**3. Biological factors in bone regeneration**

**Figure 1.** Temporal progression of bone healing. The healing response to bone injury is characterized by overlapping biological processes: immediately after bone injury, hematoma formation and inflammatory response permits the re‐ lease of pro-inflammatory cytokines and growth factors that initiate the process of wound healing. Between days 1–7, MSCs proliferate and differentiate into the osteogenic or chondrogenic lineages and increase the production of blood vessels from pre-existing vessels. New bone formation occurs through intramembranous or endochondral ossification that is finally mineralized, forming a mature bone that is continuously remodeled through the rest of his life.

The factors secreted by platelets, macrophages and bone cells include transforming growth factor beta (TGF-β), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), interleukins 1 and 6 (IL-1 and IL-6), tumor necrosis growth factor alpha (TNF-α), bone morphogenetic proteins (BMPs) [26, 27], fibroblast growth factor (FGF) and insulin-like growth factors I and II (IGF-I and IGF-II) [26]. These factors stimulate the migration of multipotent stem cells, probably originated from the periosteum, bone marrow, blood vessels and the surrounding soft tissue and induce the differentiation of cells to different mesenchymal cell

During the following days, the **construction phase** starts. This phase is characterized by the formation of new blood vessels [17], and the thrombus reorganization into granulation tissue, which is then condensed in a soft callus providing an osteoid and/or cartilage scaffold, which acts as a stabilization structure and a template for subsequent mineralization [26] (**Figure 1**).

Depending on the type of bone, the type of bone lesion, the morphology and structure of the tissue and the fixation method, bone healing can take two forms: primary healing, where osteoblasts secrete an osteoid matrix for future mineralization (intramembranous ossification); and secondary healing, which occurs through the formation of a cartilage matrix produced by chondrocytes, which is then replaced by an osteoid matrix with subsequent mineralization (endochondral ossification) [24–27]. Most common growth factors related to bone healing,

types including angioblasts, fibroblasts, chondroblasts and osteoblasts [26].

256 Advanced Techniques in Bone Regeneration

During the process of bone regeneration, the release of growth factors occurs as a series of highly time-space regulated biological events. These soluble molecules are able to regulate signaling cascades that specifically influence cellular responses such as differentiation and proliferation [28].

Biological signaling molecules function effectively by a limited window of time to get a proper result in the target cell. Therefore, it is necessary to have a precise understanding of the temporal pathways for natural bone regeneration. Biological signaling agents can be classified into the following categories: pro-inflammatory cytokines, growth and differentiation factors and angiogenic factors. Pro-inflammatory cytokines are activated immediately after bone injury and establish and maintain the acidic and hypoxic environment for the initial destruc‐ tion phase. Growth and differentiation factors function during the constructive and destructive phases, while angiogenic factors are focal points for the revascularization of the wounded area [25, 26, 35] (**Table 1**).



Essential signaling molecules in bone regeneration: their time of expression, source, target cells and their major functions (Adapted with the permission from Dimitriou et al. [25]. Copyright© 2005).

**Table 1.** Biological factors in bone regeneration.

**Signaling Molecules**

Cytokines (IL-1,IL-6, TNF-α)

Increased levels from days 1 to 3 and during bone remodeling

258 Advanced Techniques in Bone Regeneration

TGF-β Expressed from very early stages throughout fracture

healing

early stages of fracture healing

expression patterns

early stages until osteoblasts formation

throughout fracture

and endochondral ossification

healing

PDGF Released at very

BMPs Various temporal

FGFs Expressed from the

IGFs Expressed

**Expression Pattern Source Target cells function**

Mesenchymal and inflammatory cells

MSCs,

osteoprogenitor cells, osteoblasts, chondrocytes

Mesenchymal and inflammatory cells, osteoblasts

Mesenchymal and osteoprogenitor cells, osteoblasts

Mesenchymal and epithelial cells, osteoblasts and chondrocytes

MSCs, endothelial cells, osteoblasts, chondrocytes

Chemotactic effect on other inflammatory cells Stimulation of extracellular matrix synthesis, angiogenesis, recruitment of endogenous fibrogenic cells to the injury site and at later stages bone

resorption

cells

Differentiation of

into osteoblasts

α-FGF mainly effects chondrocyte proliferation β-FGF (more potent) involved in

chondrocytes

and proliferation

protein synthesis

Potent mitogenic and chemotactic for bone-forming cells, chemotactic for macrophages

Mitogenic for mesenchymal cells and osteoblasts, chemotactic for inflammatory and mesenchymal

undifferentiated mesenchymal cells into chondrocytes and osteoblasts and osteoprogenitors

Angiogenic and mitogenic for mesenchymal and epithelial cells, osteoblasts, chondrocytes

maturation and bone resorption

IGF-I: mesenchymal and osteoprogenitor cells recruitment

IGF-II: cell proliferation and

Macrophages Inflammatory cells Cells of mesenchymal

Degranulating platelets Inflammatory cells endothelium, extracellular matrix, chondrocytes,

Degranulating platelets, macrophages, monocytes (during the granulation stage) and endothelial cells, osteoblasts (at later

Osteoprogenitors and mesenchymal cells, osteoblasts, bone extracellular matrix and

Monocytes, macrophages, mesenchymal cells, osteoblasts, chondrocytes

Bone matrix, endothelial and mesenchymal cells (in granulation stage) and osteoblasts and nonhypertrophic chondrocytes (in bone and cartilage

formation)

chondrocytes

osteoblasts

stages)

origin

In the next section, we will list some of the common molecules associated with the bone regeneration process, and describe their biological significance.

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

Members of the TGF-β are the most widely studied growth factors in recent years. This family includes, among others, five isoforms of TGF-β (1–5), bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs), which participate in a complex series of molecular events that lead to mesenchymal precursors during bone morphogenesis [25, 29, 33, 36]. They originate from high molecular weight precursors and are activated by proteolytic enzymes. They act on serine/threonine kinase membrane receptor on target cells. This ligandreceptor interaction activates intracellular signaling pathways which ultimately affects gene expression in the nucleus [25].

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

The BMPs are a unique family of proteins within the TGF-β superfamily that play an essential role in regulating the formation, maintenance and bone repair [30]. To date, about 20 different proteins have been termed BMPs, but not all of them have osteogenic potential [37]. Among the BMPs with osteogenic potential we have, BMPs-2, -3 (osteogenin), -4, -6, -7 (also known as osteogenic protein-1 [OP-1]), -12 (also known as growth/differentiation factor 7 [GDF-7]) and -14 (also known as GDF-5, or cartilage-derived morphogenetic protein-1 [CDMP-1]). These proteins have been evaluated for healing and bone regeneration in clinical and preclinical models showing enhanced and accelerated bone formation [30]. In bone tissue, BMPs are produced by osteoprogenitor cells, osteoblasts, chondrocytes and platelets. Their regulatory effects depend on the target cell, stage of differentiation, local concentration, as well as interactions with other secreted proteins. BMPs induce a sequential cascade of events leading to chondrogenesis, osteogenesis, controlled angiogenesis and extracellular matrix synthesis [37]. Large number of preclinical studies has shown that BMPs are capable of inducing bone formation at ectopic sites and induce critical size defects healing [29]. It has been shown that BMPs 2, 4 and 7 play an important role in determining, migration, condensation, proliferation and apoptosis of skeletal cells. It has also been reported that BMP-4 and BMP-7 are responsible for inducing the cells of the neural crest, while BMP-2 is involved in the condensation of mesenchymal cells appearing before formation of immature bone structures during both endochondral and intramembranous ossification [33]. BMP-4 is predominantly active from days 1–5 after injury, with a peak closer to day 5. The BMP-2 is active during the bone regeneration process, culminating the bone remodeling to lamellar and haversian bone tissue, while BMP-7 is active after 14 days [23]. Target cells of BMPs include MSC, bone marrow cells, osteoblasts, myoblasts, prefibroblast and neuronal cells. The general effects on osteoblasts and cells of the periosteum involve an increase in the activity of DNA synthesis and transcription of genes involved in the synthesis of bone matrix proteins [23].

Scientific evidence of the role of BMPs in bone regeneration is overwhelming. There are a number of publications confirming that the delivery of BMP at the site of injury promotes bone regeneration in animal and human models [38–40].

BMP-2 and BMP-7 have been extensively evaluated in clinical studies of nonunion, bone defects, open tibial fractures and spinal fusion, demonstrating their efficacy in the acceleration of bone regeneration and healing of fractures [29]. In order to be used in the clinical practice, a local and controlled delivery of BMPs is required; so, it is important to consider its short halflife time. Various delivery systems have been developed to overcome this limitation [37]. Currently, there are several forms of the human recombinant proteins commercially available. For example, for rh-BMP2: InductOs® (United Kingdom) and InFUSE (United States), (Med‐ tronic Sofamor Danek, Inc., Minneapolis, MN), which are supplied in a bovine collagen sponge allowing slow release over time, and for rhBMP-7, Osigraft® (United Kingdom) and OP-1™ (United States) (Stryker Biotech, Hop-kinton, MA), in a bovine collagen granular form [34, 36, 37].

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

The five isoforms of TGF-β regulate cellular functions such as proliferation, apoptosis, differentiation and cell migration. TGF-β is produced by osteoblasts and chondrocytes, and is stored in the bone matrix [25, 41]. TGF-β is also released by platelets and TGF-β1 indeed, was the first member of the family to be described in human platelets, as a 25 kDa protein with a possible role in the healing process [42]. During the initial phase of inflammation resulting from a bone injury, platelets release TGF-β and therefore this factor seems to be involved in the initial callus formation stage [25, 41].

TGF-β is a multifunctional, secreted protein, with different functions in the cell, such as control of cell growth and proliferation, differentiation and apoptosis. TGF-β induces the proliferation of MSCs, pre-osteoblasts, osteoblasts and chondrocytes and stimulates the extracellular production of proteins such as collagen, proteoglycans, osteopontin, osteonectin and alkaline phosphatase [25, 41]. It is also a potent chemotactic agent for MSCs. During chondrogenesis and endochondral bone formation, it induces the synthesis of BMP by osteoprogenitor cells, and it inhibits the activation and promotes osteoclast apoptosis [41].

#### **3.2. Platelet-derived growth factors (PDGF)**

formation at ectopic sites and induce critical size defects healing [29]. It has been shown that BMPs 2, 4 and 7 play an important role in determining, migration, condensation, proliferation and apoptosis of skeletal cells. It has also been reported that BMP-4 and BMP-7 are responsible for inducing the cells of the neural crest, while BMP-2 is involved in the condensation of mesenchymal cells appearing before formation of immature bone structures during both endochondral and intramembranous ossification [33]. BMP-4 is predominantly active from days 1–5 after injury, with a peak closer to day 5. The BMP-2 is active during the bone regeneration process, culminating the bone remodeling to lamellar and haversian bone tissue, while BMP-7 is active after 14 days [23]. Target cells of BMPs include MSC, bone marrow cells, osteoblasts, myoblasts, prefibroblast and neuronal cells. The general effects on osteoblasts and cells of the periosteum involve an increase in the activity of DNA synthesis and transcription

Scientific evidence of the role of BMPs in bone regeneration is overwhelming. There are a number of publications confirming that the delivery of BMP at the site of injury promotes bone

BMP-2 and BMP-7 have been extensively evaluated in clinical studies of nonunion, bone defects, open tibial fractures and spinal fusion, demonstrating their efficacy in the acceleration of bone regeneration and healing of fractures [29]. In order to be used in the clinical practice, a local and controlled delivery of BMPs is required; so, it is important to consider its short halflife time. Various delivery systems have been developed to overcome this limitation [37]. Currently, there are several forms of the human recombinant proteins commercially available. For example, for rh-BMP2: InductOs® (United Kingdom) and InFUSE (United States), (Med‐ tronic Sofamor Danek, Inc., Minneapolis, MN), which are supplied in a bovine collagen sponge allowing slow release over time, and for rhBMP-7, Osigraft® (United Kingdom) and OP-1™ (United States) (Stryker Biotech, Hop-kinton, MA), in a bovine collagen granular form [34, 36,

The five isoforms of TGF-β regulate cellular functions such as proliferation, apoptosis, differentiation and cell migration. TGF-β is produced by osteoblasts and chondrocytes, and is stored in the bone matrix [25, 41]. TGF-β is also released by platelets and TGF-β1 indeed, was the first member of the family to be described in human platelets, as a 25 kDa protein with a possible role in the healing process [42]. During the initial phase of inflammation resulting from a bone injury, platelets release TGF-β and therefore this factor seems to be involved in

TGF-β is a multifunctional, secreted protein, with different functions in the cell, such as control of cell growth and proliferation, differentiation and apoptosis. TGF-β induces the proliferation of MSCs, pre-osteoblasts, osteoblasts and chondrocytes and stimulates the extracellular production of proteins such as collagen, proteoglycans, osteopontin, osteonectin and alkaline phosphatase [25, 41]. It is also a potent chemotactic agent for MSCs. During chondrogenesis

of genes involved in the synthesis of bone matrix proteins [23].

regeneration in animal and human models [38–40].

260 Advanced Techniques in Bone Regeneration

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

the initial callus formation stage [25, 41].

37].

This polypeptide growth factor has potent chemotactic and mitogenic stimulatory effects on MSCs [30], plays an important role in the differentiation of pre-osteoblasts to osteoblasts [43] with the ability to promote angiogenesis during wound healing [30]. The PDGF family includes four isoforms: PDGF-A, PDGF-B, the more recently discovered PDGF-C and PDGF-D [44]. PDGF-A and B form homodimers (AA or BB) and a heterodimer (AB) [30]. PDGF-AB and PDGF-BB are variants circulating in alpha platelet granules and are released when platelets bind to the site of injury. The PDGF-BB variant has an active role in mitogenesis and chemotaxis of cells in the injured area [15] and plays a key role in bone regeneration [23]. After bone injury, PDGF is released by macrophages and platelets and acts as a potent chemo-attractant and mitogenic factor for cells of mesenchymal lineage, recruit fibroblasts, endothelial cells, osteoblasts and cells of the immune system. PDGF is active during the first 72 hours after injury, and as a promoter of angiogenesis plays a role in revascularization of bone defects [23].

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

They constitute a family of structurally related polypeptides with a potent mitogenic effect on osteoprogenitor cells [29]. They are humoral factors originally identified by their ability to stimulate cell proliferation [33]. During bone healing, they can be secreted by monocytes, macrophages, mesenchymal cells, osteoblasts and chondrocytes in the early stages of bone fractures healing [33]. Members of the FGF family are present at the site of the wound for up to three weeks and its main activity is to stimulate endothelial cell migration and subsequent angiogenesis and mesenchymal cell mitogenesis [25, 34]. α-FGF mainly affects chondrocyte proliferation and is probably important for chondrocyte maturation, while β-FGF is expressed by osteoblasts and is generally more potent than α-FGF [45].

#### **3.4. Vascular endothelial growth factor (VEGF)**

Two separate pathways are involved in the regulation of angiogenesis during bone healing: a VEGF dependent pathway and the angiopoietin-dependent pathway [31]. VEGF is a potent angiogenic [29, 43] and vasculogenic [23] factor that not only increases the differentiation and proliferation of endothelial cells but also increases the tubular formation and mobilization and recruitment of endothelial progenitor cells [34]. VEGF is increased in response to hypoxia, ischemia and during healing of bone tissue [15, 34]. It has been shown that VEGF works synergistically with BMPs. VEGF by itself does not promote bone regeneration, but rather acts in coordination with BMPs to increase the recruitment of MSCs to the defect site and induce active differentiation of osteoblasts [46]. VEGF is expressed predominantly 14 to 21 days after the injury; and therefore, it is a candidate for early in situ application to promote mineralization and bone regeneration remodeling [23].

#### **3.5. Insulin-like growth factors (IGF)**

IGF-1 and -2 play a critical role in stimulation of organogenesis and growth during the first stages of embryogenesis as well as in regulating the functions of specific tissues and organs in later stages of development [47]. The sources of IGF-1 and IGF-2 are the bone matrix, endo‐ thelial cells, osteoblasts and chondrocytes [25]. IGF-1 promotes bone matrix formation (type I collagen and non-collagenous matrix proteins) by fully differentiated osteoblasts and is more potent than IGF-2 [45]. IGF-2 acts at a later stage of endochondral bone formation and stimulates type I collagen production, cartilage matrix synthesis and cellular proliferation [25].
