**2. Mechanism of action of growth factors (GFs)**

Growth factors (GFs) are protein molecules that have a role in controlling biological processes, such as cell growth, proliferation, differentiation and repair. GFs cannot pass through a cell's membrane; they must bind to high-affinity cell receptors in order to take effect. Many GFs stimulate several cell populations, while others are less versatile and specific to a particular cell line.

In dentistry, numerous GFs have been investigated in terms of their effect on hard and soft tissue healing and regeneration.

© 2013 Sivolella et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Whatever the tissue involved, the healing process always involves a series of molecular, biochemical and cellular events that can be grouped into three overlapping phases: inflam‐ mation, proliferation, and remodeling.

Inflammation begins spontaneously straight an after injury has occurred and lasts for 1 to 4 days. It is characterized by clotting in the wound, the release of signal molecules to recruit immune cells, and the release of specific enzymes (matrix metalloproteinases, MMPs) that clean the wound. The proliferative phase takes place between 4 and 21 days after wounding, when fibroblasts are stimulated to invade the site of the wound and produce extracellular matrix components. Highly-vascularized granulation tissue is formed and the gap is closed. The final remodeling phase can take up to a year, during which time the immature scar is converted into a stable, less vascularized tissue that exhibits good mechanical proprieties, followed by the growth of regenerated tissue.

GFs have been used in dentistry in all these phases. The most often studied GFs are probably the bone morphogenetic proteins (BMPs), discovered by Urist, who found that protein mixtures obtained from demineralized, lyophilized segments of bone were responsible for bone formation after implanting in rabbit muscle tissue [1].

BMPs are multifunctional cytokines that belong to the transforming growth factor-β (TGF-β) superfamily. They are not only involved in direct ectopic bone formation (hence their name of *bone morphogenetic proteins*), they also modulate several developmental processes, prompting numerous authors to suggest other names: for instance, Reddi suggested that they be should be called *body morphogenetic proteins*, given their extensive roles in various tissues [2].

Over 20 BMPs with various functions have been identified in humans. They have a major role in embryogenesis and in the maintenance and repair of many skeletal and non-skeletal tissues in adults [3]. BMP-1 is actually not considered a member of the BMP family, but a misnamed protein with chordinase and procollagen proteinase activities, implicated in pattern formation during the development of a number of organisms [4]. BMPs are mainly related to bone and cartilage formation, though BMPs 8b, 10 and 15 have no role in these processes, and BMPs 12, 13 and 14 are called cartilage-derived morphogenetic proteins (CDMPs) because they induce chondrogenic phenotypes rather than osteogenesis [2,5], whereas a definite bone-inducing role during bone formation has been observed for BMPs 2, 4, 6, 7 and 9 [6].

BMPs play a pivotal part in skeletal morphogenesis and repair, promoting the differentiation of mesenchymal cells into osteoblasts and inducing new bone formation. BMPs are involved in regulating mesenchymal cell differentiation and proliferation by stimulating intracellular signaling pathways. BMP signals are transmitted by the plasma membrane receptors to the nucleus through multiple signaling pathways that can be divided into two groups, the Smad and non-Smad pathways [3,7]. At the cell surface, BMP ligands bind with BMP receptors, triggering specific intracellular pathways that activate and influence gene transcription. Of the three types of receptor for the TGF-β superfamily, only types I and II appear to have significant roles in BMP binding and signaling. Five type I receptors (ALK1 [Acvrl1], ALK2 [ActRI], ALK3 [BRIa], ALK4 [ActRIb] and ALK6 [(BRIb]), and three type II receptors (BRII, ActRIIa, and ActRIIb) have been identified [8], plus a short form of BRII [9]. Type III TGF-β receptors have also been shown to have a role in BMP signaling, by mediating epithelial to mesenchymal cell conversion [10].

Whatever the tissue involved, the healing process always involves a series of molecular, biochemical and cellular events that can be grouped into three overlapping phases: inflam‐

Inflammation begins spontaneously straight an after injury has occurred and lasts for 1 to 4 days. It is characterized by clotting in the wound, the release of signal molecules to recruit immune cells, and the release of specific enzymes (matrix metalloproteinases, MMPs) that clean the wound. The proliferative phase takes place between 4 and 21 days after wounding, when fibroblasts are stimulated to invade the site of the wound and produce extracellular matrix components. Highly-vascularized granulation tissue is formed and the gap is closed. The final remodeling phase can take up to a year, during which time the immature scar is converted into a stable, less vascularized tissue that exhibits good mechanical proprieties,

GFs have been used in dentistry in all these phases. The most often studied GFs are probably the bone morphogenetic proteins (BMPs), discovered by Urist, who found that protein mixtures obtained from demineralized, lyophilized segments of bone were responsible for

BMPs are multifunctional cytokines that belong to the transforming growth factor-β (TGF-β) superfamily. They are not only involved in direct ectopic bone formation (hence their name of *bone morphogenetic proteins*), they also modulate several developmental processes, prompting numerous authors to suggest other names: for instance, Reddi suggested that they be should

Over 20 BMPs with various functions have been identified in humans. They have a major role in embryogenesis and in the maintenance and repair of many skeletal and non-skeletal tissues in adults [3]. BMP-1 is actually not considered a member of the BMP family, but a misnamed protein with chordinase and procollagen proteinase activities, implicated in pattern formation during the development of a number of organisms [4]. BMPs are mainly related to bone and cartilage formation, though BMPs 8b, 10 and 15 have no role in these processes, and BMPs 12, 13 and 14 are called cartilage-derived morphogenetic proteins (CDMPs) because they induce chondrogenic phenotypes rather than osteogenesis [2,5], whereas a definite bone-inducing role

BMPs play a pivotal part in skeletal morphogenesis and repair, promoting the differentiation of mesenchymal cells into osteoblasts and inducing new bone formation. BMPs are involved in regulating mesenchymal cell differentiation and proliferation by stimulating intracellular signaling pathways. BMP signals are transmitted by the plasma membrane receptors to the nucleus through multiple signaling pathways that can be divided into two groups, the Smad and non-Smad pathways [3,7]. At the cell surface, BMP ligands bind with BMP receptors, triggering specific intracellular pathways that activate and influence gene transcription. Of the three types of receptor for the TGF-β superfamily, only types I and II appear to have significant roles in BMP binding and signaling. Five type I receptors (ALK1 [Acvrl1], ALK2 [ActRI], ALK3 [BRIa], ALK4 [ActRIb] and ALK6 [(BRIb]), and three type II receptors (BRII, ActRIIa, and ActRIIb) have been identified [8], plus a short form of BRII [9]. Type III TGF-β receptors have

be called *body morphogenetic proteins*, given their extensive roles in various tissues [2].

during bone formation has been observed for BMPs 2, 4, 6, 7 and 9 [6].

mation, proliferation, and remodeling.

714 Regenerative Medicine and Tissue Engineering

followed by the growth of regenerated tissue.

bone formation after implanting in rabbit muscle tissue [1].

Canonical Smad-dependent TGF-β first binds to receptors type I and type II, and then signals are transduced to their Smads. Activated Smads form a complex with Smad4 and cross the nuclear membrane into the nucleus, where they regulate the expression of transcriptional factors and transcriptional coactivators that are important in osteoblasts (Dlx5, Runx2 and Osx). It has recently been demonstrated that, following TGF-β induction, the Smad and the p38 MAPK pathways converge on the Runx2 gene to control mesenchymal precursor cell differentiation [11].

As for the isolation of BMPs, after Urist's experiments, BMPs were obtained from the bones of various species, including rabbit, cow and human. Nowadays, BMPs are produced and purified using DNA recombinant technology and essentially two expression systems, in mammalian cells or bacteria [6]. Recombinant human BMP-2 (rhBMP-2) and recombinant human BMP-7 (rhBMP-7) are currently the only proteins in the group to been approved by the US Food and Drug Administration (FDA) for clinical use in humans, which explains why they are clearly the most extensively evaluated BMPs [12].

Another GF of interest in dentistry is the growth and differentiation factor (GDF), the structure of which closely resembles some BMPs, so it could be included in the BMP family. GDF-5 is also known as BMP-14, or cartilage-derived morphogenetic protein 1, because it induces chondrogenic phenotypes rather than osteogenesis [6]. GDF-5 gene mutations give rise to different types of dysplasia and can result in the autosomal recessive syndromes of brachypod in mice and Hunter-Thompson or Grebe-type chondrodysplasia in humans, involving a loss of joints in both humans and mice [13-15]. Francis-West and colleagues [14] showed that GDF-5 can modulate the initial stages of chondrogenesis by increasing cell adhesion, and can increase chondrocyte proliferation in the later stages of skeletogenesis.

The osteoinductive potential of GDF-5 has been found smaller than that of other members of the BMP family, though numerous studies have confirmed its crucial role in skeletal morpho‐ genesis. Several *in vitro* experiments have demonstrated that rhGDF-5 stimulates osteogenic differentiation and promotes angiogenic activity by increasing vascular endothelial growth factor gene expression in fat- or bone-marrow-derived stromal cells. The osteoinductive activity of rhGDF-5 has also been examined in numerous *in vivo* model systems [13].

Another GF extensively investigated for clinical applications is the platelet-derived growth factor (PDGF), which is synthesized by platelets, monocytes, macrophages, endothelial cells and osteoblasts. This is a dimeric molecule consisting of disulfide-bonded, structurally similar A- and B-polypeptide chains that combine to form homo- and heterodimers. The biologically most potent of these PDGFs is PDGF-BB, which has been thoroughly investigated. The PDGF isoforms exert their cellular effects by binding to and activating two structurally related protein tyrosine kinase receptors, called the alpha-receptor and the beta-receptor [16,17].

PDGF is stored in the alpha granules of circulating platelets and is released during blood clotting in the event of soft or hard tissue injury. Once it has been released from the platelets, PDGF binds to specific cell surface receptors and promotes rapid cell migration (chemotaxis) and proliferation (mitogenesis) at the site of injury. In particular, *in vitro* and *in vivo* studies have demonstrated that PDGF is a potent chemotactic and mitogenic factor for gingival and periodontal ligament fibroblasts, cementoblasts and osteoblasts [18].

Since the first animal study conducted by Lynch and co-workers [19], extensive *in vitro,* preclinical and clinical studies have been performed using PDGF, alone or in combination with other GFs, for incrementing bone vertically and horizontally, and for treating periodontal and peri-implant defects. The positive outcomes of these studies provide strong evidence of the safety and predictably of rhPDGF combined with specific scaffolds in periodontal and periimplant regeneration, suggesting promising clinical applications [18,20,21].

Although a large body of preclinical and clinical data has been obtained for only a few GFs, others have nonetheless been assessed for possible applications in clinical practice.

The activity and osteoinductive potential of fibroblast growth factor (FGF) have been the object of various studies [22-24]. FGF signaling reportedly interacts with BMP signaling in bone formation, showing a synergic action on osteogenesis [11].

Few studies have considered the use of parathyroid hormone (PTH) as a factor for modulating bone augmentation and healing [25]. PTH binding activates PTH1R to stimulate several downstream effectors and also drives the internalization of the PTH1R(PTH type I receptor)- TGFβRII (TGF-β type II receptor) complex, which attenuates both TGF-β and PTH signaling on bone development. The transcriptional factor/cAMP response element binding protein (CREB) mediates PTH signaling in osteoblasts, and the PTH-CREB signaling pathway serves as an effective activator of BMP-2 expression [11].

Transforming growth factor-β (TGF-β) [26-27], vascular endothelial growth factor (VEGF) [24], and insulin-like growth factor (IGF) [28] are also the object of studies regarding the biological properties of these bioactive molecules.
