Regeneration of Bones
Bone -since it is a live tissue - undertakes various insults and can modulate itself into a healthy bone. As a complex process of bone formation, the regeneration of bone involves continuous remodeling throughout adult life and can happen during normal fracture healing. The fracture healing process is modulated by the combination of a material for the new bone to grow into and cells that can allow osteoinduc- tion. In addition, the combination of cells and signals and putting down the scaffolds for the new bone is also very important [25, 26]. Unfortunately, bone damage caused by reconstructive surgery, neoplasia, trauma, and osteoporosis remains a major clinical problem. Current bone regeneration strategies include auto- or xenogeneic bone grafting, gene therapy, the application of different bioactive factors, and the use of osteoprogenitor cells, osteoconductive scaffolds, and growth factors, alone or in various combinations . Today, bone regeneration still has significant limitations.
Autologous bone grafting is considered as the ‘gold standard’ bone-grafting method, as it combines all properties required in a bonegraft material: osteoinduc- tion [bone morphogenetic proteins (BMPs) and other growth factors], osteogenesis (osteoprogenitor cells) and osteoconduction (scaffold) . But autologous bone graft has the severe shortcoming. In addition, allogeneic bone grafting has problems of immunogenicity, rejection reactions and so on. In recent years, tissue engineering and regenerative medicine has been providing exciting technologies for the development of bone-graft substitutes. Scaffolds, cells, and biological factors are the three key components of bone regeneration (see Fig. 6.2) .
Firstly, a scaffold that is similar in composition and structure to the natural bone forms the basic support frames of bone graft. A scaffold should have the following features in an ideal manner: (1) three-dimensional and highly porous; (2) biocompatible and bioresorbable with a controllable degradation and resorption rate; (3) suitable surface chemistry for advancing adhesion, growth and differentiation of cells; and (4) mechanical properties [27, 29]. Scaffolds are made of synthetic or natural biomaterials, including collagen, hydroxyapatite, tricalcium phosphate and calcium-phosphate cements, and glass ceramics . Especially nano-biomaterials could elicit a higher degree of biological plasticity compared with conventional micro-materialsdue to their high surface-to-volume ratio. Moreover, nanohydroxyapatite with chitin, chitosan, collagen, gelatin, fibrin, polylactic acid (PLA), poly (e-caprolactone) (PCL), poly (lactic-co-glycolic acid) (PLGA), polyamide (PA), polyvinyl alcohol (PVA), polyurethane (PU), and polyhydroxybutyrate (PHB) proved to be promising biomaterials for bone tissue engineering . In addition, metals including Zn, Ti, Zr, B, Sr, Mg, Ag, and Cu have the highest mechanical strength and play a major role in accelerating bone formation and promoting bone regeneration. They act as cofactors for enzymes, serving as a structural component of bone forming enzymes and proteins, increasing extra-cellular matrix synthesis, stimulating angiogenesis, promoting bone formation, and inhibiting bone resorption . Silk fibroin produced mainly by silkworms and spiders has unique mechanical properties, tunable biodegradation rate and the ability to support the differentiation
Fig. 6.2 Schematic presentation of bone regeneration by tissue engineering  (Copyright permission from Elsevier Science Ltd)
of mesenchymal stem cells (MSCs) along the osteogenic lineage. It can be processed into various scaffold forms, combined synergistically with other biomaterials to form composites and chemically modified which make it a favorable scaffold material for bone tissue engineering . Graphene and its derivatives (graphene oxide and reduced graphene oxide) have high surface area and high mechanical strength. They has the ability to promote and enhance osteogenic differentiation making it an interesting material for bone regeneration research . In addition, the combination of materials may be the most promising strategy for bone tissue regeneration .
Secondly, cells such as osteoblasts and stem cells constitute the biologically functional units of bone graft, and they have consistently been shown to promote bone formation inside scaffolds [35, 36]. Embryonic stem cells (ESCs) are an attractive stem cell source because they can self-renew over long periods of time and have a strong multilineage differentiation capability. But consideration of the ethics of using human embryos, there are a lot of controversy surrounded ESCs. In recent years, MSCs derived from cryopreserved umbilical cord blood, bone marrow, and adipose tissue have received extensive attention because they can be easily isolated, induce little immune response, have marked self-renewal properties and possess the biological capability to differentiate into osteoblasts in response to multiple environmental factors. For example, specific combinations of soluble factors including dexamethasone, ascorbic acid, and р-glycerophosphate disodium have been shown to induce osteoblastogenesis of MSCs. A variety of factors, like BMP and FGF can up-regulate expression of osteogenic related genes in MSCs. Besides chemical revulsants, physics factors such as mechanical strain, shear stress, and compressive stress also play important roles in the osteogenic differentiation of MSCs . Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of plu- ripotent stem cells. They can be generated directly from adult cells by the delivery of some genes to differentiated cells and are highly similar to ESCs. Since iPSCs can be generated directly from adult tissues without the embryo and have the strong multi-directional differentiation ability, these cells are another attractive stem cell source. Each individual could have their own pluripotent stem cell line which could be used to transplant without the risk of immune rejection. However, due to their genomic instability and tumorigenesis, further investigations are needed.
Thirdly, bioactive factors have been used to supplement bone reconstruction. Bone formation is a complex process that requires concerted function of multiple bioactive factors . To date, a large number of technologies, such as osmotic pumps, bolus injection, release of surface-adsorbed protein, and controlled release by scaffold biodegradation, have been developed to deliver bioactive factors including growth factors, genetic material, and drugs or small molecules . Growth factors have significant impacts on osteoblast behavior, and thus have been widely used in bone tissue engineering, alone and in various combinations. These factors include BMP-2, BMP-4, BMP-7, FGF-2, transforming growth factor (TGF)-p1, TGF-p2, TGF-P3, VEGF, insulin-like growth factor (IGF)-1, platelet derived growth factor (PDGF), and stromal cell-derived factor (SDF)-1 [39, 40]. However, growth factors have short half-lives, slow tissue penetration, and high toxicity, which suggest the efficiency of conventional routes is relatively low. Therefore, effective bone repair requires using multiple bioactive factors in different delivery systems .
Fourthly, bioreactor systems are key components for bone tissue engineering and medical regeneration, including perfusion, mechanical compression, and hydrostatic compression bioreactors. In particularly, perfusion bioreactors can provide dynamic environments with enhanced diffusion of nutrients and oxygen and removal of waste products [41, 42]. It is necessary to develop the automation of bioreactors to reach large-scale clinical application. Equally important is the generating of patient-specific bioreactors .
Moreover, vascularization of bone grafts exceeding a certain size is one of the main challenges of bone tissue engineering. Larger perfusable blood vessel grafts are needed for restoration of blood flow into the site of injury, while smaller microvascular beds are needed to provide thorough distribution of blood across the entire scaffold volume . Scaffolds made of hydrogels and other materials have been performed for angiogenesis due to their capacity to be loaded with cells and signals at relatively high density and their ability to sustain cell viability for relatively long periods of time. Cells such as ECs, endothelial progenitor cells (EPCs), MSCs, human umbilical vein endothelial cells (HUVECs), and ESCs, have been used to assist vascularization inside scaffolds. Growth factors including VEGF and FGF-2 have been used to enhanced tissue and vascular growth in scaffolds . Future bone grafts are hoped to have the surrounding vascular beds for complete restoration of bone function.
In addition, since results from in vitro studies can be difficult to extrapolate to the in vivo situation, animal models have been used extensively to investigate the biology of fracture healing and bone graft substitutes before clinical use in humans. There are several factors that should be considered when selecting an animal model, including availability of the animal, cost, ease of handling and care, size of the animal, the age of the animal, acceptability to society, resistance to surgery, infection and disease, biological properties analogous to humans, bone structure and composition, as well as bone modeling and remodeling characteristics. Small and large animals, including mice, rats, rabbits, dogs, pigs, goats and sheep have been used in animal experiments on bone tissue engineering and medical regeneration .