**1. Introduction**

Reconstruction and regeneration of significant skeletal defects have amazed mankind for thousands of years. Grafting techniques were employed as early as 2000 BC when Khurits employed a piece of animal bone to reconstruct a small skull defect. In the modern age, Job

© 2016 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.

van Meekeren, a Dutch surgeon, performed first documented bone graft in 1668. He utilized a xenograft to repair a skull defect in an injured soldier [1]. The understanding of orthopaedic science and bone grafts was further propelled in the seventeenth century by the work of Antoni van Leeuwenhoek who is famously known for his work on microscopy. Also, he primitively explained the microarchitecture of bone, what we now refer to as Haversian canals [2]. Hard‐ working examination of bone‐grafting criteria and outcomes surfaced in the early 1900s by Vittorio Putti who determined the principles of grafting. Putti's work presented a foundation for grafting science in the orthopaedic field. Since then, researchers and surgeons have continued to smooth the science of bone grafting to allow for the most proper surgical intervention with the best outcomes [2, 3]. The current standard treatment is harvesting autologous grafts from other positions in the body (harvested primarily from the patient's iliac crest or other locations, such as the distal femur, proximal tibia, ribs and intramedullary canal) and transplantation into the massive fractures, or the transplantation of allografts, which have many obstacles, such as donor‐site morbidity, limited tissue supply, infection, and poor integration [2, 4, 5]. Autografts are clinically approved therapies, which demonstrate the biological characteristics of osteogenesis, osteoconduction, and osteoinduction. Both grafts possess unique advantages and disadvantages; however, autografts gained desirability over allograft in the early 1900s with recognition of the advantage that vascularization provided to the integrity of the graft with the surrounding bone [6]. So, synthetic bone graft substitutes that were developed to overcome the inherent limitations of auto‐ and allograft represent an alternative strategy. These synthetic substitutes, or matrices, are made from a variety of materials, such as natural and synthetic polymers, ceramics, and composites that are designed to mimic the three‐dimensional (3D) characteristics of autograft tissue while maintaining viable cell populations. Matrices also function as delivery vehicles for factors, chemothera‐ peutic agents, and antibiotics depending on the nature of the injury to be repaired. This junction of matrices, cells, and therapeutic molecules has collectively been termed tissue engineering (TE) [7]. Clinically, a bone regenerative therapeutic to treat patients must provide fundamental criteria, including safety, predictability, and reproducibility, in providing the clinical outcome. Also, as noted earlier, a tissue‐regenerative therapy should exhibit four characteristics, including osteogenicity, osteoconductivity, osteoinductivity, and osteopromotivity [8, 9]. Osteogenesis refers to the process by which osteoprogenitor cells mature into osteoblasts, which subsequently mineralize and form bone tissue [9]. During osteoconduction process, bone formations occur on a surface. With respect to biomaterials, osteoconduction is defined by the ability of an implant to support the growth of bone at a defect site three dimensionally. Osteoinduction is the process of recruitment of immature osteoprogenitor cells to the site and the subsequent differentiation of them into osteoblasts under the influence of a diffusible bone morphogenetic factor. Finally, osteopromotion refers to the ability of a substance to enhance osteoinduction without being osteoinductive on its own [1, 9, 10].

#### **2. Bone grafts**

Fracture healing is performed based on a delicate balance between biology of fracture repair and biomechanical stability of fracture fixation, which are interrelated. Too many ‐ attempts have been developing to minimize damage to the blood supply of the fracture blocks during surgery, but the sequential activation of cells and bioactive molecules necessary for fracture healing still remains disrupted. Moreover, a non‐union often develops when this sequential activation is interfered. Some approaches suggested to overcome non‐unions and some acute fractures include bone grafts and bone graft alternatives—specifically autologous bone grafts, allografts, synthetic bone grafts, and osteoinductive proteins. The ability of grafts to promote healing depends on their osteoconductive, osteoinductive, osteogenic, and osteopromotive qualities [11–13]. Each bone graft type and its alternative own some combination of these qualities. This section is going to compare benefits and potential limitations of available grafting strategies.

van Meekeren, a Dutch surgeon, performed first documented bone graft in 1668. He utilized a xenograft to repair a skull defect in an injured soldier [1]. The understanding of orthopaedic science and bone grafts was further propelled in the seventeenth century by the work of Antoni van Leeuwenhoek who is famously known for his work on microscopy. Also, he primitively explained the microarchitecture of bone, what we now refer to as Haversian canals [2]. Hard‐ working examination of bone‐grafting criteria and outcomes surfaced in the early 1900s by Vittorio Putti who determined the principles of grafting. Putti's work presented a foundation for grafting science in the orthopaedic field. Since then, researchers and surgeons have continued to smooth the science of bone grafting to allow for the most proper surgical intervention with the best outcomes [2, 3]. The current standard treatment is harvesting autologous grafts from other positions in the body (harvested primarily from the patient's iliac crest or other locations, such as the distal femur, proximal tibia, ribs and intramedullary canal) and transplantation into the massive fractures, or the transplantation of allografts, which have many obstacles, such as donor‐site morbidity, limited tissue supply, infection, and poor integration [2, 4, 5]. Autografts are clinically approved therapies, which demonstrate the biological characteristics of osteogenesis, osteoconduction, and osteoinduction. Both grafts possess unique advantages and disadvantages; however, autografts gained desirability over allograft in the early 1900s with recognition of the advantage that vascularization provided to the integrity of the graft with the surrounding bone [6]. So, synthetic bone graft substitutes that were developed to overcome the inherent limitations of auto‐ and allograft represent an alternative strategy. These synthetic substitutes, or matrices, are made from a variety of materials, such as natural and synthetic polymers, ceramics, and composites that are designed to mimic the three‐dimensional (3D) characteristics of autograft tissue while maintaining viable cell populations. Matrices also function as delivery vehicles for factors, chemothera‐ peutic agents, and antibiotics depending on the nature of the injury to be repaired. This junction of matrices, cells, and therapeutic molecules has collectively been termed tissue engineering (TE) [7]. Clinically, a bone regenerative therapeutic to treat patients must provide fundamental criteria, including safety, predictability, and reproducibility, in providing the clinical outcome. Also, as noted earlier, a tissue‐regenerative therapy should exhibit four characteristics, including osteogenicity, osteoconductivity, osteoinductivity, and osteopromotivity [8, 9]. Osteogenesis refers to the process by which osteoprogenitor cells mature into osteoblasts, which subsequently mineralize and form bone tissue [9]. During osteoconduction process, bone formations occur on a surface. With respect to biomaterials, osteoconduction is defined by the ability of an implant to support the growth of bone at a defect site three dimensionally. Osteoinduction is the process of recruitment of immature osteoprogenitor cells to the site and the subsequent differentiation of them into osteoblasts under the influence of a diffusible bone morphogenetic factor. Finally, osteopromotion refers to the ability of a substance to enhance

4 Advanced Techniques in Bone Regeneration

osteoinduction without being osteoinductive on its own [1, 9, 10].

Fracture healing is performed based on a delicate balance between biology of fracture repair and biomechanical stability of fracture fixation, which are interrelated. Too many ‐ attempts

**2. Bone grafts**

The iliac crest bone graft (ICBG), harvested from the anterior and posterior iliac crest, is the gold standard for cancellous autografts in cases in which fracture healing rather than void filling is needed. It is corticocancellous with osteoconductive, osteoinductive, and osteogenic effects. Also, the other benefit of ICBG is the availability of large amounts of bone without structural compromise to the extremity [14]. In a study, Takemoto et al. objected to consider whether there are variations in the expression of bone morphogenetic proteins (BMPs) and their receptors in different bone‐graft‐harvesting sites. They analysed autogenous marrow aspirates obtained from the iliac crest, the proximal humerus, and the proximal tibia for the mRNA levels of BMPs and their receptors. Their results suggested that ICBG is rich in colony‐ forming cells, and the number of progenitor cells directly promotes healing [15]. Despite the relative advantages of ICBG, it is not without disadvantages. The limitations, however, have been well documented in the literature and include donor‐site morbidity, increased time in the operating room, and an increased length of hospital stay [16, 17]. So, for certain patients with compromised bone or inadequate volume for grafting, bone graft substitutes may be preferable.

Substitutes to bone grafting consist of bone bank allograft, osteoconductive materials, demin‐ eralized bone matrix (DBM), and osteoinductive proteins. The orthopaedic association has extensive experience with bone bank allograft, with the first clinical tissue bank opening in 1949 [18]. The main concerns of allografts include the risk of rejection, disease transmission, inconsistent incorporation, and late resorption. An alternative to bone bank allograft is DBM. DBM is made from an allograft with the inorganic materials removed. Researchers demon‐ strated that DBM implanted intramuscularly resulted in new bone formation [19]. Also, DBM has osteoconductive property but only weak osteoinductive feature. Furthermore, DBM offers an advantage over allografts or synthetic biomaterials that need incorporation by the host before they can support mechanical loads and would diminish the morbidity associated with harvesting autologous bone [20].

Synthetic osteoconductive materials have been widely used for bone graft in orthopaedic practice and include hydroxyapatite (HA), coralline hydroxyapatite, CaSO4 and CaPO<sup>4</sup> cements, and collagraft [21]. Hydroxyapatite has a porous structure comparable to the cancellous bone and functions as an effective osteoconductive matrix and thus replicates the biological properties of bone extracellular matrix (ECM). The nominal composition of this mixture is Ca10(PO4)6(OH)2 with an atomic ratio for calcium‐to‐phosphate of 1.67 [22, 23]. Most studies have reported the mineralization and remodelling of this material can lead to the formation of mature bone [21]. Coralline hydroxyapatite is a similar substance, in which coral is converted to pure crystalline hydroxyapatite. It has good compressive strength but has low tensile strength and limited remodelling potential. Similar to hydroxyapatite, coralline hydroxyapatite functions strictly osteoconductive, but lacking osteogenic and osteoinductive properties. Calcium‐based bone cements are osteoconductive and primarily used for filling metaphyseal defects. They possess sufficient compressive strength but lack resistance to shear and torsional forces and are very costly. They are also associated with resorption, leading to wound drainage [21]. The situations in which osteoinduction is the primary concern, BMPs are available. Detailed insights into BMPs will be provided later.

### **3. Molecular aspects of fracture healing**

Fracture healing is a complex physiological process. Cascade of complex biological events involving intracellular and extracellular molecular signalling for bone induction and conduc‐ tion remain unknown to a great extent. Indeed, it is a multistep repair process that follows a determined spatial and temporal sequence [24–26]. It was clearly demonstrated that known molecular mechanisms that regulate skeletal tissue formation during embryological develop‐ ment are replicated during the fracture‐healing process [27]. Many growth and differentiation factors (GDFs), such as cytokines, hormones, and extracellular matrix, are local and systemic regulatory factors that interact with various cell types, including bone‐ and cartilage‐forming primary cells, or even muscle mesenchymal cells, recruited at the fracture site or from the circulation. Advances in understanding cellular and molecular mechanisms will provide the tools for discovering the fracture‐healing process. This section aims to contribute to promoting and inhibiting fracture healing and to prepare awareness of the complexity of involved signalling pathways.

#### **3.1. Biology of fracture healing**

The nature of the repair phase is dependent on mechanical conditions in the fracture‐healing zone (primary or secondary bone healing) and the anatomical location of the fracture (meta‐ physeal‐epiphyseal trabecular bone healing or diaphyseal callus healing). Indeed, fracture healing is a complex process, resulting in optimal skeletal repair and restoration of skeletal function. However, it is a well‐orchestrated, regenerative process, which is initiated in response to injury. Repair process is promoted by the normal pathway of embryonic devel‐ opment repeated with the coordinated participation of several cell types [28]. Depending on several parameters involved in the fracture site, such as growth factors, nutrients, hormones, and oxygen tension, pH, the mechanical stability and the electrical environment, various components present at the injured tissue, such as the cortex, the periosteum, the external soft tissues and the bone marrow, contribute to the healing process [29–31]. Classical histology has divided fracture healing into direct (primary) and indirect (secondary) mode.

*Direct strategy* (known as primary cortical bone healing) occurs only when there is extremely low interfragmentary movement or if the bony fragments are under compression [32]. Most often compression plates and lag screws provide the required stability for direct healing [33]. Similar to the normal bone‐remodelling process, fracture surfaces in contact and under compression are bridged by Haversian systems (or osteons) when such stability is achieved. Indeed, primary process involves a direct attempt by the cortex to regenerate new Haversian systems by the formation of discrete remodelling units known as 'cutting cones', in order to restore mechanical continuity [34]. Osteoclasts digest bone, causing tunnels from one side of the fracture to the other, which provides the in‐growth of blood vessels. Subsequently, vascular endothelial cells and perivascular mesenchymal cells prepare the osteoprogenitor cells to differentiate into osteoblasts which create new osteons connecting both fragments [35, 36]. Healing by Haversian systems is slow, and notable time is necessary to gain sufficient strength by healing zone and, therefore, allow removal of load‐bearing implants. Also, because it is not associated with a major influx of inflammatory cells, primary bone healing is less affected by systemic inflammation [37].

studies have reported the mineralization and remodelling of this material can lead to the formation of mature bone [21]. Coralline hydroxyapatite is a similar substance, in which coral is converted to pure crystalline hydroxyapatite. It has good compressive strength but has low tensile strength and limited remodelling potential. Similar to hydroxyapatite, coralline hydroxyapatite functions strictly osteoconductive, but lacking osteogenic and osteoinductive properties. Calcium‐based bone cements are osteoconductive and primarily used for filling metaphyseal defects. They possess sufficient compressive strength but lack resistance to shear and torsional forces and are very costly. They are also associated with resorption, leading to wound drainage [21]. The situations in which osteoinduction is the primary concern, BMPs

Fracture healing is a complex physiological process. Cascade of complex biological events involving intracellular and extracellular molecular signalling for bone induction and conduc‐ tion remain unknown to a great extent. Indeed, it is a multistep repair process that follows a determined spatial and temporal sequence [24–26]. It was clearly demonstrated that known molecular mechanisms that regulate skeletal tissue formation during embryological develop‐ ment are replicated during the fracture‐healing process [27]. Many growth and differentiation factors (GDFs), such as cytokines, hormones, and extracellular matrix, are local and systemic regulatory factors that interact with various cell types, including bone‐ and cartilage‐forming primary cells, or even muscle mesenchymal cells, recruited at the fracture site or from the circulation. Advances in understanding cellular and molecular mechanisms will provide the tools for discovering the fracture‐healing process. This section aims to contribute to promoting and inhibiting fracture healing and to prepare awareness of the complexity of involved

The nature of the repair phase is dependent on mechanical conditions in the fracture‐healing zone (primary or secondary bone healing) and the anatomical location of the fracture (meta‐ physeal‐epiphyseal trabecular bone healing or diaphyseal callus healing). Indeed, fracture healing is a complex process, resulting in optimal skeletal repair and restoration of skeletal function. However, it is a well‐orchestrated, regenerative process, which is initiated in response to injury. Repair process is promoted by the normal pathway of embryonic devel‐ opment repeated with the coordinated participation of several cell types [28]. Depending on several parameters involved in the fracture site, such as growth factors, nutrients, hormones, and oxygen tension, pH, the mechanical stability and the electrical environment, various components present at the injured tissue, such as the cortex, the periosteum, the external soft tissues and the bone marrow, contribute to the healing process [29–31]. Classical histology has

divided fracture healing into direct (primary) and indirect (secondary) mode.

are available. Detailed insights into BMPs will be provided later.

**3. Molecular aspects of fracture healing**

6 Advanced Techniques in Bone Regeneration

signalling pathways.

**3.1. Biology of fracture healing**

Another type of fracture healing is *indirect mode* that heals the majority of fractures. This mode of fracture healing occurs by either intramembranous ossification or endochondral ossification with the subsequent formation of a callus [38, 39]. This mode is usually enhanced by motion and inhibited by rigid fixation [38].

Intramembranous ossification forms bone directly without first forming cartilage. Migrated mesenchymal stromal cells that reside in the periosteum directly differentiate into osteoblasts that synthesize and deposit bone matrix. This process results in callus formation, characterized histologically as 'hard callus' [40]. In this type of healing, the bone marrow contribute to bone formation during the early phase of healing, when endothelial cells transform into polymor‐ phic cells that subsequently express an osteoblastic phenotype [12]. Advanced studies have shown that flat bones such as bones from the skull, trabecular bones, and clavicle heal via intramembranous ossification [41].

By contrast, endochondral ossification involves the recruitment, proliferation, and differen‐ tiation of undifferentiated mesenchymal cells into a transient cartilaginous matrix, which calcifies into mature bone. This type of fracture healing is advocated to have the following identifiable stages: (1) an initial stage of haematoma formation and inflammation, (2) subse‐ quent angiogenesis and formation of cartilage, (3) cartilage calcification, (4) cartilage removal, (5) bone formation, and (6) ultimately bone remodelling [42]. Also, it is contributed from the adjacent to the fracture periosteum and the external soft tissues, providing an early bridging callus, histologically described as 'soft callus' that stabilizes the fracture fragments [40]. Many studies have shown that diaphyseal fractures heal by endochondral mechanisms, forming a cartilaginous callus intermediate [41].

The classification of fracture healing into direct and indirect forms reflects the histological events that happen during the repair process. However, it is necessary to provide a further understanding of various signalling molecules and elucidate their contribution in the initiation and control of this physiological event at the molecular level.

#### **3.2. Signalling molecules in bone regeneration and fracture repair**

Various types of signalling factors influence the fracture healing, and continuous study of these factors can lead to promising new clinical treatments for bone repairing. To date, the delivery of signalling molecules for bone regeneration has been based primarily on factors that directly affect the bone formation pathways (osteoinduction) or that apply to increase the number of bone‐forming progenitor cells. Overall, the signalling molecules can be classified into three groups, including the pro‐inflammatory cytokines, the transforming growth factor‐β (TGF‐β) superfamily and other growth factors, and the angiogenic factors [43].

#### *3.2.1. Pro‐inflammatory cytokines*

Pro‐inflammatory cytokines, such as Interleukin‐1 (IL‐1), IL‐6, IL‐11, IL‐18 and tumour necrosis factor‐α (TNF‐α), are critical for triggering the repair cascade [44]. They are secreted by macrophages, inflammatory cells, and cells of mesenchymal origin existing in the periosteum [43, 45, 46]. These molecules play key roles in the induction of downstream mediators to the fracture site by exerting a chemotactic effect on other inflammatory cells, augmenting ECM synthesis, stimulating angiogenesis, and recruiting endogenous fibrogenic cells to injury [47]. Furthermore, cytokines were found to regulate endochondral bone formation and remodelling [43, 47]. For example, TNF‐α recruits mesenchymal stem cells (MSCs), promotes the induction of apoptosis in hypertrophic chondrocytes during endochondral ossification and incites osteoclastic function. Also, IL‐1 mainly provided by osteoblasts and simplifies bone remodel‐ ling by stimulating proteases to degrade callus tissue [46]. The absence of TNF‐α results in delayed resorption of mineralized cartilage, delayed endochondral bone formation by several weeks, and impaired fracture healing. Several studies have demonstrated that TNF‐α signal‐ ling is unique to postnatal fracture repair [46].

#### *3.2.2. Growth and differentiation factors*

#### *3.2.2.1. Transforming growth factor‐β superfamily*

It is a large group of regulatory polypeptides that includes bone morphogenetic proteins (BMPs), multiple isoforms of transforming growth factor‐βs (TGF‐βs), growth and differen‐ tiation factors (GDFs), activins (ACTs), inhibins (INHs), and glial‐derived neurotrophic factors (GDNFs), as well as some proteins not included in the above families, such as Mullerian‐ inhibiting substance (MIS), also known as anti‐Mullerian hormone (AMH), left‐right determi‐ nation factor (Lefty), and nodal growth differentiation factor (Nodal) (**Figure 1**) [48, 49]. Their isolation from bone extracts and further gene identification was accomplished in the 1980s, based on the previous results by Marshall R. Urist [19]. Transforming growth factor‐β family encompasses at least 34 members in the human genome. These molecules originate from high‐ molecular‐weight precursors, which are activated by proteolytic degradation. They can activate serine/threonine kinase membrane receptor on target cells. TGF‐β ligand‐bound receptor triggers an intracellular signal transmission via a canonical signalling pathway, which ultimately affects gene expression in the nucleus [47].

**3.2. Signalling molecules in bone regeneration and fracture repair**

superfamily and other growth factors, and the angiogenic factors [43].

*3.2.1. Pro‐inflammatory cytokines*

8 Advanced Techniques in Bone Regeneration

ling is unique to postnatal fracture repair [46].

*3.2.2.1. Transforming growth factor‐β superfamily*

ultimately affects gene expression in the nucleus [47].

*3.2.2. Growth and differentiation factors*

Various types of signalling factors influence the fracture healing, and continuous study of these factors can lead to promising new clinical treatments for bone repairing. To date, the delivery of signalling molecules for bone regeneration has been based primarily on factors that directly affect the bone formation pathways (osteoinduction) or that apply to increase the number of bone‐forming progenitor cells. Overall, the signalling molecules can be classified into three groups, including the pro‐inflammatory cytokines, the transforming growth factor‐β (TGF‐β)

Pro‐inflammatory cytokines, such as Interleukin‐1 (IL‐1), IL‐6, IL‐11, IL‐18 and tumour necrosis factor‐α (TNF‐α), are critical for triggering the repair cascade [44]. They are secreted by macrophages, inflammatory cells, and cells of mesenchymal origin existing in the periosteum [43, 45, 46]. These molecules play key roles in the induction of downstream mediators to the fracture site by exerting a chemotactic effect on other inflammatory cells, augmenting ECM synthesis, stimulating angiogenesis, and recruiting endogenous fibrogenic cells to injury [47]. Furthermore, cytokines were found to regulate endochondral bone formation and remodelling [43, 47]. For example, TNF‐α recruits mesenchymal stem cells (MSCs), promotes the induction of apoptosis in hypertrophic chondrocytes during endochondral ossification and incites osteoclastic function. Also, IL‐1 mainly provided by osteoblasts and simplifies bone remodel‐ ling by stimulating proteases to degrade callus tissue [46]. The absence of TNF‐α results in delayed resorption of mineralized cartilage, delayed endochondral bone formation by several weeks, and impaired fracture healing. Several studies have demonstrated that TNF‐α signal‐

It is a large group of regulatory polypeptides that includes bone morphogenetic proteins (BMPs), multiple isoforms of transforming growth factor‐βs (TGF‐βs), growth and differen‐ tiation factors (GDFs), activins (ACTs), inhibins (INHs), and glial‐derived neurotrophic factors (GDNFs), as well as some proteins not included in the above families, such as Mullerian‐ inhibiting substance (MIS), also known as anti‐Mullerian hormone (AMH), left‐right determi‐ nation factor (Lefty), and nodal growth differentiation factor (Nodal) (**Figure 1**) [48, 49]. Their isolation from bone extracts and further gene identification was accomplished in the 1980s, based on the previous results by Marshall R. Urist [19]. Transforming growth factor‐β family encompasses at least 34 members in the human genome. These molecules originate from high‐ molecular‐weight precursors, which are activated by proteolytic degradation. They can activate serine/threonine kinase membrane receptor on target cells. TGF‐β ligand‐bound receptor triggers an intracellular signal transmission via a canonical signalling pathway, which

**Figure 1.** A schematic illustration of TGF‐β superfamily. BMPs: bone morphogenetic proteins, TGF‐β: transforming growth factor beta, GDF: growth and differentiation factor, GDNF: glial‐derived neurotrophic factors, ACT: activin, INH: inhibin, other ligands include Mullerian‐inhibiting substance (MIS) or anti‐Mullerian hormone (AMH), left‐right determination factor (Lefty), and nodal growth differentiation factor (Nodal).

Several members of the subfamilies of these morphogens including bone morphogenetic proteins (BMPs 1–8), growth and differentiation factors (GDF‐1, 5, 8, 10) and transforming factor β (TGF‐β1, TGF‐β2, TGF‐β3), have been shown to promote the various stages of intramembranous and endochondral bone ossification during fracture healing (the following parts provide details on the use of them in attempts at bone regeneration) [24]. Of course, it is difficult to determine the physiological role of many of the members of this superfamily because of their functional redundancy.

**Bone morphogenetic proteins** are secreted signalling molecules that belong to the TGF‐β superfamily, acting as potent regulators during embryogenesis and bone and cartilage formation and repair. BMP ligands are divided into at least four separate subfamilies depend‐ ing on their primary amino acid sequence similarity and functions [50]. The first group consists of BMP‐2, BMP‐4, and the second group includes BMP‐5, BMP‐6, and BMP‐7. Group three includes GDF‐5 (or BMP‐14), GDF‐6 (or BMP‐13) and GDF‐7 (or BMP‐12), and finally, group four consists of BMP‐3 (or osteogenin) and GDF‐10 (or BMP‐3b) [51, 52]. BMP‐1 does not include in this list as a member of the TGF‐β superfamily and it may carry out a role in modulating BMP functions by the proteolysis of BMP antagonists/binding proteins, such as chondrin and noggin [47, 53].

BMPs bind to type‐II serine/threonine kinase receptors and thus provoke the assembly of type‐ I and type‐II receptors in a hetero‐oligomeric complex [54]. Subsequently, the Smad‐signalling cascade is triggered into the cell. BMPs are pleiotropic morphogens and carry out an important role in regulating growth, differentiation, and apoptosis of various cell types, including osteoblasts, chondroblasts, epithelial cells, and neural cells [55]. Furthermore, it has been demonstrated that the active signalling molecule is usually formed by homodimerization through a disulphide bond [56]. However, in particular, experimental settings heterodimers have been shown to have enhanced osteoinductive activity regulating more efficiently differentiation and proliferation of mesenchymal cells to osteoblasts *in vitro* and *in vivo* than the corresponding homodimers (i.e., BMP‐2/‐5, BMP‐4/‐7, BMP‐2/‐6; BMP‐2/‐7) [57, 58]. In bone, BMPs are produced by different types of cells, including osteoprogenitors, mesenchymal cells, osteoblasts, and chondrocytes. BMPs are able to induce a sequential cascade of events for chondro‐osteogenesis, including mesenchymal and osteoprogenitor cells proliferation and differentiation, chemotaxis, angiogenesis, and controlled synthesis of extracellular matrix [53, 55].

Regulatory effect of BMPs depends on the type of the targeted cell, its differentiation stage, the local concentration of the ligand and the interaction with other circulating factors [59].

BMPs are closely related structurally and functionally; however, each has a unique role and different temporal expression pattern during the fracture healing. The researchers demon‐ strated in several studies that BMPs could have a variety of osteogenic effects, mitogenic capacities, and temporal expressions in the rat and mouse [24, 60, 61].

Cheng et al. prepared a comprehensive analysis of the osteogenic activity of 14 types of BMPs and their results suggested an osteogenic hierarchical model of BMPs. BMP‐2, BMP‐6, and BMP‐9 may act as the most potent to induce osteoblast differentiation of mesenchymal progenitor cells, while most BMPs (except BMP‐3 and BMP‐13) promote the terminal differ‐ entiation of committed osteoblastic precursors and osteoblasts [62]. Furthermore, BMPs are able to stimulate the synthesis and secretion of other bone and angiogenic growth factors such as insulin‐like growth factor (IGF) and vascular endothelial growth factor (VEGF), respectively and also stimulate bone formation by directly activating endothelial cells to stimulate angio‐ genesis [63].

Recent studies have shown that the expression of the BMP antagonists, most importantly noggin, plays an important role in fracture healing regulation [64]. They could block BMP‐2 interaction with its receptor [65].

**Transforming growth factor‐β** family includes five isoforms such as TGF‐β1, TGF‐β2, and TGF‐β3 [66, 67]. The main sources of TGF‐β existing during the bone healing are practically all cells involved in healing process, incoming blood platelets, and the surrounding ECM releasing TGF‐β following a mechanical injury causing tissue ischaemia and local change in pH, facilitating release of not only of TGF‐β, but also other growth factors, such as VEGF, platelet‐derived growth factor (PDGF), or BMP‐2 [68, 69]. Intracellular signal transduction is exerted via type‐I and type‐II serine/threonine kinase receptors, activating the Smad cascade (Smad 2 and 3) [70]. TGF‐β is a potent chemotactic stimulator of mesenchymal stem cells and it enhances proliferation of MSCs, preosteoblasts, chondrocytes, and osteoblasts. Indeed, its main role is thought to be during processes of proliferation, differentiation, and synthesis of cartilage and bone tissue, collectively mentioned as the bone‐healing process [67, 71]. Also, it is able to induce the production of extracellular proteins, such as proteoglycans, fibronectin, collagen, osteonectin, osteopontin, thrombospondin, and alkaline phosphatase [72]. Moreover, TGF‐β may trigger signalling for BMP synthesis by the osteoprogenitor cells, while it may inhibit activation, proliferation, and differentiation of osteoclasts and promote their apoptosis [60, 73].

Several studies have shown that TGF‐β2 and possibly TGF‐β3 had stronger effect in fracture‐ healing process than TGF‐β1, as their expression peak during chondrogenesis. On the other hand, Joyce et al. injected TGF‐β1 and TGF‐β2 subperiosteally to newly born rats, at doses ranging from 20 to 200 ng, and their results showed that subperiosteal MSC starts to proliferate and differentiate at the injection site, promoting chondrogenesis and osteogenesis, and that TGF‐β2 play more important roles than TGF‐β1 [74]. Moreover, Beck et al., designed an experiment concerning local administration of TGF‐β1 at doses ranging from 0.5 to 5 μg to rabbits with skull defect, caused stimulation, recruitment, and proliferation of osteoblasts at the defect site resulting in healing [75]. Despite different studies demonstrated that TGF‐β induces cellular proliferation, its osteoinductive potential is limited by concern for its unfore‐ seen side effects [71].

the corresponding homodimers (i.e., BMP‐2/‐5, BMP‐4/‐7, BMP‐2/‐6; BMP‐2/‐7) [57, 58]. In bone, BMPs are produced by different types of cells, including osteoprogenitors, mesenchymal cells, osteoblasts, and chondrocytes. BMPs are able to induce a sequential cascade of events for chondro‐osteogenesis, including mesenchymal and osteoprogenitor cells proliferation and differentiation, chemotaxis, angiogenesis, and controlled synthesis of extracellular matrix [53,

Regulatory effect of BMPs depends on the type of the targeted cell, its differentiation stage, the local concentration of the ligand and the interaction with other circulating factors [59].

BMPs are closely related structurally and functionally; however, each has a unique role and different temporal expression pattern during the fracture healing. The researchers demon‐ strated in several studies that BMPs could have a variety of osteogenic effects, mitogenic

Cheng et al. prepared a comprehensive analysis of the osteogenic activity of 14 types of BMPs and their results suggested an osteogenic hierarchical model of BMPs. BMP‐2, BMP‐6, and BMP‐9 may act as the most potent to induce osteoblast differentiation of mesenchymal progenitor cells, while most BMPs (except BMP‐3 and BMP‐13) promote the terminal differ‐ entiation of committed osteoblastic precursors and osteoblasts [62]. Furthermore, BMPs are able to stimulate the synthesis and secretion of other bone and angiogenic growth factors such as insulin‐like growth factor (IGF) and vascular endothelial growth factor (VEGF), respectively and also stimulate bone formation by directly activating endothelial cells to stimulate angio‐

Recent studies have shown that the expression of the BMP antagonists, most importantly noggin, plays an important role in fracture healing regulation [64]. They could block BMP‐2

**Transforming growth factor‐β** family includes five isoforms such as TGF‐β1, TGF‐β2, and TGF‐β3 [66, 67]. The main sources of TGF‐β existing during the bone healing are practically all cells involved in healing process, incoming blood platelets, and the surrounding ECM releasing TGF‐β following a mechanical injury causing tissue ischaemia and local change in pH, facilitating release of not only of TGF‐β, but also other growth factors, such as VEGF, platelet‐derived growth factor (PDGF), or BMP‐2 [68, 69]. Intracellular signal transduction is exerted via type‐I and type‐II serine/threonine kinase receptors, activating the Smad cascade (Smad 2 and 3) [70]. TGF‐β is a potent chemotactic stimulator of mesenchymal stem cells and it enhances proliferation of MSCs, preosteoblasts, chondrocytes, and osteoblasts. Indeed, its main role is thought to be during processes of proliferation, differentiation, and synthesis of cartilage and bone tissue, collectively mentioned as the bone‐healing process [67, 71]. Also, it is able to induce the production of extracellular proteins, such as proteoglycans, fibronectin, collagen, osteonectin, osteopontin, thrombospondin, and alkaline phosphatase [72]. Moreover, TGF‐β may trigger signalling for BMP synthesis by the osteoprogenitor cells, while it may inhibit activation, proliferation, and differentiation of osteoclasts and promote their apoptosis

capacities, and temporal expressions in the rat and mouse [24, 60, 61].

55].

10 Advanced Techniques in Bone Regeneration

genesis [63].

[60, 73].

interaction with its receptor [65].

**Platelet‐derived growth factors (PDGFs)** are homo‐ or heterodimeric polypeptides in which their A and B chains are linked by disulphide bonds. PDGF receptors exert their effect on cells by activating receptors that have tyrosine kinase activity [76]. PDGF's binding is affected by IL‐1, TNF‐a, and TGF‐β1 affect [77]. It is synthesized by numerous cell types, including platelets, monocytes, macrophages, osteoblasts, and endothelial cells and is a major mitogen for cells of mesenchymal origin such as osteoblasts, fibroblasts, glial cells, and smooth muscle cells [78–80].

PDGF is released by platelets upon activation during the early callus phase of healing and acts as a potent chemotactic for inflammatory cells and a major proliferative and migratory stimulus for MSCs and osteoblasts. It has been demonstrated that treating with PDGF increased callus density and volume in tibial osteotomies in rabbits [47, 81]. However, its therapeutic potential still remains unclear.

**Fibroblast growth factors (FGFs)** consist of nine structurally related polypeptides. The acidic and basic FGFs are the most abundant FGFs in normal adult tissue [82]. FGF effect is exerted via binding to tyrosine kinase receptors [82].

FGFs are synthesized by monocytes, macrophages, osteoblasts, mesenchymal cells, and chondrocytes during bone healing. FGFs are able to induce growth and differentiation of a variety of cells, such as fibroblasts, osteoblasts, myocytes, and chondrocytes. They function during the early stages of fracture healing and play a critical role in angiogenesis and mesen‐ chymal cell mitogenesis. α‐FGF mainly affects chondrocyte proliferation and is probably crucial for chondrocyte maturation, while β‐FGF is produced by osteoblasts and is recognized as a potent mitogen than a‐FGF [71]. In a canine tibial osteotomy model, a single injection of FGF‐2 resulted in an early increase in callus size [83].

**Insulin‐like growth factors (IGFs)** consist of IGF‐I (or somatomedin‐C) and IGF‐II (or skeletal growth factor) [84]. The sources of IGF‐I and IGF‐II are the bone matrix, osteoblasts and chondrocytes, and endothelial cells. The concentration of circulating IGF‐I is mainly regulated by the growth hormone. Also, it has been demonstrated that the biological actions of IGFs is modulated in a cell‐specific manner by IGF‐binding proteins (IGFBPs) [71, 85].

IGF‐I promotes bone matrix formation such as type‐I collagen and non‐collagenous matrix proteins by fully differentiated osteoblasts and acts more effective than IGF‐II [71, 86]. IGF‐II functions at a later stage of endochondral bone formation and incites type‐I collagen produc‐ tion, cellular proliferation cartilage matrix synthesis [87]. The findings from various animal studies assessing the influence of IGF on skeletal repair have reported different results, so further studies are required [88].

#### *3.2.3. Metalloproteinases and angiogenic factors*

Conditions of fracture healing establish a demand on the surrounding tissues to increase blood flow so that can induce bone regeneration within the callus [89]. Also, endochondral ossifica‐ tion in normal fracture healing requires the following two processes: (1) molecular mechanisms that regulate the extracellular matrix remodelling and (2) the vascular penetration of new blood vessels into the resorbing matrix [90]. Thus, angiogenesis and matrix degradation are either concurrent or correlated processes during endochondral ossification. The final stages of endochondral ossification and bone remodelling are accomplished by the action of specific matrix metalloproteinases, which degrade the cartilage and bone, allowing the invasion of the blood vessels. Angiogenesis regulation requires the coordination of both separate pathways, including a vascular endothelial growth factor (VEGF)‐dependent pathway and an angio‐ poietin‐dependent pathway [91]. Numerous types of studies reported that VEGFs are required mediators of endothelial‐cell‐specific mitogens and neo‐angiogenesis [92]. Whereas angio‐ poietin 1 and 2 are regulatory vascular morphogenetic molecules related to the formation of larger vessel and development of colateral branches from present vessels [43]. Street et al. showed that exogenous administration of VEGF can induce fracture repair [48]. Also, recent studies have reported that BMPs promote the expression of VEGF by osteoblasts and osteo‐ blast‐like cells. However, their contribution in bone repair is still not as well understood.

#### **3.3. Role of mesenchymal stem cells in bone regeneration and fracture repair**

Mesenchymal stem cells (MSCs) are non‐haematopoietic stromal stem cells capable of extensive replication without differentiation. They have many sources including bone marrow, peripheral circulation, adipose, periosteum, muscle, vessel walls, tendon, umbilical cord blood, skin, and dental tissues. MSCs have the potential to commit and differentiate along several cell lineages giving rise to those cells that form mesenchymal tissues, including cartilage, bone, muscle, ligament, tendon, and marrow stroma and fat [93, 94]. MSCs can migrate to sites of injury and have been used widely in tissue engineering, stem cell trans‐ plantation and immunotherapy. There are different sets of molecules interacting with both local cells and circulating cells to coordinate the healing cascade, such as effectors of inflam‐ mation (IL‐1, IL‐6, TNF‐a), mitogens (TGF‐β, IGF, FGF, and PDGF), morphogens (BMPs), and angiogenic factors (VEGF and angiopoietins). The effects of these molecules on the prolifera‐ tion and differentiation of MSCs have been widely investigated *in vitro* [47]. The results indicated that these signalling molecules can induce cell proliferation and differentiation, both MSC and other progenitor lineages. The temporal expression of this array of signalling molecules in models of fracture healing has been charted, but explicit data on how this microenvironment can regulate MSC activity is still needed.
