

MRI confirmed intervertebral disc degeneration at needlepunctured segments. Postmortem H & E histological analysis of Group A discs (no intervention) showed mature chondrocytes and no notochordal cells. Group B discs (needle puncture only) displayed an intact annulus fibrosus and generalized disorganization within the NP. Group C discs showed islands of notochordal cell growth (injection of ESCs).

signs on MRI. Immunostaining for

demonstrated degenerative

Aggrecan and Collagen type II expression were observed in NP after 3 and 6 months. mRNA expression of Collagen IIA, Collagen IIB, Versican, Collagen 1A, Aggrecan, and SOX9 were detected at 3 and 6 months

**Studies Model Intervention Results** 

Henriksson et al. Porcine model Three lumbar discs in each

Sheikh et al. Rabbit model 16 New Zealand white

Sakai et al. Rabbit model Stem cells labeled with


undamaged as controls, and other two segments were damaged by needle injection. Two weeks later, stem cells or saline were injected into each of the two

At 6 weeks after transplantation, the

aggrecan.

experimental group showed a significantly smaller reduction in disc height than the saline-injected group and exhibited a restoration of MRI signal intensity. Hematoxylin and eosin staining revealed a greater restoration of the inner annulus structure. There was also increased collagen type II and

Disc levels receiving stem cells more closely resembled the healthy controls as evidenced in matrix translucency,

compartmentalization of the annulus, and in cell density

regeneration of degenerated discs. GFP-positive MSCs detected in the NP region 8 weeks after transplantation expressed FasL protein.

within the nucleus pulposus. Matrix analysis showed increased Type-II collagen and aggrecan.

MSC transplantation effectively led to the

MSCs were detected in histological sections of rabbit discs up to 24 weeks after transplant with engraftment into the inner

annulus fibrosus.

segments of Sprague-Dawley rat were left

damaged segments.

partial nucleotomy were randomized to receive: (1) stem cells in hyaluronic acid carrier (Cells/HA); (2) HA only; or (3) No Intervention.

MSCs were transplanted into the degenerationinduced discs. The animals were followed for 12 weeks

when radiological, histological, biochemical, immunohistochemical, and RT-PCR analyses were

Zealand White rabbits, retrovirally transduced with the lacZ marker gene, and injected into the nucleus pulposus of the L2-3, L3-4, and L4-5 lumbar discs of 12 other NZW rabbits. Rabbits each were sacrificed at 3, 6, 12, or 24 weeks after cell implantation, and staining

performed.

**Studies Model Intervention Results** 

Jeong et al. Rat model The first coccygeal disc

Ganey et al. Canine model 3 discs that had undergone

Hiyama et al. Canine model 4 weeks after nucleotomy,

Sobajima et al. Rabbit model MSCs were isolated New


Stem Cell-Mediated Intervertebral Disc Regeneration 293

**Studies Subjects Intervention Results Study Critique** 

Clinical symptoms improved; increased T2 signal in the disc space on MRI

No clinical symptom relief Few patients

No imaging conducted

cell grafting

cell grafting

Although many laboratory and animal studies have been performed utilizing stem cells for the purposes of cell characterization and inducing chondrocyte formation, much further study is needed before human trials are undertaken on a larger scale. Several studies have already showcased the ability of ESCs to differentiate towards a chondrocytic lineage invitro and also to improve DDD in in-vivo animal and human trials, using a combination of imaging and histological analyses. Several benchtop lab studies have been performed to show that ESCs can be successfully stimulated to differentiate into chondrocyte-like cells (Hoben et al., 2009; Fecek et al., 2008; Hegert et al., 2002; Kawaguchi et al., 2005; zur Nieden et al., 2005; Kramer et al., 2000). Similar to the case with MSCs, different factors affect this process in ESCs, such as TGF-β3, BMP-2, and BMP-4 (Hegert et al., 2002; Kawaguchi et al., 2005; zur Nieden et al., 2005; Kramer et al., 2000; Sakai et al., 2005). Biological scaffolds seeded with chondrocytic cells derived from ESCs, when implanted in mice have been shown to generate cartilage tissue in-vivo (Kramer et al., 2000). Injection of ESCs in a DDDinduced rabbit model led to viable notochordal-type cells within the discs (Sheikh et al., 2009). These animal studies demonstrate the ability of ESCs to differentiate into a

Our group is currently developing chondroprogenitor stem cell lines that can restore the functional capability of the IVD (Sheikh et al., 2009). Our rationale stemmed from the idea that currently there is no biologic therapy for repairing a degenerated IVD and that ESCs have a potential to fill this role based on their regenerative potential. Studies have shown that ESCs can be induced to differentiate into specific cell lineages by using selective culture

Relying on the significant strides made by these basic science groups with regard to cell and factor characterization, our lab proceeded for further refine these methods and develop a protocol for both stem cell differentiation along a chondrocytic lineage and also for examining the utility of transplantation of these cells in a rabbit model of DDD. We initially developed a novel percutaneous animal model of disc degeneration using New Zealand white rabbits (Figure 1) and used this model to explore the possibility of ESC implantation for both structural regeneration and for the growth and continued presence of notochordal

Previous research transplanting MSCs into degenerated rabbit discs has shown consistent biochemical and radiographic (MRI) evidence of IVD restoration (Sakai et al., 2005). Human

Yoshikawa et al. 2 patients Percutaneous stem

Haufe et al. 10 patients Percutaneous stem

Table 2. Human Studies

**5. Future potential of ESCs** 

chondrocytic lineage in-vitro and in-vivo.

media and growth environments (Kawaguchi et al., 2005).

stem cells in the disc space (Sheikh et al., 2009).


Table 1. Animal Studies

Our group recently reported seminal work with regard to ESC implantation in a rabbit model (Sheikh et al., 2009). This study used a needle puncture model with appropriate controls to simulate disc injury. The effects of implanted murine ESCs were measured at 8 weeks using imaging, histological, and immunohistochemical analyses. In-vivo new notochordal cell populations were seen in ESC-injected discs, providing convincing evidence for stem-cell mediated regeneration of the IVD. Another study established the utility of stem cells implanted at 12 weeks post-injury in regenerating the IVD and maintaining perfusion to the endplate and subchondral bone in a porcine model (Bendtsen et al., 2010). Sobajima et al used a rabbit model to show that IVD cells harvested 48 weeks post-implantation revealed a restoration of both glycoprotein content and matrix characteristics (Sobajima et al., 2008). These analyses all provide further evidence that ESC transplantation does have strong potential for clinical use in regenerating the IVD and reversing the cascade of degeneration that occurs with time.

#### **4. Human studies**

To date, there have been only two studies where stem cells were injected into the IVD in humans to stimulate regeneration of the disc. Yoshikawa et al percutaneously grafted MSCs into degenerated IVDs in two women aged 67 and 70 years. After two years, both individuals had alleviation of symptoms and radiographic changes that included improvement of vacuum phenomenon on X-ray and increased signal intensity of IVDs on T2-weighted MRI (Yoshikawa et al., 2010). Another study involved intradiscal injection of hematopoietic stem cells into ten patients that had confirmed disc pain and these patients' pain was assessed at 6-month and 12-month intervals. In contrast to previous study, none of these individuals had any relief of symptoms (Haufe et al., 2006). These trials suggest that stem cells have the potential to relieve symptoms of DDD and restore normal IVD anatomy; however, more human studies are needed to truly establish this. To date, there have been no human ESC implantation studies into the IVD in humans. Further study is needed to verify safety before such work is undertaken.


Table 2. Human Studies

292 Tissue Regeneration – From Basic Biology to Clinical Application

minipigs. After 12 weeks, the animals underwent percutaneous intradiscal injection of stem cells. MRI was performed before treatment and at 24 weeks.

matrix based cell transfer after partial nucleotomy of lumbar IVDs. Segments were analyzed for retained volume of labeling particles

Our group recently reported seminal work with regard to ESC implantation in a rabbit model (Sheikh et al., 2009). This study used a needle puncture model with appropriate controls to simulate disc injury. The effects of implanted murine ESCs were measured at 8 weeks using imaging, histological, and immunohistochemical analyses. In-vivo new notochordal cell populations were seen in ESC-injected discs, providing convincing evidence for stem-cell mediated regeneration of the IVD. Another study established the utility of stem cells implanted at 12 weeks post-injury in regenerating the IVD and maintaining perfusion to the endplate and subchondral bone in a porcine model (Bendtsen et al., 2010). Sobajima et al used a rabbit model to show that IVD cells harvested 48 weeks post-implantation revealed a restoration of both glycoprotein content and matrix characteristics (Sobajima et al., 2008). These analyses all provide further evidence that ESC transplantation does have strong potential for clinical use in regenerating the IVD and

To date, there have been only two studies where stem cells were injected into the IVD in humans to stimulate regeneration of the disc. Yoshikawa et al percutaneously grafted MSCs into degenerated IVDs in two women aged 67 and 70 years. After two years, both individuals had alleviation of symptoms and radiographic changes that included improvement of vacuum phenomenon on X-ray and increased signal intensity of IVDs on T2-weighted MRI (Yoshikawa et al., 2010). Another study involved intradiscal injection of hematopoietic stem cells into ten patients that had confirmed disc pain and these patients' pain was assessed at 6-month and 12-month intervals. In contrast to previous study, none of these individuals had any relief of symptoms (Haufe et al., 2006). These trials suggest that stem cells have the potential to relieve symptoms of DDD and restore normal IVD anatomy; however, more human studies are needed to truly establish this. To date, there have been no human ESC implantation studies into the IVD in humans. Further study is needed to verify

Stem cell treated animal had increased T2 signal in the disc along with increased relative vertebral blood

There was a 90% loss of the implant material under in vivo conditions when the

annulus was not reconstructed.

flow.

**Studies Model Intervention Results** 

Bendtsen et al. Porcine model DDD was induced in 15

Omlor et al. Porcine model 6 minipigs underwent

reversing the cascade of degeneration that occurs with time.

Table 1. Animal Studies

**4. Human studies** 

safety before such work is undertaken.

### **5. Future potential of ESCs**

Although many laboratory and animal studies have been performed utilizing stem cells for the purposes of cell characterization and inducing chondrocyte formation, much further study is needed before human trials are undertaken on a larger scale. Several studies have already showcased the ability of ESCs to differentiate towards a chondrocytic lineage invitro and also to improve DDD in in-vivo animal and human trials, using a combination of imaging and histological analyses. Several benchtop lab studies have been performed to show that ESCs can be successfully stimulated to differentiate into chondrocyte-like cells (Hoben et al., 2009; Fecek et al., 2008; Hegert et al., 2002; Kawaguchi et al., 2005; zur Nieden et al., 2005; Kramer et al., 2000). Similar to the case with MSCs, different factors affect this process in ESCs, such as TGF-β3, BMP-2, and BMP-4 (Hegert et al., 2002; Kawaguchi et al., 2005; zur Nieden et al., 2005; Kramer et al., 2000; Sakai et al., 2005). Biological scaffolds seeded with chondrocytic cells derived from ESCs, when implanted in mice have been shown to generate cartilage tissue in-vivo (Kramer et al., 2000). Injection of ESCs in a DDDinduced rabbit model led to viable notochordal-type cells within the discs (Sheikh et al., 2009). These animal studies demonstrate the ability of ESCs to differentiate into a chondrocytic lineage in-vitro and in-vivo.

Our group is currently developing chondroprogenitor stem cell lines that can restore the functional capability of the IVD (Sheikh et al., 2009). Our rationale stemmed from the idea that currently there is no biologic therapy for repairing a degenerated IVD and that ESCs have a potential to fill this role based on their regenerative potential. Studies have shown that ESCs can be induced to differentiate into specific cell lineages by using selective culture media and growth environments (Kawaguchi et al., 2005).

Relying on the significant strides made by these basic science groups with regard to cell and factor characterization, our lab proceeded for further refine these methods and develop a protocol for both stem cell differentiation along a chondrocytic lineage and also for examining the utility of transplantation of these cells in a rabbit model of DDD. We initially developed a novel percutaneous animal model of disc degeneration using New Zealand white rabbits (Figure 1) and used this model to explore the possibility of ESC implantation for both structural regeneration and for the growth and continued presence of notochordal stem cells in the disc space (Sheikh et al., 2009).

Previous research transplanting MSCs into degenerated rabbit discs has shown consistent biochemical and radiographic (MRI) evidence of IVD restoration (Sakai et al., 2005). Human

Stem Cell-Mediated Intervertebral Disc Regeneration 295

A.

B.

B.

Fig. 6. Photographs of our group's rabbit model for IVD degeneration. The rabbit is positioned prone, its back is shaved and prepared for surgery (A), with a corresponding fluoroscopic view (B).

B.

Fig. 6. Photographs of our group's rabbit model for IVD degeneration. The rabbit is positioned prone, its back is shaved and prepared for surgery (A), with a corresponding

fluoroscopic view (B).

A.

B.

Stem Cell-Mediated Intervertebral Disc Regeneration 297

A.

B. Fig. 8. Photomicrographs of tissue obtained preimplanation for histological analysis of ESCs

grown in-vitro with Alcian blue staining showing 86% viability (A) and high power

magnification showed adequate GFP cell labeling (B).

Fig. 7. Sagittal T2-weighted MRI of the rabbit spine (A), with a corresponding axial view at the level of the induced disc degeneration (B) and at a separate normal control level (C).

MSCs have also been investigated for their bone-forming capabilities with good results (Jaiswal et al., 1997). Stem cells are already being used in therapeutic applications with placement of cells directly at the site of intended spinal fusion during open surgical procedures.

Our lab has developed chondroprogenitor cells lines that can restore the functional capacity of the IVD, with these cells differentiating into chondrocytes. Using our novel percutaneous model of disc degeneration in a rabbit model, we obtained MRIs preoperatively and at 2, 4, and 8 weeks postoperatively (Figure 2). Before implantation, ESCs were cultured with cisretinoic acid, TGF-beta, ascorbic acid, and insulin-like growth factor to induce differentiation along a chondrocyte lineage. After MRI confirmation of disc degeneration, the discs were then injected with murine ESCs that were labeled with mutant green fluoroscent protein (GFP). At 8 weeks post-implantation, IVDs were harvested and analyzed with hematoxylin and eosin staining along with immunohistochemical analyses (Figure 3).

Three groups were analyzed: group A consisted of control animals with nonpunctured discs; group B consisted of control animals with experimentally punctured discs; and group C consisted of animals with experimentally punctured discs that were subsequently implanted with ESCs. Gel electrophoresis was used to analyze ESCs for cartilaginous tissue formation. MRI confirmed IVD degeneration after needle puncture starting at 2 weeks postoperatively. Postmortem histological analysis of group A IVDs showed chondrocytes, but no notochordal cells. Group B disc displayed intact annulus fibrosus but disorganized

C Fig. 7. Sagittal T2-weighted MRI of the rabbit spine (A), with a corresponding axial view at the level of the induced disc degeneration (B) and at a separate normal control level (C).

MSCs have also been investigated for their bone-forming capabilities with good results (Jaiswal et al., 1997). Stem cells are already being used in therapeutic applications with placement of cells directly at the site of intended spinal fusion during open surgical

Our lab has developed chondroprogenitor cells lines that can restore the functional capacity of the IVD, with these cells differentiating into chondrocytes. Using our novel percutaneous model of disc degeneration in a rabbit model, we obtained MRIs preoperatively and at 2, 4, and 8 weeks postoperatively (Figure 2). Before implantation, ESCs were cultured with cisretinoic acid, TGF-beta, ascorbic acid, and insulin-like growth factor to induce differentiation along a chondrocyte lineage. After MRI confirmation of disc degeneration, the discs were then injected with murine ESCs that were labeled with mutant green fluoroscent protein (GFP). At 8 weeks post-implantation, IVDs were harvested and analyzed with hematoxylin and eosin staining along with immunohistochemical analyses (Figure 3). Three groups were analyzed: group A consisted of control animals with nonpunctured discs; group B consisted of control animals with experimentally punctured discs; and group C consisted of animals with experimentally punctured discs that were subsequently implanted with ESCs. Gel electrophoresis was used to analyze ESCs for cartilaginous tissue formation. MRI confirmed IVD degeneration after needle puncture starting at 2 weeks postoperatively. Postmortem histological analysis of group A IVDs showed chondrocytes, but no notochordal cells. Group B disc displayed intact annulus fibrosus but disorganized

procedures.

Fig. 8. Photomicrographs of tissue obtained preimplanation for histological analysis of ESCs grown in-vitro with Alcian blue staining showing 86% viability (A) and high power magnification showed adequate GFP cell labeling (B).

B.

Stem Cell-Mediated Intervertebral Disc Regeneration 299

have included radiographic analyses along with histological and immunohistochemical analyses have provided preliminary data that stem cell therapies are a viable option with regard to IVD regeneration (Sheikh et al., 2009). Human studies have further provided some preliminary evidence that stem cell therapy may be of clinical value (Haufe et al., 2006). The use of ESCs in regenerating IVD shows exciting new possibilities and further studies are

ESC-based regeneration of the human IVD is still in its infancy. Much progress has been made regarding laboratory research identifying the correct factors and microenvironment, and initial results from animal studies using stem cells remain promising. ESCs may be useful for repairing DDD as evidenced by their ability to differentiate into a chondrocytic lineage and yield notochordal-type cells in DDD models. ESCs need to be further studied and characterized with respect to safety, and larger human trials with appropriate clinical outcomes such as pain and disability reduction are needed to definitively establish its

The last half-century has seen an exponential rate of progress with regard to elucidating the mechanisms of degeneration of the IVD and how targeted therapies can help to alleviate this common condition. These studies have provided us with an improved understanding of the IVD and how it behaves under typical biomechanical forces and loads experienced in invivo conditions. Novel therapies are being studied, including stem cells with their potential regenerative capabilities in the spine. The development and action of these stem cells can be further modified through gene therapy and microenvironment manipulation. Immunologic markers are being used for more efficient targeting of these cells. With enhanced cell delivery and an improved understanding of the cell differentiation process, true regeneration of the IVD and surrounding supportive structures of the spine will become a

reality that can be applied to treat patients with this common, debilitating condition.

the potential for gene therapy applications. *Spine J.* 1:205-214, 2001.

[1] Chan D, Song Y, Sham P. Genetics of disc degeneration. *Eur Spine J.* 15(Suppl 3):S317-

[2] Cassinelli EH, Hall RA, Kang JD. Biochemistry of intervertebral disc degeneration and

[3] Stokes IA, Iatridis JC. Mechanical conditions that accelerate intervertebral disc degeneration: overload versus immobilization. *Spine* 29:2724-2732, 2004. [4] Ala-Kokko L. Genetic risk factors for lumbar disc disease. *Ann Med.* 34:42-47, 2002. [5] Steck E, Bertram H, Abel R, Chen B, Winter A, Richter W. Induction of intervertebral

[6] Risbud MV, Albert TJ, Guttapalli A, Vresilovic EJ, Hillibrand AS, Vaccaro AR,Shapiro

disc-like cells from adult mesenchymal stem cells. *Stem Cells*. 2005 Mar;23(3):403-11.

IM. Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy. *Spine (Phila* 

needed in humans to establish its efficacy.

clinical efficacy.

**7. Conclusions** 

**8. References** 

S325. 2006.

*Pa 1976).* 2004 Dec 1;29(23):2627-32.

fibrous tissue in the NP. Group C discs showed new notochordal cell growth, indicating survival and proper differentiation of the injected ESCs. Fluorescent microscopic analysis was positive in group C tissue, confirming the viability of GFP-labeled ESCs within the injected IVD. In addition, the notochordal cells in group C stained positive for cytokeratin and vimentin, providing further evidence of their chondrocyte origin. There was no inflammatory response in group C discs, indicating no cell-mediated immune response.

Our study provides a novel, reproducible model for the study of disc degeneration. New notochordal cell populations were seen in discs injected with ESCs. The lack of an immune response to xenograft-implanted mouse stem cells in an immune-competent rabbit suggests an immunoprivileged site within the IVD. Although preliminary, this study highlights the possible use of stem cells to promote IVD regeneration. Further ongoing studies are in the process of fully elucidating the processes involved with ESC differentiation along chondrogenic cell lines and how they may be used for new disc formation in the future. These studies will provide a good deal of evidence with regard to the future potential of ESCs for use in restoring the IVD in humans.

#### **6. Summary**

DDD is a high-morbidity condition with many modalities of treatment including surgery and more conservative measures such as pain injections, which only provide symptomatic treatment. No therapy has been developed that targets DDD at the cellular level. Recently, many biologic therapies have emerged that may be able to restore the NP and the normal cellular structure of the IVD. This restoration may in turn alleviate the symptoms of DDD through restoration of foraminal height, removing the compression of nerves. In-vitro studies have been performed to identify what cells are capable of differentiating towards a chondrocytic lineage and to best define parameters and factors that influence this differentiation. Multiple laboratory studies have been performed showing that MSCs, ADSCs, fetal cartilaginous cells, and annulus fibrosus cells all have the ability to differentiate towards a chondrocytic pathway. Factors that can induce these cells to differentiate toward a chondrocytic lineage have been identified and include TGF-β3 and BMP-2, which have a synergistic effect when used together. Other factors that may be beneficial include hypoxia, IVD-like glucose conditions (1.0 mg/mL glucose), and cell-tocell contact with NP cells; the latter negating the need for other soluble factors (i.e. TGFβ3). A major limiting factor may be the acidic pH (6.8) of the IVD, one that may be especially important as acidic pH levels are typical of increasingly degenerated discs. These studies yielded encouraging results with cells in the IVD being positive for markers of chondrocytic differentiation such as collagen type II and aggrecan. Additionally, cells exhibited NP phenotypic markers and had a close similarity to the normal biconvex structure of the NP. In-vitro studies have clearly established that ESCs are capable of differentiating into a chondrocytic lineage and have delineated some of the factors that affect this. The optimal microenvironment needs to be more accurately characterized at this time.

Animal studies of cell implantation have been performed in DDD-induction models. Weeks after injury, stem cells have been implanted and outcomes followed. These outcomes which have included radiographic analyses along with histological and immunohistochemical analyses have provided preliminary data that stem cell therapies are a viable option with regard to IVD regeneration (Sheikh et al., 2009). Human studies have further provided some preliminary evidence that stem cell therapy may be of clinical value (Haufe et al., 2006). The use of ESCs in regenerating IVD shows exciting new possibilities and further studies are needed in humans to establish its efficacy.

ESC-based regeneration of the human IVD is still in its infancy. Much progress has been made regarding laboratory research identifying the correct factors and microenvironment, and initial results from animal studies using stem cells remain promising. ESCs may be useful for repairing DDD as evidenced by their ability to differentiate into a chondrocytic lineage and yield notochordal-type cells in DDD models. ESCs need to be further studied and characterized with respect to safety, and larger human trials with appropriate clinical outcomes such as pain and disability reduction are needed to definitively establish its clinical efficacy.

#### **7. Conclusions**

298 Tissue Regeneration – From Basic Biology to Clinical Application

fibrous tissue in the NP. Group C discs showed new notochordal cell growth, indicating survival and proper differentiation of the injected ESCs. Fluorescent microscopic analysis was positive in group C tissue, confirming the viability of GFP-labeled ESCs within the injected IVD. In addition, the notochordal cells in group C stained positive for cytokeratin and vimentin, providing further evidence of their chondrocyte origin. There was no inflammatory response in group C discs, indicating no cell-mediated immune response.

Our study provides a novel, reproducible model for the study of disc degeneration. New notochordal cell populations were seen in discs injected with ESCs. The lack of an immune response to xenograft-implanted mouse stem cells in an immune-competent rabbit suggests an immunoprivileged site within the IVD. Although preliminary, this study highlights the possible use of stem cells to promote IVD regeneration. Further ongoing studies are in the process of fully elucidating the processes involved with ESC differentiation along chondrogenic cell lines and how they may be used for new disc formation in the future. These studies will provide a good deal of evidence with regard to the future potential of

DDD is a high-morbidity condition with many modalities of treatment including surgery and more conservative measures such as pain injections, which only provide symptomatic treatment. No therapy has been developed that targets DDD at the cellular level. Recently, many biologic therapies have emerged that may be able to restore the NP and the normal cellular structure of the IVD. This restoration may in turn alleviate the symptoms of DDD through restoration of foraminal height, removing the compression of nerves. In-vitro studies have been performed to identify what cells are capable of differentiating towards a chondrocytic lineage and to best define parameters and factors that influence this differentiation. Multiple laboratory studies have been performed showing that MSCs, ADSCs, fetal cartilaginous cells, and annulus fibrosus cells all have the ability to differentiate towards a chondrocytic pathway. Factors that can induce these cells to differentiate toward a chondrocytic lineage have been identified and include TGF-β3 and BMP-2, which have a synergistic effect when used together. Other factors that may be beneficial include hypoxia, IVD-like glucose conditions (1.0 mg/mL glucose), and cell-tocell contact with NP cells; the latter negating the need for other soluble factors (i.e. TGFβ3). A major limiting factor may be the acidic pH (6.8) of the IVD, one that may be especially important as acidic pH levels are typical of increasingly degenerated discs. These studies yielded encouraging results with cells in the IVD being positive for markers of chondrocytic differentiation such as collagen type II and aggrecan. Additionally, cells exhibited NP phenotypic markers and had a close similarity to the normal biconvex structure of the NP. In-vitro studies have clearly established that ESCs are capable of differentiating into a chondrocytic lineage and have delineated some of the factors that affect this. The optimal microenvironment needs to be more accurately characterized at

Animal studies of cell implantation have been performed in DDD-induction models. Weeks after injury, stem cells have been implanted and outcomes followed. These outcomes which

ESCs for use in restoring the IVD in humans.

**6. Summary** 

this time.

The last half-century has seen an exponential rate of progress with regard to elucidating the mechanisms of degeneration of the IVD and how targeted therapies can help to alleviate this common condition. These studies have provided us with an improved understanding of the IVD and how it behaves under typical biomechanical forces and loads experienced in invivo conditions. Novel therapies are being studied, including stem cells with their potential regenerative capabilities in the spine. The development and action of these stem cells can be further modified through gene therapy and microenvironment manipulation. Immunologic markers are being used for more efficient targeting of these cells. With enhanced cell delivery and an improved understanding of the cell differentiation process, true regeneration of the IVD and surrounding supportive structures of the spine will become a reality that can be applied to treat patients with this common, debilitating condition.

#### **8. References**


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D, Perez-Cruet MJ. In vivo intervertebral disc regeneration using stem cell-derived

Transplantation of mesenchymal stem cells in a canine disc degeneration model. *J* 

therapy maintains vertebral blood flow and contrast diffusion through the endplate

using marrow mesenchymal cell transplantation: a report of two case studies. *Spine* 

Chaudhry GR. Chondrogenic derivatives of embryonic stem cells seeded into 3D polycaprolactone scaffolds generated artilage tissue in vivo. *Tissue Eng Part A.* 2008

Differentiation plasticity of chondrocytes derived from mouse embryonic stem

embryonic stem cells in response to specific growth factors. *Bone.* 2005

adipogenesis in embryonic stem cells by bone morphogenetic protein-2: effect of

derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4.

to a rabbit degenerative disc model: potential and limitations for stem cell therapy

extracellular matrix production. *Spine (Phila Pa 1976).* 2010 May 1;35(10): 1033-8.


[35] Quintin A, Schizas C, Scaletta C, Jaccoud S, Applegate LA, Pioletti DP. Plasticity of fetal

[36] Quintin A, Schizas C, Scaletta C, Jaccoud S, Gerber S, Osterheld MC, Juillerat L,

[37] Jeong JH, Lee JH, Jin ES, Min JK, Jeon SR, Choi KH. Regeneration of intervertebral discs

[38] Ganey T, Hutton WC, Moseley T, Hedrick M, Meisel HJ. Intervertebral disc repair using

[39] Tan S, Jia C, Liu Z, Liu R, Yang J, Zhang L, Shou F, Ju X. Study on survival time of

[40] Jeong JH, Jin ES, Min JK, Jeon SR, Park CS, Kim HS, Choi KH. Human mesenchymal

[41] Henriksson HB, Svanvik T, Jonsson M, Hagman M, Horn M, Lindahl A, Brisby H.

[43] Zhang YG, Guo X, Xu P, Kang LL, Li J. Bone mesenchymal stem cells transplanted into

[44] Crevensten G, Walsh AJ, Ananthakrishnan D, Page P, Wahba GM, Lotz JC, Berven S.

[46] Wei A, Tao H, Chung SA, Brisby H, Ma DD, Diwan AD. The fate of transplanted

[47] Sakai D, Mochida J, Iwashina T, Watanabe T, Nakai T, Ando K, Hotta T. Differentiation

xenogeneic porcine model. *Spine (Phila Pa 1976).* 2009 Jan 15;34(2):141-8. [42] Sobajima S, Vadala G, Shimer A, Kim JS, Gilbertson LG, Kang JD. Feasibility of a stem

cartilaginous cells. *Cell Transplant.* 2010;19(10):1349-57.

foetal spine cells. *J Cell Mol Med.* 2009 Aug;13(8B):2559-69.

*Acta Neurochir (Wien).* 2010 Oct;152(10):1771-7.

*Spine (Phila Pa 1976).* 2009 Oct 1;34(21):2297-304.

1033-8.

Nov;23(11):1355-9.

Jan;(430):219-26.

10. Epub 2010 Jul 24.

2009 Mar;27(3):374-9.

*1976).* 2005 Nov 1;30(21):2379-87.

96.

*Cytotechnology.* 2009 Jan;59(1):55-64.

extracellular matrix production. *Spine (Phila Pa 1976).* 2010 May 1;35(10):

Applegate LA, Pioletti DP. Isolation and in vitro chondrogenic potential of human

in a rat disc degeneration model by implanted adipose-tissue-derived stromal cells.

adipose tissue-derived stem and regenerative cells: experiments in a canine model.

autogeneic BMSCs labeled with superparamagnetic iron oxide in rabbit intervertebral discs. *Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi.* 2009

stem cells implantation into the degenerated coccygeal disc of the rat.

Transplantation of human mesenchymal stems cells into intervertebral discs in a

cell therapy for intervertebral disc degeneration. *Spine J.* 2008 Nov-Dec;8(6):888-

rabbit intervertebral discs can increase proteoglycans. *Clin Orthop Relat Res.* 2005

Intervertebral disc cell therapy for regeneration: mesenchymal stem cell implantation in rat intervertebral discs. *Ann Biomed Eng.* 2004 Mar;32(3):430-4. [45] Yang H, Wu J, Liu J, Ebraheim M, Castillo S, Liu X, Tang T, Ebraheim NA. Transplanted

mesenchymal stem cells with pure fibrinous gelatin-transforming growth factorbeta1 decrease rabbit intervertebral disc degeneration. *Spine J.* 2010 Sep;10(9):802-

xenogeneic bone marrow-derived stem cells in rat intervertebral discs. *J Orthop Res.*

of mesenchymal stem cells transplanted to a rabbit degenerative disc model: potential and limitations for stem cell therapy in disc regeneration. *Spine (Phila Pa* 


**14** 

*Germany* 

**Towards Clinical Application of Mesenchymal** 

**Stromal Cells: Perspectives and Requirements** 

Mesenchymal stromal cells (MSC) possess a wide spectrum of interacting properties that contribute to their broad therapeutic potential: In pre- and clinical settings MSC have been demonstrated to reduce tissue damage, to activate the endogenous regenerative potential of tissues and to participate in tissue regeneration (Noort, Feye et al. 2010). Initially, MSC have been described to differentiate into derivates of the mesoderm: bone, adipose and cartilage tissue and were therefore applied to restore damaged tissue (Frohlich, Grayson et al. 2008). Subsequent analyses, however, indicated that the repair process does not only lay in the differentiation potential and plasticity of MSC. As demonstrated in later studies even if only few cells were detectable after MSC transplantation, the therapeutic effect was obvious (Fuchs, Baffour et al. 2001; Shake, Gruber et al. 2002). This could be attributed to paracrine properties with consecutive modification of the tissue microenvironment to decrease inflammatory and immune reactions. MSC are therefore beyond doubt promising candidates for cell therapy in various settings (Horwitz, Prockop et al. 2001; Le Blanc,

The broad therapeutic efficacy of MSC renders them attractive candidates for cell therapy. However, translating basic research into clinical application is a complex multistep process (Bieback, Karagianni et al. 2011). It necessitates product regulation by the regulatory authorities and accurate management of the expected therapeutic benefits with the potential risks in order to balance the speed of clinical trials with a time-consuming, cautious risk assessment (Sensebe, Bourin et al. 2011). Despite their use in clinical studies, some questions remain open: What are the deviations among the MSC from different tissue sources? How shall MSC be adequately procured, isolated and cultivated? How should their therapeutic propensity, e.g. their homing properties, the secretion of bioactive factors, the differentiation

Rasmusson et al. 2004; Prockop 2009; Pontikoglou, Deschaseaux et al. 2011).

pattern *in vivo* and their plasticity, be defined?

Both authors contributed equally

 \*

**1. Introduction** 

Marianna Karagianni\*, Torsten J. Schulze\* and Karen Bieback

*German Red Cross Blood Donor Service Baden-Württemberg – Hessen* 

**for Orthopaedic Applications** 

*Institute of Transfusion Medicine and Immunology; Medical Faculty Mannheim, Heidelberg University;* 

[62] Jaiswal N, Haynesworht S, Caplan A. Osteogenic differentiation of purified culture expanded human mesenchymal stem cells in vitro. *J Cell Biochem* 64: 295-312, 1997.

## **Towards Clinical Application of Mesenchymal Stromal Cells: Perspectives and Requirements for Orthopaedic Applications**

Marianna Karagianni\*, Torsten J. Schulze\* and Karen Bieback *Institute of Transfusion Medicine and Immunology; Medical Faculty Mannheim, Heidelberg University; German Red Cross Blood Donor Service Baden-Württemberg – Hessen Germany* 

#### **1. Introduction**

304 Tissue Regeneration – From Basic Biology to Clinical Application

[62] Jaiswal N, Haynesworht S, Caplan A. Osteogenic differentiation of purified culture

1997.

expanded human mesenchymal stem cells in vitro. *J Cell Biochem* 64: 295-312,

Mesenchymal stromal cells (MSC) possess a wide spectrum of interacting properties that contribute to their broad therapeutic potential: In pre- and clinical settings MSC have been demonstrated to reduce tissue damage, to activate the endogenous regenerative potential of tissues and to participate in tissue regeneration (Noort, Feye et al. 2010). Initially, MSC have been described to differentiate into derivates of the mesoderm: bone, adipose and cartilage tissue and were therefore applied to restore damaged tissue (Frohlich, Grayson et al. 2008). Subsequent analyses, however, indicated that the repair process does not only lay in the differentiation potential and plasticity of MSC. As demonstrated in later studies even if only few cells were detectable after MSC transplantation, the therapeutic effect was obvious (Fuchs, Baffour et al. 2001; Shake, Gruber et al. 2002). This could be attributed to paracrine properties with consecutive modification of the tissue microenvironment to decrease inflammatory and immune reactions. MSC are therefore beyond doubt promising candidates for cell therapy in various settings (Horwitz, Prockop et al. 2001; Le Blanc, Rasmusson et al. 2004; Prockop 2009; Pontikoglou, Deschaseaux et al. 2011).

The broad therapeutic efficacy of MSC renders them attractive candidates for cell therapy. However, translating basic research into clinical application is a complex multistep process (Bieback, Karagianni et al. 2011). It necessitates product regulation by the regulatory authorities and accurate management of the expected therapeutic benefits with the potential risks in order to balance the speed of clinical trials with a time-consuming, cautious risk assessment (Sensebe, Bourin et al. 2011). Despite their use in clinical studies, some questions remain open: What are the deviations among the MSC from different tissue sources? How shall MSC be adequately procured, isolated and cultivated? How should their therapeutic propensity, e.g. their homing properties, the secretion of bioactive factors, the differentiation pattern *in vivo* and their plasticity, be defined?

<sup>\*</sup> Both authors contributed equally

Towards Clinical Application of Mesenchymal Stromal Cells:

differentiating into cells or tissues (Caplan and Correa 2011).

**1.2 MSC from different tissue sources** 

Perspectives and Requirements for Orthopaedic Applications 307

precursor cells (Koc, Day et al. 2002). Later, due to their mesodermal differentiation potential, Horwitz et al. were able to perform seminal studies applying MSC to children with osteogenesis imperfecta (Horwitz, Prockop et al. 2001). MSC were then applied as immunosuppressants in patients with graft versus host disease (Le Blanc, Rasmusson et al. 2004). Further studies introduced them as promising candidates for tissue regeneration in bone and cartilage repair (Frohlich, Grayson et al. 2008), epithelial regeneration (Long, Zuk et al. 2010), cardiovascular regeneration (Noort, Feye et al. 2010; Rangappa, Makkar et al. 2010), immunomodulation in graft versus host disease (GvHD) (Ringden, Uzunel et al. 2006), and inflammatory neurological diseases (Momin, Mohyeldin et al. 2010). MSC are expected to reduce tissue damage, to activate the endogenous regenerative potential of tissues and to participate in the regeneration (Noort, Feye et al. 2010). However, in all these studies it became apparent that MSC function mainly through paracrine effects rather than

Bone marrow (BM) was the first source of MSC identified by Friedenstein and co-workers (Friedenstein, Gorskaja et al. 1976). BM-MSC are already being tested worldwide in clinical studies with currently over 1500 found in the Clinical Trials registry of the NIH (www.clinicaltrials.gov). Due to the long lasting research on BM-MSC they became the gold standard for any MSC research and therapeutic application. Nevertheless, a limitation for BM MSC clinical application is the low cell frequency in source tissue. Thus large volume bone marrow aspiration is necessary even in autologous settings, feasible only in general anaesthesia which is associated with an additional patient morbidity. In consequence, investigators have developed protocols for isolating MSC from a variety of different tissues and sources other than bone marrow. Latest studies led to the conclusion that MSC are not limited to a certain tissue source: the MSC niche is rather localized in the perivascular area of virtually all tissues (Crisan, Yap et al. 2008; da Silva Meirelles, Caplan et al. 2008). Thus numerous tissues containing MSC have been identified, for example adipose tissue (AT), cord blood (CB), fetal membranes and amniotic fluid, pancreatic islet, lung parenchyma, intestinal lamina propria, oral and nasal mucosa, eye limbus, dental tissues and synovial fluid (Jakob, Hemeda et al. ; Karaoz, Ayhan et al. ; Marynka-Kalmani, Treves et al. ; Pinchuk, Mifflin et al. ; Powell, Pinchuk et al. ; Zuk, Zhu et al. 2002; Kern, Eichler et al. 2006; Phinney and Prockop 2007; Jones, Crawford et al. 2008; Polisetty, Fatima et al. 2008; Huang, Gronthos

et al. 2009; Ilancheran, Moodley et al. 2009; Karoubi, Cortes-Dericks et al. 2009).

Among all tissue sources, AT shows several important clinical advantages compared to BM: AT procurement can be achieved via tumescent-lipoaspiration in local anaesthesia, a lower risk operating procedure. Adipose tissue is abundant even in older individuals. AT-MSC are shown to have similar functional properties to BM-MSC while their frequency is definitely higher than in BM (Zuk, Zhu et al. 2002; Kern, Eichler et al. 2006). AT-MSC are currently being applied in clinical trials, at least 33 trials can be found in the NIH registry. The high frequency of MSC in AT renders it possible to isolate the mononuclear cell fraction directly at the patients bedside without the need for expansion in a GMP facility (Duckers, Pinkernell et al. 2006). There are divergent outcomes in those studies directly comparing freshly isolated with expanded cells (Garcia-Olmo, Herreros et al. 2009). Despite the advantages of processing at the patient's bedside, direct application of the freshly isolated

It is obvious that MSC need to be further characterised in clinical studies with standardized protocols (Bieback, Karagianni et al. 2011; Sensebe, Bourin et al. 2011). Furthermore, despite immense work, still MSC cannot be identified as a distinct cell population by a set of marker proteins as CD34 defines hematopoietic stem cells. The field currently uses "minimal criteria" for MSC to describe them according to their *in vitro* behaviour (osteo-, adipo- and chondrogenic differentiation) and morphology (fibroblastoid, expressing a set of markers) (Dominici, Le Blanc et al. 2006). Nevertheless it has to be taken into account that *in vitro* data do not necessarily predict *in vivo* behaviour: MSC seem to alter their *in vitro* traits after *in vivo* transplantation and this might affect a future therapeutic outcome severely. For example MSC can express HLA-class II antigens and can therefore possibly trigger an immunreaction in the host after transplantation (Vassalli and Moccetti 2011) or may calcify spontaneously in uremic conditions and cause vessel occlusion in case of intravenous application (Kramann, Couson et al. 2011).

Using the example of bone defect regeneration, we will emphasize key parameters relevant for the translation of experimental data to clinical application. The focus on bone defect regeneration exemplifies the possibilities and challenges for MSC in combination with biomaterials in the light of regulatory frameworks in Europe, where MSC may be classified as "Advanced Therapy Medicinal Product - ATMP", or the US, where MSC fall under the term "Human Cells, Tissues, and Cellular and Tissue-Based Products -HCT/Ps". In this context, questions that need to be answered concern an adequate MSC tissue source with superior osteogenic potential compared to other tissues, the degree of cell differentiation prior to implantation and the adequate scaffold for tissue engineering (Seong, Kim et al. 2010).

#### **1.1 MSC definition**

Mesenchymal stromal cells (MSC) were initially isolated from bone marrow (BM) as described by Friedenstein and co-workers in 1968 (Friedenstein, Petrakova et al. 1968). They were identified as non hematopoietic, fibroblast-like cells adherent to plastic, with a colonyforming capacity (Friedenstein, Deriglasova et al. 1974), also as feeder cells for hematopoietic precursors (Eaves, Cashman et al. 1991; Wagner, Saffrich et al. 2008). Subsequent characterisation revealed their mesodermal differentiation and immune modulatory capacity, raising the interest in these cells (Le Blanc, Rasmusson et al. 2004; Bieback, Hecker et al. 2009; Mosna, Sensebe et al. 2010). Consequently, numerous terms for these cells were established: mesenchymal stem cells, mesenchymal stromal cells, adult stromal cells, multipotent and non hematopoietic adult precursor cells (Horwitz, Le Blanc et al. 2005; Dominici, Le Blanc et al. 2006). These conflicting nomenclature suggestions in the literature lead to a complex information exchange upon MSC (Prockop 2009). In an attempt to clarify and define the nomenclature, the ISCT (International Society for Cell Therapy) set "minimal criteria" for MSC, such as:


In the last decade there has been rapid movement from bench to bedside. Based on their stromal origin, MSC were initially applied in co-transplantation studies with hematopoietic

It is obvious that MSC need to be further characterised in clinical studies with standardized protocols (Bieback, Karagianni et al. 2011; Sensebe, Bourin et al. 2011). Furthermore, despite immense work, still MSC cannot be identified as a distinct cell population by a set of marker proteins as CD34 defines hematopoietic stem cells. The field currently uses "minimal criteria" for MSC to describe them according to their *in vitro* behaviour (osteo-, adipo- and chondrogenic differentiation) and morphology (fibroblastoid, expressing a set of markers) (Dominici, Le Blanc et al. 2006). Nevertheless it has to be taken into account that *in vitro* data do not necessarily predict *in vivo* behaviour: MSC seem to alter their *in vitro* traits after *in vivo* transplantation and this might affect a future therapeutic outcome severely. For example MSC can express HLA-class II antigens and can therefore possibly trigger an immunreaction in the host after transplantation (Vassalli and Moccetti 2011) or may calcify spontaneously in uremic conditions and cause vessel occlusion in case of intravenous

Using the example of bone defect regeneration, we will emphasize key parameters relevant for the translation of experimental data to clinical application. The focus on bone defect regeneration exemplifies the possibilities and challenges for MSC in combination with biomaterials in the light of regulatory frameworks in Europe, where MSC may be classified as "Advanced Therapy Medicinal Product - ATMP", or the US, where MSC fall under the term "Human Cells, Tissues, and Cellular and Tissue-Based Products -HCT/Ps". In this context, questions that need to be answered concern an adequate MSC tissue source with superior osteogenic potential compared to other tissues, the degree of cell differentiation prior to

implantation and the adequate scaffold for tissue engineering (Seong, Kim et al. 2010).


CD11b, CD79 alpha or CD19 and HLA-DR surface molecules,

Mesenchymal stromal cells (MSC) were initially isolated from bone marrow (BM) as described by Friedenstein and co-workers in 1968 (Friedenstein, Petrakova et al. 1968). They were identified as non hematopoietic, fibroblast-like cells adherent to plastic, with a colonyforming capacity (Friedenstein, Deriglasova et al. 1974), also as feeder cells for hematopoietic precursors (Eaves, Cashman et al. 1991; Wagner, Saffrich et al. 2008). Subsequent characterisation revealed their mesodermal differentiation and immune modulatory capacity, raising the interest in these cells (Le Blanc, Rasmusson et al. 2004; Bieback, Hecker et al. 2009; Mosna, Sensebe et al. 2010). Consequently, numerous terms for these cells were established: mesenchymal stem cells, mesenchymal stromal cells, adult stromal cells, multipotent and non hematopoietic adult precursor cells (Horwitz, Le Blanc et al. 2005; Dominici, Le Blanc et al. 2006). These conflicting nomenclature suggestions in the literature lead to a complex information exchange upon MSC (Prockop 2009). In an attempt to clarify and define the nomenclature, the ISCT (International Society for Cell Therapy) set



In the last decade there has been rapid movement from bench to bedside. Based on their stromal origin, MSC were initially applied in co-transplantation studies with hematopoietic

application (Kramann, Couson et al. 2011).

**1.1 MSC definition** 

"minimal criteria" for MSC, such as:

(Dominici, Le Blanc et al. 2006).

precursor cells (Koc, Day et al. 2002). Later, due to their mesodermal differentiation potential, Horwitz et al. were able to perform seminal studies applying MSC to children with osteogenesis imperfecta (Horwitz, Prockop et al. 2001). MSC were then applied as immunosuppressants in patients with graft versus host disease (Le Blanc, Rasmusson et al. 2004). Further studies introduced them as promising candidates for tissue regeneration in bone and cartilage repair (Frohlich, Grayson et al. 2008), epithelial regeneration (Long, Zuk et al. 2010), cardiovascular regeneration (Noort, Feye et al. 2010; Rangappa, Makkar et al. 2010), immunomodulation in graft versus host disease (GvHD) (Ringden, Uzunel et al. 2006), and inflammatory neurological diseases (Momin, Mohyeldin et al. 2010). MSC are expected to reduce tissue damage, to activate the endogenous regenerative potential of tissues and to participate in the regeneration (Noort, Feye et al. 2010). However, in all these studies it became apparent that MSC function mainly through paracrine effects rather than differentiating into cells or tissues (Caplan and Correa 2011).

#### **1.2 MSC from different tissue sources**

Bone marrow (BM) was the first source of MSC identified by Friedenstein and co-workers (Friedenstein, Gorskaja et al. 1976). BM-MSC are already being tested worldwide in clinical studies with currently over 1500 found in the Clinical Trials registry of the NIH (www.clinicaltrials.gov). Due to the long lasting research on BM-MSC they became the gold standard for any MSC research and therapeutic application. Nevertheless, a limitation for BM MSC clinical application is the low cell frequency in source tissue. Thus large volume bone marrow aspiration is necessary even in autologous settings, feasible only in general anaesthesia which is associated with an additional patient morbidity. In consequence, investigators have developed protocols for isolating MSC from a variety of different tissues and sources other than bone marrow. Latest studies led to the conclusion that MSC are not limited to a certain tissue source: the MSC niche is rather localized in the perivascular area of virtually all tissues (Crisan, Yap et al. 2008; da Silva Meirelles, Caplan et al. 2008). Thus numerous tissues containing MSC have been identified, for example adipose tissue (AT), cord blood (CB), fetal membranes and amniotic fluid, pancreatic islet, lung parenchyma, intestinal lamina propria, oral and nasal mucosa, eye limbus, dental tissues and synovial fluid (Jakob, Hemeda et al. ; Karaoz, Ayhan et al. ; Marynka-Kalmani, Treves et al. ; Pinchuk, Mifflin et al. ; Powell, Pinchuk et al. ; Zuk, Zhu et al. 2002; Kern, Eichler et al. 2006; Phinney and Prockop 2007; Jones, Crawford et al. 2008; Polisetty, Fatima et al. 2008; Huang, Gronthos et al. 2009; Ilancheran, Moodley et al. 2009; Karoubi, Cortes-Dericks et al. 2009).

Among all tissue sources, AT shows several important clinical advantages compared to BM: AT procurement can be achieved via tumescent-lipoaspiration in local anaesthesia, a lower risk operating procedure. Adipose tissue is abundant even in older individuals. AT-MSC are shown to have similar functional properties to BM-MSC while their frequency is definitely higher than in BM (Zuk, Zhu et al. 2002; Kern, Eichler et al. 2006). AT-MSC are currently being applied in clinical trials, at least 33 trials can be found in the NIH registry. The high frequency of MSC in AT renders it possible to isolate the mononuclear cell fraction directly at the patients bedside without the need for expansion in a GMP facility (Duckers, Pinkernell et al. 2006). There are divergent outcomes in those studies directly comparing freshly isolated with expanded cells (Garcia-Olmo, Herreros et al. 2009). Despite the advantages of processing at the patient's bedside, direct application of the freshly isolated

Towards Clinical Application of Mesenchymal Stromal Cells:

ongoing to develop this (Mannello and Tonti 2007).

**2.2.1 Therapeutic safety, purity and potency** 

risks and to enable to value risks against therapeutic value.

**2.2 Quality control** 

Perspectives and Requirements for Orthopaedic Applications 309

use it is necessary to define quality criteria to monitor the cell product (Bieback, Schallmoser et al. 2008; Bieback, Karagianni et al. 2011; Sensebe, Bourin et al. 2011). For expansion aiming at clinical application it is obligatory to use GMP-grade supplements and sera if available. However, these reagents are just under development. Accordingly we, amongst others, tested human blood-derived components, like human serum or platelet derivatives to replace fetal bovine serum commonly used to expand MSC (Kocaoemer, Kern et al. 2007; Mannello and Tonti 2007; Bieback, Schallmoser et al. 2008; Bieback, Hecker et al. 2009). Human blood components offer the advantage that they are both well controlled and already in clinical use for decades. Still, human serum as well as platelet lysate is a very crude protein cocktail. Essential growth factors for optimal MSC culture have not yet been defined. Platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF-ß), and insulin growth factor (IGF) have been subjected to investigation. Basic fibroblast growth factor (bFGF) has demonstrated most promising effects in expanding MSC whilst maintaining stem cell properties and reducing replicative senescence (Tsutsumi, Shimazu et al. 2001). Recently, Pytlik et al described a human serum and growth factor supplemented clinical-grade medium, which allowed high cell expansion mediated by loss of contact inhibition (Pytlik, Stehlik et al. 2009). Anyhow, the ideal solution is a chemically defined clinical-grade medium permitting both adhesion and expansion of MSC and numerous attempts are

In order to obtain a manufacturing authorization for cell therapeutics the quality criteria ought to meet the regulatory standards. Quality controls are instrumented within the manufacturing process to prove according to the set quality criteria. Essential quality criteria are the traceability of the cell product through donor identification and product labelling, the prevention of introduction and spreading of infection and communicable diseases through donor screening and aseptic cell processing and proof of the therapeutic safety, lot consistency, potency and purity of the cell product (European Parliament 2007; FDA 2010).

Safety is a key issue in cell therapy. In addition to the above mentioned aspects regarding reagents (fetal bovine serum has been elaborated on) and sterility testing (bacterial, fungal, viral, mycoplasma), cellular aspects have to be considered as well. In long term cell culture current testing methods of chromosomal aberrations and neoplastic transformation are fluoerescence in situ hybridization (FISH), karyotype analysis or detection of proto-oncogenes or activators of tumorigenesis like myc-assosiated proteins (Agrawal, Yu et al. 2010). Further lately developed testing methods are BAC-based (Bacterial Artificial Chromosome) Array to detect DNA copy number or oligonucleotide-based Array CGH (Chromosomal Comparative Genomic Hybridization) to detect small genomic regions with amplification or deletion (Wicker, Carles et al. 2007). Additionally, detection of telomerase activation is often performed, as telomerase plays a role in malignant transformation *in vitro* (Yamaoka, Hiyama et al. 2011). All these assays indicate that there is a low risk of transformation of MSC in *in vitro* expansion. However, more safety studies – especially long term follow up *in vivo* - are required to exclude

mononuclear cells in one session procedure gives no opportunity to control the clinical outcome, for an amount of diverse undefined cell populations are effective in these settings. However, this is still being exercised as autologous treatment.

Studies are being performed in order to compare BM-MSC, AT-MSC and MSC of other tissue sources. They show that MSC are not one distinct cell population. Among their tissue sources MSC differ concerning their isolating rate, their expansion potential, their differentiating capacities (Kern, Eichler et al. 2006), their immunosuppressive and migratory properties (Najar, Raicevic et al. ; Constantin, Marconi et al. 2009). These differences have probably an impact on their quality and therapeutic ability, which only can be definitely clarified in "*in vivo*" studies. Summarizing, there is a complex algorithm, which should be followed in order to find the adequate tissue source for MSC cell therapy. Very important are:


All this can rather be answered gradually applying standardized protocols. After procurement and expansion MSC have to be analysed regarding their functional properties through well defined *in vitro* potency assays. Finally functional properties have to be compared *in vivo* through animal studies and phase I clinical trials.

#### **2. MSC protocols for clinical applications**

Translating MSC into cell therapy settings requires a manufacturing process and manufacturing authorisation congruent to the local regulatory framework. Regulatory standards in the EU and USA comply with the good manufacturing practice (GMP) regulations and are set in order to control the therapeutics' safety process, e.g. tissue procurement, cell isolation, selection and expansion and have to be validated according to the quality criteria as defined by the manufacturer. Furthermore it is essential to control the quality, purity and potency of the cell product prior to their administration by well defined and validated quality control and potency assays to ensure safety.

#### **2.1 Isolation and expansion of MSC for clinical applications**

For clinical applications, MSC shall be isolated under aseptic conditions in GMP facilities. MSC are a subpopulation among the mononuclear cell fraction. They can be isolated after density gradient centrifugation or if MSC are embedded in extracellular matrix after enzymatic digestion. In general, the low frequency of human MSC within their origin tissues necessitates their expansion prior to clinical use. This raises the risk for contaminations (Bieback, Karagianni et al. 2011; Sensebe, Bourin et al. 2011). Furthermore, in long term cell culture the proliferation rate decays, the cell size increases, differentiation potential becomes affected and chromosomal instabilities and neoplastic transformation may arise (Prockop, Brenner et al.; Lepperdinger, Brunauer et al. 2008; Wagner, Horn et al. 2008) raising the risk for adverse reactions.

Similarly, the cultivation media potentially affect MSC, exposing them to pathogens and immunogens (Heiskanen, Satomaa et al. 2007; Sundin, Ringden et al. 2007; Bieback, Hecker et al. 2009). In order to achieve controlled conditions and a safe cell product for clinical use it is necessary to define quality criteria to monitor the cell product (Bieback, Schallmoser et al. 2008; Bieback, Karagianni et al. 2011; Sensebe, Bourin et al. 2011). For expansion aiming at clinical application it is obligatory to use GMP-grade supplements and sera if available. However, these reagents are just under development. Accordingly we, amongst others, tested human blood-derived components, like human serum or platelet derivatives to replace fetal bovine serum commonly used to expand MSC (Kocaoemer, Kern et al. 2007; Mannello and Tonti 2007; Bieback, Schallmoser et al. 2008; Bieback, Hecker et al. 2009). Human blood components offer the advantage that they are both well controlled and already in clinical use for decades. Still, human serum as well as platelet lysate is a very crude protein cocktail. Essential growth factors for optimal MSC culture have not yet been defined. Platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF-ß), and insulin growth factor (IGF) have been subjected to investigation. Basic fibroblast growth factor (bFGF) has demonstrated most promising effects in expanding MSC whilst maintaining stem cell properties and reducing replicative senescence (Tsutsumi, Shimazu et al. 2001). Recently, Pytlik et al described a human serum and growth factor supplemented clinical-grade medium, which allowed high cell expansion mediated by loss of contact inhibition (Pytlik, Stehlik et al. 2009). Anyhow, the ideal solution is a chemically defined clinical-grade medium permitting both adhesion and expansion of MSC and numerous attempts are ongoing to develop this (Mannello and Tonti 2007).

#### **2.2 Quality control**

308 Tissue Regeneration – From Basic Biology to Clinical Application

mononuclear cells in one session procedure gives no opportunity to control the clinical outcome, for an amount of diverse undefined cell populations are effective in these settings.

Studies are being performed in order to compare BM-MSC, AT-MSC and MSC of other tissue sources. They show that MSC are not one distinct cell population. Among their tissue sources MSC differ concerning their isolating rate, their expansion potential, their differentiating capacities (Kern, Eichler et al. 2006), their immunosuppressive and migratory properties (Najar, Raicevic et al. ; Constantin, Marconi et al. 2009). These differences have probably an impact on their quality and therapeutic ability, which only can be definitely clarified in "*in vivo*" studies. Summarizing, there is a complex algorithm, which should be followed in order

All this can rather be answered gradually applying standardized protocols. After procurement and expansion MSC have to be analysed regarding their functional properties through well defined *in vitro* potency assays. Finally functional properties have to be

Translating MSC into cell therapy settings requires a manufacturing process and manufacturing authorisation congruent to the local regulatory framework. Regulatory standards in the EU and USA comply with the good manufacturing practice (GMP) regulations and are set in order to control the therapeutics' safety process, e.g. tissue procurement, cell isolation, selection and expansion and have to be validated according to the quality criteria as defined by the manufacturer. Furthermore it is essential to control the quality, purity and potency of the cell product prior to their administration by well defined

For clinical applications, MSC shall be isolated under aseptic conditions in GMP facilities. MSC are a subpopulation among the mononuclear cell fraction. They can be isolated after density gradient centrifugation or if MSC are embedded in extracellular matrix after enzymatic digestion. In general, the low frequency of human MSC within their origin tissues necessitates their expansion prior to clinical use. This raises the risk for contaminations (Bieback, Karagianni et al. 2011; Sensebe, Bourin et al. 2011). Furthermore, in long term cell culture the proliferation rate decays, the cell size increases, differentiation potential becomes affected and chromosomal instabilities and neoplastic transformation may arise (Prockop, Brenner et al.; Lepperdinger, Brunauer et al. 2008; Wagner, Horn et al. 2008) raising the risk

Similarly, the cultivation media potentially affect MSC, exposing them to pathogens and immunogens (Heiskanen, Satomaa et al. 2007; Sundin, Ringden et al. 2007; Bieback, Hecker et al. 2009). In order to achieve controlled conditions and a safe cell product for clinical

However, this is still being exercised as autologous treatment.



**2. MSC protocols for clinical applications** 

for adverse reactions.

to find the adequate tissue source for MSC cell therapy. Very important are:


compared *in vivo* through animal studies and phase I clinical trials.

and validated quality control and potency assays to ensure safety.

**2.1 Isolation and expansion of MSC for clinical applications** 

In order to obtain a manufacturing authorization for cell therapeutics the quality criteria ought to meet the regulatory standards. Quality controls are instrumented within the manufacturing process to prove according to the set quality criteria. Essential quality criteria are the traceability of the cell product through donor identification and product labelling, the prevention of introduction and spreading of infection and communicable diseases through donor screening and aseptic cell processing and proof of the therapeutic safety, lot consistency, potency and purity of the cell product (European Parliament 2007; FDA 2010).

#### **2.2.1 Therapeutic safety, purity and potency**

Safety is a key issue in cell therapy. In addition to the above mentioned aspects regarding reagents (fetal bovine serum has been elaborated on) and sterility testing (bacterial, fungal, viral, mycoplasma), cellular aspects have to be considered as well. In long term cell culture current testing methods of chromosomal aberrations and neoplastic transformation are fluoerescence in situ hybridization (FISH), karyotype analysis or detection of proto-oncogenes or activators of tumorigenesis like myc-assosiated proteins (Agrawal, Yu et al. 2010). Further lately developed testing methods are BAC-based (Bacterial Artificial Chromosome) Array to detect DNA copy number or oligonucleotide-based Array CGH (Chromosomal Comparative Genomic Hybridization) to detect small genomic regions with amplification or deletion (Wicker, Carles et al. 2007). Additionally, detection of telomerase activation is often performed, as telomerase plays a role in malignant transformation *in vitro* (Yamaoka, Hiyama et al. 2011). All these assays indicate that there is a low risk of transformation of MSC in *in vitro* expansion. However, more safety studies – especially long term follow up *in vivo* - are required to exclude risks and to enable to value risks against therapeutic value.

Towards Clinical Application of Mesenchymal Stromal Cells:

by the directive 93/42/and the directive 90/385/ EEC.

**described by the US Food and Drug Administration (FDA)** 

medicinal products (ATMP)" that are:


2001/83/EC) and

conditions:

10.12.2007).

(www.FDA.gov).

Drug (IND) mechanism.

Perspectives and Requirements for Orthopaedic Applications 311

(EC1394/2007). Furthermore, a number of products also combine biological materials, cells and tissues with scaffolds. This regulation defines those products as "advanced therapy

Cells or tissues shall be considered 'engineered' if they fulfil at least one of the following


The scope of this regulation is to set standards for advanced therapy medicinal products which are intended to be placed on the market in European member states. It indicates the setting of manufacturing guidelines specific for ATMP as to properly reflect the particular nature of their manufacturing process. The directive 2004/23/EC amends to this regulation setting standards of quality and safety in tissue procurement and donor testing. Regarding clinical trials on ATMP, they should be conducted in accordance with the Directive 2001/20/EC. Additionally Directive 2005/28/EC laid down principles and detailed guidelines for good clinical practice as well as the requirements for authorisation of the manufacturing and importation of ATMP. Considering tissue engineered cell products, medicinal devices incorporated in the ATMP (combined medicinal products) are regulated

**2.3.2 Human cells, tissues, and cellular and tissue-based products (HCT/P's) as** 

The quality system for Food and Drug Administration (FDA) regulated products is known as current good manufacturing practices (cGMP). For globally operating pharmaceutical facilities it is mandatory to fulfil the requirements of both FDA and EU. The Code of Federal Regulation (CFR) Title 21, part 1271 has the purpose to create a unified registration and listing system for human cells, tissues, and cellular and tissue-based products (HCT/P's) and to establish donor-eligibility, current good tissue practice, and other procedures to "prevent the introduction, transmission, and spread of communicable diseases by HCT/P's"

Whereas cell products, only minimally manipulated or subjected to homologous use without systemic effect, are regulated solely by the Public Health Service (PHS) Act Section 361 and do not require to undergo premarket review (GEN Mar. 15, vol 25, no 6), they still must comply with Good Tissue practice (GTP) (Burger 2003). Clinical trials of higher-risk involving ''more-than-minimally manipulated'' HCT/P's require the Investigational New


Further aspects that are critical for the therapeutic safety and need to be analysed are the spontaneous or the induced *in vivo* differentiation potential of MSC. It has to be proven that MSC after *in vivo* application serve their therapeutic function and do not develop into unwanted cell types for example BM-MSC into adipocytes or osteocytes when intended for epithelial or myogenic regeneration. The latter could possibly lead to threatening thrombembolic incidents after intravascular application. In general, intravascular injection is associated with a higher risk than direct application into the site of injury or into the neighbouring parenchyma (Furlani, Ugurlucan et al. 2009).

MSC are not a distinct cell fraction in fresh tissue isolates. Accordingly purity is a key issue to be taken into account. To isolate MSC, mononuclear cells of fresh tissue isolates are seeded on plastic culture dishes, MSC adhere, proliferate and form colonies. Those expanded MSC should have a distinct immune phenotype, defined by the ISCT, they do not express haematopoietic markers and have a characteristic fibroblastoid morphology (Dominici, Le Blanc et al. 2006). Based on these criteria, contaminations of MSC with hematopoietic or endothelial cells can be assessed and consequently purity of the MSC cell product can be proven via flow cytometry. This is further amended by description of expanded MSC morphology and colony assays (CFU-F-assay) to quantify the precursor frequency. Quality controls of MSC expanded in scaffolds or in bioreactors vs. 2D cell culture regarding population purity is probably more complex.

MSC are applied in various clinical settings, as they possess a variety of functional properties. MSC can work as progenitor cells in tissue modelling, due to their adipo-, osteo-, chondrogenic potential, or as immunomodulatory agents in GvHD, autoimmune disease or as anti-inflammatory agents through their paracrine abilities. Due to this extremely broad range it is difficult to establish potency assays. These standardized *in vitro* functional assays have to be performed to predict the consistency of the manufacturing process and the functionality of the cell product. Quality control assays, including potency assays, have to be well established and validated to be capable of addressing the consistent quality of the cellular product. It is certainly difficult to reproduce the *in vivo* setting within *in vitro* conditions,. This is probably why *in vitro* potency assays often fail to predict the *in vivo* outcome (Sensebe, Bourin et al. 2011). Anyhow, it is a demand for the manufacturing facility to implement potency assays capable of predicting therapeutic capacity. These assays have to be quantitative and directly related to the mechanism of action. Where possible surrogate assays can replace time-consuming functional assays (e.g. cell surface marker expression, growth factor release, gene or protein expression analysis). Finally, the manufacturing process in order to conduct clinical trials in Europe and the US has to be validated and approved by the authorities in accordance to the pharmaceutical regulations.

#### **2.3 Pharmaceutical guidelines**

#### **2.3.1 Advanced therapy medicinal products as described in the Regulation (EC) No 1394/2007 of the European Parliament**

In cases where MSC are to be used in a medicinal product the donation, procurement and testing of the cells are covered in Europe by the Tissues and Cells Directive (2004/23/EC). To make innovative treatments available to patients, and to ensure that these novel treatments are safe, the EU institutions agreed on a "regulation on advanced therapies" (EC1394/2007). Furthermore, a number of products also combine biological materials, cells and tissues with scaffolds. This regulation defines those products as "advanced therapy medicinal products (ATMP)" that are:


310 Tissue Regeneration – From Basic Biology to Clinical Application

Further aspects that are critical for the therapeutic safety and need to be analysed are the spontaneous or the induced *in vivo* differentiation potential of MSC. It has to be proven that MSC after *in vivo* application serve their therapeutic function and do not develop into unwanted cell types for example BM-MSC into adipocytes or osteocytes when intended for epithelial or myogenic regeneration. The latter could possibly lead to threatening thrombembolic incidents after intravascular application. In general, intravascular injection is associated with a higher risk than direct application into the site of injury or into the

MSC are not a distinct cell fraction in fresh tissue isolates. Accordingly purity is a key issue to be taken into account. To isolate MSC, mononuclear cells of fresh tissue isolates are seeded on plastic culture dishes, MSC adhere, proliferate and form colonies. Those expanded MSC should have a distinct immune phenotype, defined by the ISCT, they do not express haematopoietic markers and have a characteristic fibroblastoid morphology (Dominici, Le Blanc et al. 2006). Based on these criteria, contaminations of MSC with hematopoietic or endothelial cells can be assessed and consequently purity of the MSC cell product can be proven via flow cytometry. This is further amended by description of expanded MSC morphology and colony assays (CFU-F-assay) to quantify the precursor frequency. Quality controls of MSC expanded in scaffolds or in bioreactors vs. 2D cell

MSC are applied in various clinical settings, as they possess a variety of functional properties. MSC can work as progenitor cells in tissue modelling, due to their adipo-, osteo-, chondrogenic potential, or as immunomodulatory agents in GvHD, autoimmune disease or as anti-inflammatory agents through their paracrine abilities. Due to this extremely broad range it is difficult to establish potency assays. These standardized *in vitro* functional assays have to be performed to predict the consistency of the manufacturing process and the functionality of the cell product. Quality control assays, including potency assays, have to be well established and validated to be capable of addressing the consistent quality of the cellular product. It is certainly difficult to reproduce the *in vivo* setting within *in vitro* conditions,. This is probably why *in vitro* potency assays often fail to predict the *in vivo* outcome (Sensebe, Bourin et al. 2011). Anyhow, it is a demand for the manufacturing facility to implement potency assays capable of predicting therapeutic capacity. These assays have to be quantitative and directly related to the mechanism of action. Where possible surrogate assays can replace time-consuming functional assays (e.g. cell surface marker expression, growth factor release, gene or protein expression analysis). Finally, the manufacturing process in order to conduct clinical trials in Europe and the US has to be validated and

neighbouring parenchyma (Furlani, Ugurlucan et al. 2009).

culture regarding population purity is probably more complex.

approved by the authorities in accordance to the pharmaceutical regulations.

**2.3.1 Advanced therapy medicinal products as described in the Regulation (EC)** 

In cases where MSC are to be used in a medicinal product the donation, procurement and testing of the cells are covered in Europe by the Tissues and Cells Directive (2004/23/EC). To make innovative treatments available to patients, and to ensure that these novel treatments are safe, the EU institutions agreed on a "regulation on advanced therapies"

**2.3 Pharmaceutical guidelines** 

**No 1394/2007 of the European Parliament** 

Cells or tissues shall be considered 'engineered' if they fulfil at least one of the following conditions:


The scope of this regulation is to set standards for advanced therapy medicinal products which are intended to be placed on the market in European member states. It indicates the setting of manufacturing guidelines specific for ATMP as to properly reflect the particular nature of their manufacturing process. The directive 2004/23/EC amends to this regulation setting standards of quality and safety in tissue procurement and donor testing. Regarding clinical trials on ATMP, they should be conducted in accordance with the Directive 2001/20/EC. Additionally Directive 2005/28/EC laid down principles and detailed guidelines for good clinical practice as well as the requirements for authorisation of the manufacturing and importation of ATMP. Considering tissue engineered cell products, medicinal devices incorporated in the ATMP (combined medicinal products) are regulated by the directive 93/42/and the directive 90/385/ EEC.

#### **2.3.2 Human cells, tissues, and cellular and tissue-based products (HCT/P's) as described by the US Food and Drug Administration (FDA)**

The quality system for Food and Drug Administration (FDA) regulated products is known as current good manufacturing practices (cGMP). For globally operating pharmaceutical facilities it is mandatory to fulfil the requirements of both FDA and EU. The Code of Federal Regulation (CFR) Title 21, part 1271 has the purpose to create a unified registration and listing system for human cells, tissues, and cellular and tissue-based products (HCT/P's) and to establish donor-eligibility, current good tissue practice, and other procedures to "prevent the introduction, transmission, and spread of communicable diseases by HCT/P's" (www.FDA.gov).

Whereas cell products, only minimally manipulated or subjected to homologous use without systemic effect, are regulated solely by the Public Health Service (PHS) Act Section 361 and do not require to undergo premarket review (GEN Mar. 15, vol 25, no 6), they still must comply with Good Tissue practice (GTP) (Burger 2003). Clinical trials of higher-risk involving ''more-than-minimally manipulated'' HCT/P's require the Investigational New Drug (IND) mechanism.

Towards Clinical Application of Mesenchymal Stromal Cells:

uncontrolled growth can also lead to benign or malign tumours.

**3.1** *In vitro* **3D culture, choice of scaffold** 

Perspectives and Requirements for Orthopaedic Applications 313

Tissue engineering aims at regenerating or replacing tissues or even organs. Therefore a complex architecture is needed, which cannot be generated by simple two-dimensional (2D) cultures. Investigation on MSC concentrates on characterization *in vitro* in a 2D culture, as mentioned above, to assess both the differentiation potential and the influence of the biomaterial surface on growth and development. MSC can be driven towards osteogenic differentiation by use of dexamethasone, β-glycerophosphate and ascorbate in addition to osteogenic basal medium (Jaiswal, Haynesworth et al. 1997; Pittenger, Mackay et al. 1999; Augello and De Bari 2010). Cells can be used as undifferentiated, pre- or terminally differentiated cells in combinations with scaffolds to achieve tissue-like conditions. Compared to 2D, 3D cultures better mimic physiological conditions. Static 3D cultures are mainly used to investigate the suitability of a certain biomaterial (Bernhardt, Lode et al. 2009). Increasing attention is recently been paid to dynamic 3D culture, assuring a more homogenous cell distribution within a scaffold, a higher number of cells and all in all less manipulation (Diederichs, Roker et al. 2009; Stiehler, Bunger et al. 2009). Flow perfusion cultures itself, even in absence of dexamethasone, may lead to differentiation into bone tissue (Holtorf, Jansen et al. 2005). Nevertheless, cell expansion of MSC in order to achieve a high cell dose prior to use in animal or humans may not always be advantageous, since

There is a broad choice of biomaterials for scaffolds for clinical applications. However, only bone cement and bone of bone banks are regularly favoured for bone defect surgeries, when available. Bone itself has become the biomaterial per se as a natural scaffold supply. Bone cement on the other hand can be stored as powder, provides immediate stability and is easily prepared and applied during an operation. Within a few minutes, the cement becomes firm (Gruner and Heller 2009). Although acellular scaffolds prove stability immediately following implantation, a better option would be to seed them with cells. For MSC application a great variety of materials, ranging from sterilised original bone to nanostructures and bioglass-collagen composites are being utilised (Karageorgiou and Kaplan 2005; Tanner 2010). Eventually, in order to approach the therapeutic effect of scaffold-MSC composites, studies are currently being performed on several stages: cell culture either in a dish or in a bioreactor, animal models and individual attempts in human (Bernstein, Bornhauser et al. 2009; Diederichs, Roker et al. 2009; Diederichs, Bohm et al. 2010). Further key parameters for the choice of the suitable biomaterial is the ability to support cell growth, cellular ingrowth, osteogenic differentiation and antimicrobial functions (Costantino, Hiltzik et al. 2002; Bernstein, Bornhauser et al. 2009). For that reason, additional osteogenic cytokines such as bone morphogenetic proteins (BMP) or bioactive

peptides that become integrated into scaffolds are of interest (Keibl, Fugl et al. 2011).

provide some standardised features (Gruner and Heller 2009).

An optimum scaffold must allow bone cells to grow into it. Pores of 300 to 500µm are requested (De Long, Einhorn et al. 2007; Stiehler, Bunger et al. 2009). Apart from this an optimum scaffold has to be adapted to bone structures. Defects in facial areas, in the skull, femur or hip require different stabilities and shapes. Only hip re-implantation seems to

Building suitable biomaterials to be combined with MSC has led to very different approaches: Collagen as a basis of any bone tissue was modified and calcified at all pore

#### **3. Example for MSC in regenerative medicine: Attempts for orthopaedic applications in bone defect healing**

Orthopaedic surgery provides a fascinating field for the application of MSC (Horwitz, Prockop et al. 2001; Le Blanc, Gotherstrom et al. 2005; Bernhardt, Lode et al. 2009; Chanda, Kumar et al. 2010; Diederichs, Bohm et al. 2010; Mosna, Sensebe et al. 2010; Parekkadan and Milwid 2010; Levi and Longaker 2011). Bone defects appear in increasing numbers in orthopaedic clinics due to aseptic loosening of hip endoprosthesis after 10 to 20 years. These defects are then covered primarily with either bone cement or acellular bone from a bone bank prior to insertion of a new endoprosthesis in order to provide primary stability - that is immediate mechanical support of a new implant (Gruner and Heller 2009).

An ideal scaffold must offer osteoinduction – induction of bone growth – and osteoconduction – providing the guiding structure that paves the way for future bone growth - and eventually osteointegration, becoming part of the bone architecture of a body (Frohlich, Grayson et al. 2008; Ferretti, Ripamonti et al. 2010). The advantages and disadvantages of bone cement have been controversially discussed regarding different rates of implant failure in follow up examinations (Kavanagh, Ilstrup et al. 1985; Izquierdo and Northmore-Ball 1994; Stromberg and Herberts 1996). Recent works suggest to proceed without use of bone cement if possible, and recommend other surgical techniques to implant a total hip endoprosthesis. Bone cement is stiff and strong with a gradual increasing resorption area at its limits. Where bone cement is placed, immediate primary stability is provided, however, at the expense of bone regeneration that does not take place anymore (Izquierdo and Northmore-Ball 1994; Gruner and Heller 2009). Depending on the localization of the bone cement and the mechanical stress, this can gradually lead to a decreased stability. In case another revision operation is needed but great bone defects and osteolysis can impede or even inhibit surgical possibilities (Kavanagh, Ilstrup et al. 1985; Izquierdo and Northmore-Ball 1994; Stromberg and Herberts 1996; Gruner and Heller 2009). Fresh autologous bone or allogenous acellular bone from a bone bank can support bone growth. These preparations are osteoconductive and are, if preserved as a cancellous bone even osteoinductive but fail to provide immediate stability alone. These scaffolds have osteoconductive potential, however regular radiological controls often demonstrate gradually increasing resorption at sites of the implanted acellular bone. In the consequence, stability may be compromised (Gruner and Heller 2009).

Given the potential of MSC to differentiate into bone, MSC became attractive candidates. For hard tissue replacement, cells alone are not adequate. Thus surgical procedures treating bone defects in which a combination of MSC and scaffolds are applied, may provide both immediate stability and permanent integration into the recipient's bone. Different techniques are described for the implantation of MSC. Still it remains unclear if implants shall carry completely osteogenically differentiated MSC, or more likely optimize adaptive possibilities within the host organism. The more differentiated the MSC the more initial stability they provide for implants in areas with high mechanical force exposure (Bernhardt, Lode et al. 2008). Less differentiated MSC on the other prove more plasticity (Niemeyer, Krause et al. 2004; Bieback, Kern et al. 2008). In the worst case, undesired differentiation or even dedifferentiation might occur. Medication, integrated drugs or even genetically engineered cells may prove a possible control *in vivo*.

#### **3.1** *In vitro* **3D culture, choice of scaffold**

312 Tissue Regeneration – From Basic Biology to Clinical Application

Orthopaedic surgery provides a fascinating field for the application of MSC (Horwitz, Prockop et al. 2001; Le Blanc, Gotherstrom et al. 2005; Bernhardt, Lode et al. 2009; Chanda, Kumar et al. 2010; Diederichs, Bohm et al. 2010; Mosna, Sensebe et al. 2010; Parekkadan and Milwid 2010; Levi and Longaker 2011). Bone defects appear in increasing numbers in orthopaedic clinics due to aseptic loosening of hip endoprosthesis after 10 to 20 years. These defects are then covered primarily with either bone cement or acellular bone from a bone bank prior to insertion of a new endoprosthesis in order to provide primary stability - that is

An ideal scaffold must offer osteoinduction – induction of bone growth – and osteoconduction – providing the guiding structure that paves the way for future bone growth - and eventually osteointegration, becoming part of the bone architecture of a body (Frohlich, Grayson et al. 2008; Ferretti, Ripamonti et al. 2010). The advantages and disadvantages of bone cement have been controversially discussed regarding different rates of implant failure in follow up examinations (Kavanagh, Ilstrup et al. 1985; Izquierdo and Northmore-Ball 1994; Stromberg and Herberts 1996). Recent works suggest to proceed without use of bone cement if possible, and recommend other surgical techniques to implant a total hip endoprosthesis. Bone cement is stiff and strong with a gradual increasing resorption area at its limits. Where bone cement is placed, immediate primary stability is provided, however, at the expense of bone regeneration that does not take place anymore (Izquierdo and Northmore-Ball 1994; Gruner and Heller 2009). Depending on the localization of the bone cement and the mechanical stress, this can gradually lead to a decreased stability. In case another revision operation is needed but great bone defects and osteolysis can impede or even inhibit surgical possibilities (Kavanagh, Ilstrup et al. 1985; Izquierdo and Northmore-Ball 1994; Stromberg and Herberts 1996; Gruner and Heller 2009). Fresh autologous bone or allogenous acellular bone from a bone bank can support bone growth. These preparations are osteoconductive and are, if preserved as a cancellous bone even osteoinductive but fail to provide immediate stability alone. These scaffolds have osteoconductive potential, however regular radiological controls often demonstrate gradually increasing resorption at sites of the implanted acellular bone. In the consequence,

Given the potential of MSC to differentiate into bone, MSC became attractive candidates. For hard tissue replacement, cells alone are not adequate. Thus surgical procedures treating bone defects in which a combination of MSC and scaffolds are applied, may provide both immediate stability and permanent integration into the recipient's bone. Different techniques are described for the implantation of MSC. Still it remains unclear if implants shall carry completely osteogenically differentiated MSC, or more likely optimize adaptive possibilities within the host organism. The more differentiated the MSC the more initial stability they provide for implants in areas with high mechanical force exposure (Bernhardt, Lode et al. 2008). Less differentiated MSC on the other prove more plasticity (Niemeyer, Krause et al. 2004; Bieback, Kern et al. 2008). In the worst case, undesired differentiation or even dedifferentiation might occur. Medication, integrated drugs or even genetically

**3. Example for MSC in regenerative medicine: Attempts for orthopaedic** 

immediate mechanical support of a new implant (Gruner and Heller 2009).

stability may be compromised (Gruner and Heller 2009).

engineered cells may prove a possible control *in vivo*.

**applications in bone defect healing** 

Tissue engineering aims at regenerating or replacing tissues or even organs. Therefore a complex architecture is needed, which cannot be generated by simple two-dimensional (2D) cultures. Investigation on MSC concentrates on characterization *in vitro* in a 2D culture, as mentioned above, to assess both the differentiation potential and the influence of the biomaterial surface on growth and development. MSC can be driven towards osteogenic differentiation by use of dexamethasone, β-glycerophosphate and ascorbate in addition to osteogenic basal medium (Jaiswal, Haynesworth et al. 1997; Pittenger, Mackay et al. 1999; Augello and De Bari 2010). Cells can be used as undifferentiated, pre- or terminally differentiated cells in combinations with scaffolds to achieve tissue-like conditions. Compared to 2D, 3D cultures better mimic physiological conditions. Static 3D cultures are mainly used to investigate the suitability of a certain biomaterial (Bernhardt, Lode et al. 2009). Increasing attention is recently been paid to dynamic 3D culture, assuring a more homogenous cell distribution within a scaffold, a higher number of cells and all in all less manipulation (Diederichs, Roker et al. 2009; Stiehler, Bunger et al. 2009). Flow perfusion cultures itself, even in absence of dexamethasone, may lead to differentiation into bone tissue (Holtorf, Jansen et al. 2005). Nevertheless, cell expansion of MSC in order to achieve a high cell dose prior to use in animal or humans may not always be advantageous, since uncontrolled growth can also lead to benign or malign tumours.

There is a broad choice of biomaterials for scaffolds for clinical applications. However, only bone cement and bone of bone banks are regularly favoured for bone defect surgeries, when available. Bone itself has become the biomaterial per se as a natural scaffold supply. Bone cement on the other hand can be stored as powder, provides immediate stability and is easily prepared and applied during an operation. Within a few minutes, the cement becomes firm (Gruner and Heller 2009). Although acellular scaffolds prove stability immediately following implantation, a better option would be to seed them with cells. For MSC application a great variety of materials, ranging from sterilised original bone to nanostructures and bioglass-collagen composites are being utilised (Karageorgiou and Kaplan 2005; Tanner 2010). Eventually, in order to approach the therapeutic effect of scaffold-MSC composites, studies are currently being performed on several stages: cell culture either in a dish or in a bioreactor, animal models and individual attempts in human (Bernstein, Bornhauser et al. 2009; Diederichs, Roker et al. 2009; Diederichs, Bohm et al. 2010). Further key parameters for the choice of the suitable biomaterial is the ability to support cell growth, cellular ingrowth, osteogenic differentiation and antimicrobial functions (Costantino, Hiltzik et al. 2002; Bernstein, Bornhauser et al. 2009). For that reason, additional osteogenic cytokines such as bone morphogenetic proteins (BMP) or bioactive peptides that become integrated into scaffolds are of interest (Keibl, Fugl et al. 2011).

An optimum scaffold must allow bone cells to grow into it. Pores of 300 to 500µm are requested (De Long, Einhorn et al. 2007; Stiehler, Bunger et al. 2009). Apart from this an optimum scaffold has to be adapted to bone structures. Defects in facial areas, in the skull, femur or hip require different stabilities and shapes. Only hip re-implantation seems to provide some standardised features (Gruner and Heller 2009).

Building suitable biomaterials to be combined with MSC has led to very different approaches: Collagen as a basis of any bone tissue was modified and calcified at all pore

Towards Clinical Application of Mesenchymal Stromal Cells:

**3.4 Clinical trials** 

**3.5 Animal model and interpretation** 

Perspectives and Requirements for Orthopaedic Applications 315

examined. Comparisons between BM-MSC and amniotic fluid derived stem cells (AFS) showed different properties in differentiation in 2D and 3D. In 2D tissue culture, AFS produce more mineralized matrix but delayed peaks in osteogenic markers. Differentiation towards bone tissue occurred faster in BM-MSC, however, after weeks mineralization slowed down. AFS differentiated more slowly but mineralized until the end of the observation period 15 weeks, producing 5 fold higher amounts of mineral matrix. Human term placenta derived MSC seem to be less prone to osteogenic differentiation than BM-MSC (Pilz, Ulrich et al. 2011). These characteristics might be of interest, when fast ingrowth is needed (Peister, Woodruff et al. 2011). As initially mentioned, for some groups AT-MSC are the most promising candidates in bone tissue engineering (Levi and Longaker 2011). Osteogenic capacity does not decrease with age in contrast to BM-MSC (Khan, Adesida et al. 2009). Also due to a relatively high and still increasing rate of obesity in the western hemisphere it can be considered that adipose tissue has a great potential as main source for MSC. So, metabolic disease can be of benefit when it comes to autologous MSC implantation (Diederichs, Bohm et al. 2010). All in all an ideal cell source has yet not been identified. Further research is important to compare the advantages of all tissue sources. Moreover, for each biomaterial the MSC differentiation properties have to be determined. The adequate

MSC will depend both on availability and differentiating / functional properties.

There is no on-going clinical trial that deals with the use of MSC and a suitable biomaterial in healing of bone defects in humans. Osteogenesis imperfecta has been successfully treated with MSC alone, even with allogenic MSC (Horwitz, Prockop et al. 2001; Le Blanc, Gotherstrom et al. 2005). The Iranian Royan Institute, Teheran, announced a clinical trial in 2008 (http://www.clinicaltrials.gov). The study aimed to establish the influence of MSC in non-union fracture healing. However, in 2011 the state of the study is still unknown and cannot be verified. One case report from 2009 refers to a clinical trial in preparation. The benefit of the use of decellularized bone and MSC was demonstrated in a case of large hip transplant loosening. Follow-up radiological exams could confirm the stable position of a new hip implant (Bernstein, Bornhauser et al. 2009). So far, no clinical trial on the use of MSC for bone fracture healing has been published. Various preclinical studies predict benefits in bone tissue healing and stability by use of MSC (Bernhardt, Lode et al. 2008; Bernhardt, Lode et al. 2009; John, Varma et al. 2009; Nair, Bernhardt et al. 2009; Nienhuijs, Walboomers et al. 2011). However the methods and more importantly the animal models to prove beneficial effects of MSC are not yet standardized. This is of great importance since the forces exerted on a fracture cannot be compared between animal species, nor can it be to humans. Comparisons between different procedures, cells and scaffolds are thus not reliable. A recent article proposes rules for comparable preclinical bone defects model that amongst others affect standardized surgical procedures and measurements. In this work tibia fracture and segmental defect models are preferred (Reichert, Saifzadeh et al. 2009).

Unfortunately, the criteria to evaluate the outcome of studies - be it *in vitro* or *in vivo* - differ considerably. Regarding the major requirement of mechanical stability, a variety of mechanical tests exist that determine stability. However, till date none of them has been defined as

sizes. Integration of MSC is easily achieved but primary stability is comparably low (Bernhardt, Lode et al. 2009; Nienhuijs, Walboomers et al. 2011). Hydroxyapatite is a ubiquitous part of the vertebrate bone. Hydroxyapatite ceramics become easily integrated and also prove enough primary stability (John, Varma et al. 2009; Nair, Bernhardt et al. 2009; Nair, Varma et al. 2009). Beta-tricalciumposphate is a completely resorbable scaffold with high purity. It is available at all sizes, all porous degrees, it can be supplied as granules or as plates and therefore serves as comparison to newly developed biomaterials (Wiedmann-Al-Ahmad, Gutwald et al. 2007). Due to its low tissue reactivity and good stability titanium based structures not only serve well as implants but also as scaffolds. Titanium or TiO2 does not become degraded or resorbed, instead as a whole it becomes very firmly integrated into any tissue (Gotman 1997; Olmedo, Tasat et al. 2009). Due to the fact that titanium is not resorbable it holds the risks of infection, be it acute or slowly increasing, so that an explantation must be performed. Since titanium becomes very well integrated into the host's body, an explantation is often associated with a great tissue loss. Application of titanium has to be carefully considered. In sum, since tissue reactions to titanium are quite well characterised as an implant it serves well as an example of future challenges and possibilities of other biomaterials. Silver nanoparticles are matter of current discussion due to their antimicrobial and toxic effects that can also be used within polymeric nanocomposites. Titanium nanostructures alone have been proven to act antimicrobially (Dallas, Sharma et al. 2011; Ercan, Taylor et al. 2011).

#### **3.2 Analysis of 3D cultures and biomaterials**

Once a 3D scaffold has been seeded, the efficiency of the seeding procedure, cell growth and differentiation must be determined, e.g. by quantifying the DNA content and mineralisation by histochemical stains or RT-PCR (Stiehler, Bunger et al. 2009; Peister, Woodruff et al. 2011). Homogeneic seeding and / or cell growth can be determined by fluorescence microscopy or µCT (Zou, Hunter et al. 2011). Mechanical tests are not standardized. For *in vitro* generated bone tissue from MSC crush tests, i.e. the use of a defined force until a scaffold breaks, are the most simple. For *in vivo* generated bone tissue shear and bending tests give additional data concerning the stability of the MSC composite within the animal's original bone. However, *in vitro* and *in vivo* experiments are only conclusive when scaffolds used are comparable in size and porosity (De Long, Einhorn et al. 2007; Stiehler, Bunger et al. 2009). The same applies to standardisation of surgical procedures and animal models used (Reichert, Saifzadeh et al. 2009).

#### **3.3 Tissue source**

As already mentioned, tissue engineering requires a scaffold next to the cells to seed it. Since MSC can be isolated from different tissue sources, the question remains: which cells are best suited? MSC derived from different tissues show different osteogenic differentiation properties: human embryonic stem cells (hESC), CB-MSC, AT-MSC, BM-MSC and even amniotic membrane-derived MSC can undergo osteogenic differentiation. Historically, most work had been performed on BM-MSC, so at least BM-MSC are the source to compare with, when MSC behaviour in a scaffold is analysed (Lindenmair, Wolbank et al. 2010; Guven, Mehrkens et al. 2011; Stockmann, Park et al. 2011; Weinand, Nabili et al. 2011). In recent studies, aspects of differentiation in 2D tissue culture and in 3D tissue culture have been examined. Comparisons between BM-MSC and amniotic fluid derived stem cells (AFS) showed different properties in differentiation in 2D and 3D. In 2D tissue culture, AFS produce more mineralized matrix but delayed peaks in osteogenic markers. Differentiation towards bone tissue occurred faster in BM-MSC, however, after weeks mineralization slowed down. AFS differentiated more slowly but mineralized until the end of the observation period 15 weeks, producing 5 fold higher amounts of mineral matrix. Human term placenta derived MSC seem to be less prone to osteogenic differentiation than BM-MSC (Pilz, Ulrich et al. 2011). These characteristics might be of interest, when fast ingrowth is needed (Peister, Woodruff et al. 2011). As initially mentioned, for some groups AT-MSC are the most promising candidates in bone tissue engineering (Levi and Longaker 2011). Osteogenic capacity does not decrease with age in contrast to BM-MSC (Khan, Adesida et al. 2009). Also due to a relatively high and still increasing rate of obesity in the western hemisphere it can be considered that adipose tissue has a great potential as main source for MSC. So, metabolic disease can be of benefit when it comes to autologous MSC implantation (Diederichs, Bohm et al. 2010). All in all an ideal cell source has yet not been identified. Further research is important to compare the advantages of all tissue sources. Moreover, for each biomaterial the MSC differentiation properties have to be determined. The adequate MSC will depend both on availability and differentiating / functional properties.

#### **3.4 Clinical trials**

314 Tissue Regeneration – From Basic Biology to Clinical Application

sizes. Integration of MSC is easily achieved but primary stability is comparably low (Bernhardt, Lode et al. 2009; Nienhuijs, Walboomers et al. 2011). Hydroxyapatite is a ubiquitous part of the vertebrate bone. Hydroxyapatite ceramics become easily integrated and also prove enough primary stability (John, Varma et al. 2009; Nair, Bernhardt et al. 2009; Nair, Varma et al. 2009). Beta-tricalciumposphate is a completely resorbable scaffold with high purity. It is available at all sizes, all porous degrees, it can be supplied as granules or as plates and therefore serves as comparison to newly developed biomaterials (Wiedmann-Al-Ahmad, Gutwald et al. 2007). Due to its low tissue reactivity and good stability titanium based structures not only serve well as implants but also as scaffolds. Titanium or TiO2 does not become degraded or resorbed, instead as a whole it becomes very firmly integrated into any tissue (Gotman 1997; Olmedo, Tasat et al. 2009). Due to the fact that titanium is not resorbable it holds the risks of infection, be it acute or slowly increasing, so that an explantation must be performed. Since titanium becomes very well integrated into the host's body, an explantation is often associated with a great tissue loss. Application of titanium has to be carefully considered. In sum, since tissue reactions to titanium are quite well characterised as an implant it serves well as an example of future challenges and possibilities of other biomaterials. Silver nanoparticles are matter of current discussion due to their antimicrobial and toxic effects that can also be used within polymeric nanocomposites. Titanium nanostructures alone have been proven to act antimicrobially

Once a 3D scaffold has been seeded, the efficiency of the seeding procedure, cell growth and differentiation must be determined, e.g. by quantifying the DNA content and mineralisation by histochemical stains or RT-PCR (Stiehler, Bunger et al. 2009; Peister, Woodruff et al. 2011). Homogeneic seeding and / or cell growth can be determined by fluorescence microscopy or µCT (Zou, Hunter et al. 2011). Mechanical tests are not standardized. For *in vitro* generated bone tissue from MSC crush tests, i.e. the use of a defined force until a scaffold breaks, are the most simple. For *in vivo* generated bone tissue shear and bending tests give additional data concerning the stability of the MSC composite within the animal's original bone. However, *in vitro* and *in vivo* experiments are only conclusive when scaffolds used are comparable in size and porosity (De Long, Einhorn et al. 2007; Stiehler, Bunger et al. 2009). The same applies to standardisation of surgical procedures and animal models

As already mentioned, tissue engineering requires a scaffold next to the cells to seed it. Since MSC can be isolated from different tissue sources, the question remains: which cells are best suited? MSC derived from different tissues show different osteogenic differentiation properties: human embryonic stem cells (hESC), CB-MSC, AT-MSC, BM-MSC and even amniotic membrane-derived MSC can undergo osteogenic differentiation. Historically, most work had been performed on BM-MSC, so at least BM-MSC are the source to compare with, when MSC behaviour in a scaffold is analysed (Lindenmair, Wolbank et al. 2010; Guven, Mehrkens et al. 2011; Stockmann, Park et al. 2011; Weinand, Nabili et al. 2011). In recent studies, aspects of differentiation in 2D tissue culture and in 3D tissue culture have been

(Dallas, Sharma et al. 2011; Ercan, Taylor et al. 2011).

**3.2 Analysis of 3D cultures and biomaterials** 

used (Reichert, Saifzadeh et al. 2009).

**3.3 Tissue source** 

There is no on-going clinical trial that deals with the use of MSC and a suitable biomaterial in healing of bone defects in humans. Osteogenesis imperfecta has been successfully treated with MSC alone, even with allogenic MSC (Horwitz, Prockop et al. 2001; Le Blanc, Gotherstrom et al. 2005). The Iranian Royan Institute, Teheran, announced a clinical trial in 2008 (http://www.clinicaltrials.gov). The study aimed to establish the influence of MSC in non-union fracture healing. However, in 2011 the state of the study is still unknown and cannot be verified. One case report from 2009 refers to a clinical trial in preparation. The benefit of the use of decellularized bone and MSC was demonstrated in a case of large hip transplant loosening. Follow-up radiological exams could confirm the stable position of a new hip implant (Bernstein, Bornhauser et al. 2009). So far, no clinical trial on the use of MSC for bone fracture healing has been published. Various preclinical studies predict benefits in bone tissue healing and stability by use of MSC (Bernhardt, Lode et al. 2008; Bernhardt, Lode et al. 2009; John, Varma et al. 2009; Nair, Bernhardt et al. 2009; Nienhuijs, Walboomers et al. 2011). However the methods and more importantly the animal models to prove beneficial effects of MSC are not yet standardized. This is of great importance since the forces exerted on a fracture cannot be compared between animal species, nor can it be to humans. Comparisons between different procedures, cells and scaffolds are thus not reliable. A recent article proposes rules for comparable preclinical bone defects model that amongst others affect standardized surgical procedures and measurements. In this work tibia fracture and segmental defect models are preferred (Reichert, Saifzadeh et al. 2009).

#### **3.5 Animal model and interpretation**

Unfortunately, the criteria to evaluate the outcome of studies - be it *in vitro* or *in vivo* - differ considerably. Regarding the major requirement of mechanical stability, a variety of mechanical tests exist that determine stability. However, till date none of them has been defined as

Towards Clinical Application of Mesenchymal Stromal Cells:

(Le Blanc, Gotherstrom et al. 2005).

cells may provide a possible control *in vivo*.

host tissues, need to be investigated further.

**4. Conclusion** 

Perspectives and Requirements for Orthopaedic Applications 317

that becomes completely integrated into bone. Osteoinduction is difficult to obtain. Local application of osteoinductive factors such as FGF, the bone morphogenic proteins BMP-2, BMP-4, BMP-7 and vascular endothelial growth factor VEGF does either not lead to results due to degradation or does lead to too strong responses since it cannot be well regulated. Recent work shows promising results in this regard. However no standard can be proposed in terms of choice of growth factor, dose and modification (Keibl, Fugl et al. 2011). Recent work demonstrated the feasibility of plasmid DNA-integration into a scaffold that lead to a higher bone differentiation ratio (Hosseinkhani, Hosseinkhani et al. 2008). Future research must also deal with possibly breaking the border between autologous and allogenic MSC in treatment, in case patients cannot donate autologous MSC of any source. Allogeneic MSC in treatment of patients with osteogenesis imperfecta defects could be recently demonstrated

The optimal degree of differentiation in culture prior to implantation in an animal model or a human remains unclear: Should implants carry completely osteogenically differentiated MSC, or more likely quite the opposite to provide an optimum of adaptive possibilities within the host organism? The more differentiated the MSC the more initial stability they provide for implants in areas in which great forces act. Less differentiated MSC on the other hand prove more plasticity. In the worst case undesired differentiation or even dedifferentiation might occur. Medication, integrated drugs or even genetically engineered

The specifications defined by the regulatory framework focussing on the clinical use of MSC are becoming increasingly detailed (Burger 2003). These are more complex when it comes to MSC and biomaterial composites as there are no standards for quality controls. *In vitro* and *in vivo* interactions between scaffolds and in-growing cells, as well as between scaffolds and

*In vitro* studies indicate that MSC possess a wide spectrum of properties in tissue regeneration as adult progenitor cells or by secreting immunomodulatory and antiinflammatory factors. Still various manufacturing protocols, cultivating media and methods hinder to correlate and interpret scientific findings. Nevertheless MSC are very promising candidates for cell therapy and have moved extremely quickly in the last ten years from the bench to the bedside. For controlled clinical trials there are several obstacles to overcome in order to define a safe and efficacious therapeutic. There is a need to determine factors that may influence the cell quality and consequently the clinical outcome in terms of the tissue source, the isolating, expansion and cultivating conditions. Above that, protocols and *in vitro* and safety animal studies need to be performed in compliance with GMP requirements. To be able to conduct clinical trials on MSC, the manufacturing process has to fulfil several regulatory standards. Advances in clinical application of MSC can be exemplified in the field of orthopaedic bone regeneration. The osteogenic potential of MSC is seen to be of great benefit in bone defect healing. However, only in rare conditions are MSC alone beneficial. The choice of a suitable biomaterial to both carry MSC and provide good primary stability is crucial for clinical applications in hard tissue regeneration. Different sources of MSC that have different differentiation properties can be used. To

standard (Hak, Makino et al. 2006; Jones, Atwood et al. 2009; Reichert, Saifzadeh et al. 2009). In animal models success criteria of implanted MSC and scaffold are restricted mainly to analysis of regenerated bone e.g. by histologiacal findings, CT-scan technology, x-ray or simply by measuring the weight of the created bone as well as by mechanical torsion tests (Zou, Hunter et al. 2011). The fate of implanted scaffold and MSC, in terms of material resorption and MSC engraftment into the host body, is rarely studied (Bernstein, Bornhauser et al. 2009). Since there is no standard in animal models, experiments are being carried out on various models. The rat model is broadly used because of availability. Bio-mechanical properties similar to humans are found in sheep, especially in hip arthroplasty (Korda, Blunn et al. 2008). Usually a fracture is induced as described by Matsumoto et al or Mifune et al (Matsumoto, Kawamoto et al. 2006; Mifune, Matsumoto et al. 2008). In a first step a tibia is fractured. Then a collagen scaffold is inserted containing saline and either BM-MSC or hESC. Then Undale et al compared the bone tissue healing properties of BM-MSC and hESC in rats after an induced fracture. BM-MSC resulted to be more efficient than hESC to bridge and heal a critical bone fracture. Moreover, in this setting hESC tended to produce benign bony tumours compromising the use of these cells in clinical settings (Undale, Fraser et al. 2011).

Bone fracture healing or integration into the animal's bone tissue can be demonstrated by follow-up conventional radiology in two weeks intervals. The limbs are both fully extended so that the broken and fractured limb can be compared. In recent studies µCT, a specialized CT for small animal structure, is used. Precise 3D models can be built from the data, allowing a comparison between the original and the newly built bone. Eight weeks after fracturing the animals can be euthanized and the limbs can be analysed histologically or biomechanically. Biomechanical stability of the fracture healing can be assessed by torsional load to evaluate normal and abnormal fracture healing (Undale, Fraser et al. 2011).

In summary, MSC from different sources appear as complementation to biomaterial implants. Depending on the tissue source and culture, different patterns of differentiation into bone, cartilage or fibre can be obtained. Depending on the precise situation different sorts of MSCbiocomposites may facilitate wound healing and functional regeneration of bone defects with high long term stability. However, the handling of biomaterial MSC composites is far more complex than conventional methods and oblige to adhere to regulatory standards: Since living cells are worked with, purity, a lack of bacterial contamination and absence of cell transformation has to be proven before clinical application. Conventional methods, that are acellular implants, may be limited because of rigidity and even lack of stability on the long run, but actually, in contrast to MSC biocomposites, they can be well compared regarding their advantages and disadvantages. MSC may differ much more as a matter of treatment, culture conditions and the cells itself need further investigation, experimental and clinical studies to evaluate their true potential at best in comparative studies. But the prospect of individual medicine with the patients' easily extractable and expandable own cells may support future research and applications in regenerative medicine.

#### **3.6 Future prospects**

Future orthopaedic research that may one day provide suitable personalized scaffolds to cover bone defects must integrate vascularisation as well. A balanced attempt to support both bone growth and blood supply must be established to create a stable long lasting graft that becomes completely integrated into bone. Osteoinduction is difficult to obtain. Local application of osteoinductive factors such as FGF, the bone morphogenic proteins BMP-2, BMP-4, BMP-7 and vascular endothelial growth factor VEGF does either not lead to results due to degradation or does lead to too strong responses since it cannot be well regulated. Recent work shows promising results in this regard. However no standard can be proposed in terms of choice of growth factor, dose and modification (Keibl, Fugl et al. 2011). Recent work demonstrated the feasibility of plasmid DNA-integration into a scaffold that lead to a higher bone differentiation ratio (Hosseinkhani, Hosseinkhani et al. 2008). Future research must also deal with possibly breaking the border between autologous and allogenic MSC in treatment, in case patients cannot donate autologous MSC of any source. Allogeneic MSC in treatment of patients with osteogenesis imperfecta defects could be recently demonstrated (Le Blanc, Gotherstrom et al. 2005).

The optimal degree of differentiation in culture prior to implantation in an animal model or a human remains unclear: Should implants carry completely osteogenically differentiated MSC, or more likely quite the opposite to provide an optimum of adaptive possibilities within the host organism? The more differentiated the MSC the more initial stability they provide for implants in areas in which great forces act. Less differentiated MSC on the other hand prove more plasticity. In the worst case undesired differentiation or even dedifferentiation might occur. Medication, integrated drugs or even genetically engineered cells may provide a possible control *in vivo*.

The specifications defined by the regulatory framework focussing on the clinical use of MSC are becoming increasingly detailed (Burger 2003). These are more complex when it comes to MSC and biomaterial composites as there are no standards for quality controls. *In vitro* and *in vivo* interactions between scaffolds and in-growing cells, as well as between scaffolds and host tissues, need to be investigated further.

#### **4. Conclusion**

316 Tissue Regeneration – From Basic Biology to Clinical Application

standard (Hak, Makino et al. 2006; Jones, Atwood et al. 2009; Reichert, Saifzadeh et al. 2009). In animal models success criteria of implanted MSC and scaffold are restricted mainly to analysis of regenerated bone e.g. by histologiacal findings, CT-scan technology, x-ray or simply by measuring the weight of the created bone as well as by mechanical torsion tests (Zou, Hunter et al. 2011). The fate of implanted scaffold and MSC, in terms of material resorption and MSC engraftment into the host body, is rarely studied (Bernstein, Bornhauser et al. 2009). Since there is no standard in animal models, experiments are being carried out on various models. The rat model is broadly used because of availability. Bio-mechanical properties similar to humans are found in sheep, especially in hip arthroplasty (Korda, Blunn et al. 2008). Usually a fracture is induced as described by Matsumoto et al or Mifune et al (Matsumoto, Kawamoto et al. 2006; Mifune, Matsumoto et al. 2008). In a first step a tibia is fractured. Then a collagen scaffold is inserted containing saline and either BM-MSC or hESC. Then Undale et al compared the bone tissue healing properties of BM-MSC and hESC in rats after an induced fracture. BM-MSC resulted to be more efficient than hESC to bridge and heal a critical bone fracture. Moreover, in this setting hESC tended to produce benign bony tumours compromising the use of these cells

Bone fracture healing or integration into the animal's bone tissue can be demonstrated by follow-up conventional radiology in two weeks intervals. The limbs are both fully extended so that the broken and fractured limb can be compared. In recent studies µCT, a specialized CT for small animal structure, is used. Precise 3D models can be built from the data, allowing a comparison between the original and the newly built bone. Eight weeks after fracturing the animals can be euthanized and the limbs can be analysed histologically or biomechanically. Biomechanical stability of the fracture healing can be assessed by torsional

In summary, MSC from different sources appear as complementation to biomaterial implants. Depending on the tissue source and culture, different patterns of differentiation into bone, cartilage or fibre can be obtained. Depending on the precise situation different sorts of MSCbiocomposites may facilitate wound healing and functional regeneration of bone defects with high long term stability. However, the handling of biomaterial MSC composites is far more complex than conventional methods and oblige to adhere to regulatory standards: Since living cells are worked with, purity, a lack of bacterial contamination and absence of cell transformation has to be proven before clinical application. Conventional methods, that are acellular implants, may be limited because of rigidity and even lack of stability on the long run, but actually, in contrast to MSC biocomposites, they can be well compared regarding their advantages and disadvantages. MSC may differ much more as a matter of treatment, culture conditions and the cells itself need further investigation, experimental and clinical studies to evaluate their true potential at best in comparative studies. But the prospect of individual medicine with the patients' easily extractable and expandable own cells may support future

Future orthopaedic research that may one day provide suitable personalized scaffolds to cover bone defects must integrate vascularisation as well. A balanced attempt to support both bone growth and blood supply must be established to create a stable long lasting graft

load to evaluate normal and abnormal fracture healing (Undale, Fraser et al. 2011).

in clinical settings (Undale, Fraser et al. 2011).

research and applications in regenerative medicine.

**3.6 Future prospects** 

*In vitro* studies indicate that MSC possess a wide spectrum of properties in tissue regeneration as adult progenitor cells or by secreting immunomodulatory and antiinflammatory factors. Still various manufacturing protocols, cultivating media and methods hinder to correlate and interpret scientific findings. Nevertheless MSC are very promising candidates for cell therapy and have moved extremely quickly in the last ten years from the bench to the bedside. For controlled clinical trials there are several obstacles to overcome in order to define a safe and efficacious therapeutic. There is a need to determine factors that may influence the cell quality and consequently the clinical outcome in terms of the tissue source, the isolating, expansion and cultivating conditions. Above that, protocols and *in vitro* and safety animal studies need to be performed in compliance with GMP requirements. To be able to conduct clinical trials on MSC, the manufacturing process has to fulfil several regulatory standards. Advances in clinical application of MSC can be exemplified in the field of orthopaedic bone regeneration. The osteogenic potential of MSC is seen to be of great benefit in bone defect healing. However, only in rare conditions are MSC alone beneficial. The choice of a suitable biomaterial to both carry MSC and provide good primary stability is crucial for clinical applications in hard tissue regeneration. Different sources of MSC that have different differentiation properties can be used. To

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**15** 

*Germany* 

**Oral Tissues as Source for Bone** 

*1University of Applied Sciences Bonn-Rhine-Sieg, Rheinbach* 

*2Oralchirurgische Praxis, Rheinbach* 

**Regeneration in Dental Implantology** 

Dilaware Khan1, Claudia Kleinfeld1, Martin Winter2 and Edda Tobiasch1

One of the most common problems in Regenerative Medicine is the regeneration of damaged bone with the aim of repairing or replacing lost or damaged bone tissue by stimulating the natural regenerative process. Particularly in the fields of orthopedic, plastic, reconstructive, maxillofacial and craniofacial surgery there is need for successful methods to restore bone. From a regenerative point of view two different bone replacement problems can be distinguished: large bone defects and small bone defects. Currently, no perfect system exists for the treatment of large bone defects. Autologous bone material from the hip or the split calvarial graft is the gold standard to repair bone defects, as it has osteoinductive and osteoconductive properties (Tessier, 1982; Tessier et al., 2005a; Laurencin et al., 2006). Unfortunately this method is associated with an additional invasive intervention that leads to an increase risk of infection, pain during recovery, morbidity and frequent long periods of convalescence due to surgical trauma. Besides, only a limited amount of tissue can be obtained and harvested (Younger & Chapman, 1989; Tessier et al., 2005b). Also, the outcome is not always satisfactory after surgical treatment using bone splits (Baltzer et al., 2000; Lietman et al., 2000; Sorger et al., 2001). Heterologous transplants on the other hand, bear the risk of infection and rejection of the donor material. If the required amount of implant material cannot be obtained, another source is bovine-derived xenografts. There is, however, a potential risk for prion infection that cannot be totally avoided. Last not least large bone defect replacement needs nutrient and oxygen supply via blood vessels, so angiogenesis must be considered. This is very different in small bone defects: here angiogenesis is not an issue, but most of the other problems addressed above do play a role here too. This chapter

will focus on small bone defects, especially those linked to dental implants.

The skeletal system is composed of bones that support the body, protect internal organs, and allow movement. Bone itself can be described as a natural composite material that consists of minerals and collagen that are merged in a complex amalgam. It consists mainly of two structures: an organic component as a matrix that contains collagen and a mineral component that is predominantly hydroxyapatite (Rho et al., 1997). The complex mineral substances give hardness to the bone and the softer organic collagen matrix causes visco-

**2. Bone structure and regulation** 

**1. Introduction** 


### **Oral Tissues as Source for Bone Regeneration in Dental Implantology**

Dilaware Khan1, Claudia Kleinfeld1, Martin Winter2 and Edda Tobiasch1 *1University of Applied Sciences Bonn-Rhine-Sieg, Rheinbach 2Oralchirurgische Praxis, Rheinbach Germany* 

#### **1. Introduction**

324 Tissue Regeneration – From Basic Biology to Clinical Application

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but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic

mesenchymal cells during proliferation in response to FGF." Biochem Biophys Res

One of the most common problems in Regenerative Medicine is the regeneration of damaged bone with the aim of repairing or replacing lost or damaged bone tissue by stimulating the natural regenerative process. Particularly in the fields of orthopedic, plastic, reconstructive, maxillofacial and craniofacial surgery there is need for successful methods to restore bone. From a regenerative point of view two different bone replacement problems can be distinguished: large bone defects and small bone defects. Currently, no perfect system exists for the treatment of large bone defects. Autologous bone material from the hip or the split calvarial graft is the gold standard to repair bone defects, as it has osteoinductive and osteoconductive properties (Tessier, 1982; Tessier et al., 2005a; Laurencin et al., 2006). Unfortunately this method is associated with an additional invasive intervention that leads to an increase risk of infection, pain during recovery, morbidity and frequent long periods of convalescence due to surgical trauma. Besides, only a limited amount of tissue can be obtained and harvested (Younger & Chapman, 1989; Tessier et al., 2005b). Also, the outcome is not always satisfactory after surgical treatment using bone splits (Baltzer et al., 2000; Lietman et al., 2000; Sorger et al., 2001). Heterologous transplants on the other hand, bear the risk of infection and rejection of the donor material. If the required amount of implant material cannot be obtained, another source is bovine-derived xenografts. There is, however, a potential risk for prion infection that cannot be totally avoided. Last not least large bone defect replacement needs nutrient and oxygen supply via blood vessels, so angiogenesis must be considered. This is very different in small bone defects: here angiogenesis is not an issue, but most of the other problems addressed above do play a role here too. This chapter will focus on small bone defects, especially those linked to dental implants.

#### **2. Bone structure and regulation**

The skeletal system is composed of bones that support the body, protect internal organs, and allow movement. Bone itself can be described as a natural composite material that consists of minerals and collagen that are merged in a complex amalgam. It consists mainly of two structures: an organic component as a matrix that contains collagen and a mineral component that is predominantly hydroxyapatite (Rho et al., 1997). The complex mineral substances give hardness to the bone and the softer organic collagen matrix causes visco-

Oral Tissues as Source for Bone Regeneration in Dental Implantology 327

lead to weakness of bones that can result in fractures. Bone defects can also occur due to trauma after accidents (Schäffler & Büchler, 2007). In addition, changes in recreational behavior especially in young adults lead to more need for bone replacement. Also, improved conditions of public health, nutrition and medicine have increased the life expectancy that resulted in an enhanced need for dental replacement. Taken together there

Studies revealed that approximately 70 % of all adults between 35 and 44 years lost at least one permanent tooth and by the age of 74 around 26 % of the adults lost all their permanent teeth (National Institutes of Health, 2001). Additionally, 45 % of the adults between 35 and 44 years and 54 % of the seniors between 65 and 74 years suffered from a middle heavy periodontitis, which is connected with a higher risk of tooth-loss (Holtfreter et al., 2010). To overcome these problems dental implants are one of the most common features to realize

In order to guarantee a long and successful osseointegration of dental implants, they should be circumferentially covered with bone. Furthermore, it seems advantageous that the intraosseous part of the fixture is longer than the extraosseous prosthetic part. At least, the length of the implant should not be shorter than the abutment. Nowadays correct implant placement is determined by esthetic and prosthetic aspects, which often cannot be realized when only the residually available bone (restoration-driven implant placement) is being

There are defects of the alveolar bone which occur as a result of trauma, inflammation, resective surgical intervention such as tumor resection, bone loss after periodontal disease or athrophia after tooth loss or agenesis. In the posterior maxilla the phenomenon of pneumatization of the sinus maxillaris increases after tooth loss, which results in a vertical compromized bone level (Fig. 2A). Thus, bone reconstruction before or simultaneously to implant placement is often necessary (Fig. 2B). To do so guided, bone regeneration with autologous material such as bone graft material or other autologous or artificial grafting

Fig. 2. A) Bone Degeneration. After tooth loss, reduced jawbone is as a result of trauma in a 24 years old male. B) Pilot pins *in situ* demonstrate the compromized bone-situation (male, 43 years old). Stable integration of implants is dependent on a thick jawbone. Stem cells could be used to fill the gaps and increase the thickness and induce osseointegration of

B

pilot pin

alveolar bone

is a growing need for bone regeneration and replacement.

**3.1 The need for bone regeneration in dental defects** 

used (Garber et al., 1995). (National Institutes of Health, 2001)

**3. Bone regeneration and replacement** 

oral prosthetic reconstruction.

implants.

A

elasticity and toughness (Hutmacher et al., 2007). Together with cartilage, connective tissue, nerves, blood vessels, and marrow, they constitute the bone.

In the mineralized organic bone matrix, living and dead cells are present. Three types are known to play a role in bone homeostasis: osteoblasts, osteocytes and osteoclasts.

Osteoblasts are derived from MSCs and are cuboidal in shape (Fig. 1). They contain prominent Golgi bodies with a well developed rough endoplasmic reticulum, which is a histological sign for prominent protein production. These cells are located on the endosteal and periosteal bone surfaces. They secrete collagen type I and the noncollagenous proteins of the organic bone matrix. These cells also synthesize the enzyme alkaline phosphatase (ALP) that regulates the mineralization of the bone matrix. Their lifetime is about three months, after which they become metabolically inactive, flattened bone lining cells (Fig. 1). Bone lining cells are found covering inactive bone surfaces where they serve as a barrier for certain ions. The osteocytes originate from metabolically inactive osteoblasts and become trapped within the newly formed bone matrix during bone formation. Osteocytes have reduced synthetic activity compared to osteoblasts but maintain their sensitivity to vitamin D while continuing to participate in calcium regulation. On the other hand osteoclasts are derived from the fusion of monocyte and macrophage lineages (Ash, 1980) (Fig. 1). They are multi-nucleated cells that resorb bone. Osteoblasts regulate the differentiation of osteoclasts and osteocytes, which secrete factors in a feedback loop that play a role in regulating the functions of osteoblasts (Hartmann, 2006) and osteoclasts (Seeman & Delmas, 2006). The formation and resorption of bone is a continuous process that is kept in balance by the regulation of these three types of cells, with emphasis on osteoblasts and osteoclasts.

Fig. 1. Development of Bone Cells. Bone marrow stem cells give rise to hematopoietic stem cells and mesenchymal stem cells. Hematopoietic stem cells give rise to osteoclasts and mesenchymal stem cells are differentiated into osteoblasts together with other cell types. Osteoblasts further develop into bone lining cells and osteocytes.

In some diseases this balance is disrupted, as in osteoporosis, where increased osteoclast activity results in more resorption of bone than formation by osteoblasts. Along with osteoporosis, other medical conditions like bone cancer and osteogenesis imperfecta can lead to weakness of bones that can result in fractures. Bone defects can also occur due to trauma after accidents (Schäffler & Büchler, 2007). In addition, changes in recreational behavior especially in young adults lead to more need for bone replacement. Also, improved conditions of public health, nutrition and medicine have increased the life expectancy that resulted in an enhanced need for dental replacement. Taken together there is a growing need for bone regeneration and replacement.

#### **3. Bone regeneration and replacement**

326 Tissue Regeneration – From Basic Biology to Clinical Application

elasticity and toughness (Hutmacher et al., 2007). Together with cartilage, connective tissue,

In the mineralized organic bone matrix, living and dead cells are present. Three types are

Osteoblasts are derived from MSCs and are cuboidal in shape (Fig. 1). They contain prominent Golgi bodies with a well developed rough endoplasmic reticulum, which is a histological sign for prominent protein production. These cells are located on the endosteal and periosteal bone surfaces. They secrete collagen type I and the noncollagenous proteins of the organic bone matrix. These cells also synthesize the enzyme alkaline phosphatase (ALP) that regulates the mineralization of the bone matrix. Their lifetime is about three months, after which they become metabolically inactive, flattened bone lining cells (Fig. 1). Bone lining cells are found covering inactive bone surfaces where they serve as a barrier for certain ions. The osteocytes originate from metabolically inactive osteoblasts and become trapped within the newly formed bone matrix during bone formation. Osteocytes have reduced synthetic activity compared to osteoblasts but maintain their sensitivity to vitamin D while continuing to participate in calcium regulation. On the other hand osteoclasts are derived from the fusion of monocyte and macrophage lineages (Ash, 1980) (Fig. 1). They are multi-nucleated cells that resorb bone. Osteoblasts regulate the differentiation of osteoclasts and osteocytes, which secrete factors in a feedback loop that play a role in regulating the functions of osteoblasts (Hartmann, 2006) and osteoclasts (Seeman & Delmas, 2006). The formation and resorption of bone is a continuous process that is kept in balance by the regulation of these three types of cells,

Fig. 1. Development of Bone Cells. Bone marrow stem cells give rise to hematopoietic stem cells and mesenchymal stem cells. Hematopoietic stem cells give rise to osteoclasts and mesenchymal stem cells are differentiated into osteoblasts together with other cell types.

mesenchymal stem cell

bone marrow stem cell

pre-osteoblast

bone lining cell

osteoblast

osteocyte

In some diseases this balance is disrupted, as in osteoporosis, where increased osteoclast activity results in more resorption of bone than formation by osteoblasts. Along with osteoporosis, other medical conditions like bone cancer and osteogenesis imperfecta can

Osteoblasts further develop into bone lining cells and osteocytes.

known to play a role in bone homeostasis: osteoblasts, osteocytes and osteoclasts.

nerves, blood vessels, and marrow, they constitute the bone.

with emphasis on osteoblasts and osteoclasts.

hematopoeitic stem cell

pre-osteoclasts

osteoclast

#### **3.1 The need for bone regeneration in dental defects**

Studies revealed that approximately 70 % of all adults between 35 and 44 years lost at least one permanent tooth and by the age of 74 around 26 % of the adults lost all their permanent teeth (National Institutes of Health, 2001). Additionally, 45 % of the adults between 35 and 44 years and 54 % of the seniors between 65 and 74 years suffered from a middle heavy periodontitis, which is connected with a higher risk of tooth-loss (Holtfreter et al., 2010). To overcome these problems dental implants are one of the most common features to realize oral prosthetic reconstruction.

In order to guarantee a long and successful osseointegration of dental implants, they should be circumferentially covered with bone. Furthermore, it seems advantageous that the intraosseous part of the fixture is longer than the extraosseous prosthetic part. At least, the length of the implant should not be shorter than the abutment. Nowadays correct implant placement is determined by esthetic and prosthetic aspects, which often cannot be realized when only the residually available bone (restoration-driven implant placement) is being used (Garber et al., 1995). (National Institutes of Health, 2001)

There are defects of the alveolar bone which occur as a result of trauma, inflammation, resective surgical intervention such as tumor resection, bone loss after periodontal disease or athrophia after tooth loss or agenesis. In the posterior maxilla the phenomenon of pneumatization of the sinus maxillaris increases after tooth loss, which results in a vertical compromized bone level (Fig. 2A). Thus, bone reconstruction before or simultaneously to implant placement is often necessary (Fig. 2B). To do so guided, bone regeneration with autologous material such as bone graft material or other autologous or artificial grafting

Fig. 2. A) Bone Degeneration. After tooth loss, reduced jawbone is as a result of trauma in a 24 years old male. B) Pilot pins *in situ* demonstrate the compromized bone-situation (male, 43 years old). Stable integration of implants is dependent on a thick jawbone. Stem cells could be used to fill the gaps and increase the thickness and induce osseointegration of implants.

Oral Tissues as Source for Bone Regeneration in Dental Implantology 329

contaminating non-mesenchymal cells such as macrophages or fibroblasts. The purity of isolated MSCs can be investigated by using the surface markers: CD73, CD90, and CD105 (should be expressed) and CD14, CD34 and CD45 (should not be expressed). These markers serve next to the adherence to plastic as a second feature for the identification and characterization of MSCs as suggested by the 'International Society for Cellular Therapy'

Another group of adult stem cells that has attracted attention are the ectomesenchymal stem cells derived from oral tissues. This stem cell group includes the dental pulp stem cells (DPSCs) and stem cells of human exfoliated deciduous teeth (SHEDs), both deriving from the pulpa, dental periodontal ligament stem cells (DPLSCs), dental follicle cells (DFCs), and stem cells from the apical papilla (SCAPs) (see Fig. 3). These cell types have the potential to differentiate into cells of all dental tissue types and bone as well. They share common

> enamel dentin dental pulp

> > gum

periodontal membrane

cement

jawbone

Fig. 3. Stem Cell Types in Tooth. From tooth different stem cell types namely, dental pulp stem cells (DPSCs), stem cells of human exfoliated deciduous teeth (SHEDs), dental periodontal ligament stem cells (DPLSCs), dental enamel derived stem cells (DESCs), stem cells from the apical papilla (SCAPs) and dental follicle cells (DFCs) can be obtained.

In comparison to other dental sources, dental follicle cells (DFCs) can be easily obtained in high amounts from young and healthy donors, since they are isolated from tooth extraction material collected during surgical removal of wisdom teeth. As these cells are derived from young donors, long telomeres extend their lifespan which makes them interesting cells for Regenerative Medicine (Shay & Wright, 2010). The dental follicle develops from ectomesenchyme. It surrounds the developing tooth germ before eruption (Ten Cate, 1997; Wise, 2002). During embryonic development, the ectomesenchyme is partly derived from migrating cells of the cranial neural crest. Therefore, the cells derivative from dental follicle differ from mesenchymal stem cells isolated from other sources (Chung et al., 2004; Slootweg, 2009). Due to having the more ectodermal character, these cells can have a differentiation potential diverse from MSCs. As expected these cells can differentiate into hard tissue such as the periodontal, cementoblastic, chondrocytic, and osteogenic lineages. ATSCs and DFCs, both show osteogenic differentiation potential and are thus suitable candidates for the use in bone regeneration for stable osseointegration of dental implants. As these cells are obtained from healthy individuals, they might be used as an autograft in

(Dominici et al., 2006).

phenotypic markers of MSCs (Alipur et al., 2010).

**DPSCs / SHEDs** 

**DPLSCs** 

**SCAPs** 

**DESCs** 

procedures are methods of choice. Nevertheless, there exist many unsolved problems such as a e.g. higher morbidity in conjunction with the second wound of the donor site.

Therefore the use of stem cells (SCs) as source material for bone regeneration could represent an interesting approach for dental implantology.

#### **3.2 Stem cells for bone regeneration**

A modern strategy in Regenerative Medicine is the approach to combine living cells and scaffold material to establish a biological alternative for the diseased organ or tissue that can restore the functions. (Sittinger et al., 1996; Vacanti & Langer, 1999; Khademhosseini et al., 2009). Some degradable polymers, ceramics, or a combination of both can provide desirable mechanical and osteoconductive properties as basic scaffold material for bone replacement (Zippel et al., 2010b). Different factors should be considered for the use of such a biomaterial scaffold. It should imitate the three dimensional environment of the extracellular matrix, it should provide stability until replaced by regrown bone tissue and serve as an extended surface area for migration, adhesion, and differentiation of cells to encourage the growth of new tissue (Schultz et al., 2000; Ringe et al., 2002; Moroni et al., 2008).

The proliferating cells cover the scaffold and can grow into three dimensional tissue within. They are also an important factor for forming new tissue through extracellular matrix synthesis (Bonassar & Vacanti, 1998). Due to the development of new blood vessels towards, and to some extent onto, the new tissue, the scaffold begins to degenerate from the outside and is reconstituted by new natural bone tissue. As tissue related cell types cannot always be obtained in an adequate number or quality, SCs are a useful alternative for tissue regeneration.

Stem cells are the precursors of all cells and are involved in the repair system of the body. They are defined by three characteristics: self sustainability, self renewal and the potential of differentiation into different tissue types. For example adipocytes, astrocytes, chondroblasts, or osteoblasts come from mesenchymal stem cells (MSCs) (Pittenger et al., 1999; Pansky et al., 2007). In several publications, it has been suggested that MSCs can differentiate towards lineages that are naturally derived from the endoderm (Zuk et al., 2002; Tobiasch, 2009). Thus, increasing their potential because of these properties, the use of SCs to heal or rebuild damaged organs may provide an approach in future Regenerative Medicine (Zippel et al., 2010a).

SCs have been isolated from embryonic sources and well developed tissues of adult organism such as bone marrow, skin, dental pulp and adipose tissue (Kern et al., 2006). In addition two other sources for SCs have been discovered: cancer stem cells and induced pluripotent stem cells (iPS) (Takahashi et al., 2007; Aoi et al., 2008). Since the higher potency of embryonic stem cells and iPS compared to adult stem cells goes together with a higher risk of tumor formation, and embryonic stem cells are ethically problematic. Therefore, adult stem cells present themselves as an interesting cell source for bone replacement.

Adult stem cells can be divided into two main subpopulations: hematopoietic and mesenchymal stem cells (MSCs). Hematopoietic stem cells derived from bone marrow have been investigated best and could be a source for osteoclasts (Ash, 1980) (see Fig. 1). MSCs have been found in umbilical cord blood, bone marrow, and adipose tissue among others (Zuk et al., 2002). Generally, the isolation of MSCs is accomplished by plastic adherence resulting in colonies that are heterogeneous in size and morphology might contain

procedures are methods of choice. Nevertheless, there exist many unsolved problems such

Therefore the use of stem cells (SCs) as source material for bone regeneration could

A modern strategy in Regenerative Medicine is the approach to combine living cells and scaffold material to establish a biological alternative for the diseased organ or tissue that can restore the functions. (Sittinger et al., 1996; Vacanti & Langer, 1999; Khademhosseini et al., 2009). Some degradable polymers, ceramics, or a combination of both can provide desirable mechanical and osteoconductive properties as basic scaffold material for bone replacement (Zippel et al., 2010b). Different factors should be considered for the use of such a biomaterial scaffold. It should imitate the three dimensional environment of the extracellular matrix, it should provide stability until replaced by regrown bone tissue and serve as an extended surface area for migration, adhesion, and differentiation of cells to encourage the growth of

The proliferating cells cover the scaffold and can grow into three dimensional tissue within. They are also an important factor for forming new tissue through extracellular matrix synthesis (Bonassar & Vacanti, 1998). Due to the development of new blood vessels towards, and to some extent onto, the new tissue, the scaffold begins to degenerate from the outside and is reconstituted by new natural bone tissue. As tissue related cell types cannot always be obtained in an adequate number or quality, SCs are a useful alternative for tissue regeneration. Stem cells are the precursors of all cells and are involved in the repair system of the body. They are defined by three characteristics: self sustainability, self renewal and the potential of differentiation into different tissue types. For example adipocytes, astrocytes, chondroblasts, or osteoblasts come from mesenchymal stem cells (MSCs) (Pittenger et al., 1999; Pansky et al., 2007). In several publications, it has been suggested that MSCs can differentiate towards lineages that are naturally derived from the endoderm (Zuk et al., 2002; Tobiasch, 2009). Thus, increasing their potential because of these properties, the use of SCs to heal or rebuild damaged

organs may provide an approach in future Regenerative Medicine (Zippel et al., 2010a).

themselves as an interesting cell source for bone replacement.

SCs have been isolated from embryonic sources and well developed tissues of adult organism such as bone marrow, skin, dental pulp and adipose tissue (Kern et al., 2006). In addition two other sources for SCs have been discovered: cancer stem cells and induced pluripotent stem cells (iPS) (Takahashi et al., 2007; Aoi et al., 2008). Since the higher potency of embryonic stem cells and iPS compared to adult stem cells goes together with a higher risk of tumor formation, and embryonic stem cells are ethically problematic. Therefore, adult stem cells present

Adult stem cells can be divided into two main subpopulations: hematopoietic and mesenchymal stem cells (MSCs). Hematopoietic stem cells derived from bone marrow have been investigated best and could be a source for osteoclasts (Ash, 1980) (see Fig. 1). MSCs have been found in umbilical cord blood, bone marrow, and adipose tissue among others (Zuk et al., 2002). Generally, the isolation of MSCs is accomplished by plastic adherence resulting in colonies that are heterogeneous in size and morphology might contain

as a e.g. higher morbidity in conjunction with the second wound of the donor site.

represent an interesting approach for dental implantology.

new tissue (Schultz et al., 2000; Ringe et al., 2002; Moroni et al., 2008).

**3.2 Stem cells for bone regeneration** 

contaminating non-mesenchymal cells such as macrophages or fibroblasts. The purity of isolated MSCs can be investigated by using the surface markers: CD73, CD90, and CD105 (should be expressed) and CD14, CD34 and CD45 (should not be expressed). These markers serve next to the adherence to plastic as a second feature for the identification and characterization of MSCs as suggested by the 'International Society for Cellular Therapy' (Dominici et al., 2006).

Another group of adult stem cells that has attracted attention are the ectomesenchymal stem cells derived from oral tissues. This stem cell group includes the dental pulp stem cells (DPSCs) and stem cells of human exfoliated deciduous teeth (SHEDs), both deriving from the pulpa, dental periodontal ligament stem cells (DPLSCs), dental follicle cells (DFCs), and stem cells from the apical papilla (SCAPs) (see Fig. 3). These cell types have the potential to differentiate into cells of all dental tissue types and bone as well. They share common phenotypic markers of MSCs (Alipur et al., 2010).

Fig. 3. Stem Cell Types in Tooth. From tooth different stem cell types namely, dental pulp stem cells (DPSCs), stem cells of human exfoliated deciduous teeth (SHEDs), dental periodontal ligament stem cells (DPLSCs), dental enamel derived stem cells (DESCs), stem cells from the apical papilla (SCAPs) and dental follicle cells (DFCs) can be obtained.

In comparison to other dental sources, dental follicle cells (DFCs) can be easily obtained in high amounts from young and healthy donors, since they are isolated from tooth extraction material collected during surgical removal of wisdom teeth. As these cells are derived from young donors, long telomeres extend their lifespan which makes them interesting cells for Regenerative Medicine (Shay & Wright, 2010). The dental follicle develops from ectomesenchyme. It surrounds the developing tooth germ before eruption (Ten Cate, 1997; Wise, 2002). During embryonic development, the ectomesenchyme is partly derived from migrating cells of the cranial neural crest. Therefore, the cells derivative from dental follicle differ from mesenchymal stem cells isolated from other sources (Chung et al., 2004; Slootweg, 2009). Due to having the more ectodermal character, these cells can have a differentiation potential diverse from MSCs. As expected these cells can differentiate into hard tissue such as the periodontal, cementoblastic, chondrocytic, and osteogenic lineages.

ATSCs and DFCs, both show osteogenic differentiation potential and are thus suitable candidates for the use in bone regeneration for stable osseointegration of dental implants. As these cells are obtained from healthy individuals, they might be used as an autograft in

Oral Tissues as Source for Bone Regeneration in Dental Implantology 331

For the isolation of ectomesenchymal stem cells, dental follicles were collected from human third molars before tooth eruption after surgical removal. The dental follicles were washed three times with 1 x PBS. Afterwards, the dental follicles were separated from the mineralized tooth and minced with a scalpel under sterile conditions. The tissue was digested in Collagenase (0.1 U / mL) and Dispase (0.8 U / mL) for 2 h at 37 °C in humidified atmosphere with 5 % CO2. The cells were passed through a 100 µm strainer to obtain singlecell suspensions and seeded in 10 cm dishes in stem cell medium (SCM) that consisted of DMEM supplemented with 10 % FCS, 2 mM L-glutamine, 100 units / mL penicillin, 100 mg / mL streptomycin and 1 % amphotericin and cultured at 37 °C in a humidified atmosphere with 5 % CO2. After 24 hours, non-adherent cells were removed by washing with 1 x PBS. The medium was changed and the plastic adherent cell fraction was cultured until 80 %

The bone chip particles were collected with a bone filter integrated into a surgical suction pipe during the implant-bed preparation to isolate primary cells. For the isolation of bone chip derived cells (BCDCs), the same procedure as described above for DFCs was

Human adipose tissue derived stem cells (ATSCs) were isolated from lipoaspirate obtained from plastic surgery. The isolation technique used during surgery was the tumescent liposuction technique. Using this particular technique, diluted epinephrine and lidocaine is infiltrated into the body fat to be removed, which leads to swelling and firmness of the targeted region, providing more accuracy during the liposuction procedure. The protocol was adjusted and modified to the procedure described by Zuk and colleagues (Zuk et al., 2001). The obtained lipoaspirate was augmented with PBS in a 1:2 ratio. After incubation for 30 minutes at room temperature (RT), two phases, a lower aqueous and upper fat phase of

The lower phase was centrifuged at 200 x g for 10 minutes at RT. The resulting pellets, comprising the cells, were pooled and washed with 1 x PBS. Remaining erythrocytes were removed by applying 10 mL erythrolysis buffer for 10 minutes at RT. After another centrifugation step, under the same conditions as mentioned before, the cells were cultured

The upper phase comprising the fat tissue was augmented with 10 mg / mL type I collagenase in 1 x PBS and incubated for 45 minutes at 37 °C with agitation. The following steps for the treatment of the upper phase were according to the treatment of the lower phase. Cells of both phases were incubated at 37 °C with 5 % CO2 in a humidified atmosphere. ATSCs were isolated due to their adherence to plastic and purified by washing

with 1 x PBS after 24 hours, to remove undesired non-adherent cells.

**4. Research methods** 

confluent for further use.

the lipoaspirate were obtained.

in 60 cm2 culture plates in SCM medium.

followed.

**4.1 Isolation of primary cells for osteo-differentiation 4.1.1 Isolation of cells from tooth extraction material** 

**4.1.2 Isolation of adipose tissue derived stem cells** 

the future. The transplantation not only of autologous but also of allogenic sources could provide benefits in comparison to other common procedures in bone regeneration. As MSCs have low immune characteristics, they appear to be suitable for allogenic therapeutic purposes, without activating the immune response in immunocompetent patients (Jung et al., 2009). In different studies the use of MSCs has been investigated to replace lost or damaged bone (Schaefer et al., 2000; Ringe et al., 2002). After tooth loss, jawbone degenerates and stable integration of dental implant needs a thick jawbone. To overcome this problem there are two different alternatives that can be considered for using SCs in dental implants. The reconstruction after bone defects with SCs to achieve a sufficient bone thickness to insert the implants and the loading of an implant or artificial tooth-root with SCs with the aim to realize a sufficient integration in the bone.

SCs have the capability to re-establish cell function, reverse cellular damage, and heal damaged tissue (Conrad and Huss, 2005). SCs could also be a source to regenerate human teeth in the future, as these cells have been successfully used to regenerate living teeth in rabbit extraction sockets (Hung et al., 2011). In some mammals like rodents, rabbits, prairie dogs, and pikas, the teeth can grow throughout life because in these mammals as the pulp cavity remains open permanently. While on the other hand in humans tooth cannot grow continuously as pulp cavity closes when the teeth are fully grown. Therefore this study cannot be adapted easily for the regeneration of teeth or teeth related tissues in humans but it at least provides interesting basic results that can be helpful for use of SCs in dental tissues.

#### **3.3 Bone chips for the stabilization of dental implants**

Another approach next to scaffold loaded with stem cells to overcome the problem of unstable dental implants is the use of particulated non-vascularized bone autografts. The particles can be collected during the implant-bed preparation in the process of drilling the hole for the implant into the bone. An advantage of the use of these bone chips is that this material can be expected to facilitate bone regeneration. However, contradictory statements were made about the quality of this material such as if it contains living cells. In addition, it is not clear how to disinfect the bone chips, which are contaminated with bacteria of the oral cavity due to the sampling process. To address these questions bone chips were collected from two different regions of bone: carticular bone and spongy bone (see Fig. 4).

Fig. 4. Schematic Structure of Lower Jaw. Bone is composed of two tissue types mainly: spongious and carticular bone. Bone chips obtained during dental surgery for implant-bed preparation is derived from both bone tissue types.

### **4. Research methods**

330 Tissue Regeneration – From Basic Biology to Clinical Application

the future. The transplantation not only of autologous but also of allogenic sources could provide benefits in comparison to other common procedures in bone regeneration. As MSCs have low immune characteristics, they appear to be suitable for allogenic therapeutic purposes, without activating the immune response in immunocompetent patients (Jung et al., 2009). In different studies the use of MSCs has been investigated to replace lost or damaged bone (Schaefer et al., 2000; Ringe et al., 2002). After tooth loss, jawbone degenerates and stable integration of dental implant needs a thick jawbone. To overcome this problem there are two different alternatives that can be considered for using SCs in dental implants. The reconstruction after bone defects with SCs to achieve a sufficient bone thickness to insert the implants and the loading of an implant or artificial tooth-root with

SCs have the capability to re-establish cell function, reverse cellular damage, and heal damaged tissue (Conrad and Huss, 2005). SCs could also be a source to regenerate human teeth in the future, as these cells have been successfully used to regenerate living teeth in rabbit extraction sockets (Hung et al., 2011). In some mammals like rodents, rabbits, prairie dogs, and pikas, the teeth can grow throughout life because in these mammals as the pulp cavity remains open permanently. While on the other hand in humans tooth cannot grow continuously as pulp cavity closes when the teeth are fully grown. Therefore this study cannot be adapted easily for the regeneration of teeth or teeth related tissues in humans but it at least provides

Another approach next to scaffold loaded with stem cells to overcome the problem of unstable dental implants is the use of particulated non-vascularized bone autografts. The particles can be collected during the implant-bed preparation in the process of drilling the hole for the implant into the bone. An advantage of the use of these bone chips is that this material can be expected to facilitate bone regeneration. However, contradictory statements were made about the quality of this material such as if it contains living cells. In addition, it is not clear how to disinfect the bone chips, which are contaminated with bacteria of the oral cavity due to the sampling process. To address these questions bone chips were collected

> spongy bone

bone chips

carticular bone

SCs with the aim to realize a sufficient integration in the bone.

interesting basic results that can be helpful for use of SCs in dental tissues.

from two different regions of bone: carticular bone and spongy bone (see Fig. 4).

Fig. 4. Schematic Structure of Lower Jaw. Bone is composed of two tissue types mainly: spongious and carticular bone. Bone chips obtained during dental surgery for implant-bed

**3.3 Bone chips for the stabilization of dental implants** 

preparation is derived from both bone tissue types.

#### **4.1 Isolation of primary cells for osteo-differentiation**

#### **4.1.1 Isolation of cells from tooth extraction material**

For the isolation of ectomesenchymal stem cells, dental follicles were collected from human third molars before tooth eruption after surgical removal. The dental follicles were washed three times with 1 x PBS. Afterwards, the dental follicles were separated from the mineralized tooth and minced with a scalpel under sterile conditions. The tissue was digested in Collagenase (0.1 U / mL) and Dispase (0.8 U / mL) for 2 h at 37 °C in humidified atmosphere with 5 % CO2. The cells were passed through a 100 µm strainer to obtain singlecell suspensions and seeded in 10 cm dishes in stem cell medium (SCM) that consisted of DMEM supplemented with 10 % FCS, 2 mM L-glutamine, 100 units / mL penicillin, 100 mg / mL streptomycin and 1 % amphotericin and cultured at 37 °C in a humidified atmosphere with 5 % CO2. After 24 hours, non-adherent cells were removed by washing with 1 x PBS. The medium was changed and the plastic adherent cell fraction was cultured until 80 % confluent for further use.

The bone chip particles were collected with a bone filter integrated into a surgical suction pipe during the implant-bed preparation to isolate primary cells. For the isolation of bone chip derived cells (BCDCs), the same procedure as described above for DFCs was followed.

#### **4.1.2 Isolation of adipose tissue derived stem cells**

Human adipose tissue derived stem cells (ATSCs) were isolated from lipoaspirate obtained from plastic surgery. The isolation technique used during surgery was the tumescent liposuction technique. Using this particular technique, diluted epinephrine and lidocaine is infiltrated into the body fat to be removed, which leads to swelling and firmness of the targeted region, providing more accuracy during the liposuction procedure. The protocol was adjusted and modified to the procedure described by Zuk and colleagues (Zuk et al., 2001). The obtained lipoaspirate was augmented with PBS in a 1:2 ratio. After incubation for 30 minutes at room temperature (RT), two phases, a lower aqueous and upper fat phase of the lipoaspirate were obtained.

The lower phase was centrifuged at 200 x g for 10 minutes at RT. The resulting pellets, comprising the cells, were pooled and washed with 1 x PBS. Remaining erythrocytes were removed by applying 10 mL erythrolysis buffer for 10 minutes at RT. After another centrifugation step, under the same conditions as mentioned before, the cells were cultured in 60 cm2 culture plates in SCM medium.

The upper phase comprising the fat tissue was augmented with 10 mg / mL type I collagenase in 1 x PBS and incubated for 45 minutes at 37 °C with agitation. The following steps for the treatment of the upper phase were according to the treatment of the lower phase. Cells of both phases were incubated at 37 °C with 5 % CO2 in a humidified atmosphere. ATSCs were isolated due to their adherence to plastic and purified by washing with 1 x PBS after 24 hours, to remove undesired non-adherent cells.

Oral Tissues as Source for Bone Regeneration in Dental Implantology 333

these cells isolate them from various tissue sources by following different protocols and characterizing these cells by different markers. Therefore, to set a standard, the minimal criteria for the definition of human MSCs were suggested by the 'Mesenchymal and Tissue Stem Cell Committee of The International Society for Cellular Therapy' (Dominici et al., 2006). The multipotent character of the isolated adipose tissue derived stem cells, ectomesenchymal dental follicle cells and bone chip derived cells was tested according to these criteria. MSCs were isolated from human adult adipose tissue of different aged female donors. DFCs were isolated from dental follicles and BCDCs from the bone chips collected during implant-bed preparation of male and female donors. The enrichment of specific stem cells was achieved due to their property of plastic adherence that is the first criterion for the testing of aMSCs character (Dominici et al., 2006). Isolated mesenchymal and ectomesenchymal cells of all donors showed a morphology similar to fibroblasts,

According to the above mentioned criteria the isolated cells should express the stem cell specific surface markers CD73, CD90, and CD105, and should not express CD14, CD34, and CD45. All isolated SC types expressed the expected markers (CD73, CD90 and CD105) as assessed by RT-PCR. The mesenchymal character of ATSCs and DFCs was also confirmed using FACS analysis for the presence of CD90, CD105, and in addition CD44. Furthermore, the cell types ATSCs and DFCs did not show the expression of leukocyte marker CD45 and macrophage marker CD14. ATSCs were positive and DFCs were negative for CD34. The presence of the expression of CD34 on ATSCs is controversial discussed. Some studies confirm the absence of CD34 expression on ATSCs (Zuk et al., 2002; Lee et al., 2004; Wagner et al., 2005) while other investigations showed ATSCs expressing CD34 (Mitchell et al., 2006; Yoshimura et al., 2006; De Francesco et al., 2009). These differences could be due to different stem cell isolation protocols, passage number or a different gating strategy during FACS

analysis. In this study a subpopulation of ATSCs was stained positive for CD34.

Another typical MSCs character is the multilineage differentiation potential towards various lineages such as adipocytes, chondroblasts and osteoblasts. ATSCs showed a strong adipogenic differentiation potential whereas DFCs and BCDCs could not differentiate towards adipocytes. However, Kémoun and colleagues reported DFCs to differentiate towards the adipogenic lineage (Kémoun et al., 2007). The differences during isolation and precipitation in cell population might be possible reasons for this discrepancy. Also, DFCs can be different in their potency because these cells are derived from ectomesenchyme that

According to all the findings mentioned above, the isolated ATSCs can be considered to belong to the population of multipotent MSCs, whereas the DFCs and BCDCs have a limited differentiation potential. Haddouti and colleagues showed that DFCs have a strong commitment towards the osteogenic lineage and show a more quantitative osteogenic differentiation (Haddouti et al., 2009). Thus, DFCs and BCDCs seem to be more committed

Taken together all these stem cell types are good candidates for bone regeneration. But material from the oral cavity for isolation of primary cells such as DFCs and BCDCs cannot be obtained without microbial contamination. The question arises if this is a draw back on

which is typical for these stem cells (Yoshimura et al., 2006).

is more committed toward hard tissue as tooth enamel.

towards osteogenic lineage.

the use of these stem cells.

#### **4.2 Fluorescence activated cell sorting**

The percentages of ATSCs or DFCs positive for the mesenchymal stem cell markers CD44, CD90 and CD105 and negative for CD14, CD45 and CD34 were measured using FACS analysis. The stem cells were trypsinized, centrifuged at 200 x g for 5 min and counted. 1 x 106 cells were resuspended in 1 mL 0.1 % PBSB and passed through a 100 μM cell strainer to obtain a single cell solution. 100 μL of the cell solution (100.000 cells) were incubated for 20 min in the dark with either the isotype control or the antibodies. Cells were washed with 2 mL 0.1 % (w / v) PBSB, centrifuged at 200 x g for 5 min and resuspended in 1 mL 0.1 % (w / v) PBSB. The cytometer settings and cell gates were adjusted to the isotype control, followed by measurement of the stem cell markers using the same conditions.

#### **4.3 Adipogenic differentiation**

For adipogenic induction, the isolated cells were seeded in a density of 2.8 x 103 cells / cm2 in SCM. After one day, the medium was changed to adipogenic differentiation medium (AM), containing 1 μM dexamethasone, 1 μM insulin and 200 μM indomethacin. The cells were grown in AM for four weeks at 37 °C with 5 % CO2 under humidified conditions. The AM was changed once a week. After four weeks, adipogenic differentiation was visualized with Oil Red O after fixing cells for 90 min with formalin (4 %) at 37 °C.

#### **4.4 Osteogenic differentiation**

The isolated cells were seeded in a density of 1.3 x 103 cells / cm2 in 6 cm2 and 12 well plates for osteogenic differentiation. After one day SCM was replaced with osteogenic medium (OM) containing dexamethasone, ascorbic acid and β-glycerophosphate. ATSCs were grown in OM for 4 weeks at 37 °C with 5 % CO2 under humidified conditions. The OM medium was changed once a week. After four weeks, osteogenic differentiation was visualized by staining with Alizarin Red S after fixing cells for 5 min with formalin (4 %) at 37 °C.

#### **4.5 Microbiological testing**

Directly after surgery the obtained dental follicles were transferred into cold sodium chloride (0.9 % w / v) for determining possible microbial contaminations. The samples were kept cold until processing.

The samples were rolled over the surface of Columbia blood agar (CBA) and fastidious anaerobe agar (FAA) plates to isolate microorganisms. In addition the transport solution was put onto CBA and FAA plates. Under aerobic and anaerobic conditions the incubations were conducted over night at 37 °C. The Gas PakTM100-system was used for the incubation under anaerobic conditions. Single colonies were picked and isolated with respect to their morphological differences. Gram stainings, catalase- and oxidase-tests were used for the first characterization. API test strips were used to determine the exact bacteria species.

#### **5. Comparison of stem cell sources**

#### **5.1 The characterization of ATSCs, DFCs and BCDCs for bone regeneration**

The high plasticity of mesenchymal stem cells has resulted in an increased interest for their use in a variety of cellular therapies. However, different laboratories working with

The percentages of ATSCs or DFCs positive for the mesenchymal stem cell markers CD44, CD90 and CD105 and negative for CD14, CD45 and CD34 were measured using FACS analysis. The stem cells were trypsinized, centrifuged at 200 x g for 5 min and counted. 1 x 106 cells were resuspended in 1 mL 0.1 % PBSB and passed through a 100 μM cell strainer to obtain a single cell solution. 100 μL of the cell solution (100.000 cells) were incubated for 20 min in the dark with either the isotype control or the antibodies. Cells were washed with 2 mL 0.1 % (w / v) PBSB, centrifuged at 200 x g for 5 min and resuspended in 1 mL 0.1 % (w / v) PBSB. The cytometer settings and cell gates were adjusted to the isotype control, followed

For adipogenic induction, the isolated cells were seeded in a density of 2.8 x 103 cells / cm2 in SCM. After one day, the medium was changed to adipogenic differentiation medium (AM), containing 1 μM dexamethasone, 1 μM insulin and 200 μM indomethacin. The cells were grown in AM for four weeks at 37 °C with 5 % CO2 under humidified conditions. The AM was changed once a week. After four weeks, adipogenic differentiation was visualized

The isolated cells were seeded in a density of 1.3 x 103 cells / cm2 in 6 cm2 and 12 well plates for osteogenic differentiation. After one day SCM was replaced with osteogenic medium (OM) containing dexamethasone, ascorbic acid and β-glycerophosphate. ATSCs were grown in OM for 4 weeks at 37 °C with 5 % CO2 under humidified conditions. The OM medium was changed once a week. After four weeks, osteogenic differentiation was visualized by

Directly after surgery the obtained dental follicles were transferred into cold sodium chloride (0.9 % w / v) for determining possible microbial contaminations. The samples were

The samples were rolled over the surface of Columbia blood agar (CBA) and fastidious anaerobe agar (FAA) plates to isolate microorganisms. In addition the transport solution was put onto CBA and FAA plates. Under aerobic and anaerobic conditions the incubations were conducted over night at 37 °C. The Gas PakTM100-system was used for the incubation under anaerobic conditions. Single colonies were picked and isolated with respect to their morphological differences. Gram stainings, catalase- and oxidase-tests were used for the first characterization. API test strips were used to determine the exact bacteria species.

The high plasticity of mesenchymal stem cells has resulted in an increased interest for their use in a variety of cellular therapies. However, different laboratories working with

staining with Alizarin Red S after fixing cells for 5 min with formalin (4 %) at 37 °C.

**5.1 The characterization of ATSCs, DFCs and BCDCs for bone regeneration** 

by measurement of the stem cell markers using the same conditions.

with Oil Red O after fixing cells for 90 min with formalin (4 %) at 37 °C.

**4.2 Fluorescence activated cell sorting** 

**4.3 Adipogenic differentiation** 

**4.4 Osteogenic differentiation** 

**4.5 Microbiological testing** 

kept cold until processing.

**5. Comparison of stem cell sources** 

these cells isolate them from various tissue sources by following different protocols and characterizing these cells by different markers. Therefore, to set a standard, the minimal criteria for the definition of human MSCs were suggested by the 'Mesenchymal and Tissue Stem Cell Committee of The International Society for Cellular Therapy' (Dominici et al., 2006). The multipotent character of the isolated adipose tissue derived stem cells, ectomesenchymal dental follicle cells and bone chip derived cells was tested according to these criteria. MSCs were isolated from human adult adipose tissue of different aged female donors. DFCs were isolated from dental follicles and BCDCs from the bone chips collected during implant-bed preparation of male and female donors. The enrichment of specific stem cells was achieved due to their property of plastic adherence that is the first criterion for the testing of aMSCs character (Dominici et al., 2006). Isolated mesenchymal and ectomesenchymal cells of all donors showed a morphology similar to fibroblasts, which is typical for these stem cells (Yoshimura et al., 2006).

According to the above mentioned criteria the isolated cells should express the stem cell specific surface markers CD73, CD90, and CD105, and should not express CD14, CD34, and CD45. All isolated SC types expressed the expected markers (CD73, CD90 and CD105) as assessed by RT-PCR. The mesenchymal character of ATSCs and DFCs was also confirmed using FACS analysis for the presence of CD90, CD105, and in addition CD44. Furthermore, the cell types ATSCs and DFCs did not show the expression of leukocyte marker CD45 and macrophage marker CD14. ATSCs were positive and DFCs were negative for CD34. The presence of the expression of CD34 on ATSCs is controversial discussed. Some studies confirm the absence of CD34 expression on ATSCs (Zuk et al., 2002; Lee et al., 2004; Wagner et al., 2005) while other investigations showed ATSCs expressing CD34 (Mitchell et al., 2006; Yoshimura et al., 2006; De Francesco et al., 2009). These differences could be due to different stem cell isolation protocols, passage number or a different gating strategy during FACS analysis. In this study a subpopulation of ATSCs was stained positive for CD34.

Another typical MSCs character is the multilineage differentiation potential towards various lineages such as adipocytes, chondroblasts and osteoblasts. ATSCs showed a strong adipogenic differentiation potential whereas DFCs and BCDCs could not differentiate towards adipocytes. However, Kémoun and colleagues reported DFCs to differentiate towards the adipogenic lineage (Kémoun et al., 2007). The differences during isolation and precipitation in cell population might be possible reasons for this discrepancy. Also, DFCs can be different in their potency because these cells are derived from ectomesenchyme that is more committed toward hard tissue as tooth enamel.

According to all the findings mentioned above, the isolated ATSCs can be considered to belong to the population of multipotent MSCs, whereas the DFCs and BCDCs have a limited differentiation potential. Haddouti and colleagues showed that DFCs have a strong commitment towards the osteogenic lineage and show a more quantitative osteogenic differentiation (Haddouti et al., 2009). Thus, DFCs and BCDCs seem to be more committed towards osteogenic lineage.

Taken together all these stem cell types are good candidates for bone regeneration. But material from the oral cavity for isolation of primary cells such as DFCs and BCDCs cannot be obtained without microbial contamination. The question arises if this is a draw back on the use of these stem cells.

Oral Tissues as Source for Bone Regeneration in Dental Implantology 335

ATSCs Human adipose tissue derived mesenchymal stem cells

**8. List of abbreviation** 

ALP Alkaline phosphatase AM Adipogenic medium

BCDCs Bone chip derived cells BMP2 Bone morphogenetic protien 2

DESCs Dental enamel derived stem cells

DMEM Dulbecco's modified Eagle medium DPLSCs Dental periodontal ligament stem cells

FACs Fluorescence activated cell sorting

Runx2 Runt-related transcription factor 2 SCAPs Stem cells from the apical papilla

PPARγ Peroxisome proliferator-activated receptor gamma

RT-PCR Reverse transcriptase polymerase chain reaction

SHEDs Stem cells of human exfoliated deciduous teeth

Alipur, R., Sadeghi, F., Hashemi-Beni, B., Zarkesh-Esfahani, S.H., Heydari, F., Mousavi, S.B.,

Adib, M., Narimani, M., & Esmaeili, N. (2010). Phenotypic characterizations and

iPS Induced pluripotent stem cells IGF-1 Insulin-like growth factor 1

°C Degree centigrade CBA Columbia blood agar CD14 Cluster of differentiation 14 CD34 Cluster of differentiation 34 CD45 Cluster of differentiation 45 CD73 Cluster of differentiation 73 CD90 Cluster of differentiation 90 CD105 Cluster of differentiation 105

cm Centimeter CO2 Carbon dioxide

DFCs Dental follicle cells

FCS Fetal calf serum

LPL Lipoprotein lipase

OM Osteogenic medium PBS Phosphate buffer saline

RT Room temperature

SCM Stem cell medium

w / v Weight per volume x g Relative centrifugal force

SCs Stem cells

**9. References** 

mL Milliliter mM Millimolar µL Microliter

DPSCs Dental pulp stem cells ECSs Embryonic stem cells FAA Fastidious anaerobe agar

#### **5.2 Microbial load of the oral tissue material**

In order to evaluate the quality of the cells derived from oral tissues, microbiological investigations were performed. Our results revealed that all samples contained microbial species. Pre-treatment of patients with the antibiotics chlorhexidine (0.2 %), which is done anyway to decrease the chances of inflammation after surgery, reduced the number of microorganisms to less than 5 % but did not suffice to eliminate all bacteria. On the other hand pre-surgical, antibiotic treatment seemed to be negative for cell-outgrowth. To reduce contamination of the harvested cell-material, an optimized surgical procedure is more important than pre-surgical irrigation with chlorhexidine (0.2 %), and the use of a stringent dual suction pipe procedure. The predominantly found species were gram-positive cocci being either catalase-positive and oxidase-negative or catalase- and oxidase-negative. Most microorganisms belonged to the families of *Streptococcaceae* and *Staphylococcaceae*. The detected microorganisms did not interfere with cell growth and differentiation. They can be easily suppressed with standard antibiotics, applied routinely in patient treatment during the implantation procedure. Thus, these stem cells can be used for bone regeneration in dental implants.

#### **6. Conclusion**

The stability of dental implants is associated with a successful osseointegration into thick jawbone. Due to bone defects, bone regeneration is often needed before an implant can be inserted. For this stem cells can be a suitable candidates.

The stem cells isolated from adipose tissue, dental follicle and bone chips share mainly the multipotent character of mesenchymal stem cells. ATSCs can be successfully differentiated towards adipogenic and osteogenic lineages while DFCs and BCDCs did not show adipogenic differentiation. However, these cell types showed stronger commitment and differentiation towards osteogenic lineage. Therefore all three cell types are promising candidates for the treatment of various bone defects, and therefore also for the incorporation of tooth implants. They can be used to reconstruct jawbone defects to achieve enough bone thickness for the insertion of dental implants. It might be possible to load these cells on a dental implant or an artificial tooth root to increase its integration stability with the bone.

DFCs might be an ideal option if there will be a bank of donor material for these cells in the future, similar to those banks already existing as umbilical cord blood stem cells. If DFCs and BCDCs are not available for a specific patient, ATSCs are a reasonable option as they can differentiate towards the osteogenic lineage and be obtained from the patient itself as well, reducing the risk for rejection. Taken together all these tested stem cell types are suitable to improve the conditions for dental implants. Patients could preserve their dental follicle cells for later use in the future or their stem cells could be isolated from fat tissue directly before use. If a stem cell bank is arranged in the future, stem cells from other stem cell donors for dental follicle and fat tissue derived SCs could be used.

#### **7. Acknowledgements**

This work was supported by the BMBF-AIF, AdiPaD; FKZ: 1720X06 for ET and the Higher Education Commission of Pakistan, DAAD Germany for DK.

#### **8. List of abbreviation**

334 Tissue Regeneration – From Basic Biology to Clinical Application

In order to evaluate the quality of the cells derived from oral tissues, microbiological investigations were performed. Our results revealed that all samples contained microbial species. Pre-treatment of patients with the antibiotics chlorhexidine (0.2 %), which is done anyway to decrease the chances of inflammation after surgery, reduced the number of microorganisms to less than 5 % but did not suffice to eliminate all bacteria. On the other hand pre-surgical, antibiotic treatment seemed to be negative for cell-outgrowth. To reduce contamination of the harvested cell-material, an optimized surgical procedure is more important than pre-surgical irrigation with chlorhexidine (0.2 %), and the use of a stringent dual suction pipe procedure. The predominantly found species were gram-positive cocci being either catalase-positive and oxidase-negative or catalase- and oxidase-negative. Most microorganisms belonged to the families of *Streptococcaceae* and *Staphylococcaceae*. The detected microorganisms did not interfere with cell growth and differentiation. They can be easily suppressed with standard antibiotics, applied routinely in patient treatment during the implantation procedure. Thus, these stem cells can be used for bone regeneration in

The stability of dental implants is associated with a successful osseointegration into thick jawbone. Due to bone defects, bone regeneration is often needed before an implant can be

The stem cells isolated from adipose tissue, dental follicle and bone chips share mainly the multipotent character of mesenchymal stem cells. ATSCs can be successfully differentiated towards adipogenic and osteogenic lineages while DFCs and BCDCs did not show adipogenic differentiation. However, these cell types showed stronger commitment and differentiation towards osteogenic lineage. Therefore all three cell types are promising candidates for the treatment of various bone defects, and therefore also for the incorporation of tooth implants. They can be used to reconstruct jawbone defects to achieve enough bone thickness for the insertion of dental implants. It might be possible to load these cells on a dental implant or an artificial tooth root to increase its integration stability with the bone.

DFCs might be an ideal option if there will be a bank of donor material for these cells in the future, similar to those banks already existing as umbilical cord blood stem cells. If DFCs and BCDCs are not available for a specific patient, ATSCs are a reasonable option as they can differentiate towards the osteogenic lineage and be obtained from the patient itself as well, reducing the risk for rejection. Taken together all these tested stem cell types are suitable to improve the conditions for dental implants. Patients could preserve their dental follicle cells for later use in the future or their stem cells could be isolated from fat tissue directly before use. If a stem cell bank is arranged in the future, stem cells from other stem

This work was supported by the BMBF-AIF, AdiPaD; FKZ: 1720X06 for ET and the Higher

cell donors for dental follicle and fat tissue derived SCs could be used.

Education Commission of Pakistan, DAAD Germany for DK.

**5.2 Microbial load of the oral tissue material** 

inserted. For this stem cells can be a suitable candidates.

dental implants.

**6. Conclusion** 

**7. Acknowledgements** 


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**16** 

*Brazil* 

**Technologies Applied to Stimulate** 

Arnaldo Rodrigues Santos Jr.1, Christiane Bertachini Lombello2

Regenerative medicine constantly encounters situations where specific tissues do not have sufficient regenerative capacity to cope with lesions. Routine techniques involve varied surgical procedures, and often involve mechanical, functional and/or aesthetic discomfort. The need to develop alternative techniques for reducing these inconveniences is therefore

Techniques that stimulate normal tissue repair represent a major advance in biology and regenerative medicine. Frequently applied to the repair of bone lesions and reconstructive surgery, these new biomedical technologies and procedures have afforded technical simplification, elimination of some surgical processes, ease in handling, availability, good levels of predictability and effectiveness, and cost reduction. All this results in an

All the components of the skeletal system, bones and cartilage as well as the connective tissue present in tendons and ligaments, are capable of repair after an injury. During morphogenesis, bone is formed in a particular sequence of events. First, mesenchymal cells proliferate and differentiate into chondroblasts. This process leads to the production of cartilaginous skeleton. In the following stages, cartilage hypertrophy is observed with mineralization of the cartilaginous matrix. The cartilage cells then replaced by osteoblasts. Vascular invasion is necessary for this stage. The bone is then remodeled. Such events are seen during bone morphogenesis and, in the adult, during fracture consolidation (Reddi,

A bone fracture results in the loss of mechanical stability, discontinuity of the bone tissue and partial destruction of its blood supply. Repair is a complex process of tissue regeneration, resulting in stabilization of the fragments, consolidation through bone union,

improvement in the quality of life of the patients involved.

**1. Introduction** 

2001; Tsonis, 2002).

necessary.

**Bone Regeneration** 

and Selma Candelária Genari3

*1Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC, Santo André, SP;* 

*Universidade Federal do ABC, Santo André, SP;* 

*2Centro de Engenharia e Ciências Sociais Aplicadas (CECS),* 

*3Centro Estadual de Educação Tecnológica Paula Souza, Faculdade de Tecnologia de Bauru (FATEC), Bauru, SP;* 

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Slootweg, P.J. (2009). Lesions of the jaws. Histopathology, 54, 401-418


## **Technologies Applied to Stimulate Bone Regeneration**

Arnaldo Rodrigues Santos Jr.1, Christiane Bertachini Lombello2 and Selma Candelária Genari3 *1Centro de Ciências Naturais e Humanas (CCNH), Universidade Federal do ABC, Santo André, SP; 2Centro de Engenharia e Ciências Sociais Aplicadas (CECS), Universidade Federal do ABC, Santo André, SP; 3Centro Estadual de Educação Tecnológica Paula Souza, Faculdade de Tecnologia de Bauru (FATEC), Bauru, SP;* 

*Brazil* 

#### **1. Introduction**

338 Tissue Regeneration – From Basic Biology to Clinical Application

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regenerative medicine. Recent. Pat. Biotechnol, 4, 1-22

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& Tobiasch, E. (2010a) Purinergic Receptors Influence the Differentiation of Human Mesenchymal Stem Cells," Stem cells & Development. Epub ahead of print

Benhaim, P. & Hedrick, M. (2002). Human adipose tissue is a source of multipotent

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derived unit. Periodontology, 13, 9-19

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aspirates. J. Cell. Physiol, 208, 64-76

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doi:10.1089/scd.2010.0576.

338, Spring Verlag, ISBN: 978-3-7908-2126-0

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stem cells from fibroblast cultures. Nat. Protoc, 2, 3081-3089

74

Surg, 116, 6-24

engineering and autologous transplant formation: practical approaches with resorbable biomaterials and new cell culture techniques. Biomaterials, 17, 237-242

Mankin, H.J. (2001). Allograft fractures revisited. Clin. Orthop. Relat. Res, 382, 66-

Regenerative medicine constantly encounters situations where specific tissues do not have sufficient regenerative capacity to cope with lesions. Routine techniques involve varied surgical procedures, and often involve mechanical, functional and/or aesthetic discomfort. The need to develop alternative techniques for reducing these inconveniences is therefore necessary.

Techniques that stimulate normal tissue repair represent a major advance in biology and regenerative medicine. Frequently applied to the repair of bone lesions and reconstructive surgery, these new biomedical technologies and procedures have afforded technical simplification, elimination of some surgical processes, ease in handling, availability, good levels of predictability and effectiveness, and cost reduction. All this results in an improvement in the quality of life of the patients involved.

All the components of the skeletal system, bones and cartilage as well as the connective tissue present in tendons and ligaments, are capable of repair after an injury. During morphogenesis, bone is formed in a particular sequence of events. First, mesenchymal cells proliferate and differentiate into chondroblasts. This process leads to the production of cartilaginous skeleton. In the following stages, cartilage hypertrophy is observed with mineralization of the cartilaginous matrix. The cartilage cells then replaced by osteoblasts. Vascular invasion is necessary for this stage. The bone is then remodeled. Such events are seen during bone morphogenesis and, in the adult, during fracture consolidation (Reddi, 2001; Tsonis, 2002).

A bone fracture results in the loss of mechanical stability, discontinuity of the bone tissue and partial destruction of its blood supply. Repair is a complex process of tissue regeneration, resulting in stabilization of the fragments, consolidation through bone union,

Technologies Applied to Stimulate Bone Regeneration 341

however, the movement is excessive and the vascular supply limited, the establishment of

In the *regeneration of a fracture without loss of bone mass*, the repair process occurs in a biologically determined order. The first priority is stabilization and consolidation through callus formation on the edges and between the fragments, followed by its remodeling, besides revascularization and substitution of the necrotic areas. External factors can deeply affect the regeneration process, but the tissues act according to biological rules that control proliferation and cell differentiation as well as the production of matrix, which may occur

However, *fractures with loss of bone mass* call for the use of grafts or implants. The latter serve as a support to bone regeneration, interacting with the interface of the receptor fragments and stimulating the tissue restoration process. These devices developed to be implanted are currently known as biomaterials (Hench, 1998), and will be addressed subsequently over the course of this text, constituting the basis of procedures such as guided tissue regeneration and tissue engineering. We will also address other technologies applied to bone

The bone repair process can be characterized by 6 physiological stages: impact, induction, inflammation, formation of cartilaginous callus, formation of bone callus and remodeling

*Impact* consists of the period of energy absorption until the fracture. The quantity of energy absorbed depends on the bone volume and is related to the loading rate. The impact stage of

The *induction* stage involves modulation and differentiation of cellular elements required during the regeneration process. In fractures there is always local hemorrhage caused by injury to the blood vessels of the bone and of the periosteum, besides destruction of the matrix and death of the bone cells adjacent to the fractured site (Fawcett, 1986). This process triggers the inflammatory stage that will persist until the remodeling stage, with phagocytic activity of macrophages that will remove tissue and clot remnants. Cells from the periosteum and from the endosteum, close to the fractured area, will be activated (induction stage) and will respond with intense proliferation of their fibroblasts. Mesenchymal tissues, undifferentiated osteogenic and chondrogenic cells will differentiate into functional osteoblasts and chondrocytes, respectively. The stimulus for this induction can be electrical, low oxygen tension, low pH, release of lysosomal enzymes, release of cytosine and the presence of a series of inductor proteins, including bone morphogenetic proteins (BMP) and cartilage growth factors (Reddi, 1981; Canalis, 1983; Zellin *et al*., 1996; Lieberman *et al*., 2002; Oringer, 2002). The induction stage actually occurs through a series of sub-stages in which the inducing phenomena for each subsequent repair stage can be quite distinct, with unique characteristics toward specific target cells (for example: chondrocytes in the cartilaginous

During the *cartilaginous callus formation* stage, there is a considerable increase of vascularity and cellularity, and of the production of collagen, proteoglycans and lipids.

local fibrous connective tissue (fibrosis) is probable (Kierszenbaum, 2004).

regardless of, yet influenced by, external interferences.

regeneration, seeking optimization and acceleration of the process.

**2. Regeneration biology without loss of bone mass** 

the fracture occurs until energy dissipation.

callus and osteoblasts in the bone callus).

(Heppenstall, 1980).

reconstruction of the tips of the avascular and partially necrotic fragments and, finally, internal and external remodeling of the newly formed tissue.

Hemorrhage caused by the blood vessel lesion, destruction of matrix and death of bone cells occurs at the site of a bone fracture. For the repair to begin, the blood clot and the cellular remnants from the matrix must be removed by the macrophages. The *periosteum* and the *endosteum* that are close to the fractured area respond with intense proliferation, forming tissue that is very rich in osteoprogenitor cells which constitute a collar around the fracture that will penetrate between the ruptured bone extremities, making a ring or collar that is located between the fractured bone extremities. This leads to the appearance of immature bone tissue, both through endochondral ossification of small pieces of cartilage that form there and through intramembranous ossification (Kierszenbaum, 2004; Junqueira & Carneiro, 2008). Areas of cartilage, areas of intramembranous ossification and areas of endochondral ossification can be found at the repair site. This process evolves with the appearance after a while of a bone callus covering the extremity of the fractured bones. The callus is formed of immature bone tissue that will temporarily join together the extremities of the fractured bone (Kierszenbaum, 2004; Junqueira & Carneiro, 2008).

Bone repair is also mediated by Mesenchymal stem cells (MSCs) (Bruder et al., 1994). These cells can be stimulated to differentiate into osteoblasts, cultivating them in the presence of serum, dexamethasone, beta-glycerophosphate and ascorbic acid. Moreover, MSCs can differentiate into osteoblasts due to the influence of vitamin D and BMP-2 (Pittenger et al, 1999). Human adipose tissue also contains stroma cells that are able to differentiate into chondrocytes and osteoblasts (Halvorsen et al, 2001).

The traction and pressure applied to the bone during fracture repair, and soon after the patient resumes normal activities, cause the remodeling of the bone callus and its complete substitution with lamellar bone tissue. When these tractions and pressures are identical to those applied to the bone before the fracture, the bone structure returns to its previous state; unlike other connective tissues, bone tissue, despite its rigidity, heals without scar formation (Kessel, 2001; Junqueira & Carneiro, 2008). Bone formation depends on the existence of an extensive vascular network and on the stability of the fracture focus that facilitates local vascularization, giving rise to the differentiation of the osteoprogenitor cells into osteoblasts; moreover, it is well established that the osteoblasts only synthesize the bone matrix in the presence of high oxygen tension.

The mechanical instability of the fracture hinders local vascularization and under these conditions the osteoprogenitor cells differentiate preferentially into chondroblasts. Accordingly, the fracture focus will initially be filled with cartilage (avascular tissue), which will provide a certain degree of stability to this focus, subsequently favoring vascularization of the site (Kierszenbaum, 2004). The quantity of cartilage formed during embryogenesis and at the site of a fracture (bone granulation tissue) is inversely proportional to the quantity of osteoprogenitor cells and of blood capillaries present at the site. If the bone callus tissue and the capillaries develop at the same time, the osteoprogenitor cells will differentiate into a vascularized environment and will consequently form bone. If there are proportionally few vessels or movement of the fracture segments, there will be cartilage formation followed by its substitution with bone tissue (endochondral ossification). If,

reconstruction of the tips of the avascular and partially necrotic fragments and, finally,

Hemorrhage caused by the blood vessel lesion, destruction of matrix and death of bone cells occurs at the site of a bone fracture. For the repair to begin, the blood clot and the cellular remnants from the matrix must be removed by the macrophages. The *periosteum* and the *endosteum* that are close to the fractured area respond with intense proliferation, forming tissue that is very rich in osteoprogenitor cells which constitute a collar around the fracture that will penetrate between the ruptured bone extremities, making a ring or collar that is located between the fractured bone extremities. This leads to the appearance of immature bone tissue, both through endochondral ossification of small pieces of cartilage that form there and through intramembranous ossification (Kierszenbaum, 2004; Junqueira & Carneiro, 2008). Areas of cartilage, areas of intramembranous ossification and areas of endochondral ossification can be found at the repair site. This process evolves with the appearance after a while of a bone callus covering the extremity of the fractured bones. The callus is formed of immature bone tissue that will temporarily join together the extremities of the fractured bone (Kierszenbaum, 2004;

Bone repair is also mediated by Mesenchymal stem cells (MSCs) (Bruder et al., 1994). These cells can be stimulated to differentiate into osteoblasts, cultivating them in the presence of serum, dexamethasone, beta-glycerophosphate and ascorbic acid. Moreover, MSCs can differentiate into osteoblasts due to the influence of vitamin D and BMP-2 (Pittenger et al, 1999). Human adipose tissue also contains stroma cells that are able to differentiate into

The traction and pressure applied to the bone during fracture repair, and soon after the patient resumes normal activities, cause the remodeling of the bone callus and its complete substitution with lamellar bone tissue. When these tractions and pressures are identical to those applied to the bone before the fracture, the bone structure returns to its previous state; unlike other connective tissues, bone tissue, despite its rigidity, heals without scar formation (Kessel, 2001; Junqueira & Carneiro, 2008). Bone formation depends on the existence of an extensive vascular network and on the stability of the fracture focus that facilitates local vascularization, giving rise to the differentiation of the osteoprogenitor cells into osteoblasts; moreover, it is well established that the osteoblasts only synthesize the bone matrix in the

The mechanical instability of the fracture hinders local vascularization and under these conditions the osteoprogenitor cells differentiate preferentially into chondroblasts. Accordingly, the fracture focus will initially be filled with cartilage (avascular tissue), which will provide a certain degree of stability to this focus, subsequently favoring vascularization of the site (Kierszenbaum, 2004). The quantity of cartilage formed during embryogenesis and at the site of a fracture (bone granulation tissue) is inversely proportional to the quantity of osteoprogenitor cells and of blood capillaries present at the site. If the bone callus tissue and the capillaries develop at the same time, the osteoprogenitor cells will differentiate into a vascularized environment and will consequently form bone. If there are proportionally few vessels or movement of the fracture segments, there will be cartilage formation followed by its substitution with bone tissue (endochondral ossification). If,

internal and external remodeling of the newly formed tissue.

Junqueira & Carneiro, 2008).

presence of high oxygen tension.

chondrocytes and osteoblasts (Halvorsen et al, 2001).

however, the movement is excessive and the vascular supply limited, the establishment of local fibrous connective tissue (fibrosis) is probable (Kierszenbaum, 2004).

In the *regeneration of a fracture without loss of bone mass*, the repair process occurs in a biologically determined order. The first priority is stabilization and consolidation through callus formation on the edges and between the fragments, followed by its remodeling, besides revascularization and substitution of the necrotic areas. External factors can deeply affect the regeneration process, but the tissues act according to biological rules that control proliferation and cell differentiation as well as the production of matrix, which may occur regardless of, yet influenced by, external interferences.

However, *fractures with loss of bone mass* call for the use of grafts or implants. The latter serve as a support to bone regeneration, interacting with the interface of the receptor fragments and stimulating the tissue restoration process. These devices developed to be implanted are currently known as biomaterials (Hench, 1998), and will be addressed subsequently over the course of this text, constituting the basis of procedures such as guided tissue regeneration and tissue engineering. We will also address other technologies applied to bone regeneration, seeking optimization and acceleration of the process.

#### **2. Regeneration biology without loss of bone mass**

The bone repair process can be characterized by 6 physiological stages: impact, induction, inflammation, formation of cartilaginous callus, formation of bone callus and remodeling (Heppenstall, 1980).

*Impact* consists of the period of energy absorption until the fracture. The quantity of energy absorbed depends on the bone volume and is related to the loading rate. The impact stage of the fracture occurs until energy dissipation.

The *induction* stage involves modulation and differentiation of cellular elements required during the regeneration process. In fractures there is always local hemorrhage caused by injury to the blood vessels of the bone and of the periosteum, besides destruction of the matrix and death of the bone cells adjacent to the fractured site (Fawcett, 1986). This process triggers the inflammatory stage that will persist until the remodeling stage, with phagocytic activity of macrophages that will remove tissue and clot remnants. Cells from the periosteum and from the endosteum, close to the fractured area, will be activated (induction stage) and will respond with intense proliferation of their fibroblasts. Mesenchymal tissues, undifferentiated osteogenic and chondrogenic cells will differentiate into functional osteoblasts and chondrocytes, respectively. The stimulus for this induction can be electrical, low oxygen tension, low pH, release of lysosomal enzymes, release of cytosine and the presence of a series of inductor proteins, including bone morphogenetic proteins (BMP) and cartilage growth factors (Reddi, 1981; Canalis, 1983; Zellin *et al*., 1996; Lieberman *et al*., 2002; Oringer, 2002). The induction stage actually occurs through a series of sub-stages in which the inducing phenomena for each subsequent repair stage can be quite distinct, with unique characteristics toward specific target cells (for example: chondrocytes in the cartilaginous callus and osteoblasts in the bone callus).

During the *cartilaginous callus formation* stage, there is a considerable increase of vascularity and cellularity, and of the production of collagen, proteoglycans and lipids.

Technologies Applied to Stimulate Bone Regeneration 343

These devices produced to serve as implants are known as biomaterials (Hench, 1998) and should exhibit characteristics that stimulate (osteoinduction) and/or guide

*Osteoinduction* consists of a set of chemical, humoral or physical signals that initiate and sustain the various stages of the bone regeneration process and several factors may be involved (Nakagawa & Tagawa, 2000). The concept of osteoinduction was explored in 1965 by Urist, who showed ectopic bone formation when the demineralized bone matrix was implanted in muscles of rabbits, rats, mice and guinea pigs (Urist, 1965). Afterwards, it was concluded that a protein, called *bone morphogenetic protein* (BMP), was involved in the sequence of events involving chemotaxis, mitosis, bone differentiation and formation (Urist *et al*., 1979). BMP is a glycoprotein of 17,500 daltons, and one of the factors currently identified in bone formation (Adriano *et al*., 2000; Nakagawa & Tagawa, 2000; Reddi, 2000). Nowadays, several groups have shown that BMPs have the capacity to induce new formation of bone tissue by the endochondral route when implanted in ectopic sites in animals used for experimentation (Habibovic & de Groot, 2007). Besides BMP, other factors such as electromagnetic fields and direct currents also manifest inductive properties. The common result of osteoinduction is the modulation and differentiation of cells for bone

O*steoconduction,* in turn, is related to the establishment of an appropriate environment model on which osteoprogenitor cells, when adequately stimulated, can produce bone (Heppenstall, 1980). Osteoconduction also facilitates production and bone deposition in the appropriate three-dimensional arrangement and increases the ability of the regeneration process in large segmental defects. Collagen, a natural organic component of bone and of bone surfaces, is the prototype of an osteoconductive substance (Kimura *et al*., 2000; Lee *et al*., 2001). A large number of natural and manufactured substances can also stimulate a favorable environment for bone formation (Mandracchia *et al*., 2001; Pineda *et al*., 1996), as discussed below. Osteoconduction is the phenomenon in which a single vehicle physically

Many studies have investigated the inductive signals for bone morphogenesis, but the greatest emphasis has been placed on BMPs (Reddi, 2000). These proteins were found to have highly specialized patterns of expression during bone repair (Bostrom, 1998; Groeneveld & Burger, 2000). During the initial phases of consolidation, some primordial cells express BMPs in the bone callus. Expression is greater in MSCs and chondrocytes when endochondral ossification occurs. Expression decreases as the cartilaginous component of the callus matures. BMPs are expressed by osteoblasts, but decrease as the primary bone is replaced by lamellar bone. BMP-2, -3, -4, -5, -7, and -8 are responsible for the induction of bone and cartilage formation. BMP-12, -13 and -14, are cartilage derived. BMPs have also been used in clinical trials for the treatment fractures and pseudarthrosis, for example

A large number of natural substances can be extracted and/or manufactured to stimulate a favorable environment for bone formation (Mandracchi *et al*., 2001; Pineda *et al*., 1996). Among all the biomaterials of natural origin that aim to assist in bone regeneration, special emphasis is placed on those of bovine origin, where different protocols of chemical treatment of the bovine bone are evaluated with the purpose of preserving the organic

(osteoconduction) the bone regeneration process.

conducts the proliferation of osteogenic cells.

production.

(Reddi, 2001).

The callus is electronegative and the osteoclasts continue to remove the necrotic bone. The cartilage formed undergoes modifications, with hypertrophy of the chondrocytes that generate compression on the preexisting cartilaginous matrix, and consequent enlargement of its gaps, being gradually reduced to fenestrated thin septa and spicules of irregular shapes (Probst & Spiegel, 1997). The hyaline matrix from this hypertrophic region becomes calcified, and small granular aggregates and crystals of calcium phosphate are deposited on it.

The *bone callus formation* stage is marked by the substitution of calcified cartilage in primary bone tissue (Heppenstall, 1980; Probst & Spiegel, 1997; Mandracchia *et al*., 2001). Cells with osteogenic potential, originating from the endosteum and particularly from the periosteum, are activated and a thin layer of bone (a periosteal ring or collar) is deposited around the central portion of the calcified cartilage. At the same time, periosteal blood vessels grow, invading the irregular cavities of the cartilaginous matrix created by the enlargement of the chondrocytes and by the confluence of its gaps. Vessels with thin walls branch out and grow into the cavities of the cartilaginous matrix with blind bottoms. Pluripotent cells are carried to the perivascular tissue of these blood vessels and, some differentiate into hematopoietic elements of the bone marrow. Other cells differentiate into osteoblasts, which deposit an aligned layer similar to an epithelium on the irregular walls of the spicules of the calcified cartilaginous matrix, and start the production of bone matrix. The osteoblasts covered by matrix become osteocytes and start to maintain contact with one another through cytoplasmic processes in a system of canaliculi. The callus remains electronegative, while the osteoclasts finish removing the necrotic bone. The cartilaginous matrix is gradually replaced by primary bone tissue.

During *remodeling*, the conversion of the primary bone tissue into secondary or lamellar bone tissue is completed. The collagen fibers are thicker and present preferential orientation alternating between layers or lamellae. These lamellae can be compacted if deposited on a flattened or concentric surface covering a blood vessel. The collagen fibers extend between the lamellae, thus increasing the bone strength. The blood vessels are contained in central canals (the Haversian canals), which intercommunicate through Volkmann's canals. Moreover, there are several canaliculi that extend to nourish the osteocytes. This assembly is known as the *osteon* or classically as *Haversian system*. Secondary osteons are formed when part of the concentric lamellar bone is converted into Haversian systems (Fawcett, 1986). The medullary canal is reestablished and the diameter and electronegativity of the callus decrease until they disappear.

#### **3. Regeneration biology with loss of bone mass**

As mentioned previously, the natural phenomenon of bone regeneration is insufficient, on its own, to reestablish the integrity of fractures with substantial loss of bone mass. For bone regeneration process to take place, it is necessary to have four components: a) a morphogenetic signal, b) host cells that respond to the signal, c) an appropriate carrier of this signal that can deliver it to the specific sites and thus serve as support to the growth of responsive cells of the host and d) a viable and well-vascularized bed (Burg *et al*., 2000). Consequently, the need to use materials that would serve as support for the regeneration process is present and currently appears as an alternative to the use of grafts, where there are difficulties involved in obtaining bone tissue and in molding for the fracture in question.

The callus is electronegative and the osteoclasts continue to remove the necrotic bone. The cartilage formed undergoes modifications, with hypertrophy of the chondrocytes that generate compression on the preexisting cartilaginous matrix, and consequent enlargement of its gaps, being gradually reduced to fenestrated thin septa and spicules of irregular shapes (Probst & Spiegel, 1997). The hyaline matrix from this hypertrophic region becomes calcified, and small granular aggregates and crystals of calcium

The *bone callus formation* stage is marked by the substitution of calcified cartilage in primary bone tissue (Heppenstall, 1980; Probst & Spiegel, 1997; Mandracchia *et al*., 2001). Cells with osteogenic potential, originating from the endosteum and particularly from the periosteum, are activated and a thin layer of bone (a periosteal ring or collar) is deposited around the central portion of the calcified cartilage. At the same time, periosteal blood vessels grow, invading the irregular cavities of the cartilaginous matrix created by the enlargement of the chondrocytes and by the confluence of its gaps. Vessels with thin walls branch out and grow into the cavities of the cartilaginous matrix with blind bottoms. Pluripotent cells are carried to the perivascular tissue of these blood vessels and, some differentiate into hematopoietic elements of the bone marrow. Other cells differentiate into osteoblasts, which deposit an aligned layer similar to an epithelium on the irregular walls of the spicules of the calcified cartilaginous matrix, and start the production of bone matrix. The osteoblasts covered by matrix become osteocytes and start to maintain contact with one another through cytoplasmic processes in a system of canaliculi. The callus remains electronegative, while the osteoclasts finish removing the necrotic bone. The cartilaginous matrix is gradually

During *remodeling*, the conversion of the primary bone tissue into secondary or lamellar bone tissue is completed. The collagen fibers are thicker and present preferential orientation alternating between layers or lamellae. These lamellae can be compacted if deposited on a flattened or concentric surface covering a blood vessel. The collagen fibers extend between the lamellae, thus increasing the bone strength. The blood vessels are contained in central canals (the Haversian canals), which intercommunicate through Volkmann's canals. Moreover, there are several canaliculi that extend to nourish the osteocytes. This assembly is known as the *osteon* or classically as *Haversian system*. Secondary osteons are formed when part of the concentric lamellar bone is converted into Haversian systems (Fawcett, 1986). The medullary canal is reestablished and the diameter and electronegativity of the callus

As mentioned previously, the natural phenomenon of bone regeneration is insufficient, on its own, to reestablish the integrity of fractures with substantial loss of bone mass. For bone regeneration process to take place, it is necessary to have four components: a) a morphogenetic signal, b) host cells that respond to the signal, c) an appropriate carrier of this signal that can deliver it to the specific sites and thus serve as support to the growth of responsive cells of the host and d) a viable and well-vascularized bed (Burg *et al*., 2000). Consequently, the need to use materials that would serve as support for the regeneration process is present and currently appears as an alternative to the use of grafts, where there are difficulties involved in obtaining bone tissue and in molding for the fracture in question.

phosphate are deposited on it.

replaced by primary bone tissue.

decrease until they disappear.

**3. Regeneration biology with loss of bone mass** 

These devices produced to serve as implants are known as biomaterials (Hench, 1998) and should exhibit characteristics that stimulate (osteoinduction) and/or guide (osteoconduction) the bone regeneration process.

*Osteoinduction* consists of a set of chemical, humoral or physical signals that initiate and sustain the various stages of the bone regeneration process and several factors may be involved (Nakagawa & Tagawa, 2000). The concept of osteoinduction was explored in 1965 by Urist, who showed ectopic bone formation when the demineralized bone matrix was implanted in muscles of rabbits, rats, mice and guinea pigs (Urist, 1965). Afterwards, it was concluded that a protein, called *bone morphogenetic protein* (BMP), was involved in the sequence of events involving chemotaxis, mitosis, bone differentiation and formation (Urist *et al*., 1979). BMP is a glycoprotein of 17,500 daltons, and one of the factors currently identified in bone formation (Adriano *et al*., 2000; Nakagawa & Tagawa, 2000; Reddi, 2000). Nowadays, several groups have shown that BMPs have the capacity to induce new formation of bone tissue by the endochondral route when implanted in ectopic sites in animals used for experimentation (Habibovic & de Groot, 2007). Besides BMP, other factors such as electromagnetic fields and direct currents also manifest inductive properties. The common result of osteoinduction is the modulation and differentiation of cells for bone production.

O*steoconduction,* in turn, is related to the establishment of an appropriate environment model on which osteoprogenitor cells, when adequately stimulated, can produce bone (Heppenstall, 1980). Osteoconduction also facilitates production and bone deposition in the appropriate three-dimensional arrangement and increases the ability of the regeneration process in large segmental defects. Collagen, a natural organic component of bone and of bone surfaces, is the prototype of an osteoconductive substance (Kimura *et al*., 2000; Lee *et al*., 2001). A large number of natural and manufactured substances can also stimulate a favorable environment for bone formation (Mandracchia *et al*., 2001; Pineda *et al*., 1996), as discussed below. Osteoconduction is the phenomenon in which a single vehicle physically conducts the proliferation of osteogenic cells.

Many studies have investigated the inductive signals for bone morphogenesis, but the greatest emphasis has been placed on BMPs (Reddi, 2000). These proteins were found to have highly specialized patterns of expression during bone repair (Bostrom, 1998; Groeneveld & Burger, 2000). During the initial phases of consolidation, some primordial cells express BMPs in the bone callus. Expression is greater in MSCs and chondrocytes when endochondral ossification occurs. Expression decreases as the cartilaginous component of the callus matures. BMPs are expressed by osteoblasts, but decrease as the primary bone is replaced by lamellar bone. BMP-2, -3, -4, -5, -7, and -8 are responsible for the induction of bone and cartilage formation. BMP-12, -13 and -14, are cartilage derived. BMPs have also been used in clinical trials for the treatment fractures and pseudarthrosis, for example (Reddi, 2001).

A large number of natural substances can be extracted and/or manufactured to stimulate a favorable environment for bone formation (Mandracchi *et al*., 2001; Pineda *et al*., 1996). Among all the biomaterials of natural origin that aim to assist in bone regeneration, special emphasis is placed on those of bovine origin, where different protocols of chemical treatment of the bovine bone are evaluated with the purpose of preserving the organic

Technologies Applied to Stimulate Bone Regeneration 345

The autogenous graft of *spongy* or *cortical* origin, whether vascularized or not, presents good integration with the adjacent tissue (Khan et al., 2005). In general autogenous grafts are obtained from the iliac crest, due to ease of access and as the obtainment of spongy bone of good quality, which is considered more osteoconductive, osteogenic and osteoinductive than the cortical bone graft, once it favors the diffusion of nutrients and revascularization of the treated area, and presents in its structure osteoprogenitor cells and osteoinductor proteins. Cortical bone graft acts mainly as a support for bone regeneration, changing the

As regards *mechanical resistance*, the spongy graft does not offer immediate resistance at the grafting site. But the osseointegration process favors the acquisition of resistance during bone neoformation, and in an interval of 6 to 12 months it acquires resistance similar to that offered by the cortical graft (Dell et al., 1985; Stevenson, 1999). The opposite occurs with the cortical bone graft, which initially presents good mechanical resistance; however, over the first 6 months of grafting, this resistance is decreased by the presence of a mixture of newly

The different properties of spongy and cortical autogenous grafts establish the direction of their clinical use. The spongy autogenous graft can be used in cases involving difficulty in the consolidation of long bone fractures, and in the reconstruction of depressed lateral tibial plateau fractures (Marsh, 2006; Marino & Ziran, 2010; Nandi et al., 2010). A case report on autograft use in osteotomy for ulnar lengthening demonstrates the use of the trabecular autograft in functional recovery of the humeroulnar joint, resulting from difficulty in bone union. The patient presented recovery of movements in the two years of follow-up,

The cortical bone graft can be used in cases that require greater initial mechanical resistance at the graft site, even with the need for stabilization of the fracture with implants. Bone defects larger than 5 or 6cm are indications for the use of cortical grafts, as they require

In spite of clinical results demonstrating the efficacy and safety of use of autogenous grafts in bone regeneration, some *disadvantages* of their clinical application limit their use (Arrington et al., 1996). We can mention high morbidity of the graft obtainment procedure, the aesthetic discomfort of this route, complications related to the surgical technique that mainly include infection and hemorrhage, and the actual limitations of indication, such as young or elderly patients and cases of recurring surgeries (Seiler & Johnson, 2000;

As possible alternatives to autograft surgeons have allografts, demineralized bone matrix

*Allografts* have a growing clinical use, favored by the upgrading of techniques for obtaining, preparing and storing these materials. Fresh, frozen or freeze-dried grafts can be obtained, but fresh materials are used less often due to their technical difficulty and associated risks (Boyce et al., 1999; Keating & McQueen, 2001). Frozen and freeze-dried allografts are kept in specific tissue banks, properly processed and sterilized prior to storage. Their processing ends up eliminating cells naturally present in the graft, so there is no osteogenic activity here. It is considered that the allograft retains osteoinductive activity, and in spite of its

immediate mechanical support and a longer period of graft use (Nandi et al., 2010).

direction of the tissue regeneration process (Cypher & Grossman, 1996).

achieving 105of humeroulnar movement (Doornberg & Marti, 2010).

and natural or synthetic bone graft substitutes at their disposal.

Giannoudis et al., 2005).

formed bone and necrotic bone at the grafting site (Goldberg & Stevenson, 1992).

components and inorganic components, such as collagen and hydroxyapatite repectively, which results in a mixed bovine bone (MBB) with an increase of the material's mechanical resistance. The MBB scaffold used in tissue regeneration has the appearance of a porous sponge that will occupy the space of the bone defect, preventing the migration of epithelial and connective cells, so that the osteoblastic cells have access to the regenerating tissue and start to populate the scaffold. Another role of the scaffold is its use as a vehicle for drugs that induce tissue regeneration and inhibit the progression of the disease.

Autogenous bone grafts are considered very advantageous, since they avoid complications of immunological rejection and supply cells that can immediately start the regenerative process (Cunha et al., 2005, 2006). The use of bone grafts is becoming frequent in orthopedics, as a method for resolution of comminuted fractures, (fractures in which the bone is splintered or crushed) thus significantly reducing the need to amputate an affected limb (Cavassini *et al*., 2001).

One option in the treatment of partial bone defects is to perform the transportation of small bone fragments - called parietal transportation. In this technique (seldom reported in medical literature), the viable bone segment contiguous to the bone cavity is preserved. A bone fragment is created in the healthy region adjacent to the cavity and transported, according to the Llizarov method, filling a cavity of approximately 50% of the bone diameter (Rodrigues & Mercadante, 2005). The option of performing resection of viable bone occupying the complete cortical, transforming the partial defect into segmental for application of the conventional bone transportation technique, appears to us to be absolute nonsense: removing healthy bone when this is what is missing. The main advantage of parietal transportation is bone formation, even during infection.

The exact mechanism of osteoinduction by biomaterials is still largely unknown. Neither is it known whether the mechanisms of osteoinduction by BMPs and biomaterials are the same. In a recent review (Habibovic & de Groot, 2007) striking differences were shown in osteoinduction by BMPs and biomaterials, namely: (1) bone induced by biomaterials is always intramembranous, while bone induced by BMP is formed mainly by the endochondral route; (2) in small animals, just as in rodents, the bone is very rarely induced by biomaterials, but easily by BMPs; (3) bone is never observed on the edge of biomaterials but instead is always formed inside their pores, while bone formation by BMPs is regularly seen on the outside of the carrier and the soft tissue distant from the surface of this carrier.

#### **4. Bone regeneration scaffolds**

#### **4.1 Autogenous grafts and allografts**

The advantages cited for autogenous grafts in the bone regeneration process allow us to classify it as "gold standard". The presence of osteoprogenitor cells and osteoblasts confer the property of *osteogenesis*; the proteins that are contained in the bone matrix (for example, transforming growth factor (TGF, and the BMPsconfer the aspect of *osteoinduction;* and the actual mineralized bone matrix provides a structural base for the growth of newly formed tissue, favoring the lodging of cells, the growth of blood vessels, and the deposition of bone matrix, characterizing *osteoconduction* (Bauer & Muschler, 2000).

components and inorganic components, such as collagen and hydroxyapatite repectively, which results in a mixed bovine bone (MBB) with an increase of the material's mechanical resistance. The MBB scaffold used in tissue regeneration has the appearance of a porous sponge that will occupy the space of the bone defect, preventing the migration of epithelial and connective cells, so that the osteoblastic cells have access to the regenerating tissue and start to populate the scaffold. Another role of the scaffold is its use as a vehicle for drugs

Autogenous bone grafts are considered very advantageous, since they avoid complications of immunological rejection and supply cells that can immediately start the regenerative process (Cunha et al., 2005, 2006). The use of bone grafts is becoming frequent in orthopedics, as a method for resolution of comminuted fractures, (fractures in which the bone is splintered or crushed) thus significantly reducing the need to amputate an affected

One option in the treatment of partial bone defects is to perform the transportation of small bone fragments - called parietal transportation. In this technique (seldom reported in medical literature), the viable bone segment contiguous to the bone cavity is preserved. A bone fragment is created in the healthy region adjacent to the cavity and transported, according to the Llizarov method, filling a cavity of approximately 50% of the bone diameter (Rodrigues & Mercadante, 2005). The option of performing resection of viable bone occupying the complete cortical, transforming the partial defect into segmental for application of the conventional bone transportation technique, appears to us to be absolute nonsense: removing healthy bone when this is what is missing. The main advantage of

The exact mechanism of osteoinduction by biomaterials is still largely unknown. Neither is it known whether the mechanisms of osteoinduction by BMPs and biomaterials are the same. In a recent review (Habibovic & de Groot, 2007) striking differences were shown in osteoinduction by BMPs and biomaterials, namely: (1) bone induced by biomaterials is always intramembranous, while bone induced by BMP is formed mainly by the endochondral route; (2) in small animals, just as in rodents, the bone is very rarely induced by biomaterials, but easily by BMPs; (3) bone is never observed on the edge of biomaterials but instead is always formed inside their pores, while bone formation by BMPs is regularly seen on the outside of the carrier and the soft tissue distant from the

The advantages cited for autogenous grafts in the bone regeneration process allow us to classify it as "gold standard". The presence of osteoprogenitor cells and osteoblasts confer the property of *osteogenesis*; the proteins that are contained in the bone matrix (for example, transforming growth factor (TGF, and the BMPsconfer the aspect of *osteoinduction;* and the actual mineralized bone matrix provides a structural base for the growth of newly formed tissue, favoring the lodging of cells, the growth of blood vessels, and the deposition

of bone matrix, characterizing *osteoconduction* (Bauer & Muschler, 2000).

that induce tissue regeneration and inhibit the progression of the disease.

parietal transportation is bone formation, even during infection.

limb (Cavassini *et al*., 2001).

surface of this carrier.

**4. Bone regeneration scaffolds 4.1 Autogenous grafts and allografts**  The autogenous graft of *spongy* or *cortical* origin, whether vascularized or not, presents good integration with the adjacent tissue (Khan et al., 2005). In general autogenous grafts are obtained from the iliac crest, due to ease of access and as the obtainment of spongy bone of good quality, which is considered more osteoconductive, osteogenic and osteoinductive than the cortical bone graft, once it favors the diffusion of nutrients and revascularization of the treated area, and presents in its structure osteoprogenitor cells and osteoinductor proteins. Cortical bone graft acts mainly as a support for bone regeneration, changing the direction of the tissue regeneration process (Cypher & Grossman, 1996).

As regards *mechanical resistance*, the spongy graft does not offer immediate resistance at the grafting site. But the osseointegration process favors the acquisition of resistance during bone neoformation, and in an interval of 6 to 12 months it acquires resistance similar to that offered by the cortical graft (Dell et al., 1985; Stevenson, 1999). The opposite occurs with the cortical bone graft, which initially presents good mechanical resistance; however, over the first 6 months of grafting, this resistance is decreased by the presence of a mixture of newly formed bone and necrotic bone at the grafting site (Goldberg & Stevenson, 1992).

The different properties of spongy and cortical autogenous grafts establish the direction of their clinical use. The spongy autogenous graft can be used in cases involving difficulty in the consolidation of long bone fractures, and in the reconstruction of depressed lateral tibial plateau fractures (Marsh, 2006; Marino & Ziran, 2010; Nandi et al., 2010). A case report on autograft use in osteotomy for ulnar lengthening demonstrates the use of the trabecular autograft in functional recovery of the humeroulnar joint, resulting from difficulty in bone union. The patient presented recovery of movements in the two years of follow-up, achieving 105of humeroulnar movement (Doornberg & Marti, 2010).

The cortical bone graft can be used in cases that require greater initial mechanical resistance at the graft site, even with the need for stabilization of the fracture with implants. Bone defects larger than 5 or 6cm are indications for the use of cortical grafts, as they require immediate mechanical support and a longer period of graft use (Nandi et al., 2010).

In spite of clinical results demonstrating the efficacy and safety of use of autogenous grafts in bone regeneration, some *disadvantages* of their clinical application limit their use (Arrington et al., 1996). We can mention high morbidity of the graft obtainment procedure, the aesthetic discomfort of this route, complications related to the surgical technique that mainly include infection and hemorrhage, and the actual limitations of indication, such as young or elderly patients and cases of recurring surgeries (Seiler & Johnson, 2000; Giannoudis et al., 2005).

As possible alternatives to autograft surgeons have allografts, demineralized bone matrix and natural or synthetic bone graft substitutes at their disposal.

*Allografts* have a growing clinical use, favored by the upgrading of techniques for obtaining, preparing and storing these materials. Fresh, frozen or freeze-dried grafts can be obtained, but fresh materials are used less often due to their technical difficulty and associated risks (Boyce et al., 1999; Keating & McQueen, 2001). Frozen and freeze-dried allografts are kept in specific tissue banks, properly processed and sterilized prior to storage. Their processing ends up eliminating cells naturally present in the graft, so there is no osteogenic activity here. It is considered that the allograft retains osteoinductive activity, and in spite of its

Technologies Applied to Stimulate Bone Regeneration 347

Therefore in spite of the *availability* of natural materials as autogenous grafts, allografts and demineralized bone matrix, some limitations of use or clinical disadvantages of these materials drive the development of new technologies for bone tissue regeneration. *Natural and synthetic bone graft substitutes* are available to perform this role. The synthetic bone graft substitutes include ceramic and polymeric biomaterials, while biopolymers represent

*Bioceramics* are biocompatible biomaterials with a long history of clinical applications for bone regeneration. Among the advantages of these biomaterials we can cite their synthetic origin, eliminating the risk of autograft morbidity, or the risk of immunorejection and transmission of diseases of allografts or even biomaterials of human and animal origin. The structural similarity between some bioceramics, such as hydroxyapatite and beta-tricalcium phosphate, and spongy bone, allows us to classify them as biomimetic in relation to physical structure and chemical composition (Giannoudis et al., 2005). This mimicry favors the differentiation of osteoprogenitor cells and the deposition of bone matrix, characterizing bioceramics as essentially *osteoconductive*. The porous structure of bioceramics, or even the crystalline structure of calcium sulfate, also allows neoangiogenesis, which is essential in the osteoconduction process. Bioceramics of interest in the bone tissue regeneration process are those classified as temporary, since they are gradually replaced by newly formed bone (Tormala et al., 1998). Calcium sulfate and beta-tricalcium phosphate do this. Resorption time varies depending on the bioceramic in question, but is generally consistent with the bone callus formation time, sustaining tissue regeneration as osteoconductive agents. Hydroxyapatite is not considered resorbable by many authors, since the resorption process of this bioceramic averages 5 years, which corresponds to the period of natural bone remodeling of the body. Therefore it is considered that this bioceramic is integrated to the newly formed bone tissue and its resorption occurs during the intrinsic remodeling of the

*Calcium Sulfate* is one of the synthetic biomaterials with a long history of clinical use as a graft substitute for bone regeneration (Peltier et al., 1957; Tay et al., 1999). The dihydrated form of calcium sulfate, also called "gypsum", presents a crystalline structure that is not very uniform, and is currently used as a raw material in a calcination process that results in hemi-hydrated calcium sulfate (CaSO4.½ H2O), also called "Plaster of Paris" (Peltier et al., 1957). Calcium sulfate presents optimal biocompatibility, with reports of sporadic cases of inflammatory reaction after its use, with good evolution and spontaneous resolution in most cases. The length of stay in the organism is 8 weeks on average, a relatively short time, yet sufficient for bone callus formation to begin (Coetzee , 1980; Kelly et al., 2001). Calcium sulfate has ample clinical application potential, including bone defects resulting from trauma or created surgically, such as osteotomies and resection of tumors (Finkemeier, 2002; Kelly et al., 2001), as well as spinal surgery, for filling or bone fusion (Hadjipavlou et al.,

More recently there was a proposal for the expansion of the clinical use of calcium sulfate as an *antibiotic release* agent, since it ensures high local concentration of the drug, avoiding its systemic circulation (Gogia et al., 2009). Reports demonstrate the control of osteomyelitis through the application of calcium sulfate pellets with antibiotics such as tobramycin,

natural bone graft substitutes, including collagen and chitosan.

**4.3 Bioceramics** 

tissue.

2001).

processing some proteins are maintained. But its main activity is conduction of bone formation. However, even the osteoconductive activity can be affected by the material processing. The freezing, drying and sterilization stages, normally using Gamma rays, end up weakening the graft structure, reducing its mechanical properties (Pelker & Friedlaender, 1987; Henman & Finlayson, 2000).

The advantages of allograft use include immediate availability in a sufficient quantity for any treatment and in varied forms, facilitating clinical handling of this graft (Nandi et al., 2010). Li and collaborators described allograft use in the treatment of malignant humeral resection in patient treated between 2005 and 2008, with bone regeneration occurring at 26.3 weeks on average (Li et al., 2011). In another study, Virolainen and collaborators performed a survey of 10 years of allograft use for the treatment of periprosthetic fractures. This type of fracture can entail some surgical complication, and in this case the fractures occur soon after the prosthesis implant surgery, while fractures occurring at a later stage usually result from osteolytic lesions or osteoporosis. In both cases there is bone impairment at the implant site, hindering corrective surgical treatment. There were 71 patients treated between 1999 and 2008 with the use of cortical allograft and stabilization of the site with metal implants, and the patients presented a bone union rate of 91%. Allograft use was considered adequate, allowing biomechanical stability of the site (Virolainen et al., 2010).

#### **4.2 Demineralized bone matrix**

An alternative to bone tissue regeneration induction is the use of demineralized bone matrix (DBM) (Pietrzak et al., 2005). This type of biomaterial, obtained by acid hydrolysis of the bone matrix, through the action of hydrochloric acid, basically presents osteoinductive properties (Tuli & Singh, 1978; Katz et al., 2009). Its principle of action is based on preservation of the trabeculated collagen structure of the matrix and of bone formation inductor proteins, even with the processing of the tissue, obtained preferentially from human or bovine bones. The use of DBM to replace grafts should be observed with restrictions, as it does not present osteoconductive properties, due to the absence of the calcified bone matrix, and osteogenic properties, since processing for demineralization ends up killing the cells initially present in the tissue.

Nowadays there is a wide variety of available forms of DBM, either rigid or malleable. One of their main applications is the treatment of unconsolidated fractures (Pietrzak et al., 2005), in addition to the filling of bone cysts and cavities (Docquier & Delloye, 2005) and long bone fractures (Tiedeman et al., 1995; Keating & McQueen, 2001). Pieske and collaborators presented data on 20 patients with unconsolidated diaphyseal long bone fractures, treated between the years 2000 and 2006. The patients received autogenous grafts (n=10) or demineralized bone matrix (n=10), with bone formation having been observed in all the patients treated with DBM, while 20% of the patients treated with autogenous graft did not obtain the expected result (Pieske et al., 2009). The use of demineralized bone matrix has also been indicated for treatments of arthrodesis of the spinal column on account of its bone formation inducing action, and there may be an association with osteoconductive graft substitutes (Morone and Boden, 1998; Park et al., 2009). However, one of the disadvantages of demineralized bone matrix is related to the significant variability of donor sources, and corresponding variability of results obtained (Pietrzak et al., 2005).

Therefore in spite of the *availability* of natural materials as autogenous grafts, allografts and demineralized bone matrix, some limitations of use or clinical disadvantages of these materials drive the development of new technologies for bone tissue regeneration. *Natural and synthetic bone graft substitutes* are available to perform this role. The synthetic bone graft substitutes include ceramic and polymeric biomaterials, while biopolymers represent natural bone graft substitutes, including collagen and chitosan.

#### **4.3 Bioceramics**

346 Tissue Regeneration – From Basic Biology to Clinical Application

processing some proteins are maintained. But its main activity is conduction of bone formation. However, even the osteoconductive activity can be affected by the material processing. The freezing, drying and sterilization stages, normally using Gamma rays, end up weakening the graft structure, reducing its mechanical properties (Pelker & Friedlaender,

The advantages of allograft use include immediate availability in a sufficient quantity for any treatment and in varied forms, facilitating clinical handling of this graft (Nandi et al., 2010). Li and collaborators described allograft use in the treatment of malignant humeral resection in patient treated between 2005 and 2008, with bone regeneration occurring at 26.3 weeks on average (Li et al., 2011). In another study, Virolainen and collaborators performed a survey of 10 years of allograft use for the treatment of periprosthetic fractures. This type of fracture can entail some surgical complication, and in this case the fractures occur soon after the prosthesis implant surgery, while fractures occurring at a later stage usually result from osteolytic lesions or osteoporosis. In both cases there is bone impairment at the implant site, hindering corrective surgical treatment. There were 71 patients treated between 1999 and 2008 with the use of cortical allograft and stabilization of the site with metal implants, and the patients presented a bone union rate of 91%. Allograft use was considered adequate,

An alternative to bone tissue regeneration induction is the use of demineralized bone matrix (DBM) (Pietrzak et al., 2005). This type of biomaterial, obtained by acid hydrolysis of the bone matrix, through the action of hydrochloric acid, basically presents osteoinductive properties (Tuli & Singh, 1978; Katz et al., 2009). Its principle of action is based on preservation of the trabeculated collagen structure of the matrix and of bone formation inductor proteins, even with the processing of the tissue, obtained preferentially from human or bovine bones. The use of DBM to replace grafts should be observed with restrictions, as it does not present osteoconductive properties, due to the absence of the calcified bone matrix, and osteogenic properties, since processing for demineralization ends

Nowadays there is a wide variety of available forms of DBM, either rigid or malleable. One of their main applications is the treatment of unconsolidated fractures (Pietrzak et al., 2005), in addition to the filling of bone cysts and cavities (Docquier & Delloye, 2005) and long bone fractures (Tiedeman et al., 1995; Keating & McQueen, 2001). Pieske and collaborators presented data on 20 patients with unconsolidated diaphyseal long bone fractures, treated between the years 2000 and 2006. The patients received autogenous grafts (n=10) or demineralized bone matrix (n=10), with bone formation having been observed in all the patients treated with DBM, while 20% of the patients treated with autogenous graft did not obtain the expected result (Pieske et al., 2009). The use of demineralized bone matrix has also been indicated for treatments of arthrodesis of the spinal column on account of its bone formation inducing action, and there may be an association with osteoconductive graft substitutes (Morone and Boden, 1998; Park et al., 2009). However, one of the disadvantages of demineralized bone matrix is related to the significant variability of donor sources, and

allowing biomechanical stability of the site (Virolainen et al., 2010).

corresponding variability of results obtained (Pietrzak et al., 2005).

1987; Henman & Finlayson, 2000).

**4.2 Demineralized bone matrix** 

up killing the cells initially present in the tissue.

*Bioceramics* are biocompatible biomaterials with a long history of clinical applications for bone regeneration. Among the advantages of these biomaterials we can cite their synthetic origin, eliminating the risk of autograft morbidity, or the risk of immunorejection and transmission of diseases of allografts or even biomaterials of human and animal origin. The structural similarity between some bioceramics, such as hydroxyapatite and beta-tricalcium phosphate, and spongy bone, allows us to classify them as biomimetic in relation to physical structure and chemical composition (Giannoudis et al., 2005). This mimicry favors the differentiation of osteoprogenitor cells and the deposition of bone matrix, characterizing bioceramics as essentially *osteoconductive*. The porous structure of bioceramics, or even the crystalline structure of calcium sulfate, also allows neoangiogenesis, which is essential in the osteoconduction process. Bioceramics of interest in the bone tissue regeneration process are those classified as temporary, since they are gradually replaced by newly formed bone (Tormala et al., 1998). Calcium sulfate and beta-tricalcium phosphate do this. Resorption time varies depending on the bioceramic in question, but is generally consistent with the bone callus formation time, sustaining tissue regeneration as osteoconductive agents. Hydroxyapatite is not considered resorbable by many authors, since the resorption process of this bioceramic averages 5 years, which corresponds to the period of natural bone remodeling of the body. Therefore it is considered that this bioceramic is integrated to the newly formed bone tissue and its resorption occurs during the intrinsic remodeling of the tissue.

*Calcium Sulfate* is one of the synthetic biomaterials with a long history of clinical use as a graft substitute for bone regeneration (Peltier et al., 1957; Tay et al., 1999). The dihydrated form of calcium sulfate, also called "gypsum", presents a crystalline structure that is not very uniform, and is currently used as a raw material in a calcination process that results in hemi-hydrated calcium sulfate (CaSO4.½ H2O), also called "Plaster of Paris" (Peltier et al., 1957). Calcium sulfate presents optimal biocompatibility, with reports of sporadic cases of inflammatory reaction after its use, with good evolution and spontaneous resolution in most cases. The length of stay in the organism is 8 weeks on average, a relatively short time, yet sufficient for bone callus formation to begin (Coetzee , 1980; Kelly et al., 2001). Calcium sulfate has ample clinical application potential, including bone defects resulting from trauma or created surgically, such as osteotomies and resection of tumors (Finkemeier, 2002; Kelly et al., 2001), as well as spinal surgery, for filling or bone fusion (Hadjipavlou et al., 2001).

More recently there was a proposal for the expansion of the clinical use of calcium sulfate as an *antibiotic release* agent, since it ensures high local concentration of the drug, avoiding its systemic circulation (Gogia et al., 2009). Reports demonstrate the control of osteomyelitis through the application of calcium sulfate pellets with antibiotics such as tobramycin,

Technologies Applied to Stimulate Bone Regeneration 349

vascularization of the treated area, confirming the osteoconductive action of hydroxyapatite (Giannoudis et al., 2005). The regeneration of defects of critical size and defects in long bones, created surgically or resulting from trauma, are general indications for its use, either pure or in association with -TCP. Hydroxyapatite can also be used in spinal fusion procedures. The report on the use of hydroxyapatite in orthopedic lesions, including the resection of bone tumors, and the treatment of cystic lesions in rheumatoid arthritis, without the occurrence of adverse reactions and with good clinical evolution of the patients, was

Bioactive glass, or *bioglass*, is a biocompatible bioceramic that allows good integration with newly formed tissue (Hench et al., 1971). It is basically composed of silica, sodium oxide, calcium oxide and phosphates. Some factors influence the integration of bioglass with the surrounding environment, such as composition of the biomaterial, pH of the environment, temperature, and porosity, directing its osteoconductive function (Nandi et al., 2010). Bioglass is indicated for filling bone cavities in general, in reconstructive surgery, including craniofacial defects, besides spinal column fusion procedures (Asano et al., 1994; Suominen

Among the polymeric, biocompatible and bioresorbable biomaterials used for bone tissue regeneration, the poly (L-lactic acid) (PLLA), poly (glycolic acid) (PGA), polycaprolactone (PCL) polyesters, and their copolymers, such as poli (D,L-lactic-co-glycolic acid) (PLGA) (Santos & Wada, 2007; Santos, 2010) deserve special emphasis. These polymers are often associated with bone formation induction proteins, such as BMPs or even with osteoconductive bioceramics, such as hydroxyapatite. One of the advantages of the use of polymers for tissue regeneration resides in the wide variety of possible applications, not just as graft substitutes, but also as fastening elements, including screws and plates. Bone tissue regeneration is guided by the polymer structure used, whereas proliferation induction and cellular differentiation are observed in these specific scaffolds (Ishaug-Riley et al., 1998; Santos et al., 2001; Santos et al., 2004). The PLGA copolymers implanted in bones induce bone tissue neoformation at the implant site, over a variable period of time, depending on

*Polymer/bioceramic composites* have the advantage of conferring on polymers the intrinsic biomechanical property of calcium phosphates, such as hydroxyapatite, favoring osteoconductive characteristic of the biomaterial (Hutmacher et al., 2007). Osteoblast cell cultures in porous PLLA/hydroxyapatite composites (PLLA-HA) enable cell proliferation, the lodging of cells throughout the scaffold of the biomaterial and the differentiation of these cells with synthesis of mineralized matrix (Ma et al., 2001). These results are corroborated by the study of Rizzi and collaborators with the biomaterial of PLA-HA and PCL-HA (Rizzi et al. 2001). HA induces the activity of the bone cells preferentially adhered

The application of HA-PLLA to two cases of mandibular reconstruction after tumor resection was published recently (Matsuo et al., 2010). The plates were designed with the use of computed tomography. In one of the cases there was association of the composite

the ratio of polyesters present in the copolymers (Reed & Gilding, 1981).

to these particles, exposed on the surface of the composite.

published by the group of Yoshikawa and collaborators (2009).

&, Kinnunen, 1996).

**4.4 Polymeric biomaterials** 

vancomycin and gentamicin (Bibbo & Patel, 2006; Chang et al., 2007). A randomized, prospective clinical study, published in 2010, presents data on local control of chronic osteomyelitis of long bones and cases of infection at non-bone consolidation sites. Thirty patients were treated, with half receiving calcium sulfate associated with tobramycin and the other half bone cement (polymethyl methacrylate) impregnated with antibiotic. The results demonstrated the mean follow-up of the patients for 38 months (ranging between 24 and 38 months) with the resolution of 86% of the cases in both experimental groups, concluding on the efficacy of calcium sulfate application in the local control of osteomyelitis (McKee et al., 2010).

In turn, *calcium phosphates* constitute bioceramics with a nanoparticulated physical structure, porous with pores of 100µm to favor the osteoconductive aspect of the biomaterial. The pore density can range between 40% and 60%, with Ca:P stequiometric ratio similar to spongy bone, imitating it (Gautier et al., 1998; Tanaka et al., 2008; Porter et al., 2009). Osteoconduction with calcium phosphate, often used in beta conformation, as *beta-tricalcium phosphate* (-TCP, CA3(PO4)2), results in resorption of the biomaterial and osseointegration of the treated region in approximately 12 weeks. The bioresorption process occurs through a combination of dissolution and osteoclastic resorption at the implant site (Dong et al., 2002).

The persistence of the biomaterial favors the treatment of cavities resulting from bone resection, filling of osteotomy regions, defects of critical size of the bone (Gaasbeek et al., 2005; Tanaka et al., 2008) or even spinal fusion. Le Huec and collaborators reported the use of -TCP for spinal fusion in 30 patients in association with bone graft, in comparison to another 24 patients treated with cortical allograft. The authors did not report pseudarthrosis and demonstrated the formation of bone callus 6 months after the -TCP implant, with full resorption in 2 years (Le Huec et al., 1997).

The physical properties of -TCP favor its association with liquids such as blood and bone marrow aspirate. In an experimental study with dogs, Bruder and collaborators demonstrated bone formation and the refinement of the bioceramic in association with mesenchymal cells obtained from bone marrow aspirate (Bruder et al., 1998). In 2007 the same author published, together with collaborators, the result of an experimental application of -TCP grafts in sheep for posterolateral fusion (Gupta et al., 2007). In this experiment the authors compared the results of the fusion process with the use of autograft, biomaterial enriched with mesenchymal cells, biomaterial associated with total bone marrow aspirate and pure biomaterial. The radiological findings, in line with histological data, demonstrated a high rate of bone formation after 6 months in the presence of autograft (25%) and in the presence of the biomaterial enriched with cells (33%), whereas the biomaterial associated with the total bone marrow aspirate presented a low rate of bone formation (8%) and no bone formation was observed with the use of pure biomaterial, reinforcing the need for association of characteristics such as osteogenesis for guided tissue regeneration.

Unlike beta-tricalcium phosphate, *hydroxyapatite* (Ca10(PO4)6(OH)2) a bioceramic with a low resorption rate and greater mechanical resistance, is commonly used in association with beta-tricalcium phosphate, in the proportion of 60/40 to improve osseointegration of the graft substitute (Balcik et al., 2007). The porosity of the biomaterial is essential for its action, requiring pores of 100-200µm, at a density of 60 to 65% for cellular lodging and

vancomycin and gentamicin (Bibbo & Patel, 2006; Chang et al., 2007). A randomized, prospective clinical study, published in 2010, presents data on local control of chronic osteomyelitis of long bones and cases of infection at non-bone consolidation sites. Thirty patients were treated, with half receiving calcium sulfate associated with tobramycin and the other half bone cement (polymethyl methacrylate) impregnated with antibiotic. The results demonstrated the mean follow-up of the patients for 38 months (ranging between 24 and 38 months) with the resolution of 86% of the cases in both experimental groups, concluding on the efficacy of calcium sulfate application in the local control of osteomyelitis

In turn, *calcium phosphates* constitute bioceramics with a nanoparticulated physical structure, porous with pores of 100µm to favor the osteoconductive aspect of the biomaterial. The pore density can range between 40% and 60%, with Ca:P stequiometric ratio similar to spongy bone, imitating it (Gautier et al., 1998; Tanaka et al., 2008; Porter et al., 2009). Osteoconduction with calcium phosphate, often used in beta conformation, as *beta-tricalcium phosphate* (-TCP, CA3(PO4)2), results in resorption of the biomaterial and osseointegration of the treated region in approximately 12 weeks. The bioresorption process occurs through a combination of dissolution and osteoclastic resorption at the implant site (Dong et al., 2002). The persistence of the biomaterial favors the treatment of cavities resulting from bone resection, filling of osteotomy regions, defects of critical size of the bone (Gaasbeek et al., 2005; Tanaka et al., 2008) or even spinal fusion. Le Huec and collaborators reported the use of -TCP for spinal fusion in 30 patients in association with bone graft, in comparison to another 24 patients treated with cortical allograft. The authors did not report pseudarthrosis and demonstrated the formation of bone callus 6 months after the -TCP implant, with full

The physical properties of -TCP favor its association with liquids such as blood and bone marrow aspirate. In an experimental study with dogs, Bruder and collaborators demonstrated bone formation and the refinement of the bioceramic in association with mesenchymal cells obtained from bone marrow aspirate (Bruder et al., 1998). In 2007 the same author published, together with collaborators, the result of an experimental application of -TCP grafts in sheep for posterolateral fusion (Gupta et al., 2007). In this experiment the authors compared the results of the fusion process with the use of autograft, biomaterial enriched with mesenchymal cells, biomaterial associated with total bone marrow aspirate and pure biomaterial. The radiological findings, in line with histological data, demonstrated a high rate of bone formation after 6 months in the presence of autograft (25%) and in the presence of the biomaterial enriched with cells (33%), whereas the biomaterial associated with the total bone marrow aspirate presented a low rate of bone formation (8%) and no bone formation was observed with the use of pure biomaterial, reinforcing the need for association of characteristics such as osteogenesis for guided tissue

Unlike beta-tricalcium phosphate, *hydroxyapatite* (Ca10(PO4)6(OH)2) a bioceramic with a low resorption rate and greater mechanical resistance, is commonly used in association with beta-tricalcium phosphate, in the proportion of 60/40 to improve osseointegration of the graft substitute (Balcik et al., 2007). The porosity of the biomaterial is essential for its action, requiring pores of 100-200µm, at a density of 60 to 65% for cellular lodging and

(McKee et al., 2010).

regeneration.

resorption in 2 years (Le Huec et al., 1997).

vascularization of the treated area, confirming the osteoconductive action of hydroxyapatite (Giannoudis et al., 2005). The regeneration of defects of critical size and defects in long bones, created surgically or resulting from trauma, are general indications for its use, either pure or in association with -TCP. Hydroxyapatite can also be used in spinal fusion procedures. The report on the use of hydroxyapatite in orthopedic lesions, including the resection of bone tumors, and the treatment of cystic lesions in rheumatoid arthritis, without the occurrence of adverse reactions and with good clinical evolution of the patients, was published by the group of Yoshikawa and collaborators (2009).

Bioactive glass, or *bioglass*, is a biocompatible bioceramic that allows good integration with newly formed tissue (Hench et al., 1971). It is basically composed of silica, sodium oxide, calcium oxide and phosphates. Some factors influence the integration of bioglass with the surrounding environment, such as composition of the biomaterial, pH of the environment, temperature, and porosity, directing its osteoconductive function (Nandi et al., 2010). Bioglass is indicated for filling bone cavities in general, in reconstructive surgery, including craniofacial defects, besides spinal column fusion procedures (Asano et al., 1994; Suominen &, Kinnunen, 1996).

#### **4.4 Polymeric biomaterials**

Among the polymeric, biocompatible and bioresorbable biomaterials used for bone tissue regeneration, the poly (L-lactic acid) (PLLA), poly (glycolic acid) (PGA), polycaprolactone (PCL) polyesters, and their copolymers, such as poli (D,L-lactic-co-glycolic acid) (PLGA) (Santos & Wada, 2007; Santos, 2010) deserve special emphasis. These polymers are often associated with bone formation induction proteins, such as BMPs or even with osteoconductive bioceramics, such as hydroxyapatite. One of the advantages of the use of polymers for tissue regeneration resides in the wide variety of possible applications, not just as graft substitutes, but also as fastening elements, including screws and plates. Bone tissue regeneration is guided by the polymer structure used, whereas proliferation induction and cellular differentiation are observed in these specific scaffolds (Ishaug-Riley et al., 1998; Santos et al., 2001; Santos et al., 2004). The PLGA copolymers implanted in bones induce bone tissue neoformation at the implant site, over a variable period of time, depending on the ratio of polyesters present in the copolymers (Reed & Gilding, 1981).

*Polymer/bioceramic composites* have the advantage of conferring on polymers the intrinsic biomechanical property of calcium phosphates, such as hydroxyapatite, favoring osteoconductive characteristic of the biomaterial (Hutmacher et al., 2007). Osteoblast cell cultures in porous PLLA/hydroxyapatite composites (PLLA-HA) enable cell proliferation, the lodging of cells throughout the scaffold of the biomaterial and the differentiation of these cells with synthesis of mineralized matrix (Ma et al., 2001). These results are corroborated by the study of Rizzi and collaborators with the biomaterial of PLA-HA and PCL-HA (Rizzi et al. 2001). HA induces the activity of the bone cells preferentially adhered to these particles, exposed on the surface of the composite.

The application of HA-PLLA to two cases of mandibular reconstruction after tumor resection was published recently (Matsuo et al., 2010). The plates were designed with the use of computed tomography. In one of the cases there was association of the composite

Technologies Applied to Stimulate Bone Regeneration 351

fractures were stabilized with metal implants suitable for each case. There was a followup on 213 patients, and a total of 249 fractures. According to the authors the collagenbased composite had the same performance observed for the autograft as regards fracture union rate and functional measurements, and is a possible treatment alternative

*Associations* of biomaterials, initially used as *scaffolds* for the conduction of bone formation, with tissue regeneration *inductor proteins,* are not just a promise for regenerative medicine, but are already taking shape as potential and usual clinical applications. At the same time associations with *osteoprogenitor cells* or bone marrow aspirate are also consolidating for the

Cells are the essential elements during repair and regeneration, with *stem cells* playing an important role in this process, as already mentioned previously. Nowadays there are a growing number of studies seeking therapeutic strategies and applications using stem cells to minimize clinical problems caused by injury or diseases in the bone tissue (Meyer, et al., 2006; Charbord, 2010), which present increasing demand, considering the demographic growth of the population and the rise in the number of elderly citizens, where the frequency

**6. Stem cells and their application to regeneration and to bioengineering of** 

Stem cells correspond to a group of *undifferentiated cells* with the capacity for unlimited selfrenewal, as they are capable of successive divisions throughout the entire lifetime of the organism. Moreover, these cells, once stimulated by specific signals and under ideal conditions, will be able to differentiate into cell types with specialized forms and functions and that will maintain the homeostasis of the body. Therefore the proliferative capacity associated with the potential to differentiate into different specific cell types, confer immense potential for application to different areas of biomedicine including gene therapy

Thus, the success of tissue engineering depends on the use of the appropriate cells, on the ability to predict the cell response and on culture techniques for proliferation and differentiation into specific cell types. Nowadays tissue engineering applications are allowing, among others, the use of cells from the actual patient (autologous cells), from donors (allogenic), from different species (xenogeneic), from immortalized lineages (both allogeneic and xenogeneic) and fetal and adult stem cells (Parenteau, 2002); which can be cultivated on molds of biocompatible materials, and subsequently implanted to the injured tissue or inoculated directly or onto the biomaterials at the implant sites. This methodology opens vast perspectives for application in the medical area, allowing the performance of graft implants in injured tissues leading to a greater benefit to the patient, with the initial use of a small number of cells, which will be expanded *in vitro* by means of culture techniques, and also due to the fact that it will be possible to either minimize or avoid immunological problems such as rejection of non-autogenous transplants (Calvert et al.,

of diseases in the musculoskeletal system is higher (De Peppo et al., 2010).

and tissue engineering on stem cells (Kirschstein & Skirboll, 2001).

(Chapman et al., 1997).

**bone tissue** 

refinement of the functions of these scaffolds.

**5. Stem cells and bone regeneration** 

biomaterial with growth factors obtained from platelets harvested from the patient and in the second case there was a dental graft. Both cases presented good clinical evolution, without the observation of bone resorption in two years of follow-up, and with the formation of good quality bone.

#### **4.5 Other biomaterials**

Besides the synthetic bioceramics and polymeric biomaterials, some biomaterials obtained in nature present considerable potential for application in bone regeneration: coralline hydroxyapatites and chitosan.

Similar to hydroxyapatite, *coralline hydroxyapatites* have been explored recently for their osteoconductive potential. They derive from marine corals, with a calcium carbonate base, a porous structure, and pore size ranging between 100 and 500µm, suitable for the proposed function. They can be obtained directly in nature (and processed mainly for sterilization), or obtained from hydroxyapatite (Keating & McQueen, 2001). Indications for use include long bone fractures and tibial plateau fractures, presenting a behavior similar to the autogenous graft (Bucholz et al., 1989).

*Chitosan* is another biocompatible biomaterial with potential for clinical application under analysis, and is considered very promising for the area of tissue regeneration. It is a natural biopolymer, obtained from the polysaccharide chitin, common in the exoskeleton of crustaceans (such as shrimps and lobsters). It presents encouraging results demonstrating its performance as an osteoconductive biomaterial guiding osseointegration. A study published in 2003 uses chitosan glutamate associated with hydroxyapatite for the treatment of defects of critical size in rat calvaria. The results were obtained after 9 and 18 weeks. The association with osteoprogenitor cells obtained from bone marrow proved ideal for tissue regeneration according to the protocol under investigation, including with mineralization of the treated areas (Mukherjee et al., 2003).

A recently published study (Jayasuriya & Kibbe, 2010) demonstrates the preparation of chitosan microparticles on a wide scale, and the incubation of these particles in concentrated physiological fluid for the stimulation of in vitro biomineralization and subsequent incorporation of insulin-like growth factor (IGF-1). The study evidenced the release of IGF-1 over a 30-day period, characterizing the possibility of the biomaterial's use as a drug release agent.

*Collagen,* in turn, exhibits a series of possible clinical applications, such as a scaffold for the regeneration of various tissues, including skin, cartilage and bone. It is a natural biopolymer, obtained from animal tissue, generally bovine, with low toxicity and immunogenicity. It can be made available in the form of gels, films and sponges, favoring cell adhesion and resorption, driving the regenerative process. In the case of bone regeneration, collagen is often associated with osteoconductive materials such as betatricalcium phosphate or hydroxyapatite (Wahl & Czernuszka, 2006). These composites aim to reproduce the natural conditions of bone and thus to drive cell behavior, with the differentiation of osteoblasts and the synthesis of mineralized bone matrix (Zhang et al., 2010). A randomized, prospective clinical study brings data on the clinical application of collagen biomaterial associated with calcium phosphate bioceramic in the treatment of long bone fractures, having the use of autogenous grafts as a form of control. The

biomaterial with growth factors obtained from platelets harvested from the patient and in the second case there was a dental graft. Both cases presented good clinical evolution, without the observation of bone resorption in two years of follow-up, and with the

Besides the synthetic bioceramics and polymeric biomaterials, some biomaterials obtained in nature present considerable potential for application in bone regeneration: coralline

Similar to hydroxyapatite, *coralline hydroxyapatites* have been explored recently for their osteoconductive potential. They derive from marine corals, with a calcium carbonate base, a porous structure, and pore size ranging between 100 and 500µm, suitable for the proposed function. They can be obtained directly in nature (and processed mainly for sterilization), or obtained from hydroxyapatite (Keating & McQueen, 2001). Indications for use include long bone fractures and tibial plateau fractures, presenting a behavior similar to the autogenous

*Chitosan* is another biocompatible biomaterial with potential for clinical application under analysis, and is considered very promising for the area of tissue regeneration. It is a natural biopolymer, obtained from the polysaccharide chitin, common in the exoskeleton of crustaceans (such as shrimps and lobsters). It presents encouraging results demonstrating its performance as an osteoconductive biomaterial guiding osseointegration. A study published in 2003 uses chitosan glutamate associated with hydroxyapatite for the treatment of defects of critical size in rat calvaria. The results were obtained after 9 and 18 weeks. The association with osteoprogenitor cells obtained from bone marrow proved ideal for tissue regeneration according to the protocol under investigation, including with mineralization of the treated

A recently published study (Jayasuriya & Kibbe, 2010) demonstrates the preparation of chitosan microparticles on a wide scale, and the incubation of these particles in concentrated physiological fluid for the stimulation of in vitro biomineralization and subsequent incorporation of insulin-like growth factor (IGF-1). The study evidenced the release of IGF-1 over a 30-day period, characterizing the possibility of the biomaterial's use as a drug release

*Collagen,* in turn, exhibits a series of possible clinical applications, such as a scaffold for the regeneration of various tissues, including skin, cartilage and bone. It is a natural biopolymer, obtained from animal tissue, generally bovine, with low toxicity and immunogenicity. It can be made available in the form of gels, films and sponges, favoring cell adhesion and resorption, driving the regenerative process. In the case of bone regeneration, collagen is often associated with osteoconductive materials such as betatricalcium phosphate or hydroxyapatite (Wahl & Czernuszka, 2006). These composites aim to reproduce the natural conditions of bone and thus to drive cell behavior, with the differentiation of osteoblasts and the synthesis of mineralized bone matrix (Zhang et al., 2010). A randomized, prospective clinical study brings data on the clinical application of collagen biomaterial associated with calcium phosphate bioceramic in the treatment of long bone fractures, having the use of autogenous grafts as a form of control. The

formation of good quality bone.

hydroxyapatites and chitosan.

graft (Bucholz et al., 1989).

areas (Mukherjee et al., 2003).

agent.

**4.5 Other biomaterials** 

fractures were stabilized with metal implants suitable for each case. There was a followup on 213 patients, and a total of 249 fractures. According to the authors the collagenbased composite had the same performance observed for the autograft as regards fracture union rate and functional measurements, and is a possible treatment alternative (Chapman et al., 1997).

*Associations* of biomaterials, initially used as *scaffolds* for the conduction of bone formation, with tissue regeneration *inductor proteins,* are not just a promise for regenerative medicine, but are already taking shape as potential and usual clinical applications. At the same time associations with *osteoprogenitor cells* or bone marrow aspirate are also consolidating for the refinement of the functions of these scaffolds.

#### **5. Stem cells and bone regeneration**

Cells are the essential elements during repair and regeneration, with *stem cells* playing an important role in this process, as already mentioned previously. Nowadays there are a growing number of studies seeking therapeutic strategies and applications using stem cells to minimize clinical problems caused by injury or diseases in the bone tissue (Meyer, et al., 2006; Charbord, 2010), which present increasing demand, considering the demographic growth of the population and the rise in the number of elderly citizens, where the frequency of diseases in the musculoskeletal system is higher (De Peppo et al., 2010).

#### **6. Stem cells and their application to regeneration and to bioengineering of bone tissue**

Stem cells correspond to a group of *undifferentiated cells* with the capacity for unlimited selfrenewal, as they are capable of successive divisions throughout the entire lifetime of the organism. Moreover, these cells, once stimulated by specific signals and under ideal conditions, will be able to differentiate into cell types with specialized forms and functions and that will maintain the homeostasis of the body. Therefore the proliferative capacity associated with the potential to differentiate into different specific cell types, confer immense potential for application to different areas of biomedicine including gene therapy and tissue engineering on stem cells (Kirschstein & Skirboll, 2001).

Thus, the success of tissue engineering depends on the use of the appropriate cells, on the ability to predict the cell response and on culture techniques for proliferation and differentiation into specific cell types. Nowadays tissue engineering applications are allowing, among others, the use of cells from the actual patient (autologous cells), from donors (allogenic), from different species (xenogeneic), from immortalized lineages (both allogeneic and xenogeneic) and fetal and adult stem cells (Parenteau, 2002); which can be cultivated on molds of biocompatible materials, and subsequently implanted to the injured tissue or inoculated directly or onto the biomaterials at the implant sites. This methodology opens vast perspectives for application in the medical area, allowing the performance of graft implants in injured tissues leading to a greater benefit to the patient, with the initial use of a small number of cells, which will be expanded *in vitro* by means of culture techniques, and also due to the fact that it will be possible to either minimize or avoid immunological problems such as rejection of non-autogenous transplants (Calvert et al.,

Technologies Applied to Stimulate Bone Regeneration 353

and there is a growing number of tissues and organs identified as carriers of the so-called mesenchymal stem cells (MSCs), including the bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelium of the skin and of the digestive system, cornea, retina, liver, pancreas and others, whereas the umbilical cord and the placenta are also carriers of cells similar to the mesenchymal stem cells. In spite of the fact that they have similar characteristics, MSCs of different origins present varied differentiation and gene expression potentials. Bone marrow is known to present considerable potential for obtaining stem cells and they have been studied with clinical and therapeutic objectives for fractures with substantial loss of bone mass and metabolic

Fig. 2. Summary diagram showing the capacity of mesenchymal stem cells (MSCs) to differentiate into bone, cartilage, muscle, tendons/ligaments and other tissues. Each stage of this differentiation and maturation process involves the control and the interaction of

The pioneer studies that evidenced the separation of a population of cells with the capacity to differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes and hematopoietic cells, were carried out by Friedenstein *et a*l, in the sixties (Friedenstein et. al., 1968). Their studies demonstrated the existence of precursor *mesenchymal cells*, with the potential to differentiate into osteoblasts and fibrous tissue (Figure 2) (Charbord, 2010;

diseases involving the bone tissue.

growth factors and cytokines (Caplan, 2010).

**9. Bone marrow stem cells** 

Hidalgo-Bastida et al., 2010).

2000; Temenoff & Mikos, 2000a,b). To this effect, several strategies are being applied to improve the efficiency of tissue engineering such as growth factors and recombinant differentiation factors, use of autologous cells, gene therapy through the incorporation of vectors and genetic engineering of cells (Satija et al., 2007).

#### **7. Embryonic stem cells**

The self-renewal capacity of *human embryonic stem cells (ESCs)* over prolonged periods and their ability to differentiate into different tissues from the three embryonic layers, were characterized by Thomson and collaborators (Thomson et al., 1998). These oocyte-derived cells fertilized in the morule phase or derived from the inner cell mass of embryos in the blastula phase, are able to divide in an unlimited manner, keeping their original characteristics and genetic information, besides being pluripotent, that is, they can differentiate into practically all cell types, derived from the three embryonic germ layers, mesoderm, ectoderm and endoderm (Figure 1) (Doetschman et al., 1985; Smith, 2001).

Fig. 1. Diagram showing pluripotent property of embryonic stem cells and their capacity to originate cell types from the three embryonic germ layers.

Although there is immense potential for the use of embryonic stem cells, due to their pluripotency, in practical terms their use is still very limited due to problems including cell regulation, immunological incompatibility, and possible development of neoplasias upon their administration (Passier & Mummery, 2003). These complications are also accentuated by ethical and religious issues and government regulations vis-à-vis the use of human embryonic cells in research (Zuk et al., 2001; Lee et al., 2003, Undale et al. 2009). Such factors led scientists to seek options with greater application potential, such as adult stem cells.

#### **8. Adult stem cells**

Although they decrease with age, *adult stem cells* are present in a wide variety of tissues throughout the lifetime of an individual. These cells, like the ESCs, also have the capacity for unlimited self-renewal and the potential to differentiate into cell types with specific morphologic and functional characteristics. This differentiation process generally involves intermediate cell types called precursor or progenitor cells that, although with a reduced self-renewal capacity, can split up to produce specific cell types (Robey, 2000; Gamradt & Lieberman, 2004). Accordingly, adult stem cells are being identified by different methods

2000; Temenoff & Mikos, 2000a,b). To this effect, several strategies are being applied to improve the efficiency of tissue engineering such as growth factors and recombinant differentiation factors, use of autologous cells, gene therapy through the incorporation of

The self-renewal capacity of *human embryonic stem cells (ESCs)* over prolonged periods and their ability to differentiate into different tissues from the three embryonic layers, were characterized by Thomson and collaborators (Thomson et al., 1998). These oocyte-derived cells fertilized in the morule phase or derived from the inner cell mass of embryos in the blastula phase, are able to divide in an unlimited manner, keeping their original characteristics and genetic information, besides being pluripotent, that is, they can differentiate into practically all cell types, derived from the three embryonic germ layers, mesoderm, ectoderm and endoderm (Figure 1) (Doetschman et al., 1985; Smith, 2001).

Fig. 1. Diagram showing pluripotent property of embryonic stem cells and their capacity to

Although there is immense potential for the use of embryonic stem cells, due to their pluripotency, in practical terms their use is still very limited due to problems including cell regulation, immunological incompatibility, and possible development of neoplasias upon their administration (Passier & Mummery, 2003). These complications are also accentuated by ethical and religious issues and government regulations vis-à-vis the use of human embryonic cells in research (Zuk et al., 2001; Lee et al., 2003, Undale et al. 2009). Such factors led scientists to seek options with greater application potential, such as adult stem cells.

Although they decrease with age, *adult stem cells* are present in a wide variety of tissues throughout the lifetime of an individual. These cells, like the ESCs, also have the capacity for unlimited self-renewal and the potential to differentiate into cell types with specific morphologic and functional characteristics. This differentiation process generally involves intermediate cell types called precursor or progenitor cells that, although with a reduced self-renewal capacity, can split up to produce specific cell types (Robey, 2000; Gamradt & Lieberman, 2004). Accordingly, adult stem cells are being identified by different methods

vectors and genetic engineering of cells (Satija et al., 2007).

originate cell types from the three embryonic germ layers.

**7. Embryonic stem cells** 

**8. Adult stem cells** 

and there is a growing number of tissues and organs identified as carriers of the so-called mesenchymal stem cells (MSCs), including the bone marrow, peripheral blood, brain, spinal cord, dental pulp, blood vessels, skeletal muscle, epithelium of the skin and of the digestive system, cornea, retina, liver, pancreas and others, whereas the umbilical cord and the placenta are also carriers of cells similar to the mesenchymal stem cells. In spite of the fact that they have similar characteristics, MSCs of different origins present varied differentiation and gene expression potentials. Bone marrow is known to present considerable potential for obtaining stem cells and they have been studied with clinical and therapeutic objectives for fractures with substantial loss of bone mass and metabolic diseases involving the bone tissue.

Fig. 2. Summary diagram showing the capacity of mesenchymal stem cells (MSCs) to differentiate into bone, cartilage, muscle, tendons/ligaments and other tissues. Each stage of this differentiation and maturation process involves the control and the interaction of growth factors and cytokines (Caplan, 2010).

#### **9. Bone marrow stem cells**

The pioneer studies that evidenced the separation of a population of cells with the capacity to differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes and hematopoietic cells, were carried out by Friedenstein *et a*l, in the sixties (Friedenstein et. al., 1968). Their studies demonstrated the existence of precursor *mesenchymal cells*, with the potential to differentiate into osteoblasts and fibrous tissue (Figure 2) (Charbord, 2010; Hidalgo-Bastida et al., 2010).

Technologies Applied to Stimulate Bone Regeneration 355

The expression of growth factors from the TGF family is crucial for bone repair in adults and has been described during the embryonic phase as essential for the development of cartilage and bones. TGF1 promotes the specific gene expression, initializing chain events with the participation of the SMAD proteins, which lead to the process of chondrogenesis

BMPs are important morphogens involved in the regulation of chondrogenesis and osteogenesis during normal embryonic development (Hogan, 1996). The effects of BMPs on MSCs has been investigated, demonstrating that the culture of MSCs in the presence of BMP2 increases alkaline phosphatase activity and osteocalcin expression, both indicators of osteoblast differentiation, whereas this effect is intensified in the presence of dexamethasone. Other factors are also known to influence the differentiation of MSCs, interacting at different levels with the metabolism of the Wnts and/or TGF/BMP. One of these factors is FGF-2 (fibroblast growth factor - 2), which promotes cell proliferation and maintains the populations of MSCs undifferentiated for prolonged periods of time (Martin

Different characteristics of MSCs, including their availability, potential for autologous use and absence of immunological rejection, make them very promising for clinical and therapeutic applications, especially in fractures with significant bone loss and metabolic diseases (Caplan, 2010). In spite of the major advances that have occurred in orthopedic surgery, fractures that involve considerable bone loss and non-union still represent a very important clinical problem. During the normal regeneration of a fracture, as seen previously, undifferentiated MSCs, with the assistance of BMPs and regulatory cytokines, proliferate and differentiate into chondrocytes and osteoblasts, which will form bone tissue reconstituting the lesion. Although related to the site where they occur, around 5 to 20% or more of fractures present failure in regeneration and consolidation (Kimelman et al., 2007; Undale, et al, 2009). Experiments on animal models using autologous MSCs and different scaffolds have resulted in bone regeneration (Arinzeh et al, 2003; Bruder et al. 1998a, b; Kon et al, 2000; Petite et al, 2000). Clinical studies on humans with the use of MSCs aspirated from the iliac crest and subsequently expanded in cultures on different biomaterials (Quarto et al., 2001; Marcacci et al, 2007), or percutaneously injected (Hernigou et al., 2005), have also been conducted, indicating clinical success positively correlated with greater capacity for *in vitro* formation of colonies and concentration of injected MSCs. Clinical applications in humans have also been described in patients with metabolic diseases of the bone tissue such as osteogenesis imperfecta and hypophosphatasia. Cultures of allogeneic MSCs and intravenous administration have mainly been used in these diseases, demonstrating the ability of these stem cells to stimulate bone mineralization and regeneration (Undale et al.,

Stem cells play a crucial role for the body's homeostasis, as they maintain the functional state of the tissues and also replace cells killed by injury or disease. These cells are very rare in the adult (Kirschstein & Skirboll, 2001). For example, it is estimated that in the bone

and differentiation of the MSCs (Tuli et al., 2003).

**10. Applications and clinical potential** 

**11. Mesenchymal stem cells of the adipose tissue** 

et al., 1997).

2009)

These stem cells in the muridae are frequently obtained from femoral or tibial flushing, obtaining the bone marrow mononuclear cells (BMMNCs) that are isolated by density gradient centrifugation then cultivated *in vitro*. On the other hand, human cells are aspirated from the iliac crest and cultivated directly, since gradient centrifugation techniques have not been seen to increase the separation efficiency and frequently present contamination with hematopoietic cells. This methodology is used to obtain populations that undergo a cloning process, and are characterized by the presence of both positive markers (Stro-1, CD29, CD73, CD90, CD105, CD166 and CD44) and negative markers (CD43, CD45, CD14, CD11b, CD19, CD79a and HLA-DR). The positive expression for Stro-1, identifies cells with osteogenic potential and expression of the three markers for osteoblast differentiation: alkaline phosphatase, 1,25-dihydroxy-vitamin D that has induction dependent on the specific bone protein: osteocalcin and hydroxyapatite production (mineralized matrix). Recent studies have pointed to other markers present in mesenchymal stem cells of the bone marrow that present osteogenic potential (Undale et al., 2009).

The molecular regulatory mechanism involved in the MSC differentiation control process has been extensively studied *in vitro*, whereas *in vivo* control is little known due to the difficulties inherent in the study process. However, the properties of the MSCs *in vivo* and *in vitro* vary according to the method of removal of these cells from their natural environment and the use of chemical and physical factors to keep them in culture, which can lead to alterations in their characteristics. Heterogeneity and diversity of types of MSCs and their ability to undergo phenotypic rearrangements in culture, modifies the expression of markers and hinders the comparison of data or renders it unfeasible in some situations (Augello & De Bari, 2010).

*In vitro*, the classical methodology to induce osteogenic differentiation in human MSCs consists of incubation with bovine fetal serum, in a medium supplemented with ascorbic acid, -glycerophosphate and dexamethasone, which leads to the increase of alkaline phosphatase and calcium deposition (Jaiswal et al, 1997; Pittenger et al. 1999).

On the other hand, *in vivo*, the information obtained from studies of embryonic development indicates that different signaling routes and transcription factors may play a critical role in the differentiation of MSCs. Different molecules have been described in the regulation of MSC differentiation, including Wnt and the TGF- superfamily.

Wnt proteins, coded by a family of 19 genes in humans and in mice, are involved in cell proliferation, differentiation and apoptosis. They act directly on MSCs, and are crucial for embryonic development and regeneration of different tissues in adults, including bone. Etheridge and collaborators (2004) demonstrated that MSCs express a series of ligands including Wnt2, Wnt4, Wnt5a, Wnt11, and Wnt16, and different Wnt receptors, FZD2, 3, 4, 5, and 6, as well as several co-receptors and inhibitors.

Several studies have shown that osteogenic differentiation *in vitro* is upregulated by some molecules related to the Wnt family and downregulated by others. For example, the administration of exogenous Wnt3 leads to osteogenesis repression. The TGF superfamily represents a set of growth factors and morphogens that play a role in skeletogenesis and in postnatal skeletal homeostasis. The TGF superfamily of ligands includes BMPs, growth and differentiation factors (GDFs), anti-mullerian hormone (AMH), activin, nodal, and TGF (Piek et al., 1999; Derynck & Miyazono, 2008).

These stem cells in the muridae are frequently obtained from femoral or tibial flushing, obtaining the bone marrow mononuclear cells (BMMNCs) that are isolated by density gradient centrifugation then cultivated *in vitro*. On the other hand, human cells are aspirated from the iliac crest and cultivated directly, since gradient centrifugation techniques have not been seen to increase the separation efficiency and frequently present contamination with hematopoietic cells. This methodology is used to obtain populations that undergo a cloning process, and are characterized by the presence of both positive markers (Stro-1, CD29, CD73, CD90, CD105, CD166 and CD44) and negative markers (CD43, CD45, CD14, CD11b, CD19, CD79a and HLA-DR). The positive expression for Stro-1, identifies cells with osteogenic potential and expression of the three markers for osteoblast differentiation: alkaline phosphatase, 1,25-dihydroxy-vitamin D that has induction dependent on the specific bone protein: osteocalcin and hydroxyapatite production (mineralized matrix). Recent studies have pointed to other markers present in mesenchymal stem cells of the bone marrow that

The molecular regulatory mechanism involved in the MSC differentiation control process has been extensively studied *in vitro*, whereas *in vivo* control is little known due to the difficulties inherent in the study process. However, the properties of the MSCs *in vivo* and *in vitro* vary according to the method of removal of these cells from their natural environment and the use of chemical and physical factors to keep them in culture, which can lead to alterations in their characteristics. Heterogeneity and diversity of types of MSCs and their ability to undergo phenotypic rearrangements in culture, modifies the expression of markers and hinders the comparison of data or renders it unfeasible in some situations (Augello &

*In vitro*, the classical methodology to induce osteogenic differentiation in human MSCs consists of incubation with bovine fetal serum, in a medium supplemented with ascorbic acid, -glycerophosphate and dexamethasone, which leads to the increase of alkaline

On the other hand, *in vivo*, the information obtained from studies of embryonic development indicates that different signaling routes and transcription factors may play a critical role in the differentiation of MSCs. Different molecules have been described in the

Wnt proteins, coded by a family of 19 genes in humans and in mice, are involved in cell proliferation, differentiation and apoptosis. They act directly on MSCs, and are crucial for embryonic development and regeneration of different tissues in adults, including bone. Etheridge and collaborators (2004) demonstrated that MSCs express a series of ligands including Wnt2, Wnt4, Wnt5a, Wnt11, and Wnt16, and different Wnt receptors, FZD2, 3, 4,

Several studies have shown that osteogenic differentiation *in vitro* is upregulated by some molecules related to the Wnt family and downregulated by others. For example, the administration of exogenous Wnt3 leads to osteogenesis repression. The TGF superfamily represents a set of growth factors and morphogens that play a role in skeletogenesis and in postnatal skeletal homeostasis. The TGF superfamily of ligands includes BMPs, growth and differentiation factors (GDFs), anti-mullerian hormone (AMH), activin, nodal, and

phosphatase and calcium deposition (Jaiswal et al, 1997; Pittenger et al. 1999).

regulation of MSC differentiation, including Wnt and the TGF- superfamily.

5, and 6, as well as several co-receptors and inhibitors.

TGF (Piek et al., 1999; Derynck & Miyazono, 2008).

present osteogenic potential (Undale et al., 2009).

De Bari, 2010).

The expression of growth factors from the TGF family is crucial for bone repair in adults and has been described during the embryonic phase as essential for the development of cartilage and bones. TGF1 promotes the specific gene expression, initializing chain events with the participation of the SMAD proteins, which lead to the process of chondrogenesis and differentiation of the MSCs (Tuli et al., 2003).

BMPs are important morphogens involved in the regulation of chondrogenesis and osteogenesis during normal embryonic development (Hogan, 1996). The effects of BMPs on MSCs has been investigated, demonstrating that the culture of MSCs in the presence of BMP2 increases alkaline phosphatase activity and osteocalcin expression, both indicators of osteoblast differentiation, whereas this effect is intensified in the presence of dexamethasone. Other factors are also known to influence the differentiation of MSCs, interacting at different levels with the metabolism of the Wnts and/or TGF/BMP. One of these factors is FGF-2 (fibroblast growth factor - 2), which promotes cell proliferation and maintains the populations of MSCs undifferentiated for prolonged periods of time (Martin et al., 1997).

#### **10. Applications and clinical potential**

Different characteristics of MSCs, including their availability, potential for autologous use and absence of immunological rejection, make them very promising for clinical and therapeutic applications, especially in fractures with significant bone loss and metabolic diseases (Caplan, 2010). In spite of the major advances that have occurred in orthopedic surgery, fractures that involve considerable bone loss and non-union still represent a very important clinical problem. During the normal regeneration of a fracture, as seen previously, undifferentiated MSCs, with the assistance of BMPs and regulatory cytokines, proliferate and differentiate into chondrocytes and osteoblasts, which will form bone tissue reconstituting the lesion. Although related to the site where they occur, around 5 to 20% or more of fractures present failure in regeneration and consolidation (Kimelman et al., 2007; Undale, et al, 2009). Experiments on animal models using autologous MSCs and different scaffolds have resulted in bone regeneration (Arinzeh et al, 2003; Bruder et al. 1998a, b; Kon et al, 2000; Petite et al, 2000). Clinical studies on humans with the use of MSCs aspirated from the iliac crest and subsequently expanded in cultures on different biomaterials (Quarto et al., 2001; Marcacci et al, 2007), or percutaneously injected (Hernigou et al., 2005), have also been conducted, indicating clinical success positively correlated with greater capacity for *in vitro* formation of colonies and concentration of injected MSCs. Clinical applications in humans have also been described in patients with metabolic diseases of the bone tissue such as osteogenesis imperfecta and hypophosphatasia. Cultures of allogeneic MSCs and intravenous administration have mainly been used in these diseases, demonstrating the ability of these stem cells to stimulate bone mineralization and regeneration (Undale et al., 2009)

#### **11. Mesenchymal stem cells of the adipose tissue**

Stem cells play a crucial role for the body's homeostasis, as they maintain the functional state of the tissues and also replace cells killed by injury or disease. These cells are very rare in the adult (Kirschstein & Skirboll, 2001). For example, it is estimated that in the bone

Technologies Applied to Stimulate Bone Regeneration 357

The proliferation capacity of MCSs is a measure of the number of cell divisions that can occur *in vitro* after the culture has been started. Many studies suggest that MCSs have doubling capacity of up to 50 times; after this period the culture is characterized by alteration of a series of cellular characteristics and properties, followed by senescence or even cell transformation. The senescence process is characterized by modifications to morphology and increase of cell volume, reduction in surface marker expression and decrease in differentiation potential. Several molecular mechanisms have already been identified in the senescence process, including DNA injury, accumulation of the cyclindependent kinase inhibitor, oxidative stress, telomeric modifications, action of epigenetic

Accordingly, the safe and efficient clinical application of stem cells to bone tissue regeneration depends on the elucidation of mechanisms associated with senescence. Moreover, it is essential to understand the mechanisms of action and interaction with other cell types, with different biomaterials, soluble factors, extracellular matrix components (Hidalgo-Bastida et al, 2010) and biochemical and mechanical agents present in the microenvironment *in vitro* and *in vivo*, as well as to keep the proliferation of stem cells restricted to the implanted site and to know the gene control mechanism for safe induction of the desired functions (Gronthos et al., 2000; Discher et al., 2009). The identification of growth factors and the signaling mechanisms involved in the actual control of stem cell renewal and differentiation will allow the design of strategies to block senescence and to safely drive

Andriano, K.P.; Chandrashekar, B.; McEnery, K.; Dunn, R.L.; Moyer, K.; Balliu, C.M.;

puttylike polymer matrix. J. Biomed. Mater. Res., Vol. 53, No.1, pp.36-43. Arinzeh, T.L.; Peter, S.J.; Archambault, M.P.; van den Bos, C.; Gordon, S.; Kraus, K.; Smith,

Arrington, E.D.; Smith, W.J.; Chambers, H.G.; Bucknell, A.L.; Davino, N.A. (1996)

Asano, S.; Kaneda, K.; Satoh, S.; Abumi, K.; Hashimoto, T.; Fujiya, M. (1994) Reconstruction

Augello, A.; De Bari, C. (2010) The regulation of differentiation in mesenchymal stem cells.

Aust, L.; Devlin, B.; Foster, S.J.; Halvorsen, Y.D.; Hicok, K.; Du Laney, T.; Sen, A.;

Balcik, C.; Tokdemir, T.; Senköylü, A.; Koç, N.; Timuçin, M.; Akin, S, Korkusuz P.;

cells from liposuction aspirates. Cytotherapy, Vol.6, No. 1, pp.7-14.

Human Gene Therapy, Vol. 21, No. 10, pp.1226–1238.

Holland, K.M.; Garrett, S.; Huffer, W.E. (2000) Preliminary in vivo studies on the osteogenic potential of bone morphogenetic proteins delivered from an absorbable

A.; Kadiyala, S. (2003) Allogeneic mesenchymal stem cells regenerate bone in a critical-sized canine segmental defect. J. Bone Joint Surg. Am. Vol., Vol. 85-A, No.

Complications of iliac crest bone graft harvesting. Clin. Orthop. Rel. Res., Vol. 329,

of an iliac crest defect with a bioactive ceramic prosthesis. Eur. Spine J., Vol.3, No.

Willingmyre, G.D.; Gimble, J.M. (2004) Yield of human adipose-derived adult stem

Korkusuz, F. (2007) Early weight bearing of porous HA/TCP (60/40) ceramics in

factors, and others (Wagner et al, 2010).

cellular differentiation (Satija et al, 2007).

10, pp.1927-1935.

pp.300-309.

1, pp.39-44.

**13. References** 

marrow only one among ten to fifteen thousand cells is a hematopoietic source cell (Weissman, 2000).

Although the bone marrow is the place where the presence and the differentiation process of MSCs is currently best known and characterized, they are also found in other places (Gamradt & Lieberman, 2004).

Studies have indicated that MSCs are also found in animal (Lee et al., 2002) and human (Zuk et al., 2002) *adipose tissue*, and can be obtained by the lipoaspiration process. They are frequently referred to in literature as PLA (processed lipoaspirative) or ADAS (adiposederived adult stem cells). Different studies have evidenced that mesenchymal stem cells obtained from the adipose tissue, when stimulated by different factors, can also differentiate into adipose cells (Halbleib et al., 2003), osteoblasts (Hicok et al., 2004), chondroblasts, myocytes and neural cells, which means that they draw great interest for applications in regenerative medicine and in tissue engineering (Barry & Murphy, 2004; Ogawa et al., 2004a,b). Since they are easily and abundantly obtained by lipoaspirative process, which is therefore less invasive, using local anesthesia, mesenchymal cells from the adipose tissue offer advantages over the bone marrow (Mizuno & Hyakusoku, 2003; Macleod et al, 2010). In the latter, the obtainment of mesenchymal cells is generally performed using aspiration and flushing of the upper part of the iliac crest, involving a process that is extremely painful for the patient, with the risk of a general or spinal anesthesia, usually implying morbidity of the donor site, resulting in a small number of functional cells. Now the obtainment of mesenchymal cells from the adipose tissue has presented more homogeneous populations with normal karyotype, and can be kept *in vitro* for long periods, with constancy in the cell doubling time and low levels of senescence (Zuk et al., 2002; Aust et al., 2004). Comparative studies between the mesenchymal cells of the bone marrow and of adipose tissue have shown that they both exhibit similarity in their ability to differentiate into adipose cells, from the bone, cartilaginous and muscle tissues; share similarities in the kinetics of growth and senescence, with the capacity for gene transduction and also among the cell surface markers (Mizuno & Hyakusoku, 2003; De Urgate et al., 2003a,b; Mosna et al., 2010)

Thus the autologous mesenchymal stem cells of the adipose tissue are also being used in the construction of three-dimensional scaffolds and applied to patients with severe problems of bone mass loss (Gamradt & Lieberman, 2004).

#### **12. Stem cells and gene therapy: prospects of future applications**

*Genetic engineering of adult stem cells* with genes presenting osteogenic potential has gained considerable emphasis in the repair of fractures and bone tissue formation. Studies have indicated that these genetically modified cells can produce autocrine and paracrine effects on the stem cells present in the actual patient, leading to a greater response in the osteogenic effect. These strategies involve both the use of viral and non-viral vectors, presenting genes that code different BMPs, as well as genetically modified cells containing these implanted transgenes. The advance of these studies may be essential for the future prospects of clinical use of stem cells for bone regeneration (Kimelman et al, 2007), bringing more efficient solutions in the field of orthopedics.

The proliferation capacity of MCSs is a measure of the number of cell divisions that can occur *in vitro* after the culture has been started. Many studies suggest that MCSs have doubling capacity of up to 50 times; after this period the culture is characterized by alteration of a series of cellular characteristics and properties, followed by senescence or even cell transformation. The senescence process is characterized by modifications to morphology and increase of cell volume, reduction in surface marker expression and decrease in differentiation potential. Several molecular mechanisms have already been identified in the senescence process, including DNA injury, accumulation of the cyclindependent kinase inhibitor, oxidative stress, telomeric modifications, action of epigenetic factors, and others (Wagner et al, 2010).

Accordingly, the safe and efficient clinical application of stem cells to bone tissue regeneration depends on the elucidation of mechanisms associated with senescence. Moreover, it is essential to understand the mechanisms of action and interaction with other cell types, with different biomaterials, soluble factors, extracellular matrix components (Hidalgo-Bastida et al, 2010) and biochemical and mechanical agents present in the microenvironment *in vitro* and *in vivo*, as well as to keep the proliferation of stem cells restricted to the implanted site and to know the gene control mechanism for safe induction of the desired functions (Gronthos et al., 2000; Discher et al., 2009). The identification of growth factors and the signaling mechanisms involved in the actual control of stem cell renewal and differentiation will allow the design of strategies to block senescence and to safely drive cellular differentiation (Satija et al, 2007).

#### **13. References**

356 Tissue Regeneration – From Basic Biology to Clinical Application

marrow only one among ten to fifteen thousand cells is a hematopoietic source cell

Although the bone marrow is the place where the presence and the differentiation process of MSCs is currently best known and characterized, they are also found in other places

Studies have indicated that MSCs are also found in animal (Lee et al., 2002) and human (Zuk et al., 2002) *adipose tissue*, and can be obtained by the lipoaspiration process. They are frequently referred to in literature as PLA (processed lipoaspirative) or ADAS (adiposederived adult stem cells). Different studies have evidenced that mesenchymal stem cells obtained from the adipose tissue, when stimulated by different factors, can also differentiate into adipose cells (Halbleib et al., 2003), osteoblasts (Hicok et al., 2004), chondroblasts, myocytes and neural cells, which means that they draw great interest for applications in regenerative medicine and in tissue engineering (Barry & Murphy, 2004; Ogawa et al., 2004a,b). Since they are easily and abundantly obtained by lipoaspirative process, which is therefore less invasive, using local anesthesia, mesenchymal cells from the adipose tissue offer advantages over the bone marrow (Mizuno & Hyakusoku, 2003; Macleod et al, 2010). In the latter, the obtainment of mesenchymal cells is generally performed using aspiration and flushing of the upper part of the iliac crest, involving a process that is extremely painful for the patient, with the risk of a general or spinal anesthesia, usually implying morbidity of the donor site, resulting in a small number of functional cells. Now the obtainment of mesenchymal cells from the adipose tissue has presented more homogeneous populations with normal karyotype, and can be kept *in vitro* for long periods, with constancy in the cell doubling time and low levels of senescence (Zuk et al., 2002; Aust et al., 2004). Comparative studies between the mesenchymal cells of the bone marrow and of adipose tissue have shown that they both exhibit similarity in their ability to differentiate into adipose cells, from the bone, cartilaginous and muscle tissues; share similarities in the kinetics of growth and senescence, with the capacity for gene transduction and also among the cell surface

markers (Mizuno & Hyakusoku, 2003; De Urgate et al., 2003a,b; Mosna et al., 2010)

**12. Stem cells and gene therapy: prospects of future applications** 

bone mass loss (Gamradt & Lieberman, 2004).

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*Genetic engineering of adult stem cells* with genes presenting osteogenic potential has gained considerable emphasis in the repair of fractures and bone tissue formation. Studies have indicated that these genetically modified cells can produce autocrine and paracrine effects on the stem cells present in the actual patient, leading to a greater response in the osteogenic effect. These strategies involve both the use of viral and non-viral vectors, presenting genes that code different BMPs, as well as genetically modified cells containing these implanted transgenes. The advance of these studies may be essential for the future prospects of clinical use of stem cells for bone regeneration (Kimelman et al, 2007), bringing more efficient

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use of 71 cortical allografts (strut-grafts) for the treatment of periprosthetic

**17** 

**Preparation of Deproteinized Human Bone and** 

**Phosphate – Innovative Bioactive Materials for** 

Anna Ślósarczyk5, Maria Borczuch-Łączka6 and Aleksander Owczarek7

**Its Mixtures with Bio-Glass and Tricalcium** 

Magdalena Cieslik1, Jacek Nocoń2, Jan Rauch3, Tadeusz Cieslik4,

**Skeletal Tissue Regeneration** 

*1Faculty and Institute of Stomatological Materials Science,* 

*5Faculty of Glass Technology and Amorphous Coatings,* 

*AGH - Krakow University of Science and Technology, Kraków,* 

*AGH - Krakow University of Science and Technology, Kraków,* 

*7Division of Statistics, Medical University of Silesia, Katowice, Sosnowiec,* 

Repair of the skeletal system is one of the principal research problems in medical science is closely associated with the field of material engineering. The reasons for using bone implants and grafts include injuries, infections, neoplasms and other hard tissue lesions. Bone replacement materials are predominantly used in medical disciplines such as dentistry, dental surgery, maxillofacial surgery and plastic surgery, as well as in orthopedics

From a biological, immunological, and legal point of view, autogenous bone grafting still remains a very popular method in reconstruction following skeletal loss (Block, 2002; Giannoudis et al., 2005). Factors considered in the selection of the source of the bone graft include, among others, the ease of surgical access and the volume of bone mass required (Precheur, 2007). The type of autogenous bone used as a graft (cortical bone vs. cancellous bone) should also be considered. For instance, the higher content of morphogenetic proteins (BMPs) in cortical bone means that grafts of this type induce the process of bone growth more effectively than cancellous bone grafts. Nonetheless, skeletal reconstruction with autogenous bone grafts always requires additional surgical manipulations that constitute an

and traumatology (Barradas et al., 2011; Kao & Scott, 2007; Precheur, 2007).

**1. Introduction** 

*Medical University of Silesia, Katowice, Bytom, 2Private Dentistry Practice, Oberhausen,* 

*3NZOZ – Specialist Dentistry Clinic, Wadowice, 4Faculty and Clinic of Oral and Maxillofacial Surgery,* 

*Medical University of Silesia, Katowice,* 

*6Faculty of Ceramic Technology,* 

*1,3,4,5,6,7Poland 2Germany* 

### **Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration**

Magdalena Cieslik1, Jacek Nocoń2, Jan Rauch3, Tadeusz Cieslik4, Anna Ślósarczyk5, Maria Borczuch-Łączka6 and Aleksander Owczarek7 *1Faculty and Institute of Stomatological Materials Science, Medical University of Silesia, Katowice, Bytom, 2Private Dentistry Practice, Oberhausen, 3NZOZ – Specialist Dentistry Clinic, Wadowice, 4Faculty and Clinic of Oral and Maxillofacial Surgery, Medical University of Silesia, Katowice, 5Faculty of Glass Technology and Amorphous Coatings, AGH - Krakow University of Science and Technology, Kraków, 6Faculty of Ceramic Technology, AGH - Krakow University of Science and Technology, Kraków, 7Division of Statistics, Medical University of Silesia, Katowice, Sosnowiec, 1,3,4,5,6,7Poland 2Germany* 

#### **1. Introduction**

Repair of the skeletal system is one of the principal research problems in medical science is closely associated with the field of material engineering. The reasons for using bone implants and grafts include injuries, infections, neoplasms and other hard tissue lesions. Bone replacement materials are predominantly used in medical disciplines such as dentistry, dental surgery, maxillofacial surgery and plastic surgery, as well as in orthopedics and traumatology (Barradas et al., 2011; Kao & Scott, 2007; Precheur, 2007).

From a biological, immunological, and legal point of view, autogenous bone grafting still remains a very popular method in reconstruction following skeletal loss (Block, 2002; Giannoudis et al., 2005). Factors considered in the selection of the source of the bone graft include, among others, the ease of surgical access and the volume of bone mass required (Precheur, 2007). The type of autogenous bone used as a graft (cortical bone vs. cancellous bone) should also be considered. For instance, the higher content of morphogenetic proteins (BMPs) in cortical bone means that grafts of this type induce the process of bone growth more effectively than cancellous bone grafts. Nonetheless, skeletal reconstruction with autogenous bone grafts always requires additional surgical manipulations that constitute an

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 371

tricalcium phosphate (betaTCP, betaCa3(PO4)2) is another calcium phosphate that has been used successfully in bone substitution. Its mineralogical analogue is whitlockite (TCP) (Barradas et al., 2011). Similar to HAp, TCP is characterized by high biocompatibility. In comparison to hydroxyapatite materials, it has a higher solubility *in vitro* and a resultant higher susceptibility to resorption and biodegradation in the environment of the living organism (Bohner, 2010; Henkel et al., 2006). TCP is considered an osteoinductive material, which stimulates the processes of bone reconstruction (Wang et al., 2009). Many studies, mostly dealing with the gradual, controlled resorption rate of calcium phosphate ceramic, resulted in the design of the second polymorphic variant of TCP, namely αTCP (Zima et al., 2010). Similar to beta-TCP, it is formed as a result of the non-stoichiometric heating of HAp with a well-defined temperature and defined kinetics of thermal processing. Compared to beta-TCP, αTCP has an approximately five-fold higher susceptibility to resorption in living tissues, along with higher biocompatibility (Oonishi et al., 1999). The high degree of osteointegration of αTCP-based bioceramic is plausibly the result of the higher solubility of αCa3(PO4)2 than of HAp or βTCP. Delivered within the ceramic, calcium and phosphate ions constitute the material for synthesizing the layer composed of non-stoichiometric hydroxyapatite that binds the implant to bone (Kon et al., 1995). Some authors suggest that αTCP can be cytotoxic, probably due to pH changes that are induced *in vitro* (Santos et al., 2002). These suggestions, however, were not confirmed during *in vivo* studies, and αTCP is included in many commercially available bone cements. Since the 1990s, biphasic HApβTCP ceramic (BCP biphasic calcium phosphate) has also been used in the reconstruction of skeletal defects (Deculsi, 1998, Deculsi et al., 2003; Fellah et al., 2008; Schwarz et al., 2007).

Bio-glass and apatite-wollastonite glass-ceramic also play an important role amongst the bioactive materials used in the processes of bone synthesis. The biological activity of glass and glass-derived crystalline materials (glass-ceramic materials) is mostly exploited for the manufacture of surface-active implants or implants that can be resorbed in the human body (Giannoudis et al., 2005). The composition of surface-active materials is selected in order to enable interactions between the physiological environment and certain components of the implant. As a result, living tissue is bound to the implant surface. Resorbable glass is characterized by its high content of chemical elements involved in metabolic processes of the human body. In both cases, living tissues can infiltrate the implant and bind it directly to the bone. These processes result not only from the chemical composition of the implants but also from the specific nature of the contained glass substance. The binding abilities of bioactive glass and glass-ceramic implants to skeletal tissue have been the subject of many studies. These studies confirmed the usefulness of such materials in tissue engineering, where they can be used in cell culture (Ferreira, 2007; Xynos et al., 2000). Their application in reconstructive surgery is reflected by their ability to stimulate and supporting bone reconstruction. In view of their confirmed ability to directly bind to skeletal tissue, they have been used successfully in various stomatological disciplines, including implantology, dental surgery and periodontology. Moreover, modern surface engineering has allowed the coating of metal implants with bioactive glass. Such coatings either protect surfaces of metal alloys against corrosion and wear, or stimulate the processes of bone formation in their surroundings. Additionally, resorbable glass can be used as a drug carrier, providing prolonged release of an active substance. Furthermore, some attempts have been made to combine various materials with each other to manufacture glass-containing bioactive composites, used as the components of binders, among others (Bellucci et al., 2011). These

increased burden to the patient and prolong the duration of the procedure. Another very frequent limitation of using autogenous bone is its poor quality, when there is a skeletal system disorder (e.g. osteoporosis) (Bohner, 2010; Giannoudis et al., 2005). Allogenic implants derived from the structures of human bones can be one alternative to autogenous grafts (Ferreira, 2007). Mineralized (FDBA) and demineralized (DFDBA) forms of such implants, additionally subjected to lyophilization, are most frequently used in reconstructive surgery. An advantage of demineralized bone arises from the fact that the organic bone matrix (collagen fibers) has to be exposed in order to remove its mineral components, and therefore so-called matrix proteins (e.g. morphogenetic proteins) can easily diffuse into the implantation site and work osteoinductively (Barradas et al., 2011). Xenogenic implants also play an important role in reconstructive bone surgery. These implants are made of skeletal material obtained from animals (Merkx et al., 2003), in most cases from equine, bovine or porcine bones. Animal material is processed by means of thermal treatment in order to deplete it completely of its organic components (Barakat et al., 2009). As a result, implants lose their immunogenic properties and become neutral to hosts (Liu et al., 2008). Therefore, ready to use preparations for bone replacement (Bio-Oss®, Endobone®) are most frequently available in deproteinized forms (DBBM) (Accorsi-Mendonça et al., 2008). Due to their osteoconductive properties they can serve as an inactive scaffold or platform for the maturation of bone cells present within the defect. They are used in orthopedics, dental and maxillofacial surgery, as well as in periodontology and implantology, etc. (Baldini et al., 2011; Cao et al., 2009; Jian et al., 2008; Merkx et al., 2003; Precheur, 2007; von Wattenwyl et al., 2011). The limited possibilities of modulating the resorption time of such preparations during skeletal tissue reconstruction, however, can be related to the poorer quality of bone at the site of their application.

An alternative solution, eliminating the potential complications associated with the application of materials of autogenous, allogenic or xenogenic origin, is the use of alloplastic implants for the purpose of bone replacement. Such implants can be synthesized from both natural and synthetic materials (Bohner, 2010; Giannoudis et al., 2005). Bioresorbable ceramic based on calcium phosphate plays a distinct role amongst novel synthetic materials used for bone replacement (Barradas et al., 2011). Hydroxyapatite (HAp) is the principal representative of this group, with the widest application in reconstructive surgery. Due to its calcium phosphate content and natural occurrence as an inorganic substance in bones and teeth, hydroxyapatite is characterized by the highest biocompatibility and bioactivity of all currently known implant materials. Additionally, due to the osteoconductive properties of hydroxyapatite, and (to a lesser extent) its osteoinductive properties, hydroxyapatitebased implants can bind directly to bone. Numerous clinical trials, supported by the results of histological observations, have confirmed complete biotolerance to hydroxyapatite ceramic, as well as its positive effects on the process of bone healing and reconstruction. Additionally, hydroxyapatite ceramic can initiate and stimulate various processes that are associated with bone formation (Bellucci et al., 2011; Ravarian et al., 2010; Yuan et al., 2001). Contact between bioactive ceramic and living skeletal tissue induces osteogenesis. As a result, an intermediate binding layer is formed between the living tissue and the implant, serving as a kind of biological glue. The structural similarity of calcium phosphate-based ceramic and the natural mineral components of bone is crucial for infiltration of the implant by the skeletal tissue of the host. This supports the intra-tissue application of hydroxyapatite-based implants whenever long-term remodeling of bone is required. Beta-

increased burden to the patient and prolong the duration of the procedure. Another very frequent limitation of using autogenous bone is its poor quality, when there is a skeletal system disorder (e.g. osteoporosis) (Bohner, 2010; Giannoudis et al., 2005). Allogenic implants derived from the structures of human bones can be one alternative to autogenous grafts (Ferreira, 2007). Mineralized (FDBA) and demineralized (DFDBA) forms of such implants, additionally subjected to lyophilization, are most frequently used in reconstructive surgery. An advantage of demineralized bone arises from the fact that the organic bone matrix (collagen fibers) has to be exposed in order to remove its mineral components, and therefore so-called matrix proteins (e.g. morphogenetic proteins) can easily diffuse into the implantation site and work osteoinductively (Barradas et al., 2011). Xenogenic implants also play an important role in reconstructive bone surgery. These implants are made of skeletal material obtained from animals (Merkx et al., 2003), in most cases from equine, bovine or porcine bones. Animal material is processed by means of thermal treatment in order to deplete it completely of its organic components (Barakat et al., 2009). As a result, implants lose their immunogenic properties and become neutral to hosts (Liu et al., 2008). Therefore, ready to use preparations for bone replacement (Bio-Oss®, Endobone®) are most frequently available in deproteinized forms (DBBM) (Accorsi-Mendonça et al., 2008). Due to their osteoconductive properties they can serve as an inactive scaffold or platform for the maturation of bone cells present within the defect. They are used in orthopedics, dental and maxillofacial surgery, as well as in periodontology and implantology, etc. (Baldini et al., 2011; Cao et al., 2009; Jian et al., 2008; Merkx et al., 2003; Precheur, 2007; von Wattenwyl et al., 2011). The limited possibilities of modulating the resorption time of such preparations during skeletal tissue reconstruction, however, can be

related to the poorer quality of bone at the site of their application.

An alternative solution, eliminating the potential complications associated with the application of materials of autogenous, allogenic or xenogenic origin, is the use of alloplastic implants for the purpose of bone replacement. Such implants can be synthesized from both natural and synthetic materials (Bohner, 2010; Giannoudis et al., 2005). Bioresorbable ceramic based on calcium phosphate plays a distinct role amongst novel synthetic materials used for bone replacement (Barradas et al., 2011). Hydroxyapatite (HAp) is the principal representative of this group, with the widest application in reconstructive surgery. Due to its calcium phosphate content and natural occurrence as an inorganic substance in bones and teeth, hydroxyapatite is characterized by the highest biocompatibility and bioactivity of all currently known implant materials. Additionally, due to the osteoconductive properties of hydroxyapatite, and (to a lesser extent) its osteoinductive properties, hydroxyapatitebased implants can bind directly to bone. Numerous clinical trials, supported by the results of histological observations, have confirmed complete biotolerance to hydroxyapatite ceramic, as well as its positive effects on the process of bone healing and reconstruction. Additionally, hydroxyapatite ceramic can initiate and stimulate various processes that are associated with bone formation (Bellucci et al., 2011; Ravarian et al., 2010; Yuan et al., 2001). Contact between bioactive ceramic and living skeletal tissue induces osteogenesis. As a result, an intermediate binding layer is formed between the living tissue and the implant, serving as a kind of biological glue. The structural similarity of calcium phosphate-based ceramic and the natural mineral components of bone is crucial for infiltration of the implant by the skeletal tissue of the host. This supports the intra-tissue application of hydroxyapatite-based implants whenever long-term remodeling of bone is required. Betatricalcium phosphate (betaTCP, betaCa3(PO4)2) is another calcium phosphate that has been used successfully in bone substitution. Its mineralogical analogue is whitlockite (TCP) (Barradas et al., 2011). Similar to HAp, TCP is characterized by high biocompatibility. In comparison to hydroxyapatite materials, it has a higher solubility *in vitro* and a resultant higher susceptibility to resorption and biodegradation in the environment of the living organism (Bohner, 2010; Henkel et al., 2006). TCP is considered an osteoinductive material, which stimulates the processes of bone reconstruction (Wang et al., 2009). Many studies, mostly dealing with the gradual, controlled resorption rate of calcium phosphate ceramic, resulted in the design of the second polymorphic variant of TCP, namely αTCP (Zima et al., 2010). Similar to beta-TCP, it is formed as a result of the non-stoichiometric heating of HAp with a well-defined temperature and defined kinetics of thermal processing. Compared to beta-TCP, αTCP has an approximately five-fold higher susceptibility to resorption in living tissues, along with higher biocompatibility (Oonishi et al., 1999). The high degree of osteointegration of αTCP-based bioceramic is plausibly the result of the higher solubility of αCa3(PO4)2 than of HAp or βTCP. Delivered within the ceramic, calcium and phosphate ions constitute the material for synthesizing the layer composed of non-stoichiometric hydroxyapatite that binds the implant to bone (Kon et al., 1995). Some authors suggest that αTCP can be cytotoxic, probably due to pH changes that are induced *in vitro* (Santos et al., 2002). These suggestions, however, were not confirmed during *in vivo* studies, and αTCP is included in many commercially available bone cements. Since the 1990s, biphasic HApβTCP ceramic (BCP biphasic calcium phosphate) has also been used in the reconstruction of skeletal defects (Deculsi, 1998, Deculsi et al., 2003; Fellah et al., 2008; Schwarz et al., 2007).

Bio-glass and apatite-wollastonite glass-ceramic also play an important role amongst the bioactive materials used in the processes of bone synthesis. The biological activity of glass and glass-derived crystalline materials (glass-ceramic materials) is mostly exploited for the manufacture of surface-active implants or implants that can be resorbed in the human body (Giannoudis et al., 2005). The composition of surface-active materials is selected in order to enable interactions between the physiological environment and certain components of the implant. As a result, living tissue is bound to the implant surface. Resorbable glass is characterized by its high content of chemical elements involved in metabolic processes of the human body. In both cases, living tissues can infiltrate the implant and bind it directly to the bone. These processes result not only from the chemical composition of the implants but also from the specific nature of the contained glass substance. The binding abilities of bioactive glass and glass-ceramic implants to skeletal tissue have been the subject of many studies. These studies confirmed the usefulness of such materials in tissue engineering, where they can be used in cell culture (Ferreira, 2007; Xynos et al., 2000). Their application in reconstructive surgery is reflected by their ability to stimulate and supporting bone reconstruction. In view of their confirmed ability to directly bind to skeletal tissue, they have been used successfully in various stomatological disciplines, including implantology, dental surgery and periodontology. Moreover, modern surface engineering has allowed the coating of metal implants with bioactive glass. Such coatings either protect surfaces of metal alloys against corrosion and wear, or stimulate the processes of bone formation in their surroundings. Additionally, resorbable glass can be used as a drug carrier, providing prolonged release of an active substance. Furthermore, some attempts have been made to combine various materials with each other to manufacture glass-containing bioactive composites, used as the components of binders, among others (Bellucci et al., 2011). These

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

based on human bone components (Zhang et al., 2009).

conditions for the formation of full value bone.

**2. Material** 

0.3 to 0.5 mm.

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 373

toxicity. Moreover, the glass-ceramic contained in this composite was confirmed to stimulate the growth and proliferation of human fibroblasts (Yoganand et al., 2010). Attempts at bone replacement with materials obtained by combining autogenous bone and deproteinized bovine bone constitute another example of the potential optimization of biological conditions for new skeletal tissue growth within a skeletal defect. The inclusion of autogenous material with osteoinductive properties in such composites results in the enhanced formation of better quality bone (Kim et al., 2009; Pripatnanont et al., 2009; Thorwarth et al., 2006; Thuaksuban et al., 2010). *In vitro* cellular studies have confirmed that allogenic materials of human origin (demineralized bone matrix, deproteinized bone) may also have some osteoinductive activity. This activity was confirmed by an increase in alkaline phosphatase (ALP), osteocalcin (OC) and Ca2+ concentrations observed in human bone marrow stromal osteoprogenitor cells (hBMSCs) cultured for three weeks in medium

The increasing demand for bone replacement materials stimulated us to design original composite materials for use in the regeneration of skeletal defects, characterized by both osteoinductive and osteoconductive properties. In designing such composites, we have used well-described biomaterials that have been applied successfully to skeletal defect regeneration, namely bio-glass (BG) and tricalcium phosphate (TCP). Lyophilized human bone obtained from a tissue bank served as a base for designing three types of mixtures: 1) human bone/bio-glass; 2) human bone/TCP; and 3) human bone/bio-glass/TCP. Such variants of material combinations enabled us to analyze the effects of particular components on the process of bone formation, and specifically on its dynamics and on the quality of newly formed bone. Also, the selection of the components included among the analyzed materials (mixtures) was not accidental. We have assumed that both bio-glass and tricalcium phosphate will activate and stimulate osteoblasts to dynamically grow on a biological scaffold of lyophilized human bone, thereby providing optimal biological

The materials used in the study included lyophilized, deproteinized human bone (B) – group B, and its mixtures with: 1) bio-glass (BG) in a proportion of 80:20% weight ratio (B:BG) – group B+BG; 2) tricalcium phosphate (TCP) in a proportion of 80:20% weight ratio (B:TCP) – group B+TCP; 3) bio-glass and tricalcium phosphate in a proportion of 70:15:15% weight ratio (B:BG:TCP) – group B+BG+TCP. The deproteinized human bone and all other components used in the mixtures were in a granulated form with diameters ranging from

Human bone used in this study was obtained from the Tissue Bank of the Regional Center of Blood Donation and Treatment in Katowice (Poland). It was cancellous bone subjected to

The bio-glass was made from the CaO-SiO2-P2O5 system with the use of the sol-gel technology, in the laboratory of the Department of Glass and Amorphous Coatings at the AGH Science and Technology University in Cracow, Poland. Its high-calcium A2 variety was used (54% mol. CaO) with a density of 2.9082 g/cm³, a dominating glassy phase and the beginnings of apatite crystallization. The thermal treatment of the bio-glass was performed

lyophilization, deep freezing and irradiation sterilization with a dose of 35 kGy.

experiments were aimed at obtaining biomaterials with better durability parameters and optimal biological characteristics. Various types of bio-glass and glass-ceramics may differ in terms of their biological properties, depending on the technique for their synthesis (Bellucci et al., 2011; Yuan et al. 2001). Amongst various methods of synthesis, the advantages of the chemical sol-gel method are worth noting. This technique enables material whose biological activity is greater than products manufactured using other processing methods to be obtained (Ravarian et al., 2010). Moreover, the sol-gel method does not require high temperature processing. Additionally, this technique allows the production of material with a strictly defined texture and parameters. Due to the possibility of manufacturing porous forms, bio-glass and glass-derived crystalline materials are morphologically similar to bone; this facilitates their infiltration by bone cells at the implantation site. Additionally, the porous structure of the implant enables the supply of fluids and nutrients required for growth to the newly formed bone, as well as the elimination of metabolites (Yuan et al., 2001). Finally, the resorption rate of the implant material is also determined by the degree of its porosity.

In view of the complexity of the biological environment at the implantation site, one principle problem of biomaterial engineering pertains to issues associated with the biodegradation and bioresorption of implanted biomaterials. Determination of degradation time and rate, and the kinetics of this process in the human body, constitute a significant challenge in the design of new implant materials (including bioceramic) used for the purposes of skeletal tissue substitution (Daculsi et al., 2003; Giannoudis et al., 2005). The controlled degradation rate of the implant along with the associated reconstruction of skeletal tissue should result in the formation of bone tissue resembling its natural structure as closely as possible, both at the biological and physico-mechanical levels. One of many methods allowing for the controlled biodegradation of bone replacement materials is the design of implants made of various composites or mixtures. Combining two or more different materials results in the manufacture of an absolutely new composite biomaterial, which is frequently superior in terms of biological and mechanical properties. Due to its specific properties, bioceramic is very frequently used as a basic component of ceramicceramic, metal-ceramic and polymer-ceramic systems (Thomas et al., 2005). Hydroxyapatite is very frequently included in bone composites, mostly due to its chemical similarity to the natural components of bone. Some studies have confirmed the positive effects of hydroxyapatite used in combination with metals, ceramic or polymers (Abu Bakar et al., 2003; Bellucci et al., 2011; Choi et al., 2004; Daculsi et al., 2003). The results of research on HAp-containing composites based on resorbable polymers, mostly polylactide (PLA), polyglycolide (PGA), co-polymer of glycolide and lactide (PLGA) or collagen [Cieślik et al., Nagata et al., 2005], seem particularly interesting. These studies confirmed the possible application of such composites as binding and reconstructive elements in reconstructive surgery, as culture media in tissue and genetic engineering, and as drug carriers (Wei & Ma, 2004; Nagata et al., 2003). Although hydroxyapatite is characterized by high biocompatibility, its reactivity with living skeletal tissue is relatively low. In view of these findings, attempts to design composites that combine this biomaterial with markedly more biologically active bio-glass propose an interesting solution. Such a composite can stimulate osteogenesis, leading to the rapid formation of new skeletal tissue around the implant (Yuan et al., 2001; Ravarian et al., 2010). Biological tests of SiO2-CaO-Mg/natural HAp (bovine bone) composite obtained by means of thermal plasma processing have confirmed its lack of toxicity. Moreover, the glass-ceramic contained in this composite was confirmed to stimulate the growth and proliferation of human fibroblasts (Yoganand et al., 2010). Attempts at bone replacement with materials obtained by combining autogenous bone and deproteinized bovine bone constitute another example of the potential optimization of biological conditions for new skeletal tissue growth within a skeletal defect. The inclusion of autogenous material with osteoinductive properties in such composites results in the enhanced formation of better quality bone (Kim et al., 2009; Pripatnanont et al., 2009; Thorwarth et al., 2006; Thuaksuban et al., 2010). *In vitro* cellular studies have confirmed that allogenic materials of human origin (demineralized bone matrix, deproteinized bone) may also have some osteoinductive activity. This activity was confirmed by an increase in alkaline phosphatase (ALP), osteocalcin (OC) and Ca2+ concentrations observed in human bone marrow stromal osteoprogenitor cells (hBMSCs) cultured for three weeks in medium based on human bone components (Zhang et al., 2009).

The increasing demand for bone replacement materials stimulated us to design original composite materials for use in the regeneration of skeletal defects, characterized by both osteoinductive and osteoconductive properties. In designing such composites, we have used well-described biomaterials that have been applied successfully to skeletal defect regeneration, namely bio-glass (BG) and tricalcium phosphate (TCP). Lyophilized human bone obtained from a tissue bank served as a base for designing three types of mixtures: 1) human bone/bio-glass; 2) human bone/TCP; and 3) human bone/bio-glass/TCP. Such variants of material combinations enabled us to analyze the effects of particular components on the process of bone formation, and specifically on its dynamics and on the quality of newly formed bone. Also, the selection of the components included among the analyzed materials (mixtures) was not accidental. We have assumed that both bio-glass and tricalcium phosphate will activate and stimulate osteoblasts to dynamically grow on a biological scaffold of lyophilized human bone, thereby providing optimal biological conditions for the formation of full value bone.

#### **2. Material**

372 Tissue Regeneration – From Basic Biology to Clinical Application

experiments were aimed at obtaining biomaterials with better durability parameters and optimal biological characteristics. Various types of bio-glass and glass-ceramics may differ in terms of their biological properties, depending on the technique for their synthesis (Bellucci et al., 2011; Yuan et al. 2001). Amongst various methods of synthesis, the advantages of the chemical sol-gel method are worth noting. This technique enables material whose biological activity is greater than products manufactured using other processing methods to be obtained (Ravarian et al., 2010). Moreover, the sol-gel method does not require high temperature processing. Additionally, this technique allows the production of material with a strictly defined texture and parameters. Due to the possibility of manufacturing porous forms, bio-glass and glass-derived crystalline materials are morphologically similar to bone; this facilitates their infiltration by bone cells at the implantation site. Additionally, the porous structure of the implant enables the supply of fluids and nutrients required for growth to the newly formed bone, as well as the elimination of metabolites (Yuan et al., 2001). Finally, the resorption rate of the implant

In view of the complexity of the biological environment at the implantation site, one principle problem of biomaterial engineering pertains to issues associated with the biodegradation and bioresorption of implanted biomaterials. Determination of degradation time and rate, and the kinetics of this process in the human body, constitute a significant challenge in the design of new implant materials (including bioceramic) used for the purposes of skeletal tissue substitution (Daculsi et al., 2003; Giannoudis et al., 2005). The controlled degradation rate of the implant along with the associated reconstruction of skeletal tissue should result in the formation of bone tissue resembling its natural structure as closely as possible, both at the biological and physico-mechanical levels. One of many methods allowing for the controlled biodegradation of bone replacement materials is the design of implants made of various composites or mixtures. Combining two or more different materials results in the manufacture of an absolutely new composite biomaterial, which is frequently superior in terms of biological and mechanical properties. Due to its specific properties, bioceramic is very frequently used as a basic component of ceramicceramic, metal-ceramic and polymer-ceramic systems (Thomas et al., 2005). Hydroxyapatite is very frequently included in bone composites, mostly due to its chemical similarity to the natural components of bone. Some studies have confirmed the positive effects of hydroxyapatite used in combination with metals, ceramic or polymers (Abu Bakar et al., 2003; Bellucci et al., 2011; Choi et al., 2004; Daculsi et al., 2003). The results of research on HAp-containing composites based on resorbable polymers, mostly polylactide (PLA), polyglycolide (PGA), co-polymer of glycolide and lactide (PLGA) or collagen [Cieślik et al., Nagata et al., 2005], seem particularly interesting. These studies confirmed the possible application of such composites as binding and reconstructive elements in reconstructive surgery, as culture media in tissue and genetic engineering, and as drug carriers (Wei & Ma, 2004; Nagata et al., 2003). Although hydroxyapatite is characterized by high biocompatibility, its reactivity with living skeletal tissue is relatively low. In view of these findings, attempts to design composites that combine this biomaterial with markedly more biologically active bio-glass propose an interesting solution. Such a composite can stimulate osteogenesis, leading to the rapid formation of new skeletal tissue around the implant (Yuan et al., 2001; Ravarian et al., 2010). Biological tests of SiO2-CaO-Mg/natural HAp (bovine bone) composite obtained by means of thermal plasma processing have confirmed its lack of

material is also determined by the degree of its porosity.

The materials used in the study included lyophilized, deproteinized human bone (B) – group B, and its mixtures with: 1) bio-glass (BG) in a proportion of 80:20% weight ratio (B:BG) – group B+BG; 2) tricalcium phosphate (TCP) in a proportion of 80:20% weight ratio (B:TCP) – group B+TCP; 3) bio-glass and tricalcium phosphate in a proportion of 70:15:15% weight ratio (B:BG:TCP) – group B+BG+TCP. The deproteinized human bone and all other components used in the mixtures were in a granulated form with diameters ranging from 0.3 to 0.5 mm.

Human bone used in this study was obtained from the Tissue Bank of the Regional Center of Blood Donation and Treatment in Katowice (Poland). It was cancellous bone subjected to lyophilization, deep freezing and irradiation sterilization with a dose of 35 kGy.

The bio-glass was made from the CaO-SiO2-P2O5 system with the use of the sol-gel technology, in the laboratory of the Department of Glass and Amorphous Coatings at the AGH Science and Technology University in Cracow, Poland. Its high-calcium A2 variety was used (54% mol. CaO) with a density of 2.9082 g/cm³, a dominating glassy phase and the beginnings of apatite crystallization. The thermal treatment of the bio-glass was performed

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

multiple layers with Dexon 4.0 sutures.

in detoxification of the body (i.e. liver and kidneys).

conditions: 0.16 s, 7 mA and 60 kV.

Labophot-2 microscope.

**4. Methods of examination** 

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 375

proportion of 25:75% weight ratio (examined material to blood). Blood served as a binder, supplying the implanted material with organic components. Additionally, it prevented the displacement of the implanted granules from within the bone defect, and facilitated their insertion. The defect on the left side of the mandible was left to fill postoperatively with clotted blood and undergo spontaneous healing (Fig.1c), and was considered a control to the right mandibular defect in each animal (control group). The bilateral wounds were closed in

On each examination day, all guinea pigs were clinically evaluated for surgical wound healing and general condition. Additionally, radiographs were taken in order to assess the regeneration of mandibular bone defects. Newly formed skeletal tissue in the defects was also analyzed quantitatively and qualitatively in terms of bone mineral density at the implantation site (computed tomography and radiographic densitometry). Following euthanasia with Morbital (sodium pentobarbital 133.3 mg/mL and pentobarbital 26.7 mg/mL, 1-2 mL/kg b.w.), the operated area and surrounding tissues were examined macroscopically. Moreover, tissue specimens from the euthanized animals were subjected to histopathologic analysis of the skeletal tissue and bone marrow at the implantation site and at its periphery, including the healing rate of the skeletal tissue and the soft and hard tissue reactions to the implanted material. Histopathologic analysis also included organs involved

Radiographs of the mandibular trunk with the sites of the bone defects were taken with a Heliodent type MD/D – 3195 nr 051692 apparatus (SIEMENS), and AGFA DENTUS M2 CONFORT films for axial pictures (75 mm x 65mm), using the following exposure

The tissue specimens taken for the histopathologic examinations were preserved in a 10% solution of buffered formalin. The osseous tissue was decalcified either in a 10% solution of disodium versenate, or electrolytically in Romeis liquid (80 mL of hydrochloric acid + 100 mL of formic acid, diluted to make 1000 mL with water) using PW23 bone decalcifier and an electric current of 0.5A. Next, all the tissues were routinely processed in Technicon's Duo autotechnicon using the sequence of 96% alcohol, acetone and xylene. They were then embedded in paraplast. The obtained cubes were then shaved on a Microm HM335E rotating microtome. The shavings (4-6 microns in thickness) were put onto basic slides, deparaffinized, and stained with hematoxylin and eosin (H&E). They were subsequently mounted with Canada balsam. Histologic slides were analyzed under an Olympus BX50 light microscope equipped with a set for optical (Olympus SC35) and digital microphotography (Olympus Canmedia C-5050 Zoom), at 40 to 400 x magnification. Additionally, the slides were consulted by other histologists using a dual-head Nikon

Bone mineral density (BMD) was determined by means of dual-energy X-ray absorptiometry (DXA) with a DPX-L densitometer (LUNAR Radiation Corporation, Madison, USA) using Small Animal Appendicular scanning. Total bone mineral density (BMD; g/cm2) was determined on the basis of examination of the entire area of the transverse cross-section of the bone defect (28.26 mm2), analyzed as 0.5 mm2 to 3 mm2 sections. One hundred and twenty

at a temperature of 800C, and its specific surface area (calculated by BET method) amounted to 57.8166 m2/g.

Resorbable, monophasic βTCP ceramic – a salt of trialkaline orthophosphoric acid Ca3(PO4)2 – was synthesized from powdered components obtained by means of wet synthesis in the Bioceramic Laboratory of the Department of Ceramic and Fire Resistant Material Technology at the AGH Science and Technology University in Cracow, Poland (Ślósarczyk & Paszkiewicz, 2005; Zima at al., 2010). The reagents included CaO (obtained by means of calcination of Ca(OH)2 – pure for analysis; MERCK, Poland), and H3PO4 (pure for analysis; POCH, Poland).

#### **3.** *In vivo* **animal experiments**

The study was carried out on a group of 48 guinea pigs, with an equal number of both sexes, and weights ranging from 500 to 600 grams. The animals were divided randomly into four groups, which corresponded to 12 animals for each studied composite material (6 males and 6 females). The animal experiments were performed on the 7th, 14th, and 21st days of the study, as well as after the 4th, 8th and 12th experimental weeks. Two guinea pigs (one male and one female) were examined in each experiment. All animal surgical procedures were performed at the Central Experimental Animal Farm at the Medical University of Silesia and were granted permission by the university's Bioethical Board for Experimental Animals.

Before starting any surgical procedures, the animals received general anesthesia with thiopental (0.4 g/kg b.w.). Bone defects (6 mm in diameter and 3 mm in depth) were formed bilaterally on the external surface of the mandibular trunk, 2 mm below its lower edge (between the radices of the incisive and molar teeth) with the aid of a rosette burr placed in the straight hand-piece of a dental machine (Fig.1a). Depending on the experimental group, bone defects on the right side of the mandible were filled with: 1) a preparation of deproteinized human bone (group B), 2) a mixture of deproteinized human bone and bioglass (group B+BG), 3) a mixture of deproteinized human bone and tricalcium phosphate (group B+TCP), or 4) a mixture of deproteinized human bone with bio-glass and tricalcium phosphate (group B+BG+TCP). Before implantation into the mandibular bone defect, each material was mixed with the animal's blood obtained from the surgical wound (Fig. 1b) in a

Fig. 1. Bone defect in the mandibular trunk of the experimental animal: a) defect prepared for filling with implant based on deproteinized human bone; b) defect filled with the mixture of deproteinized human bone and bio-glass with animal's blood; c) control defect filled with postoperatively clotted blood

proportion of 25:75% weight ratio (examined material to blood). Blood served as a binder, supplying the implanted material with organic components. Additionally, it prevented the displacement of the implanted granules from within the bone defect, and facilitated their insertion. The defect on the left side of the mandible was left to fill postoperatively with clotted blood and undergo spontaneous healing (Fig.1c), and was considered a control to the right mandibular defect in each animal (control group). The bilateral wounds were closed in multiple layers with Dexon 4.0 sutures.

### **4. Methods of examination**

374 Tissue Regeneration – From Basic Biology to Clinical Application

at a temperature of 800C, and its specific surface area (calculated by BET method)

Resorbable, monophasic βTCP ceramic – a salt of trialkaline orthophosphoric acid Ca3(PO4)2 – was synthesized from powdered components obtained by means of wet synthesis in the Bioceramic Laboratory of the Department of Ceramic and Fire Resistant Material Technology at the AGH Science and Technology University in Cracow, Poland (Ślósarczyk & Paszkiewicz, 2005; Zima at al., 2010). The reagents included CaO (obtained by means of calcination of Ca(OH)2 – pure for analysis; MERCK, Poland), and H3PO4 (pure for analysis;

The study was carried out on a group of 48 guinea pigs, with an equal number of both sexes, and weights ranging from 500 to 600 grams. The animals were divided randomly into four groups, which corresponded to 12 animals for each studied composite material (6 males and 6 females). The animal experiments were performed on the 7th, 14th, and 21st days of the study, as well as after the 4th, 8th and 12th experimental weeks. Two guinea pigs (one male and one female) were examined in each experiment. All animal surgical procedures were performed at the Central Experimental Animal Farm at the Medical University of Silesia and were granted permission by the university's Bioethical Board for Experimental Animals.

Before starting any surgical procedures, the animals received general anesthesia with thiopental (0.4 g/kg b.w.). Bone defects (6 mm in diameter and 3 mm in depth) were formed bilaterally on the external surface of the mandibular trunk, 2 mm below its lower edge (between the radices of the incisive and molar teeth) with the aid of a rosette burr placed in the straight hand-piece of a dental machine (Fig.1a). Depending on the experimental group, bone defects on the right side of the mandible were filled with: 1) a preparation of deproteinized human bone (group B), 2) a mixture of deproteinized human bone and bioglass (group B+BG), 3) a mixture of deproteinized human bone and tricalcium phosphate (group B+TCP), or 4) a mixture of deproteinized human bone with bio-glass and tricalcium phosphate (group B+BG+TCP). Before implantation into the mandibular bone defect, each material was mixed with the animal's blood obtained from the surgical wound (Fig. 1b) in a

Fig. 1. Bone defect in the mandibular trunk of the experimental animal: a) defect prepared for filling with implant based on deproteinized human bone; b) defect filled with the mixture of deproteinized human bone and bio-glass with animal's blood; c) control defect

**a b c** 

amounted to 57.8166 m2/g.

**3.** *In vivo* **animal experiments** 

filled with postoperatively clotted blood

POCH, Poland).

On each examination day, all guinea pigs were clinically evaluated for surgical wound healing and general condition. Additionally, radiographs were taken in order to assess the regeneration of mandibular bone defects. Newly formed skeletal tissue in the defects was also analyzed quantitatively and qualitatively in terms of bone mineral density at the implantation site (computed tomography and radiographic densitometry). Following euthanasia with Morbital (sodium pentobarbital 133.3 mg/mL and pentobarbital 26.7 mg/mL, 1-2 mL/kg b.w.), the operated area and surrounding tissues were examined macroscopically. Moreover, tissue specimens from the euthanized animals were subjected to histopathologic analysis of the skeletal tissue and bone marrow at the implantation site and at its periphery, including the healing rate of the skeletal tissue and the soft and hard tissue reactions to the implanted material. Histopathologic analysis also included organs involved in detoxification of the body (i.e. liver and kidneys).

Radiographs of the mandibular trunk with the sites of the bone defects were taken with a Heliodent type MD/D – 3195 nr 051692 apparatus (SIEMENS), and AGFA DENTUS M2 CONFORT films for axial pictures (75 mm x 65mm), using the following exposure conditions: 0.16 s, 7 mA and 60 kV.

The tissue specimens taken for the histopathologic examinations were preserved in a 10% solution of buffered formalin. The osseous tissue was decalcified either in a 10% solution of disodium versenate, or electrolytically in Romeis liquid (80 mL of hydrochloric acid + 100 mL of formic acid, diluted to make 1000 mL with water) using PW23 bone decalcifier and an electric current of 0.5A. Next, all the tissues were routinely processed in Technicon's Duo autotechnicon using the sequence of 96% alcohol, acetone and xylene. They were then embedded in paraplast. The obtained cubes were then shaved on a Microm HM335E rotating microtome. The shavings (4-6 microns in thickness) were put onto basic slides, deparaffinized, and stained with hematoxylin and eosin (H&E). They were subsequently mounted with Canada balsam. Histologic slides were analyzed under an Olympus BX50 light microscope equipped with a set for optical (Olympus SC35) and digital microphotography (Olympus Canmedia C-5050 Zoom), at 40 to 400 x magnification. Additionally, the slides were consulted by other histologists using a dual-head Nikon Labophot-2 microscope.

Bone mineral density (BMD) was determined by means of dual-energy X-ray absorptiometry (DXA) with a DPX-L densitometer (LUNAR Radiation Corporation, Madison, USA) using Small Animal Appendicular scanning. Total bone mineral density (BMD; g/cm2) was determined on the basis of examination of the entire area of the transverse cross-section of the bone defect (28.26 mm2), analyzed as 0.5 mm2 to 3 mm2 sections. One hundred and twenty

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

control group increased in size when compared to previous periods.

was darker in comparison to the surrounding tissues.

colored spot at its center.

**5.3 Radiological examinations** 

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 377

tissue, which could be compressed elastically. Additionally, clearly distinguishable white granulation was visible throughout the superficial tissue in animals of the B+BG and B+BG+TCP groups. Probably, these granules corresponded to the bio-glass particles included in the material implanted in these groups. In the control group, the site of the bone defect was visible as a clear protrusion up to the 7th experimental day. On the next day of examination (the 14th day), however, the site was covered with a tissue whose coloration

After the 3rd experimental week, the protrusion visible over the implantation site in groups B+BG, B+TCP and B+BG+TCP was markedly smaller in size, and covered with a hard tissue of more compact texture. Coloration of the tissues covering the implantation site still differed from the color of normal bone. In group BG, white granulation was still visible throughout the superficial tissue. On the 21st day after surgery, the coloration of the bone defect implanted with deproteinized human bone (group B) resembled the color of the surrounding tissues more closely than in previous periods. In both group B and in the control group, clearly distinguishable small areas in the form of a dark-colored spot were visible within the defects. Additionally, tissue protrusion observed over the defects in the

Four weeks following implantation, the area of the bone defect in group B+BG was slightly smaller in size but still clearly distinguishable and differed in color from the surrounding tissues. Similar changes were observed in the B+TCP group; the implantation site in this group was concave, but still appeared hard when compressed. The tissue visible over the implantation site in group B+BG+TCP resembled the surrounding normal tissues the most closely when compared to the other groups. In group BG, white granulation was still visible throughout the superficial tissue covering the bone defect. In the control group, on the other hand, a large protrusion was still visible over the implantation site, with a small dark-

After the 8th and 12th weeks of experiment, the implantation sites of the group B animals were still clearly distinguishable on macroscopic examination; they were protruding and differed in color from the surrounding tissues. Protrusions over the implantation sites were also visible in group B+BG, but only up to the 8th week of the study. After this time, the implantation site was hardly distinguishable from the normal tissues, and only the presence of granulation, which was hardly visible throughout the superficial tissue, enabled its visual identification. In group B+BG+TCP, the implantation site could also only be localized due to the subtle appearance of white granulation as early as after 8 weeks of the experiment. In group B+TCP, no concavity was observed over the implantation site beginning from the 8th week of the study, and the tissue covering the defect only slightly differed in color from the surrounding normal bone. After 12 weeks of observation, the tissue over the bone defects in this group had an identical appearance to the surrounding tissues. On the last examination day, the site of the bone defect was distinguishable only in the control group. Although the implantation site was covered with hard tissue, minute concavities and dark spots were still visible on its surface.

Material-related differences in the rates of new skeletal tissue formation were revealed as early as after the 7th day of the study. In group B, spherical translucencies with regular

BMD measurements were taken in each group (corresponding to 20 measurements per experimental day). In most cases, reproducible results were not included in further analysis; only unique values and the most frequent reproducible values were analyzed, which corresponded to 10 BMD measurements for each day of examination. Additionally, the BMD of the normal mandibular trunk of the guinea pig was determined for the purpose of comparative analysis. Based on 10 consecutive measurements, this value was estimated at 0.51 ± 0.001 g/cm2. During the measurements, the densitometer was regularly calibrated and controlled according to the manufacturer's recommendations. Both DXA measurements and BMD result analysis were performed by the same investigator.

Additionally, computed tomography (CT) was used to determine the bone mineral density, expressed in Hounsfield units (HU). These measurements were taken using a Somatom Emotion 6 scanner (Siemens; exposure parameters: 13.4 s, 14 mA, 130 kV). Transverse crosssections of the mandible were visualized in 2 mm slices. Then, cross-sections including the area of the bone defect were selected and analyzed using Volume Viewer software. In each experimental group, six measurements were taken for each examination day. Additionally, six measurements of the normal mandible were taken for the purpose of comparative analysis. Based on these measurements, the normal bone mineral density was determined to be 1218 ± 15.2 HU.

The results of bone density are presented as mean values ± standard deviation. Variables distribution was evaluated by the Shapiro-Wilk test. Homogeneity of variance was assessed by the Levene test. ANOVA for repeated measurements with contrasts analysis were done to assess time and preparation of deproteinized human bone type interaction. The Mauchley test was done to check sphericity. Differences were considered to be statistically significant at *p<0.05*. All calculations were performed using the commercially available statistical package Statistica 9.0.

#### **5. Results**

#### **5.1 Clinical observations**

Throughout the entire study period, no complications in surgical wound healing were observed in animals of any experimental group. The guinea pigs were calm, which suggested a lack of pain. The animals ate and drank water normally, and neither scraped against the cage nor scratched their wounds during the entire postoperative period. Also, wound dehiscence was not observed in any of the groups.

Tissue edema over the surgical skin wounds resolved 3 to 5 days following surgery and was replaced by protrusions of the tissue. There were no signs of excessive fluid accumulation around the wound or of hematoma formation, but in some animals skin redness was observed around the stitches. In most animals, tissue protrusions persisted until the 21st day after surgery. The stitches were removed 10 to 14 days after surgery. Over the entire study period, the animals gained weight gradually (in a statistically insignificant manner).

#### **5.2 Macroscopic examination**

Up to 14th experimental day, the sites of the implanted bone defects were clearly distinguishable from the surrounding tissues as clear, oval protrusions covered with delicate

BMD measurements were taken in each group (corresponding to 20 measurements per experimental day). In most cases, reproducible results were not included in further analysis; only unique values and the most frequent reproducible values were analyzed, which corresponded to 10 BMD measurements for each day of examination. Additionally, the BMD of the normal mandibular trunk of the guinea pig was determined for the purpose of comparative analysis. Based on 10 consecutive measurements, this value was estimated at 0.51 ± 0.001 g/cm2. During the measurements, the densitometer was regularly calibrated and controlled according to the manufacturer's recommendations. Both DXA measurements and

Additionally, computed tomography (CT) was used to determine the bone mineral density, expressed in Hounsfield units (HU). These measurements were taken using a Somatom Emotion 6 scanner (Siemens; exposure parameters: 13.4 s, 14 mA, 130 kV). Transverse crosssections of the mandible were visualized in 2 mm slices. Then, cross-sections including the area of the bone defect were selected and analyzed using Volume Viewer software. In each experimental group, six measurements were taken for each examination day. Additionally, six measurements of the normal mandible were taken for the purpose of comparative analysis. Based on these measurements, the normal bone mineral density was determined to

The results of bone density are presented as mean values ± standard deviation. Variables distribution was evaluated by the Shapiro-Wilk test. Homogeneity of variance was assessed by the Levene test. ANOVA for repeated measurements with contrasts analysis were done to assess time and preparation of deproteinized human bone type interaction. The Mauchley test was done to check sphericity. Differences were considered to be statistically significant at *p<0.05*. All calculations were performed using the commercially available statistical

Throughout the entire study period, no complications in surgical wound healing were observed in animals of any experimental group. The guinea pigs were calm, which suggested a lack of pain. The animals ate and drank water normally, and neither scraped against the cage nor scratched their wounds during the entire postoperative period. Also,

Tissue edema over the surgical skin wounds resolved 3 to 5 days following surgery and was replaced by protrusions of the tissue. There were no signs of excessive fluid accumulation around the wound or of hematoma formation, but in some animals skin redness was observed around the stitches. In most animals, tissue protrusions persisted until the 21st day after surgery. The stitches were removed 10 to 14 days after surgery. Over the entire study

Up to 14th experimental day, the sites of the implanted bone defects were clearly distinguishable from the surrounding tissues as clear, oval protrusions covered with delicate

period, the animals gained weight gradually (in a statistically insignificant manner).

BMD result analysis were performed by the same investigator.

wound dehiscence was not observed in any of the groups.

be 1218 ± 15.2 HU.

package Statistica 9.0.

**5.1 Clinical observations** 

**5.2 Macroscopic examination** 

**5. Results** 

tissue, which could be compressed elastically. Additionally, clearly distinguishable white granulation was visible throughout the superficial tissue in animals of the B+BG and B+BG+TCP groups. Probably, these granules corresponded to the bio-glass particles included in the material implanted in these groups. In the control group, the site of the bone defect was visible as a clear protrusion up to the 7th experimental day. On the next day of examination (the 14th day), however, the site was covered with a tissue whose coloration was darker in comparison to the surrounding tissues.

After the 3rd experimental week, the protrusion visible over the implantation site in groups B+BG, B+TCP and B+BG+TCP was markedly smaller in size, and covered with a hard tissue of more compact texture. Coloration of the tissues covering the implantation site still differed from the color of normal bone. In group BG, white granulation was still visible throughout the superficial tissue. On the 21st day after surgery, the coloration of the bone defect implanted with deproteinized human bone (group B) resembled the color of the surrounding tissues more closely than in previous periods. In both group B and in the control group, clearly distinguishable small areas in the form of a dark-colored spot were visible within the defects. Additionally, tissue protrusion observed over the defects in the control group increased in size when compared to previous periods.

Four weeks following implantation, the area of the bone defect in group B+BG was slightly smaller in size but still clearly distinguishable and differed in color from the surrounding tissues. Similar changes were observed in the B+TCP group; the implantation site in this group was concave, but still appeared hard when compressed. The tissue visible over the implantation site in group B+BG+TCP resembled the surrounding normal tissues the most closely when compared to the other groups. In group BG, white granulation was still visible throughout the superficial tissue covering the bone defect. In the control group, on the other hand, a large protrusion was still visible over the implantation site, with a small darkcolored spot at its center.

After the 8th and 12th weeks of experiment, the implantation sites of the group B animals were still clearly distinguishable on macroscopic examination; they were protruding and differed in color from the surrounding tissues. Protrusions over the implantation sites were also visible in group B+BG, but only up to the 8th week of the study. After this time, the implantation site was hardly distinguishable from the normal tissues, and only the presence of granulation, which was hardly visible throughout the superficial tissue, enabled its visual identification. In group B+BG+TCP, the implantation site could also only be localized due to the subtle appearance of white granulation as early as after 8 weeks of the experiment. In group B+TCP, no concavity was observed over the implantation site beginning from the 8th week of the study, and the tissue covering the defect only slightly differed in color from the surrounding normal bone. After 12 weeks of observation, the tissue over the bone defects in this group had an identical appearance to the surrounding tissues. On the last examination day, the site of the bone defect was distinguishable only in the control group. Although the implantation site was covered with hard tissue, minute concavities and dark spots were still visible on its surface.

#### **5.3 Radiological examinations**

Material-related differences in the rates of new skeletal tissue formation were revealed as early as after the 7th day of the study. In group B, spherical translucencies with regular

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

study; b) 12th week of the study

day of the study; b) 8th week of the study

phosphate: a) 14th day of the study; b) 12th week of the study

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 379

Fig. 3. Radiographic images of the mandible in experimental animals. Bone defect healing in the presence of the mixture of deproteinized human bone with bio-glass: a) 7th day of the

**b** 

**b** 

**b** 

**b** 

**a**

Fig. 4. Radiographic images of the mandible in experimental animals. Bone defect healing in the presence of the mixture of deproteinized human bone with tricalcium phosphate: a) 21st

**a**

Fig. 5. Radiographic images of the mandible in experimental animals. Bone defect healing in the presence of the mixture of deproteinized human bone with bio-glass and tricalcium

**a**

Fig. 6. Radiographic images of the mandible in experimental animals. Bone defect healing on

**a**

the basis of clotted blood: a) 21st day of the study; b) 12th week of the study

edges were observed on radiographic images, with a size corresponding to the size of the bone defect. The initial process of bone reconstruction, manifested by a foggy appearance of the implantation site, was observed no earlier than after the second experimental week (Fig. 2a). At that same time point, initial signs of bone formation were also observed on radiographic images taken in group B+BG+TCP (Fig. 5a), while in group B+BG this phenomenon was already visible after 7 days (Fig. 3a). Irregular translucencies at the implantation site were seen in this group, but a small shadow was observed in the central zone, whose area and intensity increased with time. Radiologic findings suggested that the beginning of new tissue formation was most delayed in group B+TCP (Fig. 4a) and in the controls (Fig. 6a). In these groups, a distinct shadow was observed no earlier than after the 3rd week of the experiment; this shadow was larger and more intense in B+TCP group.

After the 4th week of the study, radiographic images taken in group B revealed the nearly complete formation of new skeletal tissue. At this point in time, indistinct translucencies were seen only at the periphery of the skeletal defect, suggesting osteogenesis was ongoing in this area. After 8 weeks of the experiment, bone reconstruction was complete in this group. The whole defect was excessively shadowed, suggesting that tissue with greater mineralization was present in this area when compared to normal bone (Fig. 2b). As with group B, in group B+TCP the mineralization of the implantation site was complete after the 8th week of the study, as suggested by a fully shadowed area of the bone defect visible on radiographic images (Fig. 4b). At the same time, a small translucency was visible in the superior-medial aspect of the bone defect in group B+BG. In this group, the process of bone formation was completed no earlier than after the 12th week of the experiment. This was confirmed by the excessive mineralization of skeletal tissue, manifested radiographically as a distinct shadow at the implantation site (Fig. 3b). In the B+BG+TCP group, a homogenous shadow was visible at the implantation site as early as after the 8th week of the experiment, and an incomplete process of skeletal tissue regeneration was suggested only by the presence of spotted translucencies. However, the site of the bone defect could not be distinguished from the surrounding tissues until at least the 12th week of the study (Fig. 5b). In the control group, the process of bone formation was markedly delayed, and after the 8th week of the study a distinct, longitudinal translucency could still be seen at the site of the bone defect. Moreover, noncalcified areas were still visible on the last examination day despite newly formed skeletal tissue present within the entire area of the bone defect (Fig. 6b).

Fig. 2. Radiographic images of the mandible in experimental animals. Bone defect healing in the presence of deproteinized human bone: a) 14th day of the study; b) 8th week of the study

edges were observed on radiographic images, with a size corresponding to the size of the bone defect. The initial process of bone reconstruction, manifested by a foggy appearance of the implantation site, was observed no earlier than after the second experimental week (Fig. 2a). At that same time point, initial signs of bone formation were also observed on radiographic images taken in group B+BG+TCP (Fig. 5a), while in group B+BG this phenomenon was already visible after 7 days (Fig. 3a). Irregular translucencies at the implantation site were seen in this group, but a small shadow was observed in the central zone, whose area and intensity increased with time. Radiologic findings suggested that the beginning of new tissue formation was most delayed in group B+TCP (Fig. 4a) and in the controls (Fig. 6a). In these groups, a distinct shadow was observed no earlier than after the 3rd week of the experiment; this shadow was larger and more intense in B+TCP

After the 4th week of the study, radiographic images taken in group B revealed the nearly complete formation of new skeletal tissue. At this point in time, indistinct translucencies were seen only at the periphery of the skeletal defect, suggesting osteogenesis was ongoing in this area. After 8 weeks of the experiment, bone reconstruction was complete in this group. The whole defect was excessively shadowed, suggesting that tissue with greater mineralization was present in this area when compared to normal bone (Fig. 2b). As with group B, in group B+TCP the mineralization of the implantation site was complete after the 8th week of the study, as suggested by a fully shadowed area of the bone defect visible on radiographic images (Fig. 4b). At the same time, a small translucency was visible in the superior-medial aspect of the bone defect in group B+BG. In this group, the process of bone formation was completed no earlier than after the 12th week of the experiment. This was confirmed by the excessive mineralization of skeletal tissue, manifested radiographically as a distinct shadow at the implantation site (Fig. 3b). In the B+BG+TCP group, a homogenous shadow was visible at the implantation site as early as after the 8th week of the experiment, and an incomplete process of skeletal tissue regeneration was suggested only by the presence of spotted translucencies. However, the site of the bone defect could not be distinguished from the surrounding tissues until at least the 12th week of the study (Fig. 5b). In the control group, the process of bone formation was markedly delayed, and after the 8th week of the study a distinct, longitudinal translucency could still be seen at the site of the bone defect. Moreover, noncalcified areas were still visible on the last examination day despite newly formed skeletal

Fig. 2. Radiographic images of the mandible in experimental animals. Bone defect healing in the presence of deproteinized human bone: a) 14th day of the study; b) 8th week of the study

**a b** 

tissue present within the entire area of the bone defect (Fig. 6b).

group.

Fig. 3. Radiographic images of the mandible in experimental animals. Bone defect healing in the presence of the mixture of deproteinized human bone with bio-glass: a) 7th day of the study; b) 12th week of the study

Fig. 4. Radiographic images of the mandible in experimental animals. Bone defect healing in the presence of the mixture of deproteinized human bone with tricalcium phosphate: a) 21st day of the study; b) 8th week of the study

Fig. 5. Radiographic images of the mandible in experimental animals. Bone defect healing in the presence of the mixture of deproteinized human bone with bio-glass and tricalcium phosphate: a) 14th day of the study; b) 12th week of the study

Fig. 6. Radiographic images of the mandible in experimental animals. Bone defect healing on the basis of clotted blood: a) 21st day of the study; b) 12th week of the study

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

changes in this group was closest to a vertical line.

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 381

experimental weeks was observed in animals of group B+BG (9.98); the time plot of BMD

Fig. 7. Time profile of changes in bone mineral density (BMD) determined radiographically

1 2 3 4 8 12 **Time [weeks]**

 B B+BG B+TCP B+BG+TCP Control

Due to non-sphericity (Mauchley test: 0.42; p<0.001) only multivariable tests were used. We confirmed that there were statistically significant changes in BMD with time (F = 894.17; p<0.001). Moreover, the interaction between time and the type of material used (group) was

**Group** |t| p **B** 17.11 **<0.001 B+BG** 9.98 **<0.001 B+TCP** 24.14 **<0.001 B+BG+TCP** 25.49 **<0.001 Control** 13.58 **<0.001**  Table 3. Comparison of bone mineral density (BMD) values between the 12th and 1st weeks

Finally, statistical analysis revealed several significant intergroup differences in BMD values determined in experimental weeks 1 and 12 (Table 4). In the earlier period, the most

**12th vs. 1st week** 

in bone defects implanted with various materials and in control defects

also statistically significant (F = 35.59; p<0.001).

0,20

0,22

0,24

0,26

0,28

0,30

**BMD [g/cm2]**

0,32

0,34

0,36

0,38

0,40

0,42

with regard to the tested material and control groups

#### **5.4 Bone Mineral Density**

#### **5.4.1 Radiographic densitometry**

A gradual increase in BMD was observed in all experimental groups and in the controls. The highest BMD value was observed after the 8th week of the study in bone defects implanted with B+TCP (0.400.05 g/cm2) and B+BG+TCP (0.390.05 g/cm2) (Table 1). However, these values were still lower than the normal BMD of the mandibular trunk determined in guinea pigs (0.51± 0.001 g/cm2).


Table 1. Bone mineral density (BMD) of skeletal defects implanted with various materials and in control defects determined radiographically at various time points in the study

The most regular increase in BMD was observed in defects implanted with B+BG+TCP, followed by B and B+BG. Some irregularities in the time profiles of BMD changes were noted, however, in animals of group B+TCP and in the controls, suggesting inhomogeneous formation of new skeletal tissue (Fig. 7).

Additionally, statistical analysis revealed intergroup differences in the time profiles of BMD changes. The most pronounced differences were observed between groups B and B+BG – 0.98, followed by B+BG vs. B+TCP – 0.79, and B vs. B+TCP – 0.78. Slight differences in the time profiles were noted between the B+BG+TCP group and other material groups, and no significant differences were observed when experimental groups were compared to the control group (Table 2).


Table 2. The p-values for comparison of bone mineral density (BMD) changes in time (profiles) between the tested material and control groups

Moreover, a relative increase in BMD between the 1st and 12th experimental weeks was calculated for each group (Table 3). The highest relative increase in BMD was observed in bone defects in group B+BG+TCP (25.49) but the increase in the B+TCP group was only slightly lower (24.14). The lowest relative increase in BMD between the 1st and 12th

A gradual increase in BMD was observed in all experimental groups and in the controls. The highest BMD value was observed after the 8th week of the study in bone defects implanted with B+TCP (0.400.05 g/cm2) and B+BG+TCP (0.390.05 g/cm2) (Table 1). However, these values were still lower than the normal BMD of the mandibular trunk determined in guinea

**Group 1st week 2nd week 3th week 4th week 8th week 12th week B** 0.25 0.01 0.27 0.01 0.28 0.01 0.33 0.02 0.35 0.03 0.35 0.03 **B+BG** 0.27 0.02 0.30 0.03 0.30 0.03 0.31 0.02 0.31 0.03 0.33 0.01 **B+TCP** 0.22 0.03 0.24 0.03 0.33 0.01 0.32 0.04 0.40 0.05 0.36 0.02 **B+BG+TCP** 0.23 0.03 0.28 0.02 0.32 0.03 0.35 0.04 0.39 0.05 0.38 0.02 **Control** 0.24 0.01 0.25 0.01 0.22 0.02 0.34 0.01 0.33 0.04 0.32 0.02 Table 1. Bone mineral density (BMD) of skeletal defects implanted with various materials and in control defects determined radiographically at various time points in the study

The most regular increase in BMD was observed in defects implanted with B+BG+TCP, followed by B and B+BG. Some irregularities in the time profiles of BMD changes were noted, however, in animals of group B+TCP and in the controls, suggesting inhomogeneous

Additionally, statistical analysis revealed intergroup differences in the time profiles of BMD changes. The most pronounced differences were observed between groups B and B+BG – 0.98, followed by B+BG vs. B+TCP – 0.79, and B vs. B+TCP – 0.78. Slight differences in the time profiles were noted between the B+BG+TCP group and other material groups, and no significant differences were observed when experimental groups were compared to the

**Profile comparison (p-values)** 

**Group Control B B+BG B+TCP B <0.05** – – – **B+BG <0.05** 0.98 – – **B+TCP <0.05** 0.78 0.79 – **B+BG+TCP <0.01** 0.20 0.21 0.32

Moreover, a relative increase in BMD between the 1st and 12th experimental weeks was calculated for each group (Table 3). The highest relative increase in BMD was observed in bone defects in group B+BG+TCP (25.49) but the increase in the B+TCP group was only slightly lower (24.14). The lowest relative increase in BMD between the 1st and 12th

Table 2. The p-values for comparison of bone mineral density (BMD) changes in time

(profiles) between the tested material and control groups

**BMD [g/cm2]** 

**5.4 Bone Mineral Density** 

pigs (0.51± 0.001 g/cm2).

**5.4.1 Radiographic densitometry** 

formation of new skeletal tissue (Fig. 7).

control group (Table 2).

experimental weeks was observed in animals of group B+BG (9.98); the time plot of BMD changes in this group was closest to a vertical line.

Fig. 7. Time profile of changes in bone mineral density (BMD) determined radiographically in bone defects implanted with various materials and in control defects

Due to non-sphericity (Mauchley test: 0.42; p<0.001) only multivariable tests were used. We confirmed that there were statistically significant changes in BMD with time (F = 894.17; p<0.001). Moreover, the interaction between time and the type of material used (group) was also statistically significant (F = 35.59; p<0.001).


Table 3. Comparison of bone mineral density (BMD) values between the 12th and 1st weeks with regard to the tested material and control groups

Finally, statistical analysis revealed several significant intergroup differences in BMD values determined in experimental weeks 1 and 12 (Table 4). In the earlier period, the most

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

by contrast, noted in animals of the B and B+BG+TCP groups (Fig. 8).

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 383

The most homogeneous increase in CT bone density values was observed in groups B+BG and B+TCP, and in the controls. Some irregularities in the time profile of bone density were,

Fig. 8. Time profile of changes in CT bone density (expressed in Hounsfield units, HU) in

Due to non-sphericity (Mauchley test: 0.0030; p<0.001) only multivariable tests were used. We confirmed that there were statistically significant changes in Hounsfield Units with time (F = 1244.88; p<0.001). Moreover, the interaction between time and the type of material used

1 2 3 4 8 12 **Time [weeks]**

B B+BG B+TCP B+BG+TCP Control

When the time profiles of CT bone density changes were compared between the studied groups, the only significant differences observed were between groups B and B+BG – 0.86,

**Profile comparison (p-values)**

**Group Control B B+BG B+TCP B <0.001** –– – **B+BG <0.001** 0.86 – – **B+TCP <0.001 <0.001 <0.001** – **B+BG+TCP <0.001 <0.001 <0.001** 0.30 Table 6. The p-values for comparison of CT bone density (expressed in Hounsfield units, HU) with changes in time (profiles) between the tested material and control groups

bone defects implanted with various materials and in control defects

(group) was also statistically significant (F = 65.35; p<0.001).

and B+TCP and B+BG+TCP – 0.30 (Table 6).

100

200

300

400

500

600

700

**TC Bone Density [ H.U.]**

800

900

1000

1100

pronounced difference was observed between groups B+BG and B+TCP (4.33), while the lowest difference pertained to groups B+TCP and B+BG+TCP. After 12 weeks of the experiment, the most pronounced difference in BMD of the bone defect was noted between the control group and the B+BG+TCP group, while the difference between the B and B+TCP groups was the lowest.


Table 4. Comparison of bone mineral density (BMD) values in the 1st and 12th weeks between the tested material and control groups

We have also observed statistically significant differences in the growth of BMD with time between the analyzed groups (F=8.15; p<0.001). The smallest changes in BMD were yielded by B+BG (0.060.01), then the control group (0.080.01) and B (0.110.02). The largest changes were observed for B+TCP (0.140.01) and B+BG+TCP (0.160.01). For all paired comparisons, statistically significant differences were noted (p<0.01).

#### **5.4.2 Computed tomography**

Table 5 summarizes the values of bone density in the experimental groups and in the controls as determined by CT. A gradual increase in the density of healing bone defects was observed in all studied groups. After 12 weeks of the experiment, the highest values of CT bone density were observed in the B+BG (1014.8±53.9 HU) and B+BG+TCP (941.2±28.9 HU) groups, while the lowest values were noted in the controls (812.3±21.8 HU). However, the highest determined values of CT bone density were still lower compared to the bone density of the normal mandible (1218 ± 15.2 HU).


Table 5. CT bone density (expressed in Hounsfield units, HU) in bone defects implanted with various materials and in control defects at various time points in the study

pronounced difference was observed between groups B+BG and B+TCP (4.33), while the lowest difference pertained to groups B+TCP and B+BG+TCP. After 12 weeks of the experiment, the most pronounced difference in BMD of the bone defect was noted between the control group and the B+BG+TCP group, while the difference between the B and B+TCP

> **Groups** |t| P |t| p **B vs. Control** 0.70 0.48 3.63 **<0.001 B+BG vs. Control** 2.48 **<0.05** 1.41 0.17 **B+TCP vs. Control** 1.85 0.07 4.54 **<0.05 B+BG+TCP vs. Control** 1.27 0.21 6.40 **<0.001 B vs. B+BG** 1.78 0.08 2.22 **<0.05 B vs. B+TCP** 2.55 **<0.05** 0.91 0.37 **B vs. B+BG+TCP** 1.98 0.05 2.78 **<0.01 B+BG vs. B+TCP** 4.33 **<0.001** 3.13 **<0.01 B+BG vs. B+BG+TCP** 3.75 **<0.001** 4.99 **<0.001 B+TCP vs. B+BG+TCP** 0.57 0.57 1.87 0.07

Table 4. Comparison of bone mineral density (BMD) values in the 1st and 12th weeks

comparisons, statistically significant differences were noted (p<0.01).

We have also observed statistically significant differences in the growth of BMD with time between the analyzed groups (F=8.15; p<0.001). The smallest changes in BMD were yielded by B+BG (0.060.01), then the control group (0.080.01) and B (0.110.02). The largest changes were observed for B+TCP (0.140.01) and B+BG+TCP (0.160.01). For all paired

Table 5 summarizes the values of bone density in the experimental groups and in the controls as determined by CT. A gradual increase in the density of healing bone defects was observed in all studied groups. After 12 weeks of the experiment, the highest values of CT bone density were observed in the B+BG (1014.8±53.9 HU) and B+BG+TCP (941.2±28.9 HU) groups, while the lowest values were noted in the controls (812.3±21.8 HU). However, the highest determined values of CT bone density were still lower compared to the bone density

**Group 1st week 2nd week 3th week 4th week 8th week 12th week B** 647.7 ± 79.1 784.0 ± 82.3 863.2 ± 13.3 763.8 ± 136.1 954.3 ± 56.3 902.8 ± 13.5 **B+BG** 597.2 ± 48.8 730.5 ± 9.8 814.3 ± 38.1 822.5 ± 99.8 933.3 ± 22.0 1014.8 ± 53.9 **B+TCP** 410.3 ± 34.0 468.2 ± 59.1 606.7 ± 41.8 804.0 ± 51.7 826.3 ± 39.3 879.2 ± 24.6 **B+BG+TCP** 454.7 ± 17.1 526.3 ± 35.6 523.2 ± 27.7 725.7 ± 36.3 923.7 ± 31.8 941.2 ± 28.9 **Control** 256.0 ± 20.4 510.3 ± 28.7 539.0 ± 38.3 597.8 ± 35.3 681.8 ± 19.4 812.3 ± 21.8 Table 5. CT bone density (expressed in Hounsfield units, HU) in bone defects implanted

with various materials and in control defects at various time points in the study

**CT Bone Density [HU]** 

between the tested material and control groups

**5.4.2 Computed tomography** 

of the normal mandible (1218 ± 15.2 HU).

**1st week 12th week** 

groups was the lowest.

The most homogeneous increase in CT bone density values was observed in groups B+BG and B+TCP, and in the controls. Some irregularities in the time profile of bone density were, by contrast, noted in animals of the B and B+BG+TCP groups (Fig. 8).

Fig. 8. Time profile of changes in CT bone density (expressed in Hounsfield units, HU) in bone defects implanted with various materials and in control defects

Due to non-sphericity (Mauchley test: 0.0030; p<0.001) only multivariable tests were used. We confirmed that there were statistically significant changes in Hounsfield Units with time (F = 1244.88; p<0.001). Moreover, the interaction between time and the type of material used (group) was also statistically significant (F = 65.35; p<0.001).

When the time profiles of CT bone density changes were compared between the studied groups, the only significant differences observed were between groups B and B+BG – 0.86, and B+TCP and B+BG+TCP – 0.30 (Table 6).


Table 6. The p-values for comparison of CT bone density (expressed in Hounsfield units, HU) with changes in time (profiles) between the tested material and control groups

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

particularly intensive around the particles of implanted material.

**5.5 Histopathologic analysis** 

polynuclear cells.

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 385

In all analyzed groups, after 7 days of the study, mandibular bone defects had filled with immature fibrous tissue. Numerous, minute granules of deproteinized human bone (with no signs of activity) were seen in this tissue (group B). Additionally, depending on the implant composition, foggy fractions of bio-glass or linearly cracked deposits of tricalcium phosphate could be seen (in groups B+BG, B+TCP and B+BG+TCP). At the same time, active reconstruction of skeletal tissue was observed at the entire periphery of the bone defects implanted with B, B+TCP and B+BG+TCP, as suggested by the presence of ground substance (osteoid) containing numerous immature bone trabeculae covered with osteoblasts. In controls, as well as in groups B and B+BG, blood clots along with the remnants of necrotic bone trabeculae could be seen in the bone marrow at the base of the bone defect. In groups where bio-glass was implanted into the bone defect (B+BG and B+BG+TCP), fragments of this material formed pseudocystic structures that were covered with a thin connective tissue capsule comprised of fibroblasts, fibrocytes and single giant

After two weeks of the experiment, the implanted bone defects were filled with mature fibrous connective tissue that contained collagen fibers. Only in group B could immature connective tissue with numerous blood vessels be seen at the periphery of the defect. Immature bone trabeculae were visible in this tissue, surrounded with osteoblasts. Depending on the implant composition, fragments of deproteinized bone granules, bio-glass and/or tricalcium phosphate were seen in the fibrous connective tissue. Around BG and TCP particles, distinct, thick-walled pseudocystic structures could be observed, containing numerous giant polynuclear cells. Additionally, giant polynuclear cells were frequently visible on the surface of deproteinized bone but they did not form distinct capsule-like linear structures in this location. Fragmentation of some TCP particles could be observed due to infiltration by cells composing the previously mentioned cystic structures. Active osteogenesis was evident in all analyzed groups, as manifested by the pronounced growth of numerous immature bone trabeculae covered with osteoblasts. This process was

In group B, advanced reconstruction of bone defects was observed after three weeks of the study. Most bone trabeculae filling the defect were mature with either no or very little osteoblastic activity. Linearly placed osteoblasts, or even osteoclasts, could be seen on the surface of remaining trabeculae (Fig. 9). A similar advancement in the bone formation process was observed in B+TCP specimens: osteoid was formed on the base of connective fibrous tissue along with the intensive growth of numerous bone trabeculae. Some of these trabeculae were mature already and showed no signs of cellular activity. Skeletal tissue regeneration in this group was particularly enhanced around the fragments of deproteinized bone and tricalcium phosphate (Fig. 10). In contrast to B particles, TCP particles showed signs of dilution and structural fragmentation. Only a few giant polynuclear cells could be seen around the implants, and the previously observed cystic structures of TCP had only residual character. After three weeks of the study, slightly less advanced processes of skeletal defect healing were observed in histologic specimens from groups B+BG and B+BG+TCP. Growth of immature skeletal tissue was observed in these groups on the "scaffold" of deproteinized bone, giving the impression of the implant being "incorporated" into the growing bone trabeculae. Only single giant polynuclear

The most evident intragroup differences in CT bone density determined in the 1st and 12th experimental weeks were observed in the control group (29.77), but the differences were only slightly less pronounced in groups B+TCP (24.10) and B+BG+TCP (22.94). The least evident differences between the two analyzed time points were noted in the B (10.67) and B+BG (16.77) groups (Table 7).


Table 7. Comparison of CT bone density values (expressed in Hounsfield units, HU) between the 12th and 1st weeks with regard to the tested material and control groups

After seven days of the experiment, the most pronounced intergroup differences in CT bone density were observed between the control group and groups B and BG. The least pronounced intergroup differences noted in this period pertained to groups B and B+BG. After 12 weeks of the study, the most evident intergroup differences in CT bone density were again observed between the controls and group B+BG, while the lowest differences were noted between groups B and B+TCP.


Table 8. Comparison of CT bone density (expressed in Hounsfield units, HU) values in the 1st and 12th weeks between tested material and control groups

We have also observed statistically significant intergroup differences in the relative increase in CT bone density (expressed in Hounsfield units, HU) (F = 39.22; p<0.001). The smallest changes in CT bone density were yielded by group B (255.267.6). Larger changes were observed for B+BG (417.725.7), B+TCP (468.843.3) and B+BG+TCP (486.516.5). The largest change was noted in the control group (556.234.4). For all paired comparisons, statistically significant differences (with p<0.05) were observed, with the only exception being a comparison between B+TCP and B+BG+TCP.

#### **5.5 Histopathologic analysis**

384 Tissue Regeneration – From Basic Biology to Clinical Application

The most evident intragroup differences in CT bone density determined in the 1st and 12th experimental weeks were observed in the control group (29.77), but the differences were only slightly less pronounced in groups B+TCP (24.10) and B+BG+TCP (22.94). The least evident differences between the two analyzed time points were noted in the B (10.67) and

> **Group** |t| p **B** 10.67 **<0.001 B+BG** 16.77 **<0.001 B+TCP** 24.10 **<0.001 B+BG+TCP** 22.94 **<0.001 Control** 29.77 **<0.001**

Table 7. Comparison of CT bone density values (expressed in Hounsfield units, HU) between the 12th and 1st weeks with regard to the tested material and control groups

After seven days of the experiment, the most pronounced intergroup differences in CT bone density were observed between the control group and groups B and BG. The least pronounced intergroup differences noted in this period pertained to groups B and B+BG. After 12 weeks of the study, the most evident intergroup differences in CT bone density were again observed between the controls and group B+BG, while the lowest differences

**Groups** |t| p |t| p **B vs. Control** 16.63 **<0.001** 4.91 **<0.001 B+BG vs. Control** 15.23 **<0.001** 10.29 **<0.001 B+TCP vs. Control** 8.48 **<0.001** 3.67 **<0.05 B+BG+TCP vs. Control** 10.36 **<0.001** 6.83 **<0.001 B vs. B+BG** 1.56 0.1325 6.01 **<0.001 B vs. B+TCP** 9.11 **<0.001** 1.39 0.1779 **B vs. B+BG+TCP** 7.00 **<0.001** 2.14 **<0.05 B+BG vs. B+TCP** 7.55 **<0.001** 7.40 **<0.001 B+BG vs. B+BG+TCP** 5.44 **<0.001** 3.87 **<0.001 B+TCP vs. B+BG+TCP** 2.11 **<0.05** 3.53 **<0.01**  Table 8. Comparison of CT bone density (expressed in Hounsfield units, HU) values in the

We have also observed statistically significant intergroup differences in the relative increase in CT bone density (expressed in Hounsfield units, HU) (F = 39.22; p<0.001). The smallest changes in CT bone density were yielded by group B (255.267.6). Larger changes were observed for B+BG (417.725.7), B+TCP (468.843.3) and B+BG+TCP (486.516.5). The largest change was noted in the control group (556.234.4). For all paired comparisons, statistically significant differences (with p<0.05) were observed, with the only exception

**12th vs. 1st week** 

**1st week 12th week** 

B+BG (16.77) groups (Table 7).

were noted between groups B and B+TCP.

1st and 12th weeks between tested material and control groups

being a comparison between B+TCP and B+BG+TCP.

In all analyzed groups, after 7 days of the study, mandibular bone defects had filled with immature fibrous tissue. Numerous, minute granules of deproteinized human bone (with no signs of activity) were seen in this tissue (group B). Additionally, depending on the implant composition, foggy fractions of bio-glass or linearly cracked deposits of tricalcium phosphate could be seen (in groups B+BG, B+TCP and B+BG+TCP). At the same time, active reconstruction of skeletal tissue was observed at the entire periphery of the bone defects implanted with B, B+TCP and B+BG+TCP, as suggested by the presence of ground substance (osteoid) containing numerous immature bone trabeculae covered with osteoblasts. In controls, as well as in groups B and B+BG, blood clots along with the remnants of necrotic bone trabeculae could be seen in the bone marrow at the base of the bone defect. In groups where bio-glass was implanted into the bone defect (B+BG and B+BG+TCP), fragments of this material formed pseudocystic structures that were covered with a thin connective tissue capsule comprised of fibroblasts, fibrocytes and single giant polynuclear cells.

After two weeks of the experiment, the implanted bone defects were filled with mature fibrous connective tissue that contained collagen fibers. Only in group B could immature connective tissue with numerous blood vessels be seen at the periphery of the defect. Immature bone trabeculae were visible in this tissue, surrounded with osteoblasts. Depending on the implant composition, fragments of deproteinized bone granules, bio-glass and/or tricalcium phosphate were seen in the fibrous connective tissue. Around BG and TCP particles, distinct, thick-walled pseudocystic structures could be observed, containing numerous giant polynuclear cells. Additionally, giant polynuclear cells were frequently visible on the surface of deproteinized bone but they did not form distinct capsule-like linear structures in this location. Fragmentation of some TCP particles could be observed due to infiltration by cells composing the previously mentioned cystic structures. Active osteogenesis was evident in all analyzed groups, as manifested by the pronounced growth of numerous immature bone trabeculae covered with osteoblasts. This process was particularly intensive around the particles of implanted material.

In group B, advanced reconstruction of bone defects was observed after three weeks of the study. Most bone trabeculae filling the defect were mature with either no or very little osteoblastic activity. Linearly placed osteoblasts, or even osteoclasts, could be seen on the surface of remaining trabeculae (Fig. 9). A similar advancement in the bone formation process was observed in B+TCP specimens: osteoid was formed on the base of connective fibrous tissue along with the intensive growth of numerous bone trabeculae. Some of these trabeculae were mature already and showed no signs of cellular activity. Skeletal tissue regeneration in this group was particularly enhanced around the fragments of deproteinized bone and tricalcium phosphate (Fig. 10). In contrast to B particles, TCP particles showed signs of dilution and structural fragmentation. Only a few giant polynuclear cells could be seen around the implants, and the previously observed cystic structures of TCP had only residual character. After three weeks of the study, slightly less advanced processes of skeletal defect healing were observed in histologic specimens from groups B+BG and B+BG+TCP. Growth of immature skeletal tissue was observed in these groups on the "scaffold" of deproteinized bone, giving the impression of the implant being "incorporated" into the growing bone trabeculae. Only single giant polynuclear

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

contained mature trabeculae.

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 387

intensive around the B particles, giving the characteristic impression of "incorporating" this material into newly formed bone. After eight weeks of the study, only some bone trabeculae in the control group showed signs of osteoblastic activity, while other areas of the defect

Fig. 9. Histopathologic specimen – 3rd week, group B – single osteoclasts on the surface of

Fig. 10. Histopathologic specimen – 3rd week, group B+TCP – regeneration of skeletal tissue around fragments of deproteinized human bone and tricalcium phosphate (H&E staining,

bone trabeculae (H&E staining, magnification 400 x)

magnification 200 x)

cells could be seen on the surface of specimens from group B+BG. Additionally, thin, linearly placed cells could be observed around some bio-glass particles, forming pseudocystic structures. These structures were visible mostly in areas directly adjacent to fibrous connective tissue. Numerous bone trabeculae were observed around the B+BG+TCP mixture particles, as well as in the fibrous connective tissue between the particles (Fig. 11). Some trabeculae were mature and showed no signs of osteoblastic activity on their surfaces. Some implant particles in this group were covered with giant polynuclear cells forming structures resembling foreign body granulomas. In the control group, all osteoid trabeculae were surrounded by osteoblasts still showing signs of osteoblastic activity.

After four weeks of the study, only the bone defects in group B were nearly completely filled with mature, compact skeletal tissue. This tissue contained numerous "incorporated" granules of deproteinized human bone. Growing bone trabeculae with surface signs of osteoblastic activity could only be seen in a narrow layer of fibrous connective tissue located between the newly formed bone and the bottom of the defect. At the same time, mature cancellous skeletal tissue could only be observed at the periphery of bone defects in groups B+TCP and B+BG+TCP. "Incorporated" fragments of the implanted material, mostly human bone, were visible in this tissue. The central part of the defects was still filled with fibrous connective tissue, showing signs of the ongoing process of bone formation. This area also contained B particles and a few particles of TCP ceramic. These fragments of tricalcium phosphate were covered with a pseudo-capsule comprised of giant polynuclear cells, and were gradually fragmented and resorbed. After four weeks of the experiment, numerous mature bone trabeculae with no signs of cellular activity were observed in specimens from group B+BG (Fig. 12). Some of these trabeculae developed around B and BG particles, giving the impression of "incorporating" implanted material into the structure of reconstructed bone. Some bone defects were still partially filled with mature fibrous connective tissue containing numerous collagen fibers along with particles of implanted material. Additionally, pseudocystic structures could be observed around the BG particles, comprised of linearly placed giant polynuclear cells.

Histopathologic examination performed after eight weeks of the study revealed the completed process of bone healing in group B. Bone structure was fully regenerated, and mature compact bone and cancellous bone could be observed at the defect site, along with normal bone marrow (Fig. 13). Observations made in the B+BG+TCP group after the 8th and 12th weeks of the experiment gave similar findings. In this group bone defects were also filled with mature compact and cancellous skeletal tissue, with "incorporated" particles of bio-glass and deproteinized bone still visible (Fig. 14). These particles showed no signs of activity, and were covered with thin fibrous capsules. In the 8th experimental week, these particles could also be observed in fibrous connective tissue. After the 8th week of the study, the process of osteogenesis was still incomplete in B+BG and B+TCP specimens. In defects implanted with bio-glass, some areas, usually peripheral ones, were still filled with fibrous connective tissue containing numerous collagen fibers. This connective tissue showed signs of ongoing bone formation: maturating or mature bone trabeculae, along with bio-glass particles forming pseudocystic structures (Fig. 15). Particles of deproteinized bone were more rarely evidenced. At the same time, continued bone formation was observed in the central part of bone defects in group B+TCP (Fig. 16). This process was particularly

cells could be seen on the surface of specimens from group B+BG. Additionally, thin, linearly placed cells could be observed around some bio-glass particles, forming pseudocystic structures. These structures were visible mostly in areas directly adjacent to fibrous connective tissue. Numerous bone trabeculae were observed around the B+BG+TCP mixture particles, as well as in the fibrous connective tissue between the particles (Fig. 11). Some trabeculae were mature and showed no signs of osteoblastic activity on their surfaces. Some implant particles in this group were covered with giant polynuclear cells forming structures resembling foreign body granulomas. In the control group, all osteoid trabeculae were surrounded by osteoblasts still showing signs of

After four weeks of the study, only the bone defects in group B were nearly completely filled with mature, compact skeletal tissue. This tissue contained numerous "incorporated" granules of deproteinized human bone. Growing bone trabeculae with surface signs of osteoblastic activity could only be seen in a narrow layer of fibrous connective tissue located between the newly formed bone and the bottom of the defect. At the same time, mature cancellous skeletal tissue could only be observed at the periphery of bone defects in groups B+TCP and B+BG+TCP. "Incorporated" fragments of the implanted material, mostly human bone, were visible in this tissue. The central part of the defects was still filled with fibrous connective tissue, showing signs of the ongoing process of bone formation. This area also contained B particles and a few particles of TCP ceramic. These fragments of tricalcium phosphate were covered with a pseudo-capsule comprised of giant polynuclear cells, and were gradually fragmented and resorbed. After four weeks of the experiment, numerous mature bone trabeculae with no signs of cellular activity were observed in specimens from group B+BG (Fig. 12). Some of these trabeculae developed around B and BG particles, giving the impression of "incorporating" implanted material into the structure of reconstructed bone. Some bone defects were still partially filled with mature fibrous connective tissue containing numerous collagen fibers along with particles of implanted material. Additionally, pseudocystic structures could be observed around the BG particles,

Histopathologic examination performed after eight weeks of the study revealed the completed process of bone healing in group B. Bone structure was fully regenerated, and mature compact bone and cancellous bone could be observed at the defect site, along with normal bone marrow (Fig. 13). Observations made in the B+BG+TCP group after the 8th and 12th weeks of the experiment gave similar findings. In this group bone defects were also filled with mature compact and cancellous skeletal tissue, with "incorporated" particles of bio-glass and deproteinized bone still visible (Fig. 14). These particles showed no signs of activity, and were covered with thin fibrous capsules. In the 8th experimental week, these particles could also be observed in fibrous connective tissue. After the 8th week of the study, the process of osteogenesis was still incomplete in B+BG and B+TCP specimens. In defects implanted with bio-glass, some areas, usually peripheral ones, were still filled with fibrous connective tissue containing numerous collagen fibers. This connective tissue showed signs of ongoing bone formation: maturating or mature bone trabeculae, along with bio-glass particles forming pseudocystic structures (Fig. 15). Particles of deproteinized bone were more rarely evidenced. At the same time, continued bone formation was observed in the central part of bone defects in group B+TCP (Fig. 16). This process was particularly

osteoblastic activity.

comprised of linearly placed giant polynuclear cells.

intensive around the B particles, giving the characteristic impression of "incorporating" this material into newly formed bone. After eight weeks of the study, only some bone trabeculae in the control group showed signs of osteoblastic activity, while other areas of the defect contained mature trabeculae.

Fig. 9. Histopathologic specimen – 3rd week, group B – single osteoclasts on the surface of bone trabeculae (H&E staining, magnification 400 x)

Fig. 10. Histopathologic specimen – 3rd week, group B+TCP – regeneration of skeletal tissue around fragments of deproteinized human bone and tricalcium phosphate (H&E staining, magnification 200 x)

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

bone.

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 389

were revealed in fibrous connective tissue (Fig. 18). In the control group, specimens obtained in the 12th week of the study contained completely matured and fully mineralized

Fig. 13. Histopathologic specimen – 8th week, group B – mature skeletal tissue and bone

Fig. 14. Histopathologic specimen – 8th week, group B+BG+TCP – mature compact and cancellous skeletal tissue with "incorporated" particles of bio-glass and human bone (H&E

marrow (H&E staining, magnification 100 x)

staining, magnification 100 x)

Fig. 11. Histopathologic specimen – 3rd week, group B+BG+TCP – numerous bone trabeculae around the implanted material visible within fibrous connective tissue (H&E staining, magnification 40 x)

Fig. 12. Histopathologic specimen – 4th week, group B+BG – mature bone trabeculae with no signs of cellular activity, along with particles of deproteinized human bone and bio-glass visible within fibrous connective tissue (H&E staining, magnification 40 x)

After 12 weeks of the study, the process of skeletal tissue regeneration in specimens from groups B+BG and B+TCP was still not complete. Although the defects were nearly filled in their entirety with mature compact and cancellous bone, mature fibrous connective tissue containing numerous collagen fibers and showing signs of ongoing osteogenesis could still be seen in the superficial zone (Fig. 17). This superficial layer contained remnants of deproteinized bone, covered with capsules comprised of giant polynuclear cells, which formed inactive, fibrous foreign body granulomas. Additionally, cystic structures containing foggy remnants of incompletely resorbed TCP (group B+TCP) or BG particles (group B+BG)

Fig. 11. Histopathologic specimen – 3rd week, group B+BG+TCP – numerous bone trabeculae around the implanted material visible within fibrous connective tissue (H&E

Fig. 12. Histopathologic specimen – 4th week, group B+BG – mature bone trabeculae with no signs of cellular activity, along with particles of deproteinized human bone and bio-glass

After 12 weeks of the study, the process of skeletal tissue regeneration in specimens from groups B+BG and B+TCP was still not complete. Although the defects were nearly filled in their entirety with mature compact and cancellous bone, mature fibrous connective tissue containing numerous collagen fibers and showing signs of ongoing osteogenesis could still be seen in the superficial zone (Fig. 17). This superficial layer contained remnants of deproteinized bone, covered with capsules comprised of giant polynuclear cells, which formed inactive, fibrous foreign body granulomas. Additionally, cystic structures containing foggy remnants of incompletely resorbed TCP (group B+TCP) or BG particles (group B+BG)

visible within fibrous connective tissue (H&E staining, magnification 40 x)

staining, magnification 40 x)

were revealed in fibrous connective tissue (Fig. 18). In the control group, specimens obtained in the 12th week of the study contained completely matured and fully mineralized bone.

Fig. 13. Histopathologic specimen – 8th week, group B – mature skeletal tissue and bone marrow (H&E staining, magnification 100 x)

Fig. 14. Histopathologic specimen – 8th week, group B+BG+TCP – mature compact and cancellous skeletal tissue with "incorporated" particles of bio-glass and human bone (H&E staining, magnification 100 x)

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 391

Fig. 17. Histopathologic specimen – 12th week, group B+BG – mature fibrous connective tissue with numerous cystic spaces filled with foggy deposits of bio-glass (H&E staining, 100 x)

Fig. 18. Histopathologic specimen – 12th week, group B+TCP – cystic structures within fibrous connective tissue containing foggy remnants of tricalcium phosphate (H&E staining,

Due to the limited regenerative ability of skeletal tissue, bone grafting or the implantation of bone derivatives or bone replacement materials is required for the complete healing of large bone defects, whether the result of surgical removal of skeletal cysts or tumors, or caused by other skeletal disorders (Precheur, 2007). The most satisfactory results in stimulating skeletal tissue regeneration have been reported after using autogenous grafts. After being implanted into the bone defect, autogenous grafts can induce all the basic mechanisms responsible for bone reconstruction, i.e. osteogenesis, osteoinduction and osteoconduction (Giannoudis et

200 x)

**6. Discussion** 

Fig. 15. Histopathologic specimen – 8th week, group B+BG – bio-glass particle forming pseudocystic structure within the mature fibrous connective tissue (H&E staining, magnification 100 x)

Fig. 16. Histopathologic specimen – 8th week, group B+TCP – fibrous connective tissue with massive osteogenesis (H&E staining, magnification 100 x)

Fig. 17. Histopathologic specimen – 12th week, group B+BG – mature fibrous connective tissue with numerous cystic spaces filled with foggy deposits of bio-glass (H&E staining, 100 x)

Fig. 18. Histopathologic specimen – 12th week, group B+TCP – cystic structures within fibrous connective tissue containing foggy remnants of tricalcium phosphate (H&E staining, 200 x)

### **6. Discussion**

390 Tissue Regeneration – From Basic Biology to Clinical Application

Fig. 15. Histopathologic specimen – 8th week, group B+BG – bio-glass particle forming pseudocystic structure within the mature fibrous connective tissue (H&E staining,

Fig. 16. Histopathologic specimen – 8th week, group B+TCP – fibrous connective tissue with

massive osteogenesis (H&E staining, magnification 100 x)

magnification 100 x)

Due to the limited regenerative ability of skeletal tissue, bone grafting or the implantation of bone derivatives or bone replacement materials is required for the complete healing of large bone defects, whether the result of surgical removal of skeletal cysts or tumors, or caused by other skeletal disorders (Precheur, 2007). The most satisfactory results in stimulating skeletal tissue regeneration have been reported after using autogenous grafts. After being implanted into the bone defect, autogenous grafts can induce all the basic mechanisms responsible for bone reconstruction, i.e. osteogenesis, osteoinduction and osteoconduction (Giannoudis et

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

4 and 6 months post-operatively (Kim et al., 2009).

and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 393

effects were observed when autogenous bone graft was used alone (Pripatnanont et al., 2009). Using a mixture of allogenic bone and deproteinized bovine bone (BioOss®/Orthoblast II®) for the purposes of maxillary sinus lift did not have results as satisfactory as with the application of deproteinized animal bone or synthetic bone (Osteon®) alone. The individual use of one of these two implant materials was associated with a higher percentage of newly formed osseous fraction collected from the lateral sinus at

In this study we have combined allogenic bone with artificially obtained biomaterials (bioglass – BG, and/or beta-tricalcium phosphate – TCP) in order to form bone replacement material with improved biological characteristics. Reference materials for comparative analysis of the studied mixtures (B+BG, B+TCP, B+BG+TCP) included lyophilized human bone (B) and the clotted blood of experimental animals. Both clinical observations and further macroscopic, radiographic and histopathologic examinations confirmed that bone defects healed normally in the presence of all studied biomaterials. However, the type of implanted mixture modulated the kinetics of bone formation and the quality of newly formed bone. Bone regeneration was induced markedly earlier whenever biologically active bio-glass was included in the implanted mixture (B+BG, B+BG+TCP). In groups where bioglass was implanted, irregular shadows were observed on radiographic images of the bone defect sites as early as after two weeks of the study. Probably, these radiographic changes resulted from ongoing reparative processes within the bone. This was additionally confirmed on histopathologic analysis, which revealed intense bone formation processes as early as three weeks after the implantation of BG-containing material. However, bone density measurements (BMD and CT bone density) taken in the early period of this study confirmed the superior quality of newly formed bone only in case of the B+BG mixture. It is plausible that the lower bone densities determined for B+BG+TCP implants resulted from the low content of bio-glass in this mixture. Moreover, as confirmed by histopathologic analysis, resorption of beta-tricalcium phosphate contained in B+BG+TCP already began in the early period of this study. Nonetheless, in the later period of this study, increases in BMD and CT bone density of B+BG implanted bone were markedly lower. As a result, after 12 weeks of the experiment, the defects filled with this mixture were characterized by the lowest BMD values, and histopathologic examination confirmed ongoing bone formation. The final result of bone regeneration was markedly better in the case of defects implanted with B+BG+TCP. In the 12th week, histopathologic analysis revealed mature skeletal tissue (both compact and cancellous bone) at the implantation sites, and this finding was confirmed on radiographic examination. Additionally, new bone formed using B+BG+TCP implantation was characterized by the highest BMD and relatively high CT bone density. Therefore, this regenerated bone most closely resembled the normal skeletal tissue of experimental animals of all mixtures examined. In the 12th week of this study, bone formation processes were still observed in B+TCP implanted defects. Although the BMD of tissue formed on the basis of this implant was higher than in the B+BG implanted bone, it was still lower than in the B+BG+TCP group. Notably, in both the 1st and 12th experimental weeks, only slight differences in BMD and CT bone density were observed between the B+TCP and B+BG+TCP mixtures. Undoubtedly, the process of bone defect regeneration was completed the earliest in group B. Histopathologic studies confirmed that bone defects in this group were filled with mature skeletal tissue with no signs of osteoblastic activity as early as after eight weeks of the study. Early completion of skeletal healing was also

al., 2005; Merkx et al., 2003). Osteogenic activity is associated with the presence of osteoprogenitor cells in the periosteum, endosteum and bone marrow. In the case of free bone grafts, some of the cells located most superficially may survive and are involved in regeneration processes. The results of bone healing stimulation are definitely most satisfactory when autogenous cancellous bone chips are implanted, since this type of bone contains a high number of osteoprogenitor cells. Osteogenic activity can only be observed in fresh bone grafts. Osteoinduction is associated with the presence of so-called bone morphogenetic proteins (BMP) in the bone matrix. These proteins are released during bone remodeling, and can stimulate minimally differentiated connective tissue cells surrounding the graft to transform into osteoblasts (Barradas et al., 2011). Both fresh autogenic bone grafts and the allogenic grafts obtained from tissue bank (especially when frozen and partly decalcified) have osteoinductive properties. Additionally, BMP preparations can be obtained by extraction from bones, and as of recent, also by biotechnological synthesis in recombinant form. Allogenic bones are very frequently lyophilized, which depletes them of BMP. Additionally, such grafts lose their immunogenic properties due to irradiation sterilization and deep freezing (Bohner, 2010; Liu et al., 2008). This process of sterilization results in decreased durability of the material, and the preserved bone matrix has only osteoconductive properties. It is degraded by osteoclasts with the simultaneous formation of woven bone, which is further transformed into lamellar bone through the process of osteoclasia. Such grafts have been shown to undergo revascularization and remodeling – similar to autogenous grafts, but at a slower rate. Apart from bone implants, organic and inorganic alloplastic bone replacement materials also have osteoconductive properties. Combined with growth factors and autogenous barrier membranes, they are frequently used as basic elements in the process of guided bone regeneration (GBR) (Kao & Scott, 2007; Schwarz et al., 2007).

For various reasons, different bone materials are frequently combined with each other or with alloplastic biomaterials. As a result, biologically improved material compositions are obtained, some of which positively influence the bone formation processes. Combination of natural hydroxyapatite with chitosane resulted in a composite with osteoconductive properties. In the presence of this composite, tibial consolidation in rabbits was observed as early as 12 weeks after implantation, and complete healing was observed after 16 weeks of the study (Yuan et al., 2008). In another study, a composite based on bovine bone with the addition of bio-glass showed no cytotoxicity to human fibroblasts. Moreover, a crystalline carbonated apatite phase was developed on the sample surface as early as 12 days after immersion in simulated body fluid (Yoganad e al., 2010). Another example of the positive effects of combining deproteinized bovine bone with autogenous bone comes from a study in which such a material was used for the regeneration of bone defects in the frontal part of the porcine skull. The presence of autogenous bone in the mixture was the basis for the osteoinductive properties of the material and the more favorable biological conditions for bone growth when compared to deproteinized bovine bone alone (Thorwarth et al., 2006). Similarly, more satisfactory clinical results were reported when deproteinized bovine bone was used in combination with autogenous bone in the management of alveoschisis in humans, instead of bone autograft alone (Thuaksuban et al., 2010). Experiments on rabbits have also given interesting results. It was revealed that the addition of deproteinized bovine bone to autogenous grafts increased the mean optical density of newly formed skeletal tissue, with a simultaneous decrease in its content in bone defects (skullcap). The opposite

al., 2005; Merkx et al., 2003). Osteogenic activity is associated with the presence of osteoprogenitor cells in the periosteum, endosteum and bone marrow. In the case of free bone grafts, some of the cells located most superficially may survive and are involved in regeneration processes. The results of bone healing stimulation are definitely most satisfactory when autogenous cancellous bone chips are implanted, since this type of bone contains a high number of osteoprogenitor cells. Osteogenic activity can only be observed in fresh bone grafts. Osteoinduction is associated with the presence of so-called bone morphogenetic proteins (BMP) in the bone matrix. These proteins are released during bone remodeling, and can stimulate minimally differentiated connective tissue cells surrounding the graft to transform into osteoblasts (Barradas et al., 2011). Both fresh autogenic bone grafts and the allogenic grafts obtained from tissue bank (especially when frozen and partly decalcified) have osteoinductive properties. Additionally, BMP preparations can be obtained by extraction from bones, and as of recent, also by biotechnological synthesis in recombinant form. Allogenic bones are very frequently lyophilized, which depletes them of BMP. Additionally, such grafts lose their immunogenic properties due to irradiation sterilization and deep freezing (Bohner, 2010; Liu et al., 2008). This process of sterilization results in decreased durability of the material, and the preserved bone matrix has only osteoconductive properties. It is degraded by osteoclasts with the simultaneous formation of woven bone, which is further transformed into lamellar bone through the process of osteoclasia. Such grafts have been shown to undergo revascularization and remodeling – similar to autogenous grafts, but at a slower rate. Apart from bone implants, organic and inorganic alloplastic bone replacement materials also have osteoconductive properties. Combined with growth factors and autogenous barrier membranes, they are frequently used as basic elements in the process of guided bone regeneration (GBR) (Kao & Scott, 2007;

For various reasons, different bone materials are frequently combined with each other or with alloplastic biomaterials. As a result, biologically improved material compositions are obtained, some of which positively influence the bone formation processes. Combination of natural hydroxyapatite with chitosane resulted in a composite with osteoconductive properties. In the presence of this composite, tibial consolidation in rabbits was observed as early as 12 weeks after implantation, and complete healing was observed after 16 weeks of the study (Yuan et al., 2008). In another study, a composite based on bovine bone with the addition of bio-glass showed no cytotoxicity to human fibroblasts. Moreover, a crystalline carbonated apatite phase was developed on the sample surface as early as 12 days after immersion in simulated body fluid (Yoganad e al., 2010). Another example of the positive effects of combining deproteinized bovine bone with autogenous bone comes from a study in which such a material was used for the regeneration of bone defects in the frontal part of the porcine skull. The presence of autogenous bone in the mixture was the basis for the osteoinductive properties of the material and the more favorable biological conditions for bone growth when compared to deproteinized bovine bone alone (Thorwarth et al., 2006). Similarly, more satisfactory clinical results were reported when deproteinized bovine bone was used in combination with autogenous bone in the management of alveoschisis in humans, instead of bone autograft alone (Thuaksuban et al., 2010). Experiments on rabbits have also given interesting results. It was revealed that the addition of deproteinized bovine bone to autogenous grafts increased the mean optical density of newly formed skeletal tissue, with a simultaneous decrease in its content in bone defects (skullcap). The opposite

Schwarz et al., 2007).

effects were observed when autogenous bone graft was used alone (Pripatnanont et al., 2009). Using a mixture of allogenic bone and deproteinized bovine bone (BioOss®/Orthoblast II®) for the purposes of maxillary sinus lift did not have results as satisfactory as with the application of deproteinized animal bone or synthetic bone (Osteon®) alone. The individual use of one of these two implant materials was associated with a higher percentage of newly formed osseous fraction collected from the lateral sinus at 4 and 6 months post-operatively (Kim et al., 2009).

In this study we have combined allogenic bone with artificially obtained biomaterials (bioglass – BG, and/or beta-tricalcium phosphate – TCP) in order to form bone replacement material with improved biological characteristics. Reference materials for comparative analysis of the studied mixtures (B+BG, B+TCP, B+BG+TCP) included lyophilized human bone (B) and the clotted blood of experimental animals. Both clinical observations and further macroscopic, radiographic and histopathologic examinations confirmed that bone defects healed normally in the presence of all studied biomaterials. However, the type of implanted mixture modulated the kinetics of bone formation and the quality of newly formed bone. Bone regeneration was induced markedly earlier whenever biologically active bio-glass was included in the implanted mixture (B+BG, B+BG+TCP). In groups where bioglass was implanted, irregular shadows were observed on radiographic images of the bone defect sites as early as after two weeks of the study. Probably, these radiographic changes resulted from ongoing reparative processes within the bone. This was additionally confirmed on histopathologic analysis, which revealed intense bone formation processes as early as three weeks after the implantation of BG-containing material. However, bone density measurements (BMD and CT bone density) taken in the early period of this study confirmed the superior quality of newly formed bone only in case of the B+BG mixture. It is plausible that the lower bone densities determined for B+BG+TCP implants resulted from the low content of bio-glass in this mixture. Moreover, as confirmed by histopathologic analysis, resorption of beta-tricalcium phosphate contained in B+BG+TCP already began in the early period of this study. Nonetheless, in the later period of this study, increases in BMD and CT bone density of B+BG implanted bone were markedly lower. As a result, after 12 weeks of the experiment, the defects filled with this mixture were characterized by the lowest BMD values, and histopathologic examination confirmed ongoing bone formation. The final result of bone regeneration was markedly better in the case of defects implanted with B+BG+TCP. In the 12th week, histopathologic analysis revealed mature skeletal tissue (both compact and cancellous bone) at the implantation sites, and this finding was confirmed on radiographic examination. Additionally, new bone formed using B+BG+TCP implantation was characterized by the highest BMD and relatively high CT bone density. Therefore, this regenerated bone most closely resembled the normal skeletal tissue of experimental animals of all mixtures examined. In the 12th week of this study, bone formation processes were still observed in B+TCP implanted defects. Although the BMD of tissue formed on the basis of this implant was higher than in the B+BG implanted bone, it was still lower than in the B+BG+TCP group. Notably, in both the 1st and 12th experimental weeks, only slight differences in BMD and CT bone density were observed between the B+TCP and B+BG+TCP mixtures. Undoubtedly, the process of bone defect regeneration was completed the earliest in group B. Histopathologic studies confirmed that bone defects in this group were filled with mature skeletal tissue with no signs of osteoblastic activity as early as after eight weeks of the study. Early completion of skeletal healing was also

Preparation of Deproteinized Human Bone and Its Mixtures with Bio-Glass

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and Tricalcium Phosphate – Innovative Bioactive Materials for Skeletal Tissue Regeneration 395

Accorsi-Mendonça, T., Conz, M.B., Barros, T.C., de Sena, L.Á., de Almeida Soares, G. &

Baldini, N., De Sanctis, M. & Ferrari, M. (2011). Deproteinized Bovine Bone in Periodontal

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confirmed by the radiographic images taken in this group. In the 12th experimental week, BMD of bone defects implanted with human bone alone was higher in comparison to defects filled with B+TCP and B+BG+TCP, in contrast to the early period of this study when BMD and CT bone density in group B were among the highest.

Results of this study suggest that the quality of bone formed in the late period of the experiment was poorest in the control group, as suggested by the low bone density of defects healing on the basis of clotted blood. Since histopathologic studies confirmed that bone formation in controls was complete in week 12, one should not expect further increases in bone density in this group. It is likely that bone density would increase further, however, with prolonged observation of the B+BG and B+TCP groups, since the last densitometric measurements in these groups were taken on incompletely matured bone.

#### **7. Conclusion**

This *in vivo* animal study revealed that both lyophilized human bone (B) and mixtures formed on its basis (B+BG, B+TCP, B+BG+TCP) are fully biocompatible materials. We have confirmed that, in the presence of these materials, there is a possibility of forming normal, mature skeletal tissue in mandibular bone defects of guinea pigs. This was possible mostly thanks to the inclusion of deproteinized human bone in analyzed mixtures. Due to its osteoconductive properties, the presence of human bone resulted in favorable biological conditions that promoted skeletal regeneration. Therefore, deproteinized bone formed a scaffold to support the growth of osteogenic cells within the defects. Furthermore, the addition of alloplastic materials, bio-glass and beta-tricalcium phosphate, markedly influenced the rate of bone formation and the quality of newly formed bone. The most satisfactory results were observed in the case of lyophilized human bone mixed with bioglass and beta-tricalcium phosphate (B+BG+TCP). The group implanted with this material was the only one in which fully matured compact and cancellous bone was observed on histopathologic examination performed after 12 weeks of the study. The particles of bioglass and beta-tricalcium phosphate included in the mixture induced the processes of bone formation and stimulated the growth of osteogenic cells. As a result of these initiated biological processes, defragmented and resorbed β-TCP was gradually replaced with newly formed skeletal structures. Such a course of bone formation process modulated the quality of newly formed bone, as confirmed by high BMD and CT bone density values determined after 12 weeks in B+BG+TCP implanted defects. As confirmed on histopathologic examination, throughout the three-month period of this study, skeletal regeneration was not completed in defects implanted with B+BG and B+TCP mixtures. This incomplete regeneration was reflected by the lower bone density values observed in these groups when compared to the B+BG+TCP group. In conclusion, the results of this study confirmed our initial assumptions. We have revealed that, in addition to a proper course of bone formation processes, another important outcome in the presence of various bone replacement materials is the high quality of newly formed bone. In our opinion, these two aforementioned results were positively achieved by the mixture of lyophilized human bone with bio-glass and tricalcium phosphate.

#### **8. References**

Abu Bakar, M.S., Cheng, M.H.W., Tang, S.M., Yu, S.C., Liao, K., Tan, C.T., Khor, K.A. & Cheang, P. (2003). Tensile Properties, Tension–tension Fatigue and Biological

confirmed by the radiographic images taken in this group. In the 12th experimental week, BMD of bone defects implanted with human bone alone was higher in comparison to defects filled with B+TCP and B+BG+TCP, in contrast to the early period of this study when

Results of this study suggest that the quality of bone formed in the late period of the experiment was poorest in the control group, as suggested by the low bone density of defects healing on the basis of clotted blood. Since histopathologic studies confirmed that bone formation in controls was complete in week 12, one should not expect further increases in bone density in this group. It is likely that bone density would increase further, however, with prolonged observation of the B+BG and B+TCP groups, since the last densitometric

This *in vivo* animal study revealed that both lyophilized human bone (B) and mixtures formed on its basis (B+BG, B+TCP, B+BG+TCP) are fully biocompatible materials. We have confirmed that, in the presence of these materials, there is a possibility of forming normal, mature skeletal tissue in mandibular bone defects of guinea pigs. This was possible mostly thanks to the inclusion of deproteinized human bone in analyzed mixtures. Due to its osteoconductive properties, the presence of human bone resulted in favorable biological conditions that promoted skeletal regeneration. Therefore, deproteinized bone formed a scaffold to support the growth of osteogenic cells within the defects. Furthermore, the addition of alloplastic materials, bio-glass and beta-tricalcium phosphate, markedly influenced the rate of bone formation and the quality of newly formed bone. The most satisfactory results were observed in the case of lyophilized human bone mixed with bioglass and beta-tricalcium phosphate (B+BG+TCP). The group implanted with this material was the only one in which fully matured compact and cancellous bone was observed on histopathologic examination performed after 12 weeks of the study. The particles of bioglass and beta-tricalcium phosphate included in the mixture induced the processes of bone formation and stimulated the growth of osteogenic cells. As a result of these initiated biological processes, defragmented and resorbed β-TCP was gradually replaced with newly formed skeletal structures. Such a course of bone formation process modulated the quality of newly formed bone, as confirmed by high BMD and CT bone density values determined after 12 weeks in B+BG+TCP implanted defects. As confirmed on histopathologic examination, throughout the three-month period of this study, skeletal regeneration was not completed in defects implanted with B+BG and B+TCP mixtures. This incomplete regeneration was reflected by the lower bone density values observed in these groups when compared to the B+BG+TCP group. In conclusion, the results of this study confirmed our initial assumptions. We have revealed that, in addition to a proper course of bone formation processes, another important outcome in the presence of various bone replacement materials is the high quality of newly formed bone. In our opinion, these two aforementioned results were positively achieved by the mixture of lyophilized human bone

Abu Bakar, M.S., Cheng, M.H.W., Tang, S.M., Yu, S.C., Liao, K., Tan, C.T., Khor, K.A. &

Cheang, P. (2003). Tensile Properties, Tension–tension Fatigue and Biological

BMD and CT bone density in group B were among the highest.

**7. Conclusion** 

with bio-glass and tricalcium phosphate.

**8. References** 

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**18** 

*The Netherlands* 

**Endochondral Bone Formation as** 

Peter J. Emans, Marjolein M.J. Caron,

Lodewijk W. van Rhijn and Tim J.M. Welting

**Blueprint for Regenerative Medicine** 

*Department of Orthopaedic Surgery, Maastricht University Medical Center,* 

**During our life** moving, walking, sport, etc., are essential for our health and quality of life. Both bones and cartilage enable us to do so. Bones support us, allow muscles to move them, and protect vital internal organs. At the end of most bones articular joints are situated. The side where 2 bones form an articular joint, the ends of these bones are covered with hyaline cartilage. This articular cartilage is able to withstand very high mechanical forces with very low friction and thereby enables easy movement. A large number of bones are formed by a process called endochondral ossification. During this process a cartilage template is replaced by bone, in contrast with the cartilage in newly formed joints which remains cartilage. Both articular cartilage and bone mature and this leads to a well organised architecture and specialisation. The arcade-like architecture of cartilage is capable to withstand an enormous amount of intensive and repetitive forces during life. However, the British surgeon William Hunter made the now famous statement that "*From Hippocrates to the present age it is universally allowed that ulcerated cartilage is a troublesome thing and that once destroyed it is not repaired*" (Hunter 1743). In contrast, bone has a very high regenerative capacity. This difference in self-healing capacity may partially be explained by the access to progenitor cells which contribute to tissue repair. For bone repair, progenitor cells of three different sources have been identified. These sources are: (i) progenitor cells form the blood stream since bone is a highly vascularised tissue, (ii) progenitor cells from the overlying periosteum and (iii) progenitor cells from the bone marrow. Cartilage is not vascularised, is not covered by periosteum, nor has a specialized tissue such as bone marrow and this might be part of the explanation for the limited self-repair capacity of cartilage. Although both tissues start from the same mesenchymal cell condensations, the contrast in self-repair is striking

From a clinical point of view there is a need for repair of both bone and cartilage. Bone and cartilage were both identified as tissues for which it was thought to be possible to recreate them in a laboratory setting, using the combination of cell isolation culture techniques and carrier materials. The science of combining cells with carrier materials to reproduce tissues in the laboratory is called Tissue Engineering (TE). The collaboration of scientists of different disciplines such as cell biology, biomaterials, biomechanics, engineering and translational

**1. Introduction** 

(Hunziker, Kapfinger et al. 2007).

*Vitro*: Implications and Applications for Bone Tissue Engineering. *Calcified Tissue International,* Vol.67, No.4, (October 2000), pp. 321-329, ISSN 1432-0827


### **Endochondral Bone Formation as Blueprint for Regenerative Medicine**

Peter J. Emans, Marjolein M.J. Caron, Lodewijk W. van Rhijn and Tim J.M. Welting *Department of Orthopaedic Surgery, Maastricht University Medical Center, The Netherlands* 

#### **1. Introduction**

398 Tissue Regeneration – From Basic Biology to Clinical Application

Yuan, H., Chen, N., Lü, X. & Zheng, B. (2008). Experimental Study of Natural

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ISSN 0272-8842

379-383, ISSN 1008-1275

4376

*Vitro*: Implications and Applications for Bone Tissue Engineering. *Calcified Tissue* 

Paraskevopoulos, K.M. & Rouabhia, M. (2010). Characterization and In vitrobioactivity of Natural Hydroxyapatite Based Bio-glass–ceramics Synthesized by Thermal Plasma Processing. *Ceramics,* Vol.36, No.6, (August 2010), pp.1757-1766,

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and Deprotenized Bone Derived from Human Avascular Necrotic Femoral Head. *Chinese Journal of Traumatology (English Edition),* Vol.12, No.6, (December 2009), pp.

Orthopaedic and Stomatological Applications – Preparation and *In Vitro* Evaluation. *Ceramic Materials,* Vol.62, No.1, (2010), pp. 51-55, ISSN 1644-3470

**During our life** moving, walking, sport, etc., are essential for our health and quality of life. Both bones and cartilage enable us to do so. Bones support us, allow muscles to move them, and protect vital internal organs. At the end of most bones articular joints are situated. The side where 2 bones form an articular joint, the ends of these bones are covered with hyaline cartilage. This articular cartilage is able to withstand very high mechanical forces with very low friction and thereby enables easy movement. A large number of bones are formed by a process called endochondral ossification. During this process a cartilage template is replaced by bone, in contrast with the cartilage in newly formed joints which remains cartilage. Both articular cartilage and bone mature and this leads to a well organised architecture and specialisation. The arcade-like architecture of cartilage is capable to withstand an enormous amount of intensive and repetitive forces during life. However, the British surgeon William Hunter made the now famous statement that "*From Hippocrates to the present age it is universally allowed that ulcerated cartilage is a troublesome thing and that once destroyed it is not repaired*" (Hunter 1743). In contrast, bone has a very high regenerative capacity. This difference in self-healing capacity may partially be explained by the access to progenitor cells which contribute to tissue repair. For bone repair, progenitor cells of three different sources have been identified. These sources are: (i) progenitor cells form the blood stream since bone is a highly vascularised tissue, (ii) progenitor cells from the overlying periosteum and (iii) progenitor cells from the bone marrow. Cartilage is not vascularised, is not covered by periosteum, nor has a specialized tissue such as bone marrow and this might be part of the explanation for the limited self-repair capacity of cartilage. Although both tissues start from the same mesenchymal cell condensations, the contrast in self-repair is striking (Hunziker, Kapfinger et al. 2007).

From a clinical point of view there is a need for repair of both bone and cartilage. Bone and cartilage were both identified as tissues for which it was thought to be possible to recreate them in a laboratory setting, using the combination of cell isolation culture techniques and carrier materials. The science of combining cells with carrier materials to reproduce tissues in the laboratory is called Tissue Engineering (TE). The collaboration of scientists of different disciplines such as cell biology, biomaterials, biomechanics, engineering and translational

Endochondral Bone Formation as Blueprint for Regenerative Medicine 401

Chondrogenesis in both the primary and secondary ossification center and growth plates is characterized by highly proliferative chondrocytes, vectorially dictated to differentiate into hypertrophic chondrocytes before dying from apoptosis. The remaining mineralized extra cellular matrix provides a scaffold for infiltrating blood vessels and for bone cells to adhere to and remodel, setting the stage for *de novo* bone deposition (Kronenberg 2003) (Figure 1). The bone forming cells, osteoblasts, arise from progenitor cells from the overlying periosteal tissue and will form the bone collar (later the cortex) and primary spongiosa (later trabecular bone). In the adult, bone and overlying articular cartilage are attached by an interface of calcified cartilage (Schenk, Eggli et al. 1986). This interface distributes forces and stresses applied during load bearing and acts as a barrier to nutrients. Nutrients for the growing epiphyseal cartilage are supplied by two sources: (i) the synovial cavity and (ii) the vascularized cartilage canals (McKibbin and Maroudas 1979; Kuettner and Pauli 1983). Cartilage and synovium merge at a transitional zone which persists in the adult and is the site of osteophyte formation (Blaney Davidson, Vitters et al. 2007). This osteophyte formation is one of the first examples of endochondral ossification which takes place after growth. Another example is endochondral ossification during fracture healing where a cartilage callus is formed which will be remodelled into new bone. Studying endochondral ossification in normal growth and in healing processes will improve our understanding of both chondrogenesis and osteogenesis and as such may serve as a blueprint for

Fig. 1. The different steps of endochondral ossification; mesenchymal progenitor cells condense and undergo chondrogenesis. After maturation these chondrocytes undergo hypertrophy and die by apoptosis leaving a scaffold as a template for bone formation (these

**Bone** can be formed by 2 different processes, while endochondral bone formation drives most of the skeletal bone formation, bone can also be formed by another process called intramembranous bone formation. During intramembranous bone formation, no cartilage phase is found and progenitor cells directly differentiate into bone. Intramembranous bone formation is largely responsible for the formation of flat bones as can be found in the skull and pelvis. Endochondral bone formation is largely responsible for the formation of bones

Regenerative Medicine purposes of these tissues.

last steps are not illustrated nor discussed in this chapter).

**2.1 Bone and bone defects** 

medicine has already led to fruitful scientific achievements. However, initial expectations of tissue engineering have not been reached completely. Although some treatments which apply to the principles of TE have reached clinical practice, TE-created tissues are not generated on a large scale (Brittberg, Lindahl et al. 1994; Oberpenning, Meng et al. 1999; Macchiarini, Jungebluth et al. 2008). In addition, time consuming and expensive culture procedures and logistics, multiple operations and quality of the repair that is initiated by TE constructs remain important drawbacks.

Upon implantation of a TE-created construct the introduction of cells, biomaterials, growth factors, etc. in the body will have an effect on the local environment and natural repair mechanisms at the implant site. Since it is largely unknown what this local effect is and how these factors contribute to it, a clear shift is observed in the attempts to repair tissue. This shift includes more specific natural stimuli which trigger and enhance the regenerative capacity of the tissue itself. Injection of stem cells or progenitor cells (cell therapies), and the induction of regeneration by biologically active molecules can all be regarded as an example of Regenerative Medicine (RM). For both TE and RM it becomes more and more evident that studying the underlying natural and developmental processes of cartilage and bone can serve as a blueprint to identify important cell sources, biochemical, biomechanical, structural stimuli and timing thereof. It is expected that insight in these biological mechanisms and the process of endochondral ossification will enhance the progress in the field of both TE and RM.

This chapter describes the first phases of endochondral ossification, bone and cartilage (defects) and current approaches in TE and RM. Parallels with RM and endochondral ossification are identified from where endochondral ossification can serve as a blueprint for future RM approaches.

#### **2. Endochondral ossification**

**Endochondral ossification** is a multistage process that determines the major part of mammalian skeletal development and starts in embryogenesis with condensation of mesenchymal stem cells. The formation of cartilage, a process called chondrogenesis, is a key event in developing limb buds beginning in the center of the condensed mesenchyme. The earliest form of cartilage development is suggested to be 300 million years ago (Urist 1976). In humans, the first skeletal rudiments develop during the 5th week of gestation. In the eight week of the embryological life relatively cell-poor intermediate zones begins to develop, which will form the joint cavities (Gray and Gardner 1950; Anderson 1962; Aydelotte and Kuettner 1992). The diaphyseal cartilage, which is located at the center of the shaft of future long bones, is replaced by bone before birth (primary ossification). However most of the cartilaginous epiphysis at the end of long bones turns into bone after birth (secondary ossification). The remaining cartilage between the primary and secondary ossification centers is called the epiphyseal plate, more commonly known as the growth plate, and it continues to form new cartilage, which is replaced by bone, a process that results in increased length of the bones. Eventually all the cartilage in the growth plate will be converted into bone leaving cartilage only at the articulating surfaces of joints. Although bone and cartilage develop from the same mesenchyme, they have completely different structures, compositions and functions.

medicine has already led to fruitful scientific achievements. However, initial expectations of tissue engineering have not been reached completely. Although some treatments which apply to the principles of TE have reached clinical practice, TE-created tissues are not generated on a large scale (Brittberg, Lindahl et al. 1994; Oberpenning, Meng et al. 1999; Macchiarini, Jungebluth et al. 2008). In addition, time consuming and expensive culture procedures and logistics, multiple operations and quality of the repair that is initiated by TE

Upon implantation of a TE-created construct the introduction of cells, biomaterials, growth factors, etc. in the body will have an effect on the local environment and natural repair mechanisms at the implant site. Since it is largely unknown what this local effect is and how these factors contribute to it, a clear shift is observed in the attempts to repair tissue. This shift includes more specific natural stimuli which trigger and enhance the regenerative capacity of the tissue itself. Injection of stem cells or progenitor cells (cell therapies), and the induction of regeneration by biologically active molecules can all be regarded as an example of Regenerative Medicine (RM). For both TE and RM it becomes more and more evident that studying the underlying natural and developmental processes of cartilage and bone can serve as a blueprint to identify important cell sources, biochemical, biomechanical, structural stimuli and timing thereof. It is expected that insight in these biological mechanisms and the process of endochondral ossification will enhance the progress in the

This chapter describes the first phases of endochondral ossification, bone and cartilage (defects) and current approaches in TE and RM. Parallels with RM and endochondral ossification are identified from where endochondral ossification can serve as a blueprint for

**Endochondral ossification** is a multistage process that determines the major part of mammalian skeletal development and starts in embryogenesis with condensation of mesenchymal stem cells. The formation of cartilage, a process called chondrogenesis, is a key event in developing limb buds beginning in the center of the condensed mesenchyme. The earliest form of cartilage development is suggested to be 300 million years ago (Urist 1976). In humans, the first skeletal rudiments develop during the 5th week of gestation. In the eight week of the embryological life relatively cell-poor intermediate zones begins to develop, which will form the joint cavities (Gray and Gardner 1950; Anderson 1962; Aydelotte and Kuettner 1992). The diaphyseal cartilage, which is located at the center of the shaft of future long bones, is replaced by bone before birth (primary ossification). However most of the cartilaginous epiphysis at the end of long bones turns into bone after birth (secondary ossification). The remaining cartilage between the primary and secondary ossification centers is called the epiphyseal plate, more commonly known as the growth plate, and it continues to form new cartilage, which is replaced by bone, a process that results in increased length of the bones. Eventually all the cartilage in the growth plate will be converted into bone leaving cartilage only at the articulating surfaces of joints. Although bone and cartilage develop from the same mesenchyme, they have completely different

constructs remain important drawbacks.

field of both TE and RM.

future RM approaches.

**2. Endochondral ossification** 

structures, compositions and functions.

Chondrogenesis in both the primary and secondary ossification center and growth plates is characterized by highly proliferative chondrocytes, vectorially dictated to differentiate into hypertrophic chondrocytes before dying from apoptosis. The remaining mineralized extra cellular matrix provides a scaffold for infiltrating blood vessels and for bone cells to adhere to and remodel, setting the stage for *de novo* bone deposition (Kronenberg 2003) (Figure 1). The bone forming cells, osteoblasts, arise from progenitor cells from the overlying periosteal tissue and will form the bone collar (later the cortex) and primary spongiosa (later trabecular bone). In the adult, bone and overlying articular cartilage are attached by an interface of calcified cartilage (Schenk, Eggli et al. 1986). This interface distributes forces and stresses applied during load bearing and acts as a barrier to nutrients. Nutrients for the growing epiphyseal cartilage are supplied by two sources: (i) the synovial cavity and (ii) the vascularized cartilage canals (McKibbin and Maroudas 1979; Kuettner and Pauli 1983). Cartilage and synovium merge at a transitional zone which persists in the adult and is the site of osteophyte formation (Blaney Davidson, Vitters et al. 2007). This osteophyte formation is one of the first examples of endochondral ossification which takes place after growth. Another example is endochondral ossification during fracture healing where a cartilage callus is formed which will be remodelled into new bone. Studying endochondral ossification in normal growth and in healing processes will improve our understanding of both chondrogenesis and osteogenesis and as such may serve as a blueprint for Regenerative Medicine purposes of these tissues.

Fig. 1. The different steps of endochondral ossification; mesenchymal progenitor cells condense and undergo chondrogenesis. After maturation these chondrocytes undergo hypertrophy and die by apoptosis leaving a scaffold as a template for bone formation (these last steps are not illustrated nor discussed in this chapter).

#### **2.1 Bone and bone defects**

**Bone** can be formed by 2 different processes, while endochondral bone formation drives most of the skeletal bone formation, bone can also be formed by another process called intramembranous bone formation. During intramembranous bone formation, no cartilage phase is found and progenitor cells directly differentiate into bone. Intramembranous bone formation is largely responsible for the formation of flat bones as can be found in the skull and pelvis. Endochondral bone formation is largely responsible for the formation of bones

Endochondral Bone Formation as Blueprint for Regenerative Medicine 403

environment to a hypoxic environment while cells residing in allografts are frozen and stored before transplantation, it is therefore likely that these cells do not contribute to the repair process (Emans, Pieper et al. 2006). Seeding of these materials with bone marrow cells is promising, however also costly, time consuming and infection prone during isolation and expansion (Shastri 2006). Among the disadvantages listed here lies the reason why this topic

**Joint motion** is possible by a both structurally and functionally truly remarkable material called hyaline cartilage (Buckwalter and Mankin 1998; Hasler, Herzog et al. 1999; Poole, Kojima et al. 2001). Hyaline cartilage is predominantly found in articular cartilage. Next to hyaline cartilage, two other types of cartilage can be found in the human body; elastic and fibrocartilage. Elastic cartilage is found in the ear, nose-tip and respiratory tract, whereas the

The only cell type found in articular cartilage is the chondrocyte. In contrast to other tissues, the chondrocyte contributes to a relatively low percentage of the cartilage volume in human (1-5 percent). Articular chondrocytes are formed by chondrogenic differentiation of chondroprogenitor cells as described above and in Figure 1, however these cells arrest in the mature chondrocyte phase and normally do not become hypertrophic cells. Each chondrocyte is a metabolically active unit which expands and maintains the extracellular matrix (ECM) in its immediate vicinity (Aydelotte, Greenhill et al. 1988). In adults chondrocytes lack cell-cell contact; therefore communication between cells has to occur via ECM. Furthermore, cartilage is characterized by the absence of blood vessels, lymphatics and nerve fibers. Due to the lack of vascularisation in cartilage the environment is dominated by low oxygen levels and therefore the chondrocytes have an anaerobic metabolism (Schenk, Eggli et al. 1986). This also implicates that chondrocytes have to obtain their nutrients and oxygen via diffusion from the synovial fluid, through the ECM and from

**Structure.** In articular cartilage four zones can be distinguished (see Figure 2), based on collagen type II orientation and chondrocyte shape and distribution (Buckwalter and Mankin 1998; Mankin, Mow et al. 2000; Poole, Kojima et al. 2001). In the superficial or tangential zone, chondrocytes are disc shaped and form a layer of several cells thick. The long axis of the cells are parallel to the joint surface and the cells are surrounded by a thin layer of ECM. Thin collagen fibers are oriented parallel with the articular surface. This orientation and the relatively low content of proteoglycans results in high tensile stiffness and the ability to distribute load over the surface. The cells in the transitional or middle zone are more spherical and appear dispersed randomly (Aydelotte and Kuettner 1992; Hunziker 1992), also collagen fibers in this zone are organized randomly. At this zone and at the deep zone, high concentrations of proteoglycans enable the tissue to bear compressive forces. In the radial or deep zone, chondrocytes are ellipsoid, grouped radially in columns of 2-6 cells with their long axes perpendicular to the joint surface. The thicker collagen fibres are also arranged perpendicular to the articular surface. In the calcified zone, chondrocytes are distributed sparsely and remain surrounded by a calcified matrix. The calcified cartilage is less stiff than the subchondral bone. At this calcified zone shear stresses are converted into compressive forces which are in turn transmitted to the subchondral bone (Radin, Martin et

is currently studied extensively by many groups worldwide.

menisci and intervertebral discs contain fibrocartilage.

**2.2 Cartilage and cartilage defects** 

the underlying bone.

of the axial skeleton. While in cartilage only one type of cell (chondrocyte) can be found, multiple cell types can be found in bone. Generally the bone forming cells are called osteoblasts and the cells which resorb bone are called osteoclasts. Osteoblasts produce the bone matrix (osteoid) which consists mainly of the organic collagen type I which is mineralized by inorganic hydroxyapatite (calcium phosphate). This gives bones a high compressive strength combined with significant elasticity. When osteoblasts become entrapped in their matrix they become osteocytes; the mature bone cells (Harada and Rodan 2003). Osteoclasts, on the other hand, are multinucleated cells that arise from the monocyte stem-cell lineage and are located at bone surfaces in Howship's lacunae. The cells are equipped with phagocytic-like mechanisms and are characterized by high expression of tartrate resistant acid phosphatase (TRAP) and cathepsin K which are able to break down bone matrix (Boyle, Simonet et al. 2003). The process of bone formation and bone resorption is able to adapt to mechanical forces and as such remodel into the desired architecture (Wolff's law). This process is mostly found in trabecular bone and while no evidence has been found that cartilage adapts/remodels after growth, bone is replaced constantly (Hunziker, Kapfinger et al. 2007). Another important function of bone resorption and formation is controlling homeostasis of important minerals such as calcium and phosphate.

**Different specialized structures** can be identified in bone; the bone attached to the joint cartilage is called subchondral bone. The zone directly beneath the subchondral bone is called the metaphysis. The metaphysis is characterized by a thin cortex and a highly vascularised trabecular bone. Within this trabecular bone bone marrow can be found. Bone marrow is also present at the inside of long bones where it enables hematopoiesis. In the center of long bones lies the diaphysial bone. Here, trabeculi become more sparse and the cortex thickens. The outer site of all bones is covered by periosteum. This periosteum is largely responsible for appositional growth of long bones as it contains a lining of osteoprogenitor cells. **Bone defects.** In the field of Orthopaedic and Trauma Surgery a large demand exists for autologous or allogenic bone. Clinical problems which fuel this demand are; large segmental bone defects (after infection, trauma or tumor resection), fracture nonunions (e.g. tibia, femur, humerus, carpal bones, and talus), bone defects in the increasing field of prosthesis related revision surgery, and spinal fusions (e.g. spondylolisthesis, discopaty, etc)(Glowacki 1998; Stevenson 1998; Huitema, van Rhijn et al. 2006). Although bone from the iliac crest is the golden standard, it is limited in source and donor site morbidity is a major concern. Alternatively, allografts are expensive and pose the risk of viral infection. While the inorganic part of bone (e.g. TriCalcium Phosphate (TCP), Hydroxyapatite (HA)) is widely explored as ceramics and combined with cells in the field of TE, this approach is not successful in generating a satisfying bone substitute (Petite, Viateau et al. 2000; Kim, Park et al. 2006; Zhao, Grayson et al. 2006). Main drawbacks are mechanical features and handling properties of these ceramics. Combining ceramic with polymers may overcome this problem, but toxic degradation products often affect healing and remodeling of the bone defect (Martin, Shastri et al. 2001; Kim, Park et al. 2006; Zhao, Grayson et al. 2006). In addition, these materials are often inert for Matrix Metallo-Proteins (MMPs) and often interfere with biomechanical signaling which is essential for repair and remodeling of loaded structures such as bone (Wolff's law). Furthermore, increased infection risk in implanted tissue-engineered devices is recently described (Kuijer, Jansen et al. 2007) and supply of oxygen and nutrients is the final aspect of concern when treating bone defects (Shastri 2006). Cells in autologous bone are transplanted from a highly vascularized environment to a hypoxic environment while cells residing in allografts are frozen and stored before transplantation, it is therefore likely that these cells do not contribute to the repair process (Emans, Pieper et al. 2006). Seeding of these materials with bone marrow cells is promising, however also costly, time consuming and infection prone during isolation and expansion (Shastri 2006). Among the disadvantages listed here lies the reason why this topic is currently studied extensively by many groups worldwide.

#### **2.2 Cartilage and cartilage defects**

402 Tissue Regeneration – From Basic Biology to Clinical Application

of the axial skeleton. While in cartilage only one type of cell (chondrocyte) can be found, multiple cell types can be found in bone. Generally the bone forming cells are called osteoblasts and the cells which resorb bone are called osteoclasts. Osteoblasts produce the bone matrix (osteoid) which consists mainly of the organic collagen type I which is mineralized by inorganic hydroxyapatite (calcium phosphate). This gives bones a high compressive strength combined with significant elasticity. When osteoblasts become entrapped in their matrix they become osteocytes; the mature bone cells (Harada and Rodan 2003). Osteoclasts, on the other hand, are multinucleated cells that arise from the monocyte stem-cell lineage and are located at bone surfaces in Howship's lacunae. The cells are equipped with phagocytic-like mechanisms and are characterized by high expression of tartrate resistant acid phosphatase (TRAP) and cathepsin K which are able to break down bone matrix (Boyle, Simonet et al. 2003). The process of bone formation and bone resorption is able to adapt to mechanical forces and as such remodel into the desired architecture (Wolff's law). This process is mostly found in trabecular bone and while no evidence has been found that cartilage adapts/remodels after growth, bone is replaced constantly (Hunziker, Kapfinger et al. 2007). Another important function of bone resorption and formation is controlling homeostasis of important minerals such as calcium and phosphate. **Different specialized structures** can be identified in bone; the bone attached to the joint cartilage is called subchondral bone. The zone directly beneath the subchondral bone is called the metaphysis. The metaphysis is characterized by a thin cortex and a highly vascularised trabecular bone. Within this trabecular bone bone marrow can be found. Bone marrow is also present at the inside of long bones where it enables hematopoiesis. In the center of long bones lies the diaphysial bone. Here, trabeculi become more sparse and the cortex thickens. The outer site of all bones is covered by periosteum. This periosteum is largely responsible for appositional growth of long bones as it contains a lining of osteoprogenitor cells. **Bone defects.** In the field of Orthopaedic and Trauma Surgery a large demand exists for autologous or allogenic bone. Clinical problems which fuel this demand are; large segmental bone defects (after infection, trauma or tumor resection), fracture nonunions (e.g. tibia, femur, humerus, carpal bones, and talus), bone defects in the increasing field of prosthesis related revision surgery, and spinal fusions (e.g. spondylolisthesis, discopaty, etc)(Glowacki 1998; Stevenson 1998; Huitema, van Rhijn et al. 2006). Although bone from the iliac crest is the golden standard, it is limited in source and donor site morbidity is a major concern. Alternatively, allografts are expensive and pose the risk of viral infection. While the inorganic part of bone (e.g. TriCalcium Phosphate (TCP), Hydroxyapatite (HA)) is widely explored as ceramics and combined with cells in the field of TE, this approach is not successful in generating a satisfying bone substitute (Petite, Viateau et al. 2000; Kim, Park et al. 2006; Zhao, Grayson et al. 2006). Main drawbacks are mechanical features and handling properties of these ceramics. Combining ceramic with polymers may overcome this problem, but toxic degradation products often affect healing and remodeling of the bone defect (Martin, Shastri et al. 2001; Kim, Park et al. 2006; Zhao, Grayson et al. 2006). In addition, these materials are often inert for Matrix Metallo-Proteins (MMPs) and often interfere with biomechanical signaling which is essential for repair and remodeling of loaded structures such as bone (Wolff's law). Furthermore, increased infection risk in implanted tissue-engineered devices is recently described (Kuijer, Jansen et al. 2007) and supply of oxygen and nutrients is the final aspect of concern when treating bone defects (Shastri 2006). Cells in autologous bone are transplanted from a highly vascularized

**Joint motion** is possible by a both structurally and functionally truly remarkable material called hyaline cartilage (Buckwalter and Mankin 1998; Hasler, Herzog et al. 1999; Poole, Kojima et al. 2001). Hyaline cartilage is predominantly found in articular cartilage. Next to hyaline cartilage, two other types of cartilage can be found in the human body; elastic and fibrocartilage. Elastic cartilage is found in the ear, nose-tip and respiratory tract, whereas the menisci and intervertebral discs contain fibrocartilage.

The only cell type found in articular cartilage is the chondrocyte. In contrast to other tissues, the chondrocyte contributes to a relatively low percentage of the cartilage volume in human (1-5 percent). Articular chondrocytes are formed by chondrogenic differentiation of chondroprogenitor cells as described above and in Figure 1, however these cells arrest in the mature chondrocyte phase and normally do not become hypertrophic cells. Each chondrocyte is a metabolically active unit which expands and maintains the extracellular matrix (ECM) in its immediate vicinity (Aydelotte, Greenhill et al. 1988). In adults chondrocytes lack cell-cell contact; therefore communication between cells has to occur via ECM. Furthermore, cartilage is characterized by the absence of blood vessels, lymphatics and nerve fibers. Due to the lack of vascularisation in cartilage the environment is dominated by low oxygen levels and therefore the chondrocytes have an anaerobic metabolism (Schenk, Eggli et al. 1986). This also implicates that chondrocytes have to obtain their nutrients and oxygen via diffusion from the synovial fluid, through the ECM and from the underlying bone.

**Structure.** In articular cartilage four zones can be distinguished (see Figure 2), based on collagen type II orientation and chondrocyte shape and distribution (Buckwalter and Mankin 1998; Mankin, Mow et al. 2000; Poole, Kojima et al. 2001). In the superficial or tangential zone, chondrocytes are disc shaped and form a layer of several cells thick. The long axis of the cells are parallel to the joint surface and the cells are surrounded by a thin layer of ECM. Thin collagen fibers are oriented parallel with the articular surface. This orientation and the relatively low content of proteoglycans results in high tensile stiffness and the ability to distribute load over the surface. The cells in the transitional or middle zone are more spherical and appear dispersed randomly (Aydelotte and Kuettner 1992; Hunziker 1992), also collagen fibers in this zone are organized randomly. At this zone and at the deep zone, high concentrations of proteoglycans enable the tissue to bear compressive forces. In the radial or deep zone, chondrocytes are ellipsoid, grouped radially in columns of 2-6 cells with their long axes perpendicular to the joint surface. The thicker collagen fibres are also arranged perpendicular to the articular surface. In the calcified zone, chondrocytes are distributed sparsely and remain surrounded by a calcified matrix. The calcified cartilage is less stiff than the subchondral bone. At this calcified zone shear stresses are converted into compressive forces which are in turn transmitted to the subchondral bone (Radin, Martin et

Endochondral Bone Formation as Blueprint for Regenerative Medicine 405

Combining technologies from material science, cell biology, and clinical needs has led to the rise of the field of TE and RM. In the 1960's researches proposed the idea of creating tissues in a laboratory which may replace damaged or diseased tissues and cell biologists observed that cells could sort themselves *in vitro* to populations with tissue-like characteristics (Steinberg 1962). Adding a structure (material) such as a collagen gel to fibroblast cultures was shown to further resemble structural characteristics of skin. Later the work of Brittberg and co-workers showed that chondrocytes could be cultured and successfully be transplanted for the repair of cartilage defects (Brittberg, Lindahl et al. 1994). This technique is entitled Autologous Chondrocyte Transplantation or Implantation (ACT or ACI). The combination of specific tissue features and the early findings of culturing and transplanting chondrocytes and fibroblasts, skin, cartilage and bone were identified as tissues which potentially could be repaired by engineering these tissues in the laboratory by combining cells and supporting scaffolds. In the beginning of ACT no artificial structures were used to keep the chondrocytes in the cartilage defect. Optimization of ACT has led to the introduction of collagen meshes to support and maintain chondrocytes which were transplanted into the defect. Already earlier, in the mid-1980s, Langer and co-workers proposed that biodegradable polymers could serve as a scaffold for the organisation and maturation of cells into the desired tissues. As such it was proposed that this approach would enable engineering of thicker and hard tissues such as cartilage. Although cell therapies based on TE for skin are commercially available, which apply to the definition of TE such as Carticel® and Epicel® of Genzyme, the initial expectations of TE and RM have not been met. Although some examples of successful treatment by engineered tissues such as bladder and trachea can be found in the clinic, engineering tissues is not performed on a

large scale (Oberpenning, Meng et al. 1999; Macchiarini, Jungebluth et al. 2008).

In the approach to engineer tissues in a laboratory setting and subsequently transplanting them into the body lies the key question; "*until what level should we engineer tissue and when should nature take over?*". It is often the aim of many researchers to engineer a mature tissue which is directly able to take over the function of the diseased tissue or organ. Per example it is often a goal that engineered cartilage and bone should be able to bear mechanical forces directly after implantation. In contrast, in nature a cascade of interactions occur during the process of tissue repair. During this process both the environment as well as the reparative tissue adapt to each other and the biomechanical requirements. In such a manner both integration of repair tissue and tissue remodelling is achieved. The capacity of a mature TE tissue to adapt to the local needs such as integration, remodelling, etc. is lower than a relatively less mature tissue. In addition, in order to create a robust and thicker tissue, the use of scaffolds, growth factors and more differentiated cells may be inevitable. However the question remains whether the local environment is able to adapt in an appropriate manner to all non-physiological stimuli which are introduced. Per example how does the normal tissue remodelling, repair and integration respond to a scaffold which alters local biomechanical stimuli which are known to be essential for tissue remodelling? How do transplanted and environmental cells respond to material properties such as material surface, breakdown products, architecture etc? How does the normal fine-tuned orchestra of tissue repair respond to transplanted cells which are normally not present at a certain phase

**3. Tissue engineering and regenerative medicine** 

al. 1984). The junction between uncalcified and calcified cartilage is called the "tidemark", a line which can be seen on histology (Figure 2). Therefore mechanical forces also change at the tidemark which provides a definite boundary for the uncalcified layer (Donohue, Buss et al. 1983; Aydelotte and Kuettner 1992).

**Cartilage defects** can arise due to trauma or cartilage degeneration. Although patient's history may differentiate between traumatic and degenerative lesions, the exact cause of cartilage defects often remains difficult to diagnose. Since cartilage has no nerve fibers, cartilage lesions often present with only (minor) effusion of the affected joint or without symptoms. Diagnosis of structures likely to be damaged upon trauma (e.g. subchondral bone, ligaments or menisci), may reveal a cartilage lesion. An X-ray indicates a cartilage lesion in the minority of the cases and Magnetic Resonance Imaging (MRI) is the best noninvasive technique available for diagnosis of cartilage lesions. Important developments are new protocols such as delayed Gadolinium Enhanced MRI of Cartilage (dGEMRIC) and sodium MRI which can visualize cartilage on the Collagen and GAG content level (Gold, Burstein et al. 2006). Overall the MRI is expected to diagnose cartilage lesions in an early stage and will become more important in evaluation of progression of cartilage degeneration and cartilage repair techniques.

As early as 1743 it was recognized that articular cartilage, once destroyed, does not heal spontaneously (Hunter 1995; Hunziker 1999). Whereas the progenitor cells of bone marrow and periosteum contribute to bone formation during fracture healing, articular cartilage is deprived of these progenitors. Although it has been shown that the superficial layer of cartilage and the synovium contain progenitor cells (Dowthwaite, Bishop et al. 2004; Park, Sugimoto et al. 2005), cartilage has a limited ability for self repair (Mankin, Mow et al. 2000; Emans, Surtel et al. 2005). Therefore cartilage and tissue engineering approaches are studied in an attempt to overcome the inability of cartilage to repair itself.

Fig. 2. Architecture of articular cartilage. Four zones can be distinguished with respect to (A) orientation of collagen fibers and (B) cell shape and orientation

#### **3. Tissue engineering and regenerative medicine**

404 Tissue Regeneration – From Basic Biology to Clinical Application

al. 1984). The junction between uncalcified and calcified cartilage is called the "tidemark", a line which can be seen on histology (Figure 2). Therefore mechanical forces also change at the tidemark which provides a definite boundary for the uncalcified layer (Donohue, Buss et

**Cartilage defects** can arise due to trauma or cartilage degeneration. Although patient's history may differentiate between traumatic and degenerative lesions, the exact cause of cartilage defects often remains difficult to diagnose. Since cartilage has no nerve fibers, cartilage lesions often present with only (minor) effusion of the affected joint or without symptoms. Diagnosis of structures likely to be damaged upon trauma (e.g. subchondral bone, ligaments or menisci), may reveal a cartilage lesion. An X-ray indicates a cartilage lesion in the minority of the cases and Magnetic Resonance Imaging (MRI) is the best noninvasive technique available for diagnosis of cartilage lesions. Important developments are new protocols such as delayed Gadolinium Enhanced MRI of Cartilage (dGEMRIC) and sodium MRI which can visualize cartilage on the Collagen and GAG content level (Gold, Burstein et al. 2006). Overall the MRI is expected to diagnose cartilage lesions in an early stage and will become more important in evaluation of progression of cartilage

As early as 1743 it was recognized that articular cartilage, once destroyed, does not heal spontaneously (Hunter 1995; Hunziker 1999). Whereas the progenitor cells of bone marrow and periosteum contribute to bone formation during fracture healing, articular cartilage is deprived of these progenitors. Although it has been shown that the superficial layer of cartilage and the synovium contain progenitor cells (Dowthwaite, Bishop et al. 2004; Park, Sugimoto et al. 2005), cartilage has a limited ability for self repair (Mankin, Mow et al. 2000; Emans, Surtel et al. 2005). Therefore cartilage and tissue engineering approaches are studied

Fig. 2. Architecture of articular cartilage. Four zones can be distinguished with respect to (A)

al. 1983; Aydelotte and Kuettner 1992).

degeneration and cartilage repair techniques.

in an attempt to overcome the inability of cartilage to repair itself.

orientation of collagen fibers and (B) cell shape and orientation

Combining technologies from material science, cell biology, and clinical needs has led to the rise of the field of TE and RM. In the 1960's researches proposed the idea of creating tissues in a laboratory which may replace damaged or diseased tissues and cell biologists observed that cells could sort themselves *in vitro* to populations with tissue-like characteristics (Steinberg 1962). Adding a structure (material) such as a collagen gel to fibroblast cultures was shown to further resemble structural characteristics of skin. Later the work of Brittberg and co-workers showed that chondrocytes could be cultured and successfully be transplanted for the repair of cartilage defects (Brittberg, Lindahl et al. 1994). This technique is entitled Autologous Chondrocyte Transplantation or Implantation (ACT or ACI). The combination of specific tissue features and the early findings of culturing and transplanting chondrocytes and fibroblasts, skin, cartilage and bone were identified as tissues which potentially could be repaired by engineering these tissues in the laboratory by combining cells and supporting scaffolds. In the beginning of ACT no artificial structures were used to keep the chondrocytes in the cartilage defect. Optimization of ACT has led to the introduction of collagen meshes to support and maintain chondrocytes which were transplanted into the defect. Already earlier, in the mid-1980s, Langer and co-workers proposed that biodegradable polymers could serve as a scaffold for the organisation and maturation of cells into the desired tissues. As such it was proposed that this approach would enable engineering of thicker and hard tissues such as cartilage. Although cell therapies based on TE for skin are commercially available, which apply to the definition of TE such as Carticel® and Epicel® of Genzyme, the initial expectations of TE and RM have not been met. Although some examples of successful treatment by engineered tissues such as bladder and trachea can be found in the clinic, engineering tissues is not performed on a large scale (Oberpenning, Meng et al. 1999; Macchiarini, Jungebluth et al. 2008).

In the approach to engineer tissues in a laboratory setting and subsequently transplanting them into the body lies the key question; "*until what level should we engineer tissue and when should nature take over?*". It is often the aim of many researchers to engineer a mature tissue which is directly able to take over the function of the diseased tissue or organ. Per example it is often a goal that engineered cartilage and bone should be able to bear mechanical forces directly after implantation. In contrast, in nature a cascade of interactions occur during the process of tissue repair. During this process both the environment as well as the reparative tissue adapt to each other and the biomechanical requirements. In such a manner both integration of repair tissue and tissue remodelling is achieved. The capacity of a mature TE tissue to adapt to the local needs such as integration, remodelling, etc. is lower than a relatively less mature tissue. In addition, in order to create a robust and thicker tissue, the use of scaffolds, growth factors and more differentiated cells may be inevitable. However the question remains whether the local environment is able to adapt in an appropriate manner to all non-physiological stimuli which are introduced. Per example how does the normal tissue remodelling, repair and integration respond to a scaffold which alters local biomechanical stimuli which are known to be essential for tissue remodelling? How do transplanted and environmental cells respond to material properties such as material surface, breakdown products, architecture etc? How does the normal fine-tuned orchestra of tissue repair respond to transplanted cells which are normally not present at a certain phase

Endochondral Bone Formation as Blueprint for Regenerative Medicine 407

ossification (also see chapter "scaffolds") as a blueprint, bone fillers need to be further optimized; next to being expensive, these aids only address one or a few aspects of the cascade of tissue responses which are necessary for bone repair. Most bone fillers are osteoconductive (supportive) and they lack the timing and onset of essential growth factors to be osteoinductive (stimulating bone growth). Growth factors by themselves have been shown to be osteoinductive but addition of one of the essential growth factors does not necessarily recapitulate the physiological, initial tissue response which leads to

Inflammation is the first and essential phase of tissue repair in general and bone repair in particular. Mimicking this inflammatory response may be a method to enhance bone fracture healing. Several clinical examples such as spondylodesis after infection of the intervertebral disc (e.g. after discography) and the method described by Masquelet confirm that inflammatory responses contribute to osteogenesis (Guyer, Collier et al. 1988; Masquelet and Begue 2010). However, in contrast, from an engineering perspective, the aim is often to create bone which has comparable mechanical features as native bone. The initial mechanical properties of currently used bone chip auto or allografts are incapable of withstanding the mechanical forces to which they are exposed. During impaction of these chips the mechanical properties of the impacted bone as a whole are capable to withstand mechanical forces in a non-loadbearing environment. After vascularisation, bone ingrowth and remodelling of the repaired bone defect adapts to finally bear full loading. As such surgical handling properties, osteoconduction, and most important osteoinduction are features one should aim for rather than engineering mature bone with biomechanical properties comparable to native bone. As mentioned before during endochondral ossification large amounts of cartilage are generated. This cartilage does not have the required mechanical features of the bone it should repair, but does have strong osteoconductive and osteoinductive features. Another challenge when aiming for creation of bone is the scale to which should be generated. During fracture healing bone defects can be repaired by deposition of large amounts of bone which is formed by endochondral ossification. In the pre-remodelling phase of endochondral ossification, the generated bone histologically resembles the metaphysial bone chips which are used on a large scale for bone impaction grafting. In conclusion, regarding endochondral ossification as a blueprint for engineering or regeneration of bone, it has the potential to generate vast amounts of bone,

with good handling properties, and is osteoinductive and osteoconductive.

when attempts are made to heal or restore cartilage.

**Treatment of damaged cartilage** can be grouped to four concepts of principle: the four R's (O'Driscoll 1998). The joint surface can be: (i) resected, (ii) relieved, (iii) replaced or (iv) restored. A joint prosthesis is an example of joint replacement; joint distraction and osteotomies can induce joint relieve. Osteotomies are used to re-align the axis of loading in patients with a malalignment of the leg. By transferring the load to the less affected cartilage (e.g. previously less loaded/damaged cartilage) the damaged part is relieved. Arthodesis is an example of joint resection. For TE and RM techniques the focus is on cartilage restoration. Restoration implies methods to heal or regenerate the joint surface with or without the subchondral bone into healthy hyaline articular cartilage. Three strategies can be considered

fracture/bone repair.

**3.2 Cartilage repair** 

of tissue repair? Finally, the use of cells in RM and TE approaches often implies the use of two surgical procedures as well as costly and time consuming culture procedures and logistics.

#### **3.1 Bone repair**

**Natural bone healing.** As described above, endochondral ossification drives skeletal growth. Similar sequential steps of endochondral ossification are largely responsible for fracture healing of long bones (Bostrom, Lane et al. 1995; Einhorn 2005). Periosteum is the main source of progenitor cells capable of creating large volumes of non-vascularized cartilage surrounding a fracture (Hall and Jacobson 1975). This first phase of endochondral bone formation is called soft callus. During the second phase chondrocytes become hypertrophic, mineralize (hard callus) (Figure 1), secrete pro-angiogenic factors such as VEGF and finally bone is deposited. In the final phase the newly formed bone is vascularized and will remodel under influence of mechanical forces. Bone healing by endochondral ossification is influenced by many regulatory mechanisms. However, while interaction of Indian hedgehog (Ihh) and Parathyroid hormone related protein (PTHrP) is one of the best known regulatory mechanism in the growth plate, such an interplay is yet unknown for fracture healing (Wu, Ishikawa et al. 1995; Vortkamp, Lee et al. 1996; Volk and Leboy 1999). The role of growth factors during bone healing processes is better studied. Chondrocytes at different stages of maturation release cytokines and growth factors such as Fibroblast Growth Factor (FGF), Transforming Growth Factor (TGF)-β, Bone Morphogenetic Proteins (BMPs) and Vascular Endothelial Growth Factor (VEGF) (Gibson 1998; Gerber, Vu et al. 1999; Blunk, Sieminski et al. 2002). For instance FGF-2 and TGF-β control endochondral ossification by inhibition of chondrocyte proliferation, hypertrophy and apoptosis (Gibson 1998) and in addition from our own findings we know that TGF-β is important for osteoand chondrogenesis both in *ex vivo* and *in vivo* models (Kuijer, Emans et al. 2003). *In vivo*, in the Osteoarthritic (OA) joint TGF-β is produced. Under the influence of this TGF-β osteophytes are formed which are derived from periosteum adjacent to the joint via endochondral bone formation (van der Kraan and van den Berg 2007). BMPs are also positively involved in ectopic cartilage and bone formation, partly by opposing the actions of the FGF pathways (Yoon and Lyons 2004; Miyazono, Maeda et al. 2005; Yoon, Pogue et al. 2006). Neo-vascularization under influence of VEGF ensures blood vessel formation which supply oxygen and nutrients to osteoblast and osteoclasts. The latter produce MMP-9 and - 13 which degrade the matrix surrounding terminally hypertrophic chondrocytes (Gerber, Vu et al. 1999). Blocking VEGF in the hypertrophic zone of the growth plate prevents degradation of this zone which in turn enlarges (Gerber, Vu et al. 1999).

**Current approaches for bone repair**. Multiple causes may lead to impaired healing of large bone defects. As mentioned before, nature has a good regenerative capacity for fractures, however from a clinical perspective the need for bone is not in fracture repair but mostly for the filling of large bone defects after revision arthroplasty and spondylodesis. These bone defects can be regarded as "non-natural" occurring bone defects and bone healing or filling is impaired at these sites because endochondral ossification does not occur. To deal with this problem a scaffold is introduced as a template for bone ongrowth, ingrowth and remodelling. Currently many bone fillers (scaffolds) and growth factors are available for treatment of bone defects. Taking the scaffold which is formed during endochondral

of tissue repair? Finally, the use of cells in RM and TE approaches often implies the use of two surgical procedures as well as costly and time consuming culture procedures and

**Natural bone healing.** As described above, endochondral ossification drives skeletal growth. Similar sequential steps of endochondral ossification are largely responsible for fracture healing of long bones (Bostrom, Lane et al. 1995; Einhorn 2005). Periosteum is the main source of progenitor cells capable of creating large volumes of non-vascularized cartilage surrounding a fracture (Hall and Jacobson 1975). This first phase of endochondral bone formation is called soft callus. During the second phase chondrocytes become hypertrophic, mineralize (hard callus) (Figure 1), secrete pro-angiogenic factors such as VEGF and finally bone is deposited. In the final phase the newly formed bone is vascularized and will remodel under influence of mechanical forces. Bone healing by endochondral ossification is influenced by many regulatory mechanisms. However, while interaction of Indian hedgehog (Ihh) and Parathyroid hormone related protein (PTHrP) is one of the best known regulatory mechanism in the growth plate, such an interplay is yet unknown for fracture healing (Wu, Ishikawa et al. 1995; Vortkamp, Lee et al. 1996; Volk and Leboy 1999). The role of growth factors during bone healing processes is better studied. Chondrocytes at different stages of maturation release cytokines and growth factors such as Fibroblast Growth Factor (FGF), Transforming Growth Factor (TGF)-β, Bone Morphogenetic Proteins (BMPs) and Vascular Endothelial Growth Factor (VEGF) (Gibson 1998; Gerber, Vu et al. 1999; Blunk, Sieminski et al. 2002). For instance FGF-2 and TGF-β control endochondral ossification by inhibition of chondrocyte proliferation, hypertrophy and apoptosis (Gibson 1998) and in addition from our own findings we know that TGF-β is important for osteoand chondrogenesis both in *ex vivo* and *in vivo* models (Kuijer, Emans et al. 2003). *In vivo*, in the Osteoarthritic (OA) joint TGF-β is produced. Under the influence of this TGF-β osteophytes are formed which are derived from periosteum adjacent to the joint via endochondral bone formation (van der Kraan and van den Berg 2007). BMPs are also positively involved in ectopic cartilage and bone formation, partly by opposing the actions of the FGF pathways (Yoon and Lyons 2004; Miyazono, Maeda et al. 2005; Yoon, Pogue et al. 2006). Neo-vascularization under influence of VEGF ensures blood vessel formation which supply oxygen and nutrients to osteoblast and osteoclasts. The latter produce MMP-9 and - 13 which degrade the matrix surrounding terminally hypertrophic chondrocytes (Gerber, Vu et al. 1999). Blocking VEGF in the hypertrophic zone of the growth plate prevents

degradation of this zone which in turn enlarges (Gerber, Vu et al. 1999).

**Current approaches for bone repair**. Multiple causes may lead to impaired healing of large bone defects. As mentioned before, nature has a good regenerative capacity for fractures, however from a clinical perspective the need for bone is not in fracture repair but mostly for the filling of large bone defects after revision arthroplasty and spondylodesis. These bone defects can be regarded as "non-natural" occurring bone defects and bone healing or filling is impaired at these sites because endochondral ossification does not occur. To deal with this problem a scaffold is introduced as a template for bone ongrowth, ingrowth and remodelling. Currently many bone fillers (scaffolds) and growth factors are available for treatment of bone defects. Taking the scaffold which is formed during endochondral

logistics.

**3.1 Bone repair** 

ossification (also see chapter "scaffolds") as a blueprint, bone fillers need to be further optimized; next to being expensive, these aids only address one or a few aspects of the cascade of tissue responses which are necessary for bone repair. Most bone fillers are osteoconductive (supportive) and they lack the timing and onset of essential growth factors to be osteoinductive (stimulating bone growth). Growth factors by themselves have been shown to be osteoinductive but addition of one of the essential growth factors does not necessarily recapitulate the physiological, initial tissue response which leads to fracture/bone repair.

Inflammation is the first and essential phase of tissue repair in general and bone repair in particular. Mimicking this inflammatory response may be a method to enhance bone fracture healing. Several clinical examples such as spondylodesis after infection of the intervertebral disc (e.g. after discography) and the method described by Masquelet confirm that inflammatory responses contribute to osteogenesis (Guyer, Collier et al. 1988; Masquelet and Begue 2010). However, in contrast, from an engineering perspective, the aim is often to create bone which has comparable mechanical features as native bone. The initial mechanical properties of currently used bone chip auto or allografts are incapable of withstanding the mechanical forces to which they are exposed. During impaction of these chips the mechanical properties of the impacted bone as a whole are capable to withstand mechanical forces in a non-loadbearing environment. After vascularisation, bone ingrowth and remodelling of the repaired bone defect adapts to finally bear full loading. As such surgical handling properties, osteoconduction, and most important osteoinduction are features one should aim for rather than engineering mature bone with biomechanical properties comparable to native bone. As mentioned before during endochondral ossification large amounts of cartilage are generated. This cartilage does not have the required mechanical features of the bone it should repair, but does have strong osteoconductive and osteoinductive features. Another challenge when aiming for creation of bone is the scale to which should be generated. During fracture healing bone defects can be repaired by deposition of large amounts of bone which is formed by endochondral ossification. In the pre-remodelling phase of endochondral ossification, the generated bone histologically resembles the metaphysial bone chips which are used on a large scale for bone impaction grafting. In conclusion, regarding endochondral ossification as a blueprint for engineering or regeneration of bone, it has the potential to generate vast amounts of bone, with good handling properties, and is osteoinductive and osteoconductive.

#### **3.2 Cartilage repair**

**Treatment of damaged cartilage** can be grouped to four concepts of principle: the four R's (O'Driscoll 1998). The joint surface can be: (i) resected, (ii) relieved, (iii) replaced or (iv) restored. A joint prosthesis is an example of joint replacement; joint distraction and osteotomies can induce joint relieve. Osteotomies are used to re-align the axis of loading in patients with a malalignment of the leg. By transferring the load to the less affected cartilage (e.g. previously less loaded/damaged cartilage) the damaged part is relieved. Arthodesis is an example of joint resection. For TE and RM techniques the focus is on cartilage restoration.

Restoration implies methods to heal or regenerate the joint surface with or without the subchondral bone into healthy hyaline articular cartilage. Three strategies can be considered when attempts are made to heal or restore cartilage.

Endochondral Bone Formation as Blueprint for Regenerative Medicine 409

**The role of endochondral ossification in cartilage repair**. When using progenitor cells for cartilage repair, ossification of the repaired tissue may impair clinical results. Examples hereof are ossification and formation of interlesional osteophytes when applying techniques such as microfracture and periosteum or perichondrium plasty (Bouwmeester, Beckers et al. 1997; Cole, Farr et al. 2011). These findings illustrate that maintaining differentiated progenitor cells in their chondrogenic state remains challenging in cartilage repair. It appears that in contrast to chondrocytes, progenitor cells have the tendency to follow the different phases of endochondral ossification towards hypertrophy and mineralisation when triggered to differentiate into cartilage. As such, locking cells in their desired differentiation state is of the utmost importance when applying these cells for RM purposes. Findings of Hendriks and co-workers showed that chondrocytes stimulate progenitor cells towards chondrogenesis when both cell types are co-cultured (Hendriks, Riesle et al. 2007). These findings were later bolstered by Fisher and co-workers showing that human articular cartilage-derived soluble factors and direct co-culture are potent means of improving chondrogenesis and suppressing the hypertrophic development of mesenchymal stem cells (Fischer, Dickhut et al. 2010). In this study and other work of the group of Richter the PTHrP is an important candidate soluble factor involved in this effect. PTHrP is primarily known as a key regulator in the process of endochondral ossification. Furthermore, we have recently shown that cyclooxygenase (COX) inhibitors are also able to decrease hypertrophy of chondrocytes (unpublished data). Thus studying the process of endochondral ossification and further unravelling how and why articular chondrocytes maintain their phenotype as well as prevention of hypertrophy may enhance cartilage repair techniques by generating stable cartilage which does not lead to intra-lesional osteophytes. Finally, cartilage defects lead to early OA, also in the process of OA more evidence is found that articular chondrocytes loose their capacity to maintain their phenotype and seem to undergo endochondrogenesis since they become hypertrophic and express collagen type X (Saito, Fukai et al. 2010). As such understanding and controlling the process of

endochondrogenesis may be of relevance for future insight and treatment of OA.

**Scaffold or carrier material** refers to a wide variety of artificial 2D or 3D structures that are designed for the purpose of tissue engineering. Scaffolds may be seeded with cells before implantation or are designed to recruit or retain cells at the desired place. (Bentley and Greer 1971; Wakitani, Kimura et al. 1989). For bone and cartilage regeneration, relevant cells are (mesenchymal) stem cells of different origins (bone marrow, adipose tissue, dental pulp, iPS etc.) as well as differentiated cells like chondrocytes. Different variables are important parameters for scaffold design: pore diameter, shape, kind of material, (bio)degradability, implantation site, functionalization, mechanical stability and others. Several materials have been and are being explored for this purpose. Generally scaffold materials can be divided in natural or synthetic. Examples of natural material-based scaffolds for cartilage and bone regeneration are: fibrin, hyaluronan, alginate, agarose, demineralized bone matrix, collagen etc. Synthetic scaffold materials include ceramics and copolymers PolyGlycolic Lactic acid (PGLA) and PolyethyleneGlycol-terephthalate/PolyButylene Terephthalate (PEGT/PBT) (Figure 3) etc. When applying a collagen as carrier material, some authors find enhanced cartilage healing, while others conclude that collagen scaffolds have a limited usefulness for chondrocyte grafting in large defects (Wakitani, Kimura et al. 1989; Nixon, Sams et al. 1993;

**3.3 Scaffolds** 


**The role of endochondral ossification in cartilage repair**. When using progenitor cells for cartilage repair, ossification of the repaired tissue may impair clinical results. Examples hereof are ossification and formation of interlesional osteophytes when applying techniques such as microfracture and periosteum or perichondrium plasty (Bouwmeester, Beckers et al. 1997; Cole, Farr et al. 2011). These findings illustrate that maintaining differentiated progenitor cells in their chondrogenic state remains challenging in cartilage repair. It appears that in contrast to chondrocytes, progenitor cells have the tendency to follow the different phases of endochondral ossification towards hypertrophy and mineralisation when triggered to differentiate into cartilage. As such, locking cells in their desired differentiation state is of the utmost importance when applying these cells for RM purposes. Findings of Hendriks and co-workers showed that chondrocytes stimulate progenitor cells towards chondrogenesis when both cell types are co-cultured (Hendriks, Riesle et al. 2007). These findings were later bolstered by Fisher and co-workers showing that human articular cartilage-derived soluble factors and direct co-culture are potent means of improving chondrogenesis and suppressing the hypertrophic development of mesenchymal stem cells (Fischer, Dickhut et al. 2010). In this study and other work of the group of Richter the PTHrP is an important candidate soluble factor involved in this effect. PTHrP is primarily known as a key regulator in the process of endochondral ossification. Furthermore, we have recently shown that cyclooxygenase (COX) inhibitors are also able to decrease hypertrophy of chondrocytes (unpublished data). Thus studying the process of endochondral ossification and further unravelling how and why articular chondrocytes maintain their phenotype as well as prevention of hypertrophy may enhance cartilage repair techniques by generating stable cartilage which does not lead to intra-lesional osteophytes. Finally, cartilage defects lead to early OA, also in the process of OA more evidence is found that articular chondrocytes loose their capacity to maintain their phenotype and seem to undergo endochondrogenesis since they become hypertrophic and express collagen type X (Saito, Fukai et al. 2010). As such understanding and controlling the process of endochondrogenesis may be of relevance for future insight and treatment of OA.

#### **3.3 Scaffolds**

408 Tissue Regeneration – From Basic Biology to Clinical Application

i. Subchondral Drilling, Abrasion or Microfracture are techniques to allow penetration of bone marrow through the subchondral bone into the damaged cartilage (Meachim and Roberts 1971; Insall 1974; Mitchell and Shepard 1976; Furukawa, Eyre et al. 1980; Vachon, Bramlage et al. 1986; Bradley and Dandy 1989; Rae and Noble 1989; Kim, Moran et al. 1991; Altman, Kates et al. 1992; Aglietti, Buzzi et al. 1994). These techniques improve the clinical well being of the patient and the joint surface defect may be healed to some extent. However the healing process is inadequate since no functional hyaline cartilage but fibrocartilage is formed (Vachon, Bramlage et al. 1986; Altman, Kates et al. 1992). Nonetheless, these methods are cheap and easy to perform and are therefore seen as the currently best option to relieve the complaints. Other clinical studies have suggested that any beneficial effect is related to the arthroscopic procedure itself. A nonspecific effect might be related to joint lavage rather than the penetration of the subchondral bone (Jackson 1986; Ogilvie-Harris and Fitsialos 1991). In conclusion, these techniques may have some benefit with regard to small defects but no effect has been proven in relation to large defects, osteoarthritic joints or older patients (Kim, Moran et

ii. Implants vary from non-degradable and degradable, cells, periosteum or perichondrium, Osteochondral Autograft Transfer System (OATS or Mosaicplasty) and Osteochondral Allografts (Elford, Graeber et al. 1992; Freed, Vunjak-Novakovic et al. 1993; Nixon, Sams et al. 1993; Hendrickson, Nixon et al. 1994; Reddi 1994; Chu, Coutts et al. 1995; Grande, Halberstadt et al. 1997). The biomaterials and periosteum can be combined with cells or growth factors. Periosteal Arthroplasty is an interesting way of treating cartilage defects since many have reported the chondrogenic potential of periosteum (O'Driscoll, Keeley et al. 1986; O'Driscoll, Keeley et al. 1988; Zarnett and Salter 1989; Nakahara, Bruder et al. 1990; Nakahara, Dennis et al. 1991; Nakahara, Goldberg et al. 1991; Nakata, Nakahara et al. 1992; Iwasaki, Nakata et al. 1993; Gallay, Miura et al. 1994; Iwasaki, Nakahara et al. 1994; Iwasaki, Nakahara et al. 1995; O'Driscoll, Saris et al. 2001; Emans, Surtel et al. 2005). Over 90 percent of collagen type II in the hyaline cartilage formed in the cartilage defects treated with periosteal grafts has been reported (O'Driscoll, Keeley et al. 1986; O'Driscoll, Keeley et al. 1988). Perichondrial Arthroplasty used for human cartilage repair was first described by Skoog et al. (Skoog and Johansson 1976). This technique has been reported to give an initial cartilage repair (Homminga, Bulstra et al. 1990; Homminga, Bulstra et al. 1991). On the long term poor results related to overgrowth of the graft and calcification are reported by Bouwmeester *et al.* (Bouwmeester, Beckers et al. 1997). These authors concluded that a better fixation of the graft might improve the results. In a study comparing periosteum with perichondrium, chondrogenesis was observed significantly more using periosteal grafts (Vachon, McIlwraith et al. 1989). This finding and the

accessibility make periosteum to be preferred over perichondrium.

Schnettler et al. 2000; Gross, Aubin et al. 2002).

iii. Osteochondral Grafts can be divided in autologous and allogenic. Mosaicplasty or OATS involves harvesting one or more osteochondral plugs from a relatively less weight-bearing region of the joint and subsequent implantation of these plugs into an articular defect. Possible donor site morbidity is bypassed if osteochondral allografts are used (Gross, McKee et al. 1983; Garrett 1986; Czitrom, Keating et al. 1990; Convery, Meyers et al. 1991; Garrett 1994; Ghazavi, Pritzker et al. 1997; Garrett 1998; Horas,

al. 1991).

**Scaffold or carrier material** refers to a wide variety of artificial 2D or 3D structures that are designed for the purpose of tissue engineering. Scaffolds may be seeded with cells before implantation or are designed to recruit or retain cells at the desired place. (Bentley and Greer 1971; Wakitani, Kimura et al. 1989). For bone and cartilage regeneration, relevant cells are (mesenchymal) stem cells of different origins (bone marrow, adipose tissue, dental pulp, iPS etc.) as well as differentiated cells like chondrocytes. Different variables are important parameters for scaffold design: pore diameter, shape, kind of material, (bio)degradability, implantation site, functionalization, mechanical stability and others. Several materials have been and are being explored for this purpose. Generally scaffold materials can be divided in natural or synthetic. Examples of natural material-based scaffolds for cartilage and bone regeneration are: fibrin, hyaluronan, alginate, agarose, demineralized bone matrix, collagen etc. Synthetic scaffold materials include ceramics and copolymers PolyGlycolic Lactic acid (PGLA) and PolyethyleneGlycol-terephthalate/PolyButylene Terephthalate (PEGT/PBT) (Figure 3) etc. When applying a collagen as carrier material, some authors find enhanced cartilage healing, while others conclude that collagen scaffolds have a limited usefulness for chondrocyte grafting in large defects (Wakitani, Kimura et al. 1989; Nixon, Sams et al. 1993;

Endochondral Bone Formation as Blueprint for Regenerative Medicine 411

**The endochondral scaffold.** During endochondral ossification nature creates its own scaffold. Hypertrophic chondrocytes die and leave a large scaffold. During this process multiple growth factors are released in an orchestrated manner. The scaffold itself is used as a template for invading cells to deposite bone and provide vascularisation. The scaffold itself is resorbed by osteoclasts which in turn respond to biomechanical and biochemical stimuli. As such the scaffold degrades and simultaneously the proper factors are released. The repair tissue remodels to the appropriate architecture as defined by Wolff's law. Studying this process in detail reveals the challenge when "artificial" scaffolds are designed from a material science point of view, and so far no scaffolds have been created with the

same properties capable to dictate the same processes of endochondral remodelling.

Cells for "orthopaedic" tissues, such as bone and cartilage, originate from the mesenchymal cell lineage and may be derived from different autologous or allogenic sources. Interestingly, cells used for bone regeneration are almost always progenitor cells, whereas for cartilage regeneration also differentiated cells are used, next to progenitor cells. Some authors prefer the use of chondrocytes for transplantation while others prefer the use of undifferentiated multipotent cells (Skoog and Johansson 1976; O'Driscoll, Keeley et al. 1986; Homminga, Bulstra et al. 1991; Brittberg, Lindahl et al. 1994; Lindahl, Brittberg et al. 2003; Nathan, Das De et al. 2003; Emans, Surtel et al. 2005; Park, Sugimoto et al. 2005).Mature chondrocytes can be released from their cartilaginous matrix, selected and expanded *in vitro*. In this way a relatively small amount of autologous tissue can be used as an appropriate cell source. Both chondrocytes and progenitor cells originating from different cell sources have been studied in combination with various biomaterials (Bentley and Greer 1971; Haynesworth, Baber et al. 1992; Freed, Marquis et al. 1993; Freed, Vunjak-Novakovic et al. 1993; Iwasaki, Nakata et al. 1993; Brittberg, Lindahl et al. 1994; Bruder, Fink et al. 1994; Freed, Grande et al. 1994; Gallay, Miura et al. 1994; Wakitani, Goto et al. 1994; Iwasaki, Nakahara et al. 1995). Bone marrow, adipose tissue, synovium, dental pulp, perichondrium and periosteum can serve as a source for multipotent cells (Skoog and Johansson 1976; Homminga, Bulstra et al. 1991; Bouwmeester, Beckers et al. 1997; Chu, Dounchis et al. 1997; O'Driscoll, Saris et al. 2001; Nathan, Das De et al. 2003; Emans, Surtel et al. 2005; Park, Sugimoto et al. 2005). Numerous publications described subpopulations of progenitor cells in these donor tissues that might be more optimal cell sources than e.g. whole cell pool isolates. However, lots of these studies were only performed *in vitro* and one can question whether selection of subtypes based on cell surface markers may bias the outcome of the intervention in a difficult to predict way. The involvement of subchondral bone may play a role in cell source selection as chondrocytes are capable of producing cartilage under the appropriate conditions, but in a situation where simultaneous bone formation is required (involvement of the subchondral bone), multipotent cells might be a better cell source. After selection and expansion, the main challenge is to keep these cells in the damaged area of the joint and this challenge becomes even bigger in larger defects (Bentley and Greer 1971). Grande *et al.* reported that only 8 percent of the total number of cells in the healing tissue originated from transplanted chondrocytes (Grande, Pitman et al. 1989). Chondrocytes can be maintained in the defect by suturing a periosteal flap or a collagen mesh over the defect (Grande, Pitman et al. 1989; Brittberg, Lindahl et al. 1994; Bartlett, Skinner et al. 2005). As discussed above, chondrocytes can also be seeded in a matrix or scaffold. This matrix can be

**3.4 Cells** 

Sams, Minor et al. 1995; Sams and Nixon 1995). The use of fibrin as carrier material was reported to give superior cartilage healing compared to controls (empty defect) (Hendrickson, Nixon et al. 1994). Within time an ideal scaffold should degrade or allow the populated cells to take over functionality of the artificial tissue implant. Breakdown products and biomechanical features of the scaffold should not negatively interfere with differentiation towards this tissue. It is therefore challenging to design a scaffold with all the optimal characteristics; proper initial mechanical stability, timed release of required growth factors, timed degradation which allows biomechanical stimuli to remodel the formed tissue, no release of degradation products which interfere with tissue repair, good handling properties, etc. Next generation scaffolds will be so called "smart scaffolds". These scaffolds will be loaded with bioactive factors (e.g. TGF-β1 and members of its superfamily such as BMPs) that can directly influence the differentiation pathways (Sellers, Peluso et al. 1997; Sellers, Zhang et al. 2000; Huang, Goh et al. 2002). Effort is being put in e.g. functionalized scaffolds with specific affinity peptides to retain cells (Dong, Wei et al. 2009). Also, the release of e.g. growth factors may be regulated by "on demand" smart systems that depend on incorporated microspheres or proteolytic degradation of linker-peptides. Unfortunately, an ideal material for artificial scaffolds for cartilage and bone regeneration has not been identified yet, as the biological processes involved are far more complex than anticipated.

Fig. 3. A PolyethyleneGlycol-terephthalate/PolyButylene Terephthalate (PEGT/PBT) scaffold produced by using a three dimensional rapid prototyping technique.

**The endochondral scaffold.** During endochondral ossification nature creates its own scaffold. Hypertrophic chondrocytes die and leave a large scaffold. During this process multiple growth factors are released in an orchestrated manner. The scaffold itself is used as a template for invading cells to deposite bone and provide vascularisation. The scaffold itself is resorbed by osteoclasts which in turn respond to biomechanical and biochemical stimuli. As such the scaffold degrades and simultaneously the proper factors are released. The repair tissue remodels to the appropriate architecture as defined by Wolff's law. Studying this process in detail reveals the challenge when "artificial" scaffolds are designed from a material science point of view, and so far no scaffolds have been created with the same properties capable to dictate the same processes of endochondral remodelling.

#### **3.4 Cells**

410 Tissue Regeneration – From Basic Biology to Clinical Application

Sams, Minor et al. 1995; Sams and Nixon 1995). The use of fibrin as carrier material was reported to give superior cartilage healing compared to controls (empty defect) (Hendrickson, Nixon et al. 1994). Within time an ideal scaffold should degrade or allow the populated cells to take over functionality of the artificial tissue implant. Breakdown products and biomechanical features of the scaffold should not negatively interfere with differentiation towards this tissue. It is therefore challenging to design a scaffold with all the optimal characteristics; proper initial mechanical stability, timed release of required growth factors, timed degradation which allows biomechanical stimuli to remodel the formed tissue, no release of degradation products which interfere with tissue repair, good handling properties, etc. Next generation scaffolds will be so called "smart scaffolds". These scaffolds will be loaded with bioactive factors (e.g. TGF-β1 and members of its superfamily such as BMPs) that can directly influence the differentiation pathways (Sellers, Peluso et al. 1997; Sellers, Zhang et al. 2000; Huang, Goh et al. 2002). Effort is being put in e.g. functionalized scaffolds with specific affinity peptides to retain cells (Dong, Wei et al. 2009). Also, the release of e.g. growth factors may be regulated by "on demand" smart systems that depend on incorporated microspheres or proteolytic degradation of linker-peptides. Unfortunately, an ideal material for artificial scaffolds for cartilage and bone regeneration has not been identified yet, as the biological processes involved are far more complex than anticipated.

Fig. 3. A PolyethyleneGlycol-terephthalate/PolyButylene Terephthalate (PEGT/PBT)

scaffold produced by using a three dimensional rapid prototyping technique.

Cells for "orthopaedic" tissues, such as bone and cartilage, originate from the mesenchymal cell lineage and may be derived from different autologous or allogenic sources. Interestingly, cells used for bone regeneration are almost always progenitor cells, whereas for cartilage regeneration also differentiated cells are used, next to progenitor cells. Some authors prefer the use of chondrocytes for transplantation while others prefer the use of undifferentiated multipotent cells (Skoog and Johansson 1976; O'Driscoll, Keeley et al. 1986; Homminga, Bulstra et al. 1991; Brittberg, Lindahl et al. 1994; Lindahl, Brittberg et al. 2003; Nathan, Das De et al. 2003; Emans, Surtel et al. 2005; Park, Sugimoto et al. 2005).Mature chondrocytes can be released from their cartilaginous matrix, selected and expanded *in vitro*. In this way a relatively small amount of autologous tissue can be used as an appropriate cell source. Both chondrocytes and progenitor cells originating from different cell sources have been studied in combination with various biomaterials (Bentley and Greer 1971; Haynesworth, Baber et al. 1992; Freed, Marquis et al. 1993; Freed, Vunjak-Novakovic et al. 1993; Iwasaki, Nakata et al. 1993; Brittberg, Lindahl et al. 1994; Bruder, Fink et al. 1994; Freed, Grande et al. 1994; Gallay, Miura et al. 1994; Wakitani, Goto et al. 1994; Iwasaki, Nakahara et al. 1995). Bone marrow, adipose tissue, synovium, dental pulp, perichondrium and periosteum can serve as a source for multipotent cells (Skoog and Johansson 1976; Homminga, Bulstra et al. 1991; Bouwmeester, Beckers et al. 1997; Chu, Dounchis et al. 1997; O'Driscoll, Saris et al. 2001; Nathan, Das De et al. 2003; Emans, Surtel et al. 2005; Park, Sugimoto et al. 2005). Numerous publications described subpopulations of progenitor cells in these donor tissues that might be more optimal cell sources than e.g. whole cell pool isolates. However, lots of these studies were only performed *in vitro* and one can question whether selection of subtypes based on cell surface markers may bias the outcome of the intervention in a difficult to predict way. The involvement of subchondral bone may play a role in cell source selection as chondrocytes are capable of producing cartilage under the appropriate conditions, but in a situation where simultaneous bone formation is required (involvement of the subchondral bone), multipotent cells might be a better cell source. After selection and expansion, the main challenge is to keep these cells in the damaged area of the joint and this challenge becomes even bigger in larger defects (Bentley and Greer 1971). Grande *et al.* reported that only 8 percent of the total number of cells in the healing tissue originated from transplanted chondrocytes (Grande, Pitman et al. 1989). Chondrocytes can be maintained in the defect by suturing a periosteal flap or a collagen mesh over the defect (Grande, Pitman et al. 1989; Brittberg, Lindahl et al. 1994; Bartlett, Skinner et al. 2005). As discussed above, chondrocytes can also be seeded in a matrix or scaffold. This matrix can be

Endochondral Bone Formation as Blueprint for Regenerative Medicine 413

differentiation processes during skeletal development by endochondral ossification. Resemblances are: chondrocyte proliferation, chondrocyte hypertrophic marker expression (e.g. Collagen type X and MMP-13), vascularisation and focal calcification of joint cartilage. This suggests that during OA the articular cartilage is terminally differentiating via "normal" endochondral pathways. However, how the mature articular cartilage is kept in its cartilaginous state and why it starts a terminal differentiation program in OA is currently poorly understood. In the final stage of endochondral bone formation secretion of proangiogenic factors such as VEGF is essential. Sox9 and RunX2 are important transcription factors. Sox9 is the master regulator of chondrogenesis and acts as a negative regulator for chondrocyte hypertrophy, cartilage vascularisation and bone marrow formation (Hattori, Muller et al. 2010). Amongst others it does this via negatively regulating expression of RunX2 via Nkx3.2 (also known as BapX1) (Yamashita, Andoh 2009). RunX2 is a central regulator for the transition from proliferating to hypertrophic chondrocytes, as it drives the transcription of Collagen type X. Interestingly, RunX2 also drives multiple osteogenic developmental programs. Inflammatory pathways are other key players in endochondral ossification (Einhorn, Majeska et al. 1995; Mountziaris and Mikos 2008). Bone fracture healing by endochondral ossification depends on a haematoma-induced inflammatory environment (Grundnes and Reikeras 1993) and several inflammatory molecules (e.g. IL-6, TNFα, COX-2 and iNOS) are involved in bone fracture repair (Einhorn, Majeska et al. 1995; Mountziaris and Mikos 2008) by influencing chondrocyte maturation and osteogenic development. An important chondrogenic growth factor is Insulin Growth Factor 1 (IGF-1). Together with its receptors and several IGF binding proteins it determines chondrocyte proliferation and differentiation. Importantly IGF-1 appears to play a role in preventing chondrocyte apoptosis. Hence, it determines the pace of hypertrophic differentiation and thus growth plate development and fracture callus maturation. It was shown that IGF-1 exerts its action via NF-κB/p65 signaling (Wu, Gong et al. 2008). Furthermore, IGF-1 also directly influences osteocyte biology. It has been reported that IGF-1 stimulates cancellous bone formation and increases the activity of resident osteoblasts (Zhao, Monier-Faugere et al. 2000). RANK is crucially important for bone homeostasis and remodelling. Activation of RANKL on monocytic cells by RANK on osteoblasts induces osteoclastogenesis of committed monocytic cells. Multinucleation is induced, ultimately leading to the generation of mature bone resorbing osteoclasts (Novack and Faccio 2011). This process is counterbalanced by the soluble factor osteoprotegerin (OPG), thereby preventing bone loss due to osteoclast activation. Activation of the RANKL system is potentiated by prostaglandins. PGE2, one of the main cyclooxygenase metabolites is reported to increase

In conclusion, the process of endochondral ossification is dictated by spatiotemporal expression and use of variable interacting growth factors and other molecules. It is clear that mimicking this complex, yet incompletely known, tissue formation in an *in vitro* setting on the same scale as TE was expected to do is quite challenging. Several findings such as endochondral ossification after subcutaneous injection of BMPs show that, *in vivo*, this process may be triggered using stimuli which trigger and enhance the regenerative capacity of the tissue itself. In such an approach the amount of unknown stimuli is expected to be limited and the body's own regenerative capacity is used to generate cartilage or bone, which in turn can be transplanted into the damaged site. As such, this approach applies more to the principles of RM than to the principles of TE. The application of a specific *in vivo*

bone resorption.

implanted in a cartilage defect. This Matrix Assisted Chondrocyte Transplantation (MACT) is technically less demanding and has shown identical results compared to Autologous Chondrocyte Transplantation on the short term (Bartlett, Skinner et al. 2005) The use or allogenic chondrocytes has been reported to be successful in rabbits but experiments in horses do not support this finding (Wakitani, Kimura et al. 1989; Freed, Grande et al. 1994; Sams, Minor et al. 1995; Sams and Nixon 1995). Immunological rejection or allogenic chondrocytes upon implantation in rabbits has been reported and this remains a major concern when applying allogenic cells. The use of periosteal tissue or cells has been suggested by several authors and is one of the most clear examples of the use of endochondral ossification as a blueprint for cartilage and bone regenerative medicine. The periosteum is populated with mesenchymal progenitor cells that normally contribute to endochondral bone fracture healing. The differentiation capacity of these cells can also be used to create cartilage or bone for regenerative purposes. This principle is further explained below (see paragraph 3.6).

When using differentiated or undifferentiated cells for cartilage, bone or osteochondral repair it is a challenge to differentiate these cells into their desired state and maintain their desired phenotype. As cells from the mesenchymal lineage, once differentiated into chondrocytes, have a natural tendency for terminal differentiation via the endochondral pathway. This is a big concern for regenerative applications. Much effort is being put in technologies that prevent hypertrophic differentiation of transplanted chondrocytes, while on the other hand for bone regeneration hypertrophic differentiation may be a prerequisite for success (Fischer, Dickhut et al. 2010; Scotti, Tonnarelli et al. 2010).

#### **3.5 Biochemical signaling pathways**

**Growth factors.** In growth plate development, homeostasis of articular cartilage as well as bone formation and maintenance, several signaling pathways are interacting or shared between the different tissues. Indian hedgehog (Ihh) and Parathyroid hormone related peptide (PTHrP) coordinate chondrocyte proliferation and differentiation in the so-called PTHrP-Ihh feedback loop (Kronenberg 2003). This coordination influences the length of proliferative chondrocyte columns as well as chondrocyte hypertrophy. Next to the Ihh and PTHrP loop, fibroblast growth factor crucially regulates chondrocyte proliferation and differentiation. Many of the 22 distinct FGF genes and their four receptor genes are expressed at every stage of endochondral bone formation (Ornitz and Marie 2002). Also Bone Morphogenic Proteins (BMPs) have multiple roles during bone and cartilage formation, as well as growth plate development. Interestingly, BMPs were discovered because of their remarkable ability to induce endochondral bone formation when injected subcutaneously in mice. In a cartilage context, BMPs are involved in early chondrogenesis, cartilage maintenance and hypertrophic differentiation. In a bone context they drive differentiation of progenitor cells to osteocytes and induce alkaline phosphatase activity in osteocytes. TGF- isoforms are also involved in similar processes and interestingly were found to trigger the formation of osteophytes upon intra-articular injection and during OA (Elford, Graeber et al. 1992; van Beuningen, van der Kraan et al. 1993; van Beuningen, van der Kraan et al. 1994; Hunziker 2001). As osteophyte formation itself is an example of endochondral ossification, the role of TGF- isoforms in endochondral ossification is supported by this finding. Remarkably, some characteristics of OA resemble chondrocyte

implanted in a cartilage defect. This Matrix Assisted Chondrocyte Transplantation (MACT) is technically less demanding and has shown identical results compared to Autologous Chondrocyte Transplantation on the short term (Bartlett, Skinner et al. 2005) The use or allogenic chondrocytes has been reported to be successful in rabbits but experiments in horses do not support this finding (Wakitani, Kimura et al. 1989; Freed, Grande et al. 1994; Sams, Minor et al. 1995; Sams and Nixon 1995). Immunological rejection or allogenic chondrocytes upon implantation in rabbits has been reported and this remains a major concern when applying allogenic cells. The use of periosteal tissue or cells has been suggested by several authors and is one of the most clear examples of the use of endochondral ossification as a blueprint for cartilage and bone regenerative medicine. The periosteum is populated with mesenchymal progenitor cells that normally contribute to endochondral bone fracture healing. The differentiation capacity of these cells can also be used to create cartilage or bone for regenerative purposes. This principle is further explained

When using differentiated or undifferentiated cells for cartilage, bone or osteochondral repair it is a challenge to differentiate these cells into their desired state and maintain their desired phenotype. As cells from the mesenchymal lineage, once differentiated into chondrocytes, have a natural tendency for terminal differentiation via the endochondral pathway. This is a big concern for regenerative applications. Much effort is being put in technologies that prevent hypertrophic differentiation of transplanted chondrocytes, while on the other hand for bone regeneration hypertrophic differentiation may be a prerequisite

**Growth factors.** In growth plate development, homeostasis of articular cartilage as well as bone formation and maintenance, several signaling pathways are interacting or shared between the different tissues. Indian hedgehog (Ihh) and Parathyroid hormone related peptide (PTHrP) coordinate chondrocyte proliferation and differentiation in the so-called PTHrP-Ihh feedback loop (Kronenberg 2003). This coordination influences the length of proliferative chondrocyte columns as well as chondrocyte hypertrophy. Next to the Ihh and PTHrP loop, fibroblast growth factor crucially regulates chondrocyte proliferation and differentiation. Many of the 22 distinct FGF genes and their four receptor genes are expressed at every stage of endochondral bone formation (Ornitz and Marie 2002). Also Bone Morphogenic Proteins (BMPs) have multiple roles during bone and cartilage formation, as well as growth plate development. Interestingly, BMPs were discovered because of their remarkable ability to induce endochondral bone formation when injected subcutaneously in mice. In a cartilage context, BMPs are involved in early chondrogenesis, cartilage maintenance and hypertrophic differentiation. In a bone context they drive differentiation of progenitor cells to osteocytes and induce alkaline phosphatase activity in osteocytes. TGF- isoforms are also involved in similar processes and interestingly were found to trigger the formation of osteophytes upon intra-articular injection and during OA (Elford, Graeber et al. 1992; van Beuningen, van der Kraan et al. 1993; van Beuningen, van der Kraan et al. 1994; Hunziker 2001). As osteophyte formation itself is an example of endochondral ossification, the role of TGF- isoforms in endochondral ossification is supported by this finding. Remarkably, some characteristics of OA resemble chondrocyte

for success (Fischer, Dickhut et al. 2010; Scotti, Tonnarelli et al. 2010).

below (see paragraph 3.6).

**3.5 Biochemical signaling pathways** 

differentiation processes during skeletal development by endochondral ossification. Resemblances are: chondrocyte proliferation, chondrocyte hypertrophic marker expression (e.g. Collagen type X and MMP-13), vascularisation and focal calcification of joint cartilage. This suggests that during OA the articular cartilage is terminally differentiating via "normal" endochondral pathways. However, how the mature articular cartilage is kept in its cartilaginous state and why it starts a terminal differentiation program in OA is currently poorly understood. In the final stage of endochondral bone formation secretion of proangiogenic factors such as VEGF is essential. Sox9 and RunX2 are important transcription factors. Sox9 is the master regulator of chondrogenesis and acts as a negative regulator for chondrocyte hypertrophy, cartilage vascularisation and bone marrow formation (Hattori, Muller et al. 2010). Amongst others it does this via negatively regulating expression of RunX2 via Nkx3.2 (also known as BapX1) (Yamashita, Andoh 2009). RunX2 is a central regulator for the transition from proliferating to hypertrophic chondrocytes, as it drives the transcription of Collagen type X. Interestingly, RunX2 also drives multiple osteogenic developmental programs. Inflammatory pathways are other key players in endochondral ossification (Einhorn, Majeska et al. 1995; Mountziaris and Mikos 2008). Bone fracture healing by endochondral ossification depends on a haematoma-induced inflammatory environment (Grundnes and Reikeras 1993) and several inflammatory molecules (e.g. IL-6, TNFα, COX-2 and iNOS) are involved in bone fracture repair (Einhorn, Majeska et al. 1995; Mountziaris and Mikos 2008) by influencing chondrocyte maturation and osteogenic development. An important chondrogenic growth factor is Insulin Growth Factor 1 (IGF-1). Together with its receptors and several IGF binding proteins it determines chondrocyte proliferation and differentiation. Importantly IGF-1 appears to play a role in preventing chondrocyte apoptosis. Hence, it determines the pace of hypertrophic differentiation and thus growth plate development and fracture callus maturation. It was shown that IGF-1 exerts its action via NF-κB/p65 signaling (Wu, Gong et al. 2008). Furthermore, IGF-1 also directly influences osteocyte biology. It has been reported that IGF-1 stimulates cancellous bone formation and increases the activity of resident osteoblasts (Zhao, Monier-Faugere et al. 2000). RANK is crucially important for bone homeostasis and remodelling. Activation of RANKL on monocytic cells by RANK on osteoblasts induces osteoclastogenesis of committed monocytic cells. Multinucleation is induced, ultimately leading to the generation of mature bone resorbing osteoclasts (Novack and Faccio 2011). This process is counterbalanced by the soluble factor osteoprotegerin (OPG), thereby preventing bone loss due to osteoclast activation. Activation of the RANKL system is potentiated by prostaglandins. PGE2, one of the main cyclooxygenase metabolites is reported to increase

In conclusion, the process of endochondral ossification is dictated by spatiotemporal expression and use of variable interacting growth factors and other molecules. It is clear that mimicking this complex, yet incompletely known, tissue formation in an *in vitro* setting on the same scale as TE was expected to do is quite challenging. Several findings such as endochondral ossification after subcutaneous injection of BMPs show that, *in vivo*, this process may be triggered using stimuli which trigger and enhance the regenerative capacity of the tissue itself. In such an approach the amount of unknown stimuli is expected to be limited and the body's own regenerative capacity is used to generate cartilage or bone, which in turn can be transplanted into the damaged site. As such, this approach applies more to the principles of RM than to the principles of TE. The application of a specific *in vivo*

bone resorption.

Endochondral Bone Formation as Blueprint for Regenerative Medicine 415

Chondrocytes

Mesenchymal

Collagen II, Proteoglycans, GAGs and Hyaluronic acid

Native cartilage Expensive

Host-vs-graft reaction

Donor site morbidity

(allografts) Infection

Fixation Cell morbidity during storage

Fibrocartilage

Infection Time consuming Expansion of cells

Fixation Expensive

Calcification of cartilage Fixation

Toxic degradation products

Superficial layer cartilage? Synovium?

Activation of bone marrow Very effective Cheap and easy

Good integration Native cartilage

Can be loaded

with cells/growth factors Initial cartilage repair

**Bone Cartilage Function** Weight bearing Weight bearing and joint articulation

No Yes, important

Via vascularisation Via diffusion

Complete Has to stop at chondrocyte-phase

**l grafts** 

**Advantages Disadvantages Advantages Disadvantages** 

**Osteochondra**

**Subchondral drilling, abrasion, microfracture**

**ACI/ACT MACT** 

**Implants/ biomaterials** 

**Cell-cell contact** Yes and important No, 'communication' via ECM

**Remodelling** Constant (Wolff's law) Low grade of remodelling?

**Vascularisation** Yes No, hypoxic tissue

Bloodstream, Periosteum,

Expensive Host-vs-graft reaction Infection Freezing cells? Donor site morbidity

Not osteoinductive Host-vs-graft reaction Infection

Not osteoinductive Mechanical features Handling properties Interference with biomechanical signalling

Osteoinductive Not osteoconductive Expensive Overload of growth

> factor Ectopic bone formation

**Challenges in Tissue Engineering** 

**Cells** Osteoblasts/osteocytes

**ECM** Collagen I and Calcium

**Origin** Mesenchymal and monocyte lineage

phosphate

**Regenerative capacity** High Low

Bone marrow

**Bone Cartilage** 

Osteoconductive Osteoinductive? Native bone

Osteoconductive Resembles native bone

Osteoconductive Resembles native bone properties Can be loaded

with cells/growth factors

and osteoclasts

**Tissue characteristics** 

**Functional ECM water content** 

**Nutrient/oxygen supply** 

**Access to progenitor** 

**Current approaches in TE**

**Endochondral ossification** 

**Auto- and Allografts** 

**Decellularized bone** 

**HA/TCP/ Bonefillers** 

**Growth factors** 

**cells** 

trigger to stimulate endochondral bone formation has many advantages; no expensive culture procedures, no more harvesting of cells, and no introduction of factors which possibly conflict with the natural tissue repair and integration. Table 1 summarises the differences in tissue features, currently applied (TE) techniques for restoration, and remaining challenges.

#### **3.6 Examples of endochondral ossification as blueprint for regenerative medicine**

Currently for TE purposes cells are harvested during the first operation and the implantation of the graft/cells is performed during the second procedure. A question that remains is the amount of cells that survives the transplantation. It has been shown that periosteal cells show a much poorer survival compared to chondrocytes after transplantation into the hostile environment of a fresh osteochondral defect (Emans, Pieper et al. 2006). However, the disadvantage of using chondrocytes is the fact that the joint is further damaged. It would be ideal to generate cartilage in an ectopical place which does not further interfere with the joint homeostasis, survives the transplantation and is capable to adapt and repair the defect. In line with this, an interesting variation for cartilage repair is a reported by Takahashi *et al.* who used the early fracture callus, induced at the iliac crest (Takahashi, Oka et al. 1995). The early fracture callus was implanted into osteochondral defects of rabbit knees with excellent results. A paper of our group also reported excellent results after transplantation of periosteum derived cartilage callus into osteochondral defects (Emans, van Rhijn et al. 2010). Stevens *et al.* published an interesting paper on inducing chondrogenesis by subperiosteal injection of a hyaluronan-based gel containing the antiangiogenic factor Suramin. The resulting tissue also resembled cartilage of early fracture callus (Stevens, Marini et al. 2005). The main advantage of this approach is that the body is used as its own "*in situ* incubator"; cells provide their own matrix and complex and costly isolation, selection and culturing procedures are bypassed. After this first report focussing on bone, we aimed to control the local environment by injecting a gel into the space between bone and periosteum which would initiate endochondrogenesis. Both agarose and a gel loaded with TGF-β1 were successful to trigger endochondrogenesis. This tissue was harvested during its first chondrogenic phase and successfully implanted into an osteochondral defect where an excellent lateral integration and no calcification of the cartilage adjacent to the joint was observed (Emans, van Rhijn et al. 2010).

It was recognised by the group of Martin that TE and RM attempts to create bone using the intramembranous pathway (Scotti, Tonnarelli et al. 2010). In contrast, during development most bones are formed by endochondral ossification and the parts that do not ossify forms articular cartilage. In addition, during fracture healing bone gaps and defects are often repaired by endochondral bone formation, during which large amounts of callus can be formed. Depending on the phase in which specific tissue is generated by endochondrogenesis, this tissue can be harvested for different purposes. If tissue in the early chondrogenic phase is harvested this may be ideal to heal both bone and cartilage. If this tissue is harvested at a later stage it resembles trabecular bone which has the potential to be used for bone impaction grafting. Compared to the frequently used TE approach to create bone directly (intramembranous), it seems more logical that endochondral bone formation which is capable to produce large amounts of cartilage and bone, even in an ectopic site, may fuel further research

trigger to stimulate endochondral bone formation has many advantages; no expensive culture procedures, no more harvesting of cells, and no introduction of factors which possibly conflict with the natural tissue repair and integration. Table 1 summarises the differences in tissue features, currently applied (TE) techniques for restoration, and

**3.6 Examples of endochondral ossification as blueprint for regenerative medicine** 

cartilage adjacent to the joint was observed (Emans, van Rhijn et al. 2010).

It was recognised by the group of Martin that TE and RM attempts to create bone using the intramembranous pathway (Scotti, Tonnarelli et al. 2010). In contrast, during development most bones are formed by endochondral ossification and the parts that do not ossify forms articular cartilage. In addition, during fracture healing bone gaps and defects are often repaired by endochondral bone formation, during which large amounts of callus can be formed. Depending on the phase in which specific tissue is generated by endochondrogenesis, this tissue can be harvested for different purposes. If tissue in the early chondrogenic phase is harvested this may be ideal to heal both bone and cartilage. If this tissue is harvested at a later stage it resembles trabecular bone which has the potential to be used for bone impaction grafting. Compared to the frequently used TE approach to create bone directly (intramembranous), it seems more logical that endochondral bone formation which is capable to produce large amounts of cartilage and bone, even in an ectopic site, may fuel further research

Currently for TE purposes cells are harvested during the first operation and the implantation of the graft/cells is performed during the second procedure. A question that remains is the amount of cells that survives the transplantation. It has been shown that periosteal cells show a much poorer survival compared to chondrocytes after transplantation into the hostile environment of a fresh osteochondral defect (Emans, Pieper et al. 2006). However, the disadvantage of using chondrocytes is the fact that the joint is further damaged. It would be ideal to generate cartilage in an ectopical place which does not further interfere with the joint homeostasis, survives the transplantation and is capable to adapt and repair the defect. In line with this, an interesting variation for cartilage repair is a reported by Takahashi *et al.* who used the early fracture callus, induced at the iliac crest (Takahashi, Oka et al. 1995). The early fracture callus was implanted into osteochondral defects of rabbit knees with excellent results. A paper of our group also reported excellent results after transplantation of periosteum derived cartilage callus into osteochondral defects (Emans, van Rhijn et al. 2010). Stevens *et al.* published an interesting paper on inducing chondrogenesis by subperiosteal injection of a hyaluronan-based gel containing the antiangiogenic factor Suramin. The resulting tissue also resembled cartilage of early fracture callus (Stevens, Marini et al. 2005). The main advantage of this approach is that the body is used as its own "*in situ* incubator"; cells provide their own matrix and complex and costly isolation, selection and culturing procedures are bypassed. After this first report focussing on bone, we aimed to control the local environment by injecting a gel into the space between bone and periosteum which would initiate endochondrogenesis. Both agarose and a gel loaded with TGF-β1 were successful to trigger endochondrogenesis. This tissue was harvested during its first chondrogenic phase and successfully implanted into an osteochondral defect where an excellent lateral integration and no calcification of the

remaining challenges.


Endochondral Bone Formation as Blueprint for Regenerative Medicine 417

for generating both bone and cartilage. Creating cartilage or bone by triggering endochondrogenesis in an ectopic site bypasses expensive and time consuming culture techniques, logistics, and when triggered by injection of a specific stimulus may even limit

From nature it is known that vast amounts of cartilage are formed in the process of endochondrogenesis. Chondrocytes in this cartilage tissue are replaced by a matrix deposited by hypertrophic chondrocytes which die by apoptosis. This matrix is used as an active scaffold for cells that contribute to bone formation. Following embryonic joint formation and post natal growth, the adult skeleton maintains the cellularity and phenotype of articular cartilage, whereas growth plate cartilage completely disappears. This process entitled endochondral ossification can be recapitulated in other places than growth plates. Examples hereof are fracture healing, osteophyte formation and peri-articular ossifications. Even in the process of OA endochondrogenesis plays a role. Next to the formation of osteophytes in OA, evidence has been reported that during the process of OA, articular chondrocytes are triggered to follow the final phase of endochondral ossification (Saito,

A scaffold which serves as a template for tissue generation has also been introduced in the field of TE. Thusfar TE has not met initial expectations. Materials used as a scaffold to engineer bone are often engineered to be biocompatible and have good initial biomechanical properties. These properties may interfere with biomechanical stimuli needed for tissue organisation and degradation products from these artificial scaffolds may interfere with the natural healing response. In contrast to a natural endochondral scaffold, artificial scaffolds

Periosteum seems to play an important role in postnatal endochondrogenesis. However subcutaneous injection of growth factors leads to generation of bone via the endochondral pathway. The first examples of successful generation of bone and cartilage by triggering the progenitor cells of periosteum are found in literature (Emans, Surtel et al. 2005; Emans, van Rhijn et al. 2010). Also repair of cartilage and bone has been reported to be successful in animal studies using this approach. Using the postnatal endochondrogenic capacity for generation of cartilage and bone has many advantages: expensive culture procedures and logistics are bypassed and sufficient amounts of tissue are likely to be generated. Depending on the stage in which endochondral tissue is harvested, different clinical needs could be treated varying from (osteo)chondral defects to bone defects (Scotti, Tonnarelli et al. 2010). Finally, studying the process of endochondrogenesis may not only be a logical direction for tissue generation, but is also expected to provide useful information how to lock progenitors

in the desired phase and will contribute to our understanding of diseases like OA.

The authors wish to acknowledge the Dutch Arthritis Association and Dutch Stichting Annafonds, as well as Prof. V. Prasad Shastri for the collaboration which has led to further

do not orchestrate ingress of progenitor cells, vascularisation etc.

the total approach to one operation.

**4. Conclusion** 

Fukai et al. 2010).

**5. Acknowledgment** 

insight into periosteal chondrogenesis.


Table 1. Differences in: tissue characteristics, currently applied Tissue Engineering, and remaining challenges for bone and cartilage.

for generating both bone and cartilage. Creating cartilage or bone by triggering endochondrogenesis in an ectopic site bypasses expensive and time consuming culture techniques, logistics, and when triggered by injection of a specific stimulus may even limit the total approach to one operation.

### **4. Conclusion**

416 Tissue Regeneration – From Basic Biology to Clinical Application

Scaffold design: pore diameter and shape (bio)Material: natural or synthetic

Release of growth factors at right time Interference with tissue environment **Cartilage:** Nutrient supply in scaffold Allow ECM formation, but prevent mineralisation

provides in essential growth factors and vascularisation at appropriate time points.

Keep growth factors at damaged area Effect on tissue *in vivo* incompletely known

If allografts: host-vs-graft reaction Keep cells at damaged area

**Bone:** Nutrient and oxygen supply

Vascularisation

Donor site morbidity

consuming and expensive

death or dedifferentiation If allografts: allogenic reaction Keep cells at damaged area **Cartilage:** Nutrient supply

hypertrophic chondrocytes

**Cartilage:** Nutrient and oxygen supply

Cell isolation and culturing is still time

Does not recapitulate total physiological repair

Have to differentiate and remain differentiated into

Have to stop differentiating at chondrocyte

Cells are out of natural environment, can lead to cell

Prevent further differentiation towards

Breakdown products Integration / fixation

loading and load damping **Bone:** Nutrient and oxygen supply Stimulate vascularisation Allow bone mineralisation Support high mechanical loading **Endochondral:** progenitors differentiate to hypertrophic chondrocytes which leave a natural 'scaffold' for bone cells to adhere and remodel and

response

Still expensive

required tissue Infection

phase

Table 1. Differences in: tissue characteristics, currently applied Tissue Engineering, and

Biodegradability and degradation at right time

Allow articulation, repetitive mechanical

**Current advantages Remaining challenges** 

Can be loaded with cells and/or growth factors to recruit, retain and/

Immediate initial mechanical stability

Can regulate differentiation of cells

High potential to differentiate into

Various origins (bone marrow, dental pulp, adipose tissue, periosteum,

or differentiate the cells

**Challenges** 

**Growth factors** (BMPs, TGFβs, PTHrP, VEGF, etc)

**Progenitor cells** 

Inductive

required tissue

blood etc.)

**Adult cells** Inductive of natural tissue

Only for cartilage cells?

remaining challenges for bone and cartilage.

Easy

**Scaffolds** Conductive

From nature it is known that vast amounts of cartilage are formed in the process of endochondrogenesis. Chondrocytes in this cartilage tissue are replaced by a matrix deposited by hypertrophic chondrocytes which die by apoptosis. This matrix is used as an active scaffold for cells that contribute to bone formation. Following embryonic joint formation and post natal growth, the adult skeleton maintains the cellularity and phenotype of articular cartilage, whereas growth plate cartilage completely disappears. This process entitled endochondral ossification can be recapitulated in other places than growth plates. Examples hereof are fracture healing, osteophyte formation and peri-articular ossifications. Even in the process of OA endochondrogenesis plays a role. Next to the formation of osteophytes in OA, evidence has been reported that during the process of OA, articular chondrocytes are triggered to follow the final phase of endochondral ossification (Saito, Fukai et al. 2010).

A scaffold which serves as a template for tissue generation has also been introduced in the field of TE. Thusfar TE has not met initial expectations. Materials used as a scaffold to engineer bone are often engineered to be biocompatible and have good initial biomechanical properties. These properties may interfere with biomechanical stimuli needed for tissue organisation and degradation products from these artificial scaffolds may interfere with the natural healing response. In contrast to a natural endochondral scaffold, artificial scaffolds do not orchestrate ingress of progenitor cells, vascularisation etc.

Periosteum seems to play an important role in postnatal endochondrogenesis. However subcutaneous injection of growth factors leads to generation of bone via the endochondral pathway. The first examples of successful generation of bone and cartilage by triggering the progenitor cells of periosteum are found in literature (Emans, Surtel et al. 2005; Emans, van Rhijn et al. 2010). Also repair of cartilage and bone has been reported to be successful in animal studies using this approach. Using the postnatal endochondrogenic capacity for generation of cartilage and bone has many advantages: expensive culture procedures and logistics are bypassed and sufficient amounts of tissue are likely to be generated. Depending on the stage in which endochondral tissue is harvested, different clinical needs could be treated varying from (osteo)chondral defects to bone defects (Scotti, Tonnarelli et al. 2010). Finally, studying the process of endochondrogenesis may not only be a logical direction for tissue generation, but is also expected to provide useful information how to lock progenitors in the desired phase and will contribute to our understanding of diseases like OA.

#### **5. Acknowledgment**

The authors wish to acknowledge the Dutch Arthritis Association and Dutch Stichting Annafonds, as well as Prof. V. Prasad Shastri for the collaboration which has led to further insight into periosteal chondrogenesis.

Endochondral Bone Formation as Blueprint for Regenerative Medicine 419

Dong, X., Wei, X., Yi, W. (2009). "RGD-modified acellular bovine pericardium as a bioprosthetic scaffold for tissue engineering." *J Mater Sci Mater Med*. Donohue, J. M., Buss, D., Oegema, T. R., Jr. (1983). "The effects of indirect blunt trauma on

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**19** 

*Shanghai, China* 

**Tissue Engineering in Low** 

Chao Feng and Yue-min Xu

**Urinary Tract Reconstruction** 

*Department of Urology, Shanghai Jiaotong University-Affiliated 6th People's Hospital,* 

Acquired and congenital abnormalities of the lower urinary tract often require eventual reconstruction. Traditionally, different types of autologous tissue can be chosen for surgery, depending on which organ requires reconstruction. Bladder reconstruction, for example, is usually performed with intestinal tissue while urethral reconstruction can us buccal mucosa, lingual mucosa, colonic mucosa or prepuce skin. However, the problems of a shortage of patients' own tissues, and of nmany complications related to surgery, have not yet been resolved. There is therefore an effort to obtain sufficient tissue resources, to involve fewer complications, to reduce surgery to relatively minor invasion and to achieve better surgical

outcomes. These goals may be attainable by the use of tissue engineering techniques.

engineering technologies in lower urinary tract reconstruction.

**2. Basic knowledge of tissue engineering in low urinary tract** 

Over the last 50 years, tissue engineering techniques for low urinary tract regeneration have been applied successfully in a variety of animal models and clinical patients. Rapid advancement has been made in this field, which has broadened the theoretical options for the future of low urinary tract reconstruction. These developments include improvements in cell culture techniques, such as the development of cell resources and identification of markers to isolate and characterize specific cell types. Many new types of natural and synthetic biomaterials for use as scaffold components have been created (1). In addition to these, the applications of nanotechnology and bioreactors have been strengthened within recent decades. Here, we review the literature on the basic principles and latest developments of tissue

Because epithelial cells are one of the most important components of the lower urinary tract, optimizing sources for them have always been a popular focus of investigators. Traditionally, urothelial cells obtained from bladder or urethra have often been used in previous studies (Fig 1a) (2,3). Although this technique exploits homotypy between the graft cells and host, it involves injury to the genitourinary tract and the operation is complicated.

**1. Introduction** 

**2.1 Cell sources** 

**2.1.1 Autologous stromal cells** 

chondrocytes: evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization." *J Cell Biochem* 57(2): 218-37.

