**2. Physiological process of bone regeneration**

ical disorders, congenital disorders or abnormal skeletal development are the major causes of

One of the goals of treating a bone defect is to restore the normal morphology and function of the affected structure. Specific surgical techniques such as distraction osteogenesis, implanta‐ tion of biomaterials (bone substitutes) and implants of bone grafting have been developed to reach bone regeneration [1, 2]. Demand for bone grafts is considerable and represents the second most common procedure after blood transplants, with more than 2.2 million bone grafts

Despite advances in bone regeneration and the availability of many treatments, most clinicians and researchers continue to come to the same conclusion: autologous bone grafting remains the "gold standard," compared to other reconstructive procedures [4–9]. Bone from the same patient lacks immunogenicity and contains all the elements necessary to effectively induce tissue regeneration. It has osteoprogenitor cells which go directly to the implant site, cytokines and extracellular matrix [5], providing the three classic elements of an ideal bone graft: osteogenesis, osteoconduction and osteoinduction [5–7, 9, 10]. However, autologous bone grafts have several important limitations, including high risk of morbidity in the donor site [5, 6, 11], with disadvantages in terms of costs, time of surgical procedure, discomfort for the

Additionally, many times the volume of tissue available for the procedure is not sufficient to fill or cover a defect, given the limited availability of autologous tissue [4, 10], and the quality of the autograft is highly variable and is influenced by age and metabolic abnormalities of the patient [7]. To overcome these limitations, a variety of exogenous substitutes, including allografts, xenografts and alloplastic materials, have been introduced into clinical practice in the past three decades [4]. However, these substitutes have less osteogenic and osteoinductive properties [6, 12] and a greater possibility of transmission of infectious diseases [6, 8], restrict‐

In order to successfully overcome the shortcomings of current approaches for bone regenera‐ tion, tissue engineering emerged as a discipline that provides the necessary tools for bone regeneration and restoration. The presence of cell populations that orchestrate the release of growth factors, the maintenance of a stable matrix and the stimulation of angiogenesis are key factors to successful regeneration of bone tissue, because they play a decisive role in the healing process [13, 14]. The technologies developed recently based on tissue engineering, such as gene therapy, stem cell therapy and the application of osteoinductive growth factors, looking for the control of the dynamics of these elements to enable more predictable bone regeneration

Cell-based therapy for the regeneration of bone tissue has been extensively investigated. Several cell types have been used as alternatives for the reconstruction of bone tissue, including osteoblasts, embryonic stem cells, periostium derived-progenitor cells (a specialized cell type that covers bone surfaces and have the potential to differentiate into multiple mesenchymal tissues, including bone) and mesenchymal stem cells, also known as multipotential stromal

functional disability, and esthetic and psychological trauma for patients.

performed annually worldwide in orthopedics and dentistry [3].

patient and possible complications.

254 Advanced Techniques in Bone Regeneration

surge as a significant promise in clinical practice [15].

ing their use [8].

cells (MSC) [16].

For the development of new therapeutic tools for restoring bone defects exceeding the critical size, it is necessary to look at the prototype model of physiological bone regeneration. This process, involving a coordinated interaction of cells, growth factors and extracellular matrix, consists of multiple and well-orchestrated stages that start immediately after the injury occurs, with a local inflammatory response followed by the mobilization of hematopoietic and mesenchymal stem cells to the site of injury to form new vascular networks, soft tissue matrix, cartilage and/or bone and finally inducing mature bone formation [22–24]. All four compo‐ nents involved in the site of injury, including cortical bone, periosteum, bone marrow and external soft tissue, contribute to a different extent in the healing process, depending on various parameters such as growth factors, hormones and nutrients, pH, oxygen tension, the electrical environment and mechanical stability [25].

Immediately after bone trauma, damage of the local vasculature at the site of injury is respon‐ sible for producing a blood clot or hematoma [24, 26, 27]. This hematoma is a localized collection of blood products, including platelets, leukocytes, macrophages, fibrin, soluble growth factors and cytokines, which in turn provides a matrix that allows the migration of inflammatory cells, endothelial cells and fibroblasts [24] (**Figure 1**).

This first stage of fracture healing is the beginning of the so-called **inflammatory phase**, which begins within the first 12 to 14 hours, has its peak during the first 24 hours and is completed around 7 days after the injury. It is characterized by a destructive phase, with a local acute inflammatory response and hypoxia. The first cells to arrive at the site of injury are neutrophils, and subsequently macrophages and lymphocytes. Macrophages not only phagocyte necrotic tissue but also release a number of growth factors and cytokines that initiate the healing process of bone wound [26] (**Figure 1**).

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

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, osteoinduction and osteoconduction are: PDGF, BMPs [15, 28, 29], IGFs [28, 30], TGF-β [15, 28], FGF [24, 29] and VEGF [15, 24, 29].

Local vascularization at the site of injury has been identified as one of the most important parameters that influence the healing process [14, 31, 32]. Bone formation can only proceed successfully if the tissue is adequately vascularized [15]; therefore, angiogenesis is a key component in bone repair. The new blood vessels carry oxygen and nutrients to the metabol‐ ically active callus, allowing gas exchange and the output of waste products and serve as a route for inflammatory cells, and cartilage and bone precursor cells [33, 34], and also provide the gateway of systemically circulating factors that can modify the bone healing process [34]. Vascularization is needed for both the formation of intramembranous and endochondral bone. During the formation of endochondral bone, cartilage avascular environment is invaded by blood vessels that allow the osteoblastic, chondroblastic and progenitor cells, to deposit new bone on the surface of the islands of cartilage. During intramembranous ossification, vascu‐ larization is also needed to allow the arrival of osteoblast precursor cells [34].

Angiogenesis and migration of vascular endothelial cells are stimulated by pro-angiogenic factors such as VEGF, BMPs, TGF-β, FGF and angiopoietins (especially angiopoyectina I and II) [26].

Finally, over the course of months to years, the third stage, the **remodeling phase** of bone healing occurs, whose main objective is to reshape the bone in order to restore its original structure and strength. During this phase, osteoclasts reabsorb recently formed bone tissue, due to the stimulation of growth factors and cytokines that promote osteoclastogenesis as TNFα, TGF and BMPs. Osteoblasts deposit more osteoid and calcium phosphate in the newly regenerated bone, increasing the density of mineralized matrix. Therefore, the transverse diameter of the bone decreases but the density of internal structure increases, closer and closer to the architecture of the intact bone. As this stage keeps going, cellularity is gradually reduced and bone density is enhanced [26].
