**3. Bone healing**

Bone is a living tissue responsible for the structural support and calcium metabolism in the body [11]. It is constituted by bone cells and bone matrix. Bone cells

are of three main types' viz. osteoblasts, osteocytes and osteoclasts. These cells are differentiated by the mesenchymal cells, (during growth, at the time of trauma or general remodeling), to become osteoprogenitor cells and later take up the function of specific bone cells.

Bone matrix is composed of 35% organic and 65% inorganic components. Calcium and phosphate ions mainly constitute the inorganic part. Type I collagen forms the major part (90%) of the bone matrix which is impregnated with minerals and calcium salts. Minerals are in form of hydroxyapatites. Bone matrix also consists of non- collagenous proteins (10%) like osteocalcin, osteonectin, osteopondin etc. which function to regulate mineralization and interaction of collagenous and non-collagenous proteins, mediate cell to matrix binding.

Besides the bone matrix also consists of small amount of potent regulatory proteins that are produced by osteoblasts and incorporated by the extracellular matrix during bone formation. These are called as Growth Factors [5].

These are protein molecules that regulate osteoblast and osteoclast metabolism during bone remodeling and/or initiate and control healing response after bone trauma. They can exhibit their effects in the local environment only. Thus, to facilitate cell proliferation and matrix production at the site, they effect by paracrine or autocrine mechanism.

The extensive number of growth factors being discovered and researched lays consideration on the significance of their existence. When we try to understand bone metabolism it is empirical to read about the hormones that effect changes in bone matrix. It has been researched and found that the growth factors mediate the response of these systemic hormones, locally by augmenting the cell replication and initiating the cell differentiation by binding to membrane bound receptors [5].

#### **3.1 Important growth factors in bone healing**

Here is a list of the growth factors (**Table 2**) majorly required at a healing site. Apart from the main ones there are various other factors required by different tissues of the body during healing. We will discuss in detail the factors affecting bone healing.

#### *3.1.1 Transforming growth factor-beta (TGF-ß)*

This multifactorial cytokine basically regulates growth and differentiation of the cells. It stimulates the cells of mesenchymal origin and inhibits those of ectodermal origin. Although almost all body cells posses this factor but bone and platelets have approximately 100 times more TGF-ß than others and osteoblasts bear highest number of TGF-ß receptors [12]. TGF- ß 1, TGF- ß 2, and TGF- ß3 are found in mammals, but TGF- ß 1 predominates in cutaneous wound healing.

TGF- ß 1 facilitates the recruitment of additional inflammatory cells and augments macrophage mediated tissue debridement. It deactivates superoxide production from macrophages in vitro protecting the surrounding healthy tissue and prepares the wound for granulation tissue formation. When overexpressed, TGF- ß 1 has been shown to stimulate connective tissue growth factor (CTGF) also shown to play an important role in the development of hypertrophic and keloid scars [1].

TGF- ß 2 is also involved in recruiting inflammatory cells and fibroblasts to the wound site. In vivo experiments show that TGF- ß 2 stimulates the formation of granulation tissue by inducing angiogenesis. 121,122 During matrix formation and *Growth Factors and Dental Implantology DOI: http://dx.doi.org/10.5772/intechopen.101082*


#### **Table 2.**

*Important bone forming growth factors.*

remodeling, TGF- ß 2 increases protein, DNA, and collagen production. By stimulating recruitment of fibroblasts to the wound site, the combined result is increased collagen deposition (particularly type I and III) and scar formation in vivo.

TGF- ß 3 promote wound healing by recruiting inflammatory cells and fibroblasts to the wound site and by facilitating keratinocyte migration. Furthermore, it has been demonstrated that TGF- ß 3 is a potent inhibitor of DNA synthesis in human keratinocytes. TGF- ß 3 inhibits scarring and promotes better collagen organization in vivo [1].

Due to abundance of TGF-ß in body cells, it is necessary to regulate its action in specific sites at the time of need. Hence it is released in a biologically inactive form i.e. latent TGF-ß. Its activation occurs in acidic environment through an enzymatic reaction [13] which probably regulates the exhibition of its effects.

#### *3.1.2 Bone morphogenetic proteins (BMP)*

BMP's were first discovered with formation of a completely mineralized woven bone with marrow, ectopically. It was achieved with the experiments done by Marshall Urist [14] using demineralized bone matrix placed in the subcutaneous tissue.

BMPs are currently well known to induce expression of osteoblast markers and stimulate bone formation in vivo [15]. It is believed that BMP are the most potent proteins to stimulate the mesenchymal stem cell differentiation in a chondroblastic and osteoblastic origin. They work by stimulating bone formation from the periphery of the implant, while matrix is laid down towards the center until it is entirely replaced by trabecular bone [16]. Their release is initiated from traumatized bone tissue during early-stage o fracture healing.

Today 12 types of BMP's have been isolated which exert their effects through specific receptor complexes. To utilize the action of these protein, they should be produced in large amounts. Also, a special carrier protein can be used to exhibit their action in low doses [5]. The function of the carrier protein is to immobilize the bone inducing protein at a site for the required time. E.g., Collagen matrix, demineralized bone matrix, synthetic polysaccharide matrices.

A recent prospective, randomized, controlled clinical trial showed the ability of recombinant human BMP-2 (rhBMP-2) applied to a collagen sponge to accelerate fracture and wound healing in patients with open tibia fractures [17].

#### *3.1.3 Platelet derived growth factors (PDGF)*

A powerful chemotactic factor, that is responsible for mitogenesis, angiogenesis and chemotaxis of fibroblasts and osteoblasts at the wound site. Glycoprotein by nature it has a crucial role in bone formation which was evident when reduced intramembranous bone was recorded on usage of PDGF inhibitors. Rat calvaria defect studies also demonstrate new bone formation in 2 weeks when PDGF is used with a poly L-lactide membrane.

#### *3.1.4 Insulin growth factors (IGF)*

Earlier designated as somatomedin-C and skeletal growth factor, these peptides are found to be synthesized by several tissue of body including bone. The secretion of IGF in bone tissue is controlled by parathyroid hormone and growth hormone. These hormones regulate the longitudnal growth and metabolism of cartilage by stimulating the chondroblastic IGF [18, 19].

The subtype IGF-II is found 10–20 times more than IGF-I in the bone matrix. Both subtypes stimulate osteoblast replication thereby increasing the bone matrix synthesizing cells. This in turn lead to new bone formation. However IGF-I is 4–7 times more potent than IGF-II. Apart from these, IGF also stimulates collagen production and inhibits collagen degradation.

It is observed in animal studies, that a single subcutaneous injection of growth hormone resulted in increase in serum IGF-1 during fracture healing in pig tibias [20]. Mesenchymal stromal cells transfected with IGF-1 and administered to a mouse femur fracture site, increased bone healing [21].

#### *3.1.5 Fibroblast growth factors (FGF)*

This polypeptide growth factor exerts proliferative effect on osteoblasts thus contributing in increasing the bone collagen. They are recognized as mitogenic factors for cells of mesenchymal and neuroectodermal origin and also play significant role in angiogenesis during the healing phase. Out of the 7 members of the FGF family, FGF-1 is acidic and FGF-2 is basic. Basic FGF (bFGF) is considered more potent and is believed that it has stimulatory effect on TGF-ß secretion by osteoblasts. Various animal studies have also concurred with this fact.

#### *3.1.6 Vascular endothelial growth factors (VEGF)*

VEGF is a powerful angiogenic growth factor that has been studied extensively in the oncologic and wound healing literature [22, 23]. It is released from endothelial cells, platelets, megakaryocytes, lymphocytes, and plasma cells. The major functions it takes care, are angiogenesis, neovascularization, and wound healing [24]. It is also mitogenic to endothelial cells, increases vascular permeability, and increases tissue oxygenation [25]. Individual administration of these growth factors shows the improvement of fracture healing and the potential for use in alveolar bone defects in preparation for implant placement [10].

## **4. Osseointegration and role of growth factors**

"Osseointegration, (as defined by Zarb &Albrektsson) is a time dependent healing process whereby clinically asymptomatic rigid fixation of alloplastic materials is achieved, and maintained, in bone during functional loading" [26].

Clinically it has been demonstrated that the implants were anchored in bone without intervening fibrous tissue. Experimentally this data was researched at the ultrastructural level. Collagen filaments approaching the titanium oxide surface were seen that were separated only by a 20–40 nm thick Proteoglycan layer [27].

Branemark and Albrektsson [28] in their study evaluated the outcome of all implants inserted during 1 year and then followed them up for 5 years. They found an implant success rate of 96.5% in the mandible. This improved success rate compared to the data published by Adell et al. [29] reflects a true improvement in the outcome. This success was attributed to meticulous surgical and prosthodontic techniques.

Direct bone healing occurs in defects, primary fracture healing and in osseointegration. It is activated by any lesion of the pre-existing bone matrix. Once activated; the process continues in a biologically determined program. Thus, osseointegration also is programmed healing of the bone by developing a direct structural and functional connection between ordered, living bone and the surface of a load-bearing implant [30].

Osseointegration is facilitated on a precise fitting (anatomical reduction), primary stability (stable fixation) and adequate loading during the healing period. Osseointegration requires a bioinert or bioactive material and surface configurations that are conducive for bone deposition (osteophilic) [31].

#### **4.1 Stages of osseointegration**

On an event of trauma, when the bone matrix is exposed to extra cellular fluid, non-collagenous proteins and growth factors are set free and activate bone repair [32]. These facilitate chemotaxis of osteoprogenitor cells of the bone marrow and from the endocortical and periosteal bone envelopes. They proliferate and differentiate into osteoblast precursors and osteoblasts that begin bone opposition from the defect wall proceeding towards implant surface.

As the process gets initiated, it proceeds into a well-planned cascade in 3 stages:


## *4.1.1 Incorporation by woven bone formation*

The first bone tissue formed is a primitive type, characterized by randomly oriented collagen fibrils, numerous, irregularly shaped osteocytes developing into an initially low-density bone: the woven bone. Its major role is to provide a scaffold of rods and plates thereby spreading out into the surrounding at a rapid rate. Simultaneous growth of elaborate vascular nets forming primary spongiosa bridging gaps rom bone to implant takes place for next 4–6 weeks.
