**4. Tissue engineering strategies for bone regeneration**

As it was defined by Laurencin, tissue engineering (TE) is 'the application of biological, chemical, and engineering principles toward the repair, restoration, or regeneration of living tissue by using biomaterials, cells, and factors alone or in combination' [95].

Bone tissue engineering (BTE) is a dynamic and complex process that includes migration and recruitment of osteoprogenitor cells, followed by their proliferation, differentiation, ma‐ trix formation along with remodelling of the bone. In this section, we consider BTE as three interplaying components: (a) the extracellular matrix/scaffold, (b) the cells that reside in the matrix/scaffold, and (c) the environment that hosts the cells. However, major advances in BTE with scaffolds are achieved through biochemical factors, such as growth factors, genes, proteins, and drugs. Bone scaffolds are typically made of porous‐degradable materials that prepare the mechanical support during repair and regeneration of diseased or damaged bone [7]. Also, physical factors, including substrate topography, stiffness, shear stress, and electrical forces, are other stimuli that have been proposed as one of the principal mediators of de novo tissue formation [96]. Box 1 highlights requirements for an ideal scaffold.

#### **4.1. Biomolecule delivery**

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

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**

microenvironment can regulate MSC activity is still needed.

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

further studies are required [88].

12 Advanced Techniques in Bone Regeneration

*3.2.3. Metalloproteinases and angiogenic factors*

The strategy of concurrently modulating the chemical environments of the fracture site *in vivo* via controlled delivery/elution of biomolecule agents from an orthopaedic implant rep‐ resents an elegant method of targeted therapeutics in bone regeneration [97, 98]. This strat‐ egy enables higher local concentration (localized delivery) of the bioactive agent to the fracture site, while the favourable bulk properties of the orthopaedic implant are un‐ changed. It also provides the chance to maximize the local growth‐inducing potentials of bi‐ oactive agents at a desired rate without any local and systematic toxic effects to the host tissue that is attributed to other routes of delivery such as systemic or non‐controllable local delivery. Soluble biochemical molecules that are integrated into scaffolds include proteins/ growth factors, such as TGF‐β, BMP, VEGF, IGF, and FGF, which have attracted much atten‐ tion because of their potency in bone tissue repair. As described earlier, these growth factors are able to control osteogenesis, bone tissue regeneration, and ECM formation via recruiting and differentiating MSCs (osteoprogenitor) to specific lineages [99]. Therefore, various growth factors and other biomolecules are of special interest for bone tissue engineering and effective incorporation of them in scaffolds could reduce fracture healing time and thus fa‐ cilitate in patient recovery [100, 101]. Also, bone is a highly vascularized tissue; therefore, the performance of a scaffold in bone engineering can be affected by its ability to induce new blood vessel formation. Because insufficient vascularization can lead to oxygen and nu‐ trient deficiency, this may result in improper cell integration and cell death [102, 103]. On the other hand, in the *in vivo* conditions, supply of oxygen and nutrients are essential for the survival of growing cells and tissues within scaffolds. So, VEGF is used to induce a complex network of blood vessels throughout a scaffold [104].

#### **Box 1.** Requirements for an ideal scaffold

**Biocompatibility** is one of the primary requirements of bone scaffolds. It is a term that has been defined in many ways. Biocompatibility can be principally defined as the ability of scaffold to support normal cellular activity, such as molecular signalling pathways, without any local and systematic toxic effects to the host tissue [105]. An ideal bone scaffold must act as an osteoconductive substrate such that it permits the bone cells to adhere, proliferate, and form ECM on its surface and pores. Furthermore, the scaffold needs to induce bone formation within the defect through signalling systems and recruiting progenitor cells, a feature known as osteoinduction. Also, an ideal scaffold should be able to serve as a platform for formation of blood vessels in or around the implant during few weeks of implantation to promote nutrients and metabolic waste transportation [106].

**Mechanical properties**: An ideal bone scaffold should yield a close match to the host bone properties and also convenient load transfer is important. Mechanical properties of bone vary widely from cancellous to cortical bone. Cortical bone exhibits a Young's modulus between 15 and 20 GPa and that of cancellous bone is between 0.1 and 2 GPa. Compressive strength of cortical bone is between 100 and 200 MPa, and between 2 and 20 MPa for cancellous bone. Because of the large variation in mechanical property and geometry, it is difficult to design an 'ideal scaffold' for BTE [106].

**Pore size** and closed void volumes may concurrently play important roles in scaffold degradation patterns and associated bone healing [107]. It should be approximately 100 μm in diameter for successful cellular infiltration and nutrient and oxygen supply for cell survivability [102]. However, scaffolds with pore sizes between 200 and 350 μm are indicated to be optimum for bone tissue in‐growth [108]. Moreover, recent papers have reported that multi‐scale porous scaffolds which involve both micro‐ and macroporosities can act better than only macroporous scaffolds [109]. Unfortunately, porosity can reduce mechanical properties, such as compressive strength, and also increase the complexity for reproducible scaffold making. Researchers have developed porous scaffolds using polymers, ceramics, metals, and composites. Strength of different polymers matches close to the cancellous bone and dense bioceramic materials to that of cortical bone. However, scaffolds manufacturing ceramic‐polymer composite are typically weaker than bone. Porous metallic scaffolds provide the mechanical necessities of bone, but fail to meet the required implant‐tissue integration and also, there is potential concern regarding metal ion leaching [110].

**Bioresorbability** is another crucial requirement for scaffolds in BTE [105]. In addition to similar me‐ chanical properties that of the host tissue, an ideal scaffold should be able to degrade with time *in vivo* by cellular and enzymatic activity, preferably at a controlled resorption rate in parallel with the produc‐ tion of new bone matrix. The degradation behaviour of the scaffolds is determined based on their appli‐ cations; for example, 3–6 months for scaffolds in cranio‐maxillofacial applications or 9 months or more for scaffolds in spinal fusion. Recently, design and development of multi‐scale porous scaffolds having ideal composition, including related bioresorbability, targeted biomolecules, and mechanical properties are some challenging areas of research [106, 111].

#### **4.2. Stem/progenitor cells applicable to bone tissue engineering**

#### *4.2.1. Mesenchymal stem cells*

**Box 1.** Requirements for an ideal scaffold

14 Advanced Techniques in Bone Regeneration

and metabolic waste transportation [106].

geometry, it is difficult to design an 'ideal scaffold' for BTE [106].

are some challenging areas of research [106, 111].

**Biocompatibility** is one of the primary requirements of bone scaffolds. It is a term that has been defined in many ways. Biocompatibility can be principally defined as the ability of scaffold to support normal cellular activity, such as molecular signalling pathways, without any local and systematic toxic effects to the host tissue [105]. An ideal bone scaffold must act as an osteoconductive substrate such that it permits the bone cells to adhere, proliferate, and form ECM on its surface and pores. Furthermore, the scaffold needs to induce bone formation within the defect through signalling systems and recruiting progenitor cells, a feature known as osteoinduction. Also, an ideal scaffold should be able to serve as a platform for formation of blood vessels in or around the implant during few weeks of implantation to promote nutrients

**Mechanical properties**: An ideal bone scaffold should yield a close match to the host bone properties and also convenient load transfer is important. Mechanical properties of bone vary widely from cancellous to cortical bone. Cortical bone exhibits a Young's modulus between 15 and 20 GPa and that of cancellous bone is between 0.1 and 2 GPa. Compressive strength of cortical bone is between 100 and 200 MPa, and between 2 and 20 MPa for cancellous bone. Because of the large variation in mechanical property and

**Pore size** and closed void volumes may concurrently play important roles in scaffold degradation patterns and associated bone healing [107]. It should be approximately 100 μm in diameter for successful cellular infiltration and nutrient and oxygen supply for cell survivability [102]. However, scaffolds with pore sizes between 200 and 350 μm are indicated to be optimum for bone tissue in‐growth [108]. Moreover, recent papers have reported that multi‐scale porous scaffolds which involve both micro‐ and macroporosities can act better than only macroporous scaffolds [109]. Unfortunately, porosity can reduce mechanical properties, such as compressive strength, and also increase the complexity for reproducible scaffold making. Researchers have developed porous scaffolds using polymers, ceramics, metals, and composites. Strength of different polymers matches close to the cancellous bone and dense bioceramic materials to that of cortical bone. However, scaffolds manufacturing ceramic‐polymer composite are typically weaker than bone. Porous metallic scaffolds provide the mechanical necessities of bone, but fail to meet the required implant‐tissue integration and also, there is potential concern regarding metal ion leaching [110].

**Bioresorbability** is another crucial requirement for scaffolds in BTE [105]. In addition to similar me‐ chanical properties that of the host tissue, an ideal scaffold should be able to degrade with time *in vivo* by cellular and enzymatic activity, preferably at a controlled resorption rate in parallel with the produc‐ tion of new bone matrix. The degradation behaviour of the scaffolds is determined based on their appli‐ cations; for example, 3–6 months for scaffolds in cranio‐maxillofacial applications or 9 months or more for scaffolds in spinal fusion. Recently, design and development of multi‐scale porous scaffolds having ideal composition, including related bioresorbability, targeted biomolecules, and mechanical properties

Mesenchymal stem cells have been isolated from a diverse host tissues throughout the adult organism including bone marrow [94] and an array of other postnatal tissues, such as adipose tissue [112], periodontal ligaments [113], synovium [114], blood [115] and the lung [116]. As the ultimate aim of regenerative medicine is to avoid *in vitro* expansion of cells and the associated complications, the adipose‐derived stem cell indicates an ideal progenitor cell in bone tissue engineering.

Intriguingly, several studies have reported that 6 × 10<sup>6</sup> nucleated cells can be isolated from 1 mL bone marrow of which 0.001–0.01% are considered to be stem cells [94]. Contrastingly, adipose tissue aspiration yields 2 × 10<sup>6</sup> nucleated cells per 1 g, of which 10% are stem cells. Thus, one can easily distinguish the potential clinical implications of this abundant source of MSCs [117, 118]. In a study, researchers compared the in vivo osteogenic potential of adipose‐ derived, bone marrow‐derived, and periosteal‐derived MSCs in a guided bone regeneration model in pig calvarial defects to identify if there is a more desirable site from which to harvest MSCs for bone tissue engineering. They reported that regardless of the tissue source of MSCs, the speed and pattern of bone healing after cell transplantations into monocortical bone defects were comparable, indicating that the performance of autologous adipose‐derived MSCs, periosteal‐derived MSC, and bone marrow‐derived MSC (BM‐MSCs) following *ex vivo* cell expansion was not considerably different for the guided regeneration of bone defects [119].

#### *4.2.2. Endothelial progenitor cells*

Vascularization is a vital process for the survival of the implanted cells on the carrier material after implantation. Many studies demonstrated that close spatial and temporal association between blood vessels and bone cells is necessary to maintain skeletal integrity. Several studies have shown that new bone formation in porous scaffolds was considerably increased by the insertion of a vascular pedicle in the scaffold, while others have shown that fracture healing and new bone formation could be prohibited by the administration of angiogenesis inhibitors. Such that previous reports illustrated that the rate of delayed union or non‐union of fracture can be as high as 46% in fracture patients with concomitant vascular injuries [120]. Because adequate vascularization making it possible to stem cells reach the site of tissue repair and allows the delivery of nutrients, oxygen, and morphogens and the removal of waste [121–124].

In 1997, Asahara and colleagues identified endothelial progenitor cells (EPCs) in the peripheral blood and reported their ability to initiate neovascularization [125]. EPC derived from purified hematopoietic progenitor cells, express endothelial‐associated markers (i.e., cluster of differ‐ entiation molecule, CD34) and display endothelial phenotypical characteristics. They can enhance neovascularization by incorporation and differentiation, and by the secretion of angiogenic factors affecting resident endothelium [126].

The major role of EPCs in the ability of EPCs to proliferate and differentiate into endothelial cells and new vessel formation present them as an ideal therapeutic strategy for recovery of the ischemic environment of a critical‐sized bone defect in bone tissue engineering. Further‐ more, a research group reported that the frequency of EPCs increased in the bone marrow and peripheral blood in the early stages of fracture repair and further illustrated incorporation of EPCs into developing blood vessels at the site of bone injury. Further histological results demonstrated that neovascularization did not exclusively involve the EPC population; however, supporting the hypothesis that paracrine signalling from EPCs may also contribute to neovascularization at the ischemic site [127].

#### *4.2.3. Induced pluripotent stem cells*

Induced pluripotent stem (iPS) cells, a discovery that resulted in a Nobel Prize in 2012, are somatic cells from embryonic or adult fibroblasts that are reprogrammed with defined classical transcription factors (Oct4, Sox2, Klf4, and c‐Myc) [121, 128]. By forcing expression of these transcription factors, iPS cells retain the capacities of embryonic stem cells, including self‐ renewal and pluripotentiality to differentiate into all three germ layers [129]. Using these biological properties, iPS cells with an incorporation of gene therapy will be able to not only treat degenerative syndromes and genetic disorders but also appear as a promising candidate for autologous cell transplantation in bone defects. [129, 130]. Also, iPS cells, without the challenges of immunological rejection and ethical controversy, are preferable to embryonic stem cells and seem to be a potential alternative stem cell source for bone tissue engineering.
