**4. Direction by growth factors**

Growth factors (GFs) serve a critical role in Regenerative Medicine, facilitating tissue growth *in vitro* and repair *in vivo.* In the case of skeletal tissues, they are being using to regulate chemotaxis, proliferation, and differentiation of MSCs. Also, selected hormones, cytokines, and nutrients are potentially useful in controlling MSCs growth.

A GF is a signaling biomolecule, commonly polypeptide, that is not a nutrient. Typically they act as signaling molecules via binding to specific receptors on the same cells that secrete the factors (autocrine signaling) or on neighboring cells (paracrine signaling). The binding of the receptor initiates a cascade of cellular reactions, often involving the activation of specific gene transcription. These cellular activities lead to alterations in cell proliferation, differentiation, maturation, and production of other GFs and ECM, all of which result in the formation of specific tissues. Unlike hormones, which act on cells distant from the source (endocrine signaling), GFs have a local (nonsystemic) effect and are often secreted at low concentrations (Fig. 6).

#### **4.1. Transforming growth factor-beta (TGF-β) superfamily**

Members of this superfamily, as bone BMPs, growth differentiation factors (GDFs) and TGFβs, are involved in the different stages of repair bone (intramembranous and endochondral bone ossification) during bone repair (Gerstenfeld et al., 2003).

#### *4.1.1. Transforming growth factor-beta (TGF-β)*

The term transforming growth factor beta is applied to the superfamily of length well-known growth factors involved generally with connective tissue repair and bone regeneration present in many types of tissue (Lieberman et al., 2002). TGF-β exists as five isoforms, three of them have received the most attention regarding fracture repair and proliferation of MSCs (TGF-β1, TGF-β2, TGF-β3), although TGF-β3 has the most pronounced effect on increases proliferation

BM is generally considered milieu plentiful for various cell types, collectively referred to as stromal cells. Amongst these, the multipotent subset of MSCs comprises a small fraction (<0.01) (Dazzi et al., 2006), yet despite their small numbers, the relative ease with which MSCs can be harvested has propelled their experimental use. Researchers have pioneered the creation of stable animal models aimed at mimicking human conditions to study the therapeutic capacity of these BM-derived cells (Kadiyala et al., 1997). Because of their ubiquity, tolerance of expansion, paracrine capabilities, and multipotency, the potential for clinical applications of

The first problem that arises when Cell Therapy methods are used to rebuild bone tissue is how to obtain a sufficiently large number of osteocompetent cells for the intervention to be successful. Hence, the idea of using SCs, which are self-renewing and differentiate into various

Growth factors (GFs) serve a critical role in Regenerative Medicine, facilitating tissue growth *in vitro* and repair *in vivo.* In the case of skeletal tissues, they are being using to regulate chemotaxis, proliferation, and differentiation of MSCs. Also, selected hormones, cytokines,

A GF is a signaling biomolecule, commonly polypeptide, that is not a nutrient. Typically they act as signaling molecules via binding to specific receptors on the same cells that secrete the factors (autocrine signaling) or on neighboring cells (paracrine signaling). The binding of the receptor initiates a cascade of cellular reactions, often involving the activation of specific gene transcription. These cellular activities lead to alterations in cell proliferation, differentiation, maturation, and production of other GFs and ECM, all of which result in the formation of specific tissues. Unlike hormones, which act on cells distant from the source (endocrine signaling), GFs have a local (nonsystemic) effect and are often secreted at low concentrations

Members of this superfamily, as bone BMPs, growth differentiation factors (GDFs) and TGFβs, are involved in the different stages of repair bone (intramembranous and endochondral

The term transforming growth factor beta is applied to the superfamily of length well-known growth factors involved generally with connective tissue repair and bone regeneration present in many types of tissue (Lieberman et al., 2002). TGF-β exists as five isoforms, three of them have received the most attention regarding fracture repair and proliferation of MSCs (TGF-β1, TGF-β2, TGF-β3), although TGF-β3 has the most pronounced effect on increases proliferation

MSCs in the orthopaedic realm is countless (Becerra et al., 2011).

and nutrients are potentially useful in controlling MSCs growth.

**4.1. Transforming growth factor-beta (TGF-β) superfamily**

bone ossification) during bone repair (Gerstenfeld et al., 2003).

*4.1.1. Transforming growth factor-beta (TGF-β)*

tissues, surfaced.

(Fig. 6).

**4. Direction by growth factors**

624 Regenerative Medicine and Tissue Engineering

**Figure 6.** Growth factor regulation of BM-derived MSCs differentiation. Among the classes of bioactive factors, such as matrix ligand, mechanical stimulation, and cell shape, GFs exert strong effects on the regulation of the lineage dif‐ ferentiation of MSCs. Boxed GFs and hormones are used to control bone differentiation *in vitro.* Factors under the ar‐ row have been implicated in promoting regulation differentiation. Pictures represent two histological sections, stained with Sirius red and observed under light (left, femur segmental resection) and polarized (right, profile of a hy‐ droxyapatite implant) microscopes. New bone tissue appears in red. *Results obtained in LABRET-UMA*

of MSCs and chondrogenesis (Weiss et al., 2010). All TGF-β members superfamily are synthe‐ sized as large precursors which are proteolytically cleaved to yield mature protein dimers (Massague et al., 1994). TGF-β signaling that involves two receptor types, TGF-β receptor type I and type II, occurs when factors from the family bind a type II serine/threonine kinase receptor, recruiting another similar transmembrane protein (receptor I). Receptor I phosphor‐ ylates the primary intracellular superfamily signal effector molecules, SMADs, causing their translocation into the nucleus and specific gene transcription (Valcourt et al., 2002). TGF-β and members of this growth factor family can also signal via the mitogen activated protein tyrosine kinase (MAPK), Rho GTPase and phosphoinositide 3kinase (PI3K) pathways (Zhang, 2009).

Like PDGF, they are synthesized and found in platelets and macrophages, as well as MSCs and some other cell types (Barnes et al., 1999), acting as paracrine and autocrine fashion (Fig. 7). TGF-βs inhibit osteoclast formation and bone resorption, thus favoring bone formation over resorption by two different mechanisms (Mohan & Baylink, 1991). The TGF-β activates fibroblasts and preosteoblasts to increase their numbers, as well as promoting their differen‐ tiation toward mature functioning osteoblasts. It influences the osteoblasts to lay down bone matrix and the fibroblast to lay down collagen matrix to support capillary growth (Marx et al., 1996). They also play a role in osteogenesis, its actions are diverse and it is thought to influence the activity of BMPs (Salgado et al., 2004). TGF-β1 plays a pivotal role in the process and site of fracture healing where appears elevated levels in humans, as well as in other mammals, as it enhances the proliferation and differentiation of MSCs and is chemotaxis on bone cells (Sarahrudi et al., 2011).

**Figure 7.** The signal cascade inside the cell after the receptor binding of GFs involves in bone repair. Taken and modi‐ *fied from L. Barnes et al. 1999, and Rodrigues et al. 2010, with permission*

Also, our group have demonstrated that osteogenic precursor cells can be selected from a mixed population of BM MSCs by virtue of their distinctive survival responses in the presence of a recombinant human TGF-β1 fusion protein (Andrades et al., 1999a; 1999b; Andrades and Becerra, 2002a; Andrades et al., 2003; Becerra et al., 2006; Claros et al., in press), engineered to contain an auxiliary collagen binding domain (rhTGF-β1-F2) (Fig. 8), and further, that these selected cells exhibit unique properties in the chodroosteogenic lineage that can ultimately be utilized to therapeutic advantage

#### *4.1.2. Bone morphogenetic proteins (BMPs)*

The first BMP was identified by Urist (1965). He observed the ability from demineralized bone matrix (DBM), to induce ectopic bone formation when implanted under the skin of rodents, and showed that there was a recapitulation of all the events that taking place during skeletal

**Figure 8.** Schematic representation of the genetically engineered fusion construct, containing a histidine purification tag, a protease site, an auxiliary von Willebrand Factor collagen binding domain. Recombinant hTGF-β1-F2 applied to a bovine collagen matrix as vehicle and delivery system could be of advantage in promoting the survival, proliferation, differentiation, and colony mineralization of the osteogenic precursor cell population. It plays a crucial role in early stages of osteogenic commitment and differentiation. *Results obtained in LABRET-UMA*

APC

Dsh

**Wnt5 a**

**6** R1

**Wnt3 a**

P

R2 R1

P R2 R1

> Sma d2

Sma d3

Smad 3

P Smad P 2

kinase receptors

VEGF BMP TGF-

**IGFBP**

Tirosine kinase receptors Serine/Threonine

**Grb2 SOS**

**PI3K** P

**PIP 3**

> **Smad2 Smad3 Smad4** P P

IL-6 TNF- IL-1

**Gsk3** B catenin

C-myc C-myc

**CREB**

**Figure 7.** The signal cascade inside the cell after the receptor binding of GFs involves in bone repair. Taken and modi‐

Also, our group have demonstrated that osteogenic precursor cells can be selected from a mixed population of BM MSCs by virtue of their distinctive survival responses in the presence of a recombinant human TGF-β1 fusion protein (Andrades et al., 1999a; 1999b; Andrades and Becerra, 2002a; Andrades et al., 2003; Becerra et al., 2006; Claros et al., in press), engineered to contain an auxiliary collagen binding domain (rhTGF-β1-F2) (Fig. 8), and further, that these selected cells exhibit unique properties in the chodroosteogenic lineage that can ultimately be

The first BMP was identified by Urist (1965). He observed the ability from demineralized bone matrix (DBM), to induce ectopic bone formation when implanted under the skin of rodents, and showed that there was a recapitulation of all the events that taking place during skeletal

**CREB**

P

P

**Raf MEK 1/2 E r kR s k**

**Ras GTP**

**MEK Erk Rs k**

P P

P

<sup>P</sup>**Erk**<sup>P</sup>

Myt1

*fied from L. Barnes et al. 1999, and Rodrigues et al. 2010, with permission*

utilized to therapeutic advantage

*4.1.2. Bone morphogenetic proteins (BMPs)*

**Tcf/Lef** ? - catenin

**Tcf /Lef** ? - catenin

Sma d4

Smad 4

Akt

**Grb 2**

P

**SO S**

P

**PDGFR FGFR**

**VEGFR** PDGF FGF IGF

**PI3KGrb2 Grb2**

**SOS**

P

**Grb2**

**SOS**

P

**PI3K**

**PIP3**

626 Regenerative Medicine and Tissue Engineering

Wee 1 p27Ki p

Dsh

**FRIZZLE D LPR 5/6**

> Axin Gsk3

Axin Gsk3 B APC

? - catenin

development. In 1971, it was named as the responsible factors BMPs. More lately, others searchers, as Reddi and Huggins (1972) demonstrated that these molecules are important during development. Even present, at least many than 30 BMPs have been identified and BMP ´s functions have been studied by means of analysis of mutant genes and knockout experiments in mice. Different BMPs, among others member of the TGF-βs superfamily, trigger a serine/ threonine kinasa cascade of events that induce the formation of cartilage and bone (Fig.7).

During fracture repair, BMPs are produced by MSCs, osteoblasts, and chondrocytes, and bind to cells by direct interaction or are accumulated and subsequently delivered of ECM to promote the bone generation. These proteins induce a cascade of cellular pathways that promote cell growth, migration and differentiation of MSCs to repair the injury, stimulates angiogenesis, as well as synthesis of ECM and play a regulatory role in tissue homeostasis (Reddi, 2001). The different BMPs act in different temporal scale during bone repair. In studies of fracture healing, BMP-2 mRNA expression showed maximal levels within 24 hrs of injury, suggesting that this BMP plays a role in initiating the repair cascade. Other *in vitro* studies examining marrow MSCs differentiation have shown that BMP-2 controls the expression of several other BMPs, and when its activity is blocked, marrow MSCs fail to differentiate into osteoblasts (Edgar et al., 2007).

BMP-3, BMP-4, BMP-7, and BMP-8 are expressed during bone repair, from days 14 to 21, when the resorption of calcified cartilage and osteoblastic recruitment are most active, and bone formation takes place. Our group has demonstrated that BMP-7 is capable of selecting a cell population from BM which, in a three dimensional collagen type I gel, achieves skeletogenic potential under *in vitro* and *in vivo* environments (Andrades et al., 2001; Andrades and Becerra, 2002b; Andrades et al., 2003). BMP-5 and BMP-6 and other members of the TGF-βs superfamily are constitutively expressed from days 3-21 during fracture in mice, suggesting that they have a regulatory effect on both intramembranous and endochondral ossification. BMP-2 to BMP-8 show high osteogenic potencial, however BMP-2, BMP-6, and BMP-9 may be the most potent inducers of MSCs differentiation to osteoblasts, while the others, stimulate the maturation of osteoblasts (Cheng et al., 2003).

The first BMP extracted in a highly purified recombinant form was BMP-2. In preclinical models, BMP-2 has the ability to induce bone formation and heal bone defects and promote the maturation and consolidation of regenerated bone. Recombinant human BMP-7 and BMP-2 are among the first growth factor based products available for clinical use to treat patients afflicted with bone diseases. A large number of studies have been performed to determine appropriate carriers for BMPs (Cheng et al., 2003).

*In vitro* cultures, MSCs and osteoblasts exhibit a high number of BMP receptors and synthesize the BMP antagonist's noggin, which are capable of blocking osteogenesis as MSCs differentiate into osteoblasts. BMP antagonists are important in normal bone turnover and regulation. The expression of the BMP antagonists, as noggin, which blocks BMP-2, BMP-4, and BMP-7 interaction with its receptor, also is modulated during bone repair (Balemans et al. 2002).

#### *4.1.3. Wnt proteins*

The Wnt pathway was initially identified as a proto-oncogene in mammary tumors that was activated by integration of the mouse mammary virus (Nusse & Varmus, 1982). Since then, it has been the subject of many studies. It knows Wnt proteins are secreted cysteine-rich glycosylated family proteins to share a highly conserved pattern of 23–24 cysteine residues and several asparagines-linked glycosylation sites (Li et al., 2006). In mature tissues, Wnt pathway play a regulator role of osteogenesis and stem/progenitor cells self-renewal, it is involved in bone formation, and also cellular adhesion and migration through their indirect interactions with the cadherin pathway (Arnsdorf et al., 2009).

Wnt proteins are divided towars to activate one of two main signaling pathways that consist of the Wnt1 class, also called Wnt/β-catenin or canonical Wnt pathway and Wnt5a class, Wnt/ Ca2+ or non-canonical pathway. Several lines of evidence have demonstrated the importance of canonical Wnt signaling in promoting osteogenesis *in vitro* and *in vivo* (Chung et al., 2009). Wnt signaling is a prime target for bone active drugs and the approaches include inhibition of Wnt antagonist like Dkk1, sclerostin, and Sfrp1 with neutralizing antibodies and inhibition of glycogen synthase kinase 3β (GSK-3β), which promotes phosphorylation and degradation of β-catenin. Enhancement of Wnt signaling either by Wnt overexpression or deficiency of Wnt antagonists (ten Dijke et al., 2008) is associated with increased bone formation in mice and humans. Gain of function mutations of *LRP5/6* that lead to impaired binding of Dkk-1 (Dickkopf-1 is a secreted Wnt antagonist that binds LRP5/6) to this Wnt coreceptor are associated with increased bone mass (Boyden et al., 2002).

In spite of osteogenic inhibitory function of canonical Wnts, this pathway plays a positive role in bone homeostasis *in vivo* (Liu et al., 2009). Canonical Wnt signaling in osteoblast differen‐ tiation is modulated by Runx2 and osterix transcription factors (Hill et al., 2005). Quarto et al. (2010) have shown canonical Wnt signaling can either inhibit or promote osteogenic differen‐ tiation depending on the status of cell (cellular differentiation degree undifferentiated vs. differentiated), the threshold levels of its activation (existence of a differential activation of canonical Wnt signaling between an undifferentiated MSC and osteoblast), and Wnt ligands concentration showing *in vitro* and *in vivo* data correlated results for Wnt3a treatment of calvarial defects created in juvenile mice where rise activation of canonical Wnt signaling inhibited osteogenic differentiation of undifferentiated MSCs, whereas increased the miner‐ alization of differentiated osteoblasts.

#### **4.2. Growth hormone and insulin-like growth factors (GH and IGF)**

In clinic, the patients that present short stature are treated with the Growth Hormone (GH); for this reason, many researcher study the effects of GH in the treatment for osteoporosis and repair bone fracture. It is released by pituitary gland and travels through the circulation to the liver, where target cells are stimulated to release IGF. There are two IGFs identified: IGF-I and IGF-II. Various studies have shown that both IGF-I and IGF-II (Swolin et al., 1996;) are delivered by osteoblasts, chondrocytes, endotelial cells, and bone matrix, and they are detected by recruitment MSCs and bone cells in a paracrine/autocrine manner thanks to the presence of six insulin growth factor-binding proteins (IGFBPs), which modulate their action by intracellular tisone kinase cascade.

IGF-II is the most abundant GF in bone matrix. However, IGF-I is 4 to 7 times more potent in synthesis of bone matrix (type I collagen and non-collagen matrix proteins) (Lind, 1996). IGF-II acts on phase of endochondral bone formation and induces type I collagen production, stimulates cartilage matrix synthesis, and cellular proliferation. Both factors have been localized in bone studies of animals and humans with GH-deficient levels. The expression and secretion of IGFBPs, IGF-I and IGF-II (Birnbaum et al., 1995) changes during *in vitro* MSCs cultures. Prisell et al. (1993) showed that IGF-I mRNA was expressed during the MSCs recruitment and proliferation; however IGF-II mRNA expression happened later, during endochondral bone formation by osteoblasts and chondrocytes. IGF production is not only under the control of GH, is also regulated by estrogen, PTH, cortisol (inhibits IGF-I synthesis), local GFs and cytokines (Ohlsson et al., 1998). This abundant supply of IGFs is necessary to promote bone formation, bone repair, and MSCs cell proliferation and differentiation.

#### **4.3. Fibroblast growth factor (FGF)**

inducers of MSCs differentiation to osteoblasts, while the others, stimulate the maturation of

The first BMP extracted in a highly purified recombinant form was BMP-2. In preclinical models, BMP-2 has the ability to induce bone formation and heal bone defects and promote the maturation and consolidation of regenerated bone. Recombinant human BMP-7 and BMP-2 are among the first growth factor based products available for clinical use to treat patients afflicted with bone diseases. A large number of studies have been performed to determine

*In vitro* cultures, MSCs and osteoblasts exhibit a high number of BMP receptors and synthesize the BMP antagonist's noggin, which are capable of blocking osteogenesis as MSCs differentiate into osteoblasts. BMP antagonists are important in normal bone turnover and regulation. The expression of the BMP antagonists, as noggin, which blocks BMP-2, BMP-4, and BMP-7 interaction with its receptor, also is modulated during bone repair (Balemans et al. 2002).

The Wnt pathway was initially identified as a proto-oncogene in mammary tumors that was activated by integration of the mouse mammary virus (Nusse & Varmus, 1982). Since then, it has been the subject of many studies. It knows Wnt proteins are secreted cysteine-rich glycosylated family proteins to share a highly conserved pattern of 23–24 cysteine residues and several asparagines-linked glycosylation sites (Li et al., 2006). In mature tissues, Wnt pathway play a regulator role of osteogenesis and stem/progenitor cells self-renewal, it is involved in bone formation, and also cellular adhesion and migration through their indirect

Wnt proteins are divided towars to activate one of two main signaling pathways that consist of the Wnt1 class, also called Wnt/β-catenin or canonical Wnt pathway and Wnt5a class, Wnt/ Ca2+ or non-canonical pathway. Several lines of evidence have demonstrated the importance of canonical Wnt signaling in promoting osteogenesis *in vitro* and *in vivo* (Chung et al., 2009). Wnt signaling is a prime target for bone active drugs and the approaches include inhibition of Wnt antagonist like Dkk1, sclerostin, and Sfrp1 with neutralizing antibodies and inhibition of glycogen synthase kinase 3β (GSK-3β), which promotes phosphorylation and degradation of β-catenin. Enhancement of Wnt signaling either by Wnt overexpression or deficiency of Wnt antagonists (ten Dijke et al., 2008) is associated with increased bone formation in mice and humans. Gain of function mutations of *LRP5/6* that lead to impaired binding of Dkk-1 (Dickkopf-1 is a secreted Wnt antagonist that binds LRP5/6) to this Wnt coreceptor are

In spite of osteogenic inhibitory function of canonical Wnts, this pathway plays a positive role in bone homeostasis *in vivo* (Liu et al., 2009). Canonical Wnt signaling in osteoblast differen‐ tiation is modulated by Runx2 and osterix transcription factors (Hill et al., 2005). Quarto et al. (2010) have shown canonical Wnt signaling can either inhibit or promote osteogenic differen‐ tiation depending on the status of cell (cellular differentiation degree undifferentiated vs. differentiated), the threshold levels of its activation (existence of a differential activation of

osteoblasts (Cheng et al., 2003).

628 Regenerative Medicine and Tissue Engineering

*4.1.3. Wnt proteins*

appropriate carriers for BMPs (Cheng et al., 2003).

interactions with the cadherin pathway (Arnsdorf et al., 2009).

associated with increased bone mass (Boyden et al., 2002).

FGF is a secreted glycoproteins family whose signals are implicated in wound healing and angiogenesis, which influence in cellular proliferation, differentiation, migration, survival and polarity transduced through their receptors (FGFR1, FGFR2, FGFR3 and FGFR4). These receptors are constituted of extracellular immunoglobulin-like (Ig-like) domains and cyto‐ plasmic tyrosine kinase activity domain. FGF proliferation signals occur through the tyrosine kinase cascade in various target cell types (Ng et al., 2008).

The various FGF receptors display varying affinities for each of the members of the FGF family and are expressed in a wide variety of tissues including indeed, bone. As with many of the tyrosine kinase receptors, activation of the intrinsic tyrosine kinase activity occurs through receptor dimerization in response to ligand binding. An additional com‐ plexity may be added to the receptor-ligand association through the binding of FGF li‐ gand by either secreted or membrane-bound proteoglycans, heparin-like proteoglycans in particular because their high affinity (Givol & Yayon, 1992). Nine members of the FGF family have been identified of which, the most abundant in human tissue are FGF-1 (acid character) and FGF-2 (basic character) (Lieberman et al., 2002). FGFs are important regu‐ lators of fracture repair expressed by MSCs, maturing chondrocytes and osteoblasts and have been demonstrated to enhance TGF-β expression in osteoblastic cells (Bolander, 1998). They play a role in maintaining the balance between bone-forming cells and boneresorbing cells and promote angiogenesis. Specifically, FGF-2 not only maintains MSCs proliferation potential, it also retains a slight osteogenic, adipogenic and chondrogenic differentiation potentials through the early mitogenic cycles; eventually, however, all of the MSCs differentiate into the chondrogenic line (Yanada et al., 2006).

#### **4.4. Platelet derived growth factor (PDGF)**

PDGFs are potent mitogens of MSCs (Ng et al., 2008) which express all forms of the GF: PDGF-A and PDGF-C at higher levels, and PDGF-B and PDGF-D at lower levels, such as both receptors type PDGFRα and PDGFRβ through which PDGF signaling is transduced (Tokunaga et al., 2008). PDGF is a dimeric molecule can exist either as a homodimeric (PDGF-AA, PDGF-BB, etc) or a heterodimeric form (PDGF-AB) according to the relative levels of each subunit generating a level of ligand complexity in cells in which both polypeptides are expressed. The different PDGF isoforms exert their effect on target cells by binding with different specificity to two structurally related protein tyrosine kinase receptors, denoted as the α-receptors and β-receptors, which are autophosphorylate ligand bound (Tokunaga et al., 2008). Several groups have found PDGF-BB to induce both proliferation and migration in MSCs (Fierro et al., 2007). While PDGFRβ inhibits osteogenesis, however, PDGFRα has been observed to induce osteogenesis. Akt signaling has been proposed to mediate both the suppression and induction of osteogenesis by PDGFR signaling (Tokunaga et al., 2008).

These molecules acts as paracrine manner stimulating mitogenesis of the marrow SCs and endosteal osteoblasts transferred in grafts to increase their numbers by several orders of magnitude. It also begins an angiogenesis of capillary budding into the graft by induc‐ ing endothelial cell mitosis and macrophage activator effect. It is known to emerge from degranulating platelets at the time of injury. PDGF also increased hMSC proliferation like Wnt (Liu et al., 2009). PDGF recruits MSCs and promotes chemotaxis and angiogenesis (Salgado et al., 2004).

### **5. Biomaterials as support**

Natural bone consists of an ECM with nanosized apatitic minerals and collagen fibers that support bone cell functions. It is advantageous for a synthetic biomimetic scaffold to: (1) contain nano-apatite crystals similar to those in bone, together with fibers to form a matrix that supports cell attachment; (2) have mechanical properties similar to those of bone; and (3) encapsulate and support cells for osteogenic differentiation and bone regeneration. The success in regenerating a damaged tissue using the tissue engineering approach depends on the various types of interactions between the cells, scaffolds, and GFs. Besides, an understanding of the phenomena of cell adhesion and, beyond, the function of the proteins involved in cell adhesion on contact with the materials and the purpose depends of supramolecular assembly (scaffolding) of biomimetic biomaterials such as collagens, proteoglycans, and cell adhesion glycoproteins such as fibronectins and laminin.

gand by either secreted or membrane-bound proteoglycans, heparin-like proteoglycans in particular because their high affinity (Givol & Yayon, 1992). Nine members of the FGF family have been identified of which, the most abundant in human tissue are FGF-1 (acid character) and FGF-2 (basic character) (Lieberman et al., 2002). FGFs are important regu‐ lators of fracture repair expressed by MSCs, maturing chondrocytes and osteoblasts and have been demonstrated to enhance TGF-β expression in osteoblastic cells (Bolander, 1998). They play a role in maintaining the balance between bone-forming cells and boneresorbing cells and promote angiogenesis. Specifically, FGF-2 not only maintains MSCs proliferation potential, it also retains a slight osteogenic, adipogenic and chondrogenic differentiation potentials through the early mitogenic cycles; eventually, however, all of

PDGFs are potent mitogens of MSCs (Ng et al., 2008) which express all forms of the GF: PDGF-A and PDGF-C at higher levels, and PDGF-B and PDGF-D at lower levels, such as both receptors type PDGFRα and PDGFRβ through which PDGF signaling is transduced (Tokunaga et al., 2008). PDGF is a dimeric molecule can exist either as a homodimeric (PDGF-AA, PDGF-BB, etc) or a heterodimeric form (PDGF-AB) according to the relative levels of each subunit generating a level of ligand complexity in cells in which both polypeptides are expressed. The different PDGF isoforms exert their effect on target cells by binding with different specificity to two structurally related protein tyrosine kinase receptors, denoted as the α-receptors and β-receptors, which are autophosphorylate ligand bound (Tokunaga et al., 2008). Several groups have found PDGF-BB to induce both proliferation and migration in MSCs (Fierro et al., 2007). While PDGFRβ inhibits osteogenesis, however, PDGFRα has been observed to induce osteogenesis. Akt signaling has been proposed to mediate both the suppression and

These molecules acts as paracrine manner stimulating mitogenesis of the marrow SCs and endosteal osteoblasts transferred in grafts to increase their numbers by several orders of magnitude. It also begins an angiogenesis of capillary budding into the graft by induc‐ ing endothelial cell mitosis and macrophage activator effect. It is known to emerge from degranulating platelets at the time of injury. PDGF also increased hMSC proliferation like Wnt (Liu et al., 2009). PDGF recruits MSCs and promotes chemotaxis and angiogenesis

Natural bone consists of an ECM with nanosized apatitic minerals and collagen fibers that support bone cell functions. It is advantageous for a synthetic biomimetic scaffold to: (1) contain nano-apatite crystals similar to those in bone, together with fibers to form a matrix that supports cell attachment; (2) have mechanical properties similar to those of bone; and (3) encapsulate and support cells for osteogenic differentiation and bone regeneration. The success

the MSCs differentiate into the chondrogenic line (Yanada et al., 2006).

induction of osteogenesis by PDGFR signaling (Tokunaga et al., 2008).

**4.4. Platelet derived growth factor (PDGF)**

630 Regenerative Medicine and Tissue Engineering

(Salgado et al., 2004).

**5. Biomaterials as support**

Osteogenesis is highly dependent on the substrate carrier used, which has to provide a favorable environment into which bone cells can migrate before proliferating, differentiating, and depositing bone matrix (i.e., osteoconduction) (Ono et al., 1999). At the cell level, substrates of this kind must have specific biochemical (molecular) properties, physicochemical charac‐ teristics (surface free energy, charge, hydrophobicity, and so on), and a specific geometric conformation (they must be three dimensional and show interconnected porosity) (Jin, 2000). From the biomaterial point of view, the scaffolds used for bone engineering purposes have to meet a number of criteria, including (1) biocompatibility (nonimmunogenicity and nontoxic‐ ity); (2) resorbability (showing resorption rates commensurate with the bone formation rates); (3) preferably radiolucency (to allow the new bone to be distinguished radiographycally from the implant); (4) osteoconductivity; (5) mechanical properties to match those of the tissues at the site of implantation; (6) easy to manufacture and sterilize; and they must be (7) easy to handle in the operating theater, preferably without requiring any preparatory procedures (in order to limit the risk of infection).

The bone substitute materials intended to replace the need for autologous or allogeneic bone, consist of bioactive ceramics, bioactive glasses, biological or synthetic polymers, and compo‐ sites of these. Biological polymers, such as collagen and hyaluronic acid provide guidance to cells that favors cell attachment and promotes chemotactic responses, but, a disadvantage is immunogenicity for the potential risk of disease transmission. On the other hand, other alternative is synthetic polymers such as polyfumarates, polylactic acid (PLA), polyglycolic acid (PGA), copolymers of PLA and PGA (PLGA), and polycaprolactone. Nevertheless, there are a wide range of bioactive inorganic materials similar in composition to the mineral phase of bone, for example, tricalcium phosphate, HA, bioactive glasses, and their combinations; and all of these can be tailored to deliver ions such as Si at levels capable of activating complex gene transduction pathways, leading to enhanced cell differentiation and osteogenesis. Hydrogels, such as polyethylene glycol or alginate-based, are to provide a three-dimensional cellular microenvironment with high water content, this is suitable for cartilage regeneration. Polyethylene glycol (PEG) hydrogels were investigated as encapsulation matrices for osteo‐ blasts to assess their applicability in promoting bone tissue engineering. Non-adhesive hydrogels were modified with adhesive Arg-Gly-Asp (RGD) peptide sequences to facilitate the adhesion, spreading, and, consequently, cytoskeletal organization of osteoblasts. Finally, mineral deposits were seen in all hydrogels after 4 weeks of *in vitro* culture, but a significant increase in mineralization was observed upon introduction of adhesive peptides throughout the network. Potentially, the cell suspension could be injected into the body through a needle and photopolymerized through the skin to provide a non-invasive technique to enhance bone regeneration.

**Figure 9.** Overview of nanoparticle applications in bone regeneration

Biomaterials such as polymers, ceramics, and metals are widely used in bone for regenerative therapies, including in bone grafts and in Tissue Engineering as well as for temporary or permanent implants to stabilize fractures (Navarro et al., 2008). In recent years, biomaterials in general and bone-related implant materials in particular have been considerably refined, with the objective of developing functionalized materials, so-called smart materials, containing bioactive molecules to directly influence cell behaviour (Mieszawska and Kaplan, 2010). Rapid developments in nanotechnology have yielded many clinical benefits, in particular in the field of bone tissue engineering. The main advantage in that several novel biomaterials can be fabricated into nanostructures that closely mimic the bone in structure and composition. The optimization in the surface features of biomaterials has strongly improved cell behaviour in terms of adhesion, proliferation, differentiation and tissue formation in three dimensions. In this context, nanoparticles that are in the same size range as integral parts of natural bone, such as HA crystals or cellular compartments, are promising candidates for local applications. In bone, locally applied nanoparticles may be suitable for numerous potential uses with respect to the improvement of tissue regeneration, the enhanced osseointegration of implants, and the prevention of infections.

Increasingly refined nanoparticles are being developed for a wide range of applications (Fig. 9). These include cell labelling to broaden research possibilities as well as to improve and noninvasively monitor cell therapy approaches (Bhirde et al., 2011; Andrades et al., in press (b). Moreover, drug delivery systems with improved pharmacologic characteristics are being developed. They promote enhanced therapeutic outcome by providing controlled release of bioactive molecules, such as growth factors or anticancer drugs (Allen and Cullis, 2004). In addition, gene therapy concepts with good prospects are required for future treatment options based on intracellular manipulation (Evans, 2011).

The heterogeneous picture of research on the interactions of nanoparticles with MSCs makes it difficult to draw general conclusions. However, it becomes clear that parameters such as chemistry, size, and shape in some cases greatly affect the particle uptake behaviour of MSCs as well as their natural differentiation potential. Different strategies for nanoparticle applica‐

**Figure 10.** Bone fracture repair and regeneration is a question of balance among cells, growth factors and bioma‐ terials.

tion in bone (i.e., as cell-labeling agents and for drug or gene delivery) have great potential for monitoring and supporting tissue regeneration.
