**2. Stem cells and bone regeneration**

Bone regenerates through complex and organized biological events of bone induction and conduction. This process involves a number of cell types and molecular signalling pathways in a defined sequence to maximize the repair and regeneration of the skeletal tissue. The organic matrix of the bone tissue is composed of collagen type I fibres (approximately 95%) proteoglycans and numerous non-collagenous proteins (5%) [7]. Non-collagenous proteins participate in the process of matrix maturation, mineralization and may regulate the func‐ tional activity of bone cells. Primarily, the functional integrity of bone tissue is maintained by the cell types such as osteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells).

During the phase of bone formation, the osteoblasts are recruited from mesenchymal stem cells (MSCs) present in bone marrow [8]. On the other hand, osteoclasts are derived from haematopoietic stem cells through committed osteoclast progenitors that fuse to form mature multinucleated cells [9]. The regeneration process occurs through osteogenesis initiated by skeletal stem cell also known as mesenchymal stem cells. During embryogenesis, the devel‐ opment of the skeletal tissue occurs by intramembranous and endochondral ossification. Bone formations begin with aggregation of MSCs to form condensations and within the mesenchy‐ mal condensation core; cells differentiate into chondrocytes in endochondral ossification or directly into osteoblasts in the intramembranous bone formation pathway [10]. Therefore, stem cells play a key role in bone regeneration and are considered to have the potential to treat bone defects either through cell-based therapies or tissue engineering. Stem cell-mediated bone regeneration provides a number of potential therapeutic advantages as compared to the use of autograft tissues. As such, therapeutic uses of stem cells are being explored extensively for bone tissue regeneration applications. MSCs and adipose-derived stem cells (ADSCs) have received considerable attention in this regard and have been extensively evaluated for bone regeneration.

significant impacts on the health and lifestyle of individuals. In the USA alone, more than half a million patients experience problems due to bone defects each year, with medical cost associated with these defects being more than \$2.5 billion/annum and this figure is expected to double by 2020. It is estimated that about 2.2 million bone graft procedures are performed around the world annually [1–3]. The current strategies used for augmenting bone regenera‐ tion include different bone-grafting methods, such as autologous bone grafts and allografts [4]. Autologous bone grafts have relatively successful clinical outcomes; however, donor site morbidity, limited supply and the complicated surgical procedures associated with bone harvests hinder the efficacy of such procedures. On the other hand, allogenic bone grafts are excellent in terms of sourcing large quantities of donor tissue required for treating large bone defects; however, the issues related to immunogenicity, rejection reaction and disease transmission render this treatment less ideal [4, 5]. The shortcomings associated with these treatments have led to exploring tissue engineering approaches and stem cell-based thera‐

There is great promise for stem cell-based therapeutics for the treatment of numerous diseases and injuries; as such, substantial investment has been made over the past decade for new therapies. Stem cells play a critical role in tissue regeneration and repair, maintenance, turnover and the control of haematopoiesis in the bone marrow. They are considered as an attractive cell population for bone repair due to their proliferation, osteogenic potential and secretion of potent endogenous trophic factors to enhance local vascularization. These cells have an incredible ability to differentiate into specific cell types like osteoblasts, chondro‐ cytes or myocytes and to develop bone, cartilage or muscle tissues. It is believed that the stem cells can help in repairing the damaged tissue not only by direct differentiation process but also indirectly through the secretion of their bioactive (trophic) factor [6]. In case of any tissue damage, the stem cells can be attracted to the damage site wherein they secrete bioactive factors

In this chapter, we discuss about the (1) role of the stem cells in bone regeneration and their trophic factors and (2) the influence of stem cell microenvironment on the secretion of trophic factors and their effects on bone regeneration. The stem cell-based therapies using trophic

Bone regenerates through complex and organized biological events of bone induction and conduction. This process involves a number of cell types and molecular signalling pathways in a defined sequence to maximize the repair and regeneration of the skeletal tissue. The organic matrix of the bone tissue is composed of collagen type I fibres (approximately 95%) proteoglycans and numerous non-collagenous proteins (5%) [7]. Non-collagenous proteins participate in the process of matrix maturation, mineralization and may regulate the func‐ tional activity of bone cells. Primarily, the functional integrity of bone tissue is maintained by the cell types such as osteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells).

that can function to trophically assist the repair and regeneration process.

factors may have profound clinical applications.

**2. Stem cells and bone regeneration**

pies for bone repair.

358 Advanced Techniques in Bone Regeneration

Numerous studies in animal models clearly demonstrate that stem cells have the potential to treat critical-sized segmental defects, mandibular defects and effective spinal fusion to name a few. More recently, Liu et al. showed that systemic injection of MSCs into mandibular defects of dogs can increase new bone formation as compared to the defect without any cells [11]. Some clinical reports also suggest that MSCs and ADSCs can be used for treating fractures of the distal tibia, osteonecrosis ofthe femoral head and maxillary defects [12–16]. Although these results are promising, the efficacy of translating these outcomes into clinical practice at a large scale is still in infancy.

In addition to the cell-based therapies, stem cells are combined with biomaterials and im‐ planted into the defect site and this tissue engineering approach is considered to be a prom‐ ising strategy to treat bone defects. Numerous small animal studies have shown that treating the bone defects with a combination of biomaterials and MSCs can augment bone regenera‐ tion. Human bone marrow MSCs and macroporous calcium phosphate cement were com‐ bined and transplanted into critical-sized cranial defects in rats. The constructs generated much more new bone and blood vessels than the control calcium phosphate cement without cells [16]. Porous tantalum rods were implanted with or without autologous bone marrow stromal cells (BMSCs) on hind legs in dogs and the scaffold combined with cells enhanced new bone formation after 12 weeks of implantation [17]. These studies indicate that the combina‐ tion of scaffolds with stem cells can enhance bone regeneration to greater extent. Likewise, composite scaffolds consisting of polycaprolactone and tricalcium phosphate (TCP) com‐ bined with autologous MSCs orrecombinant human bone morphogenetic protein 7 was trans‐ planted into critical-sized defects of the long bones of the sheep. The composite scaffold loaded with growth factor and MSCs was able to induce enhanced bone formation, indicating the importance of soluble factor in effective bone regeneration [18]. The osteogenic capability of ADSCs cells in healing critical-size mouse calvarial defects showed that implantation of apa‐ tite-coated poly lactic-co-glycolic acid scaffolds seeded with ADSCs can heal critical-size skel‐ etal defects without genetic manipulation orthe addition of exogenous growth factors [19, 20]. These animal studies with a combination of scaffolds and stem cells have shown great prom‐ ise with excellent bone regeneration capabilities; however, translation of these into clinical use is limited. A study by Kawate et al. used a tissue engineering approach and transplanted β-TCP with MSCs and a free vascularized fibula into three young patients with steroid-in‐ duced osteonecrosis of the femoral head. Two out of the three patients showed healing of the defect with new bone formation and vascularization within 27 months of implantation. Although these studies with a tissue engineering approach was promising, problems still persist in terms of validating the source of the stem cells, the safety, the cost involved and more importantly understanding the molecular mechanisms involved and these questions has to be addressed before any clinical application can be achieved [21].
