**4. Regenerative therapies**

Clinical failure of bone tissue is defined as a discontinuity of the integrity of bone resulting from trauma, congenital malformation or surgical recession. Particularly, bone deficiency "critical size" is the bone defect that cannot regenerate spontaneously during the lifespan of the patient and, therefore, requires a surgical intervention for recovery [23].

The processes that drive the biology and biomechanics of bone regeneration remain largely unknown. During regeneration of bone tissue, many highly complex interactions between multiple cell types are mediated by soluble and insoluble factors and they have not been sufficiently characterized. The challenge for tissue engineering and regenerative medicine is to rebuild the regenerative healing process of bone tissue and then join the components to produce osteoangiogenic and, therefore, osteoregenerative therapies that fulfill the biome‐ chanical parameters for the healing of a bone defect that exceeds the critical size.

We must remember that the bone has an inherent capacity for regeneration, so it is important to not only design therapies that do not interfere with the natural regenerative processes but also complement them and work synergistically with the endogenous bone healing process.

Regenerative therapy of bone tissue should include the three essential elements of bone regeneration: osteogenesis, osteoinduction and osteoconduction. Osteogenesis refers to the ability to produce new bone by bone-forming cells. Osteoinduction is the process whereby the presence of biological mediators stimulates the recruitment of mesenchymal stem cells to the wound site and their subsequent differentiation into mature bone cells, and osteoconduction is the physical property of providing a matrix facilitating the invasion of blood vessels and the new bone formation [48, 49].

Based on these fundamental principles, the main goal of regenerative medicine in clinical treatment is to reduce surgical morbidity by applying biological signals or cellular components that allow the reconstruction and restoration of lost tissue without autologous tissue transfer.

#### **4.1. Mesenchymal stem cells-based therapy**

Bone cell-based therapies seek to create viable tissue equivalents, providing live and meta‐ bolically active cells to repair the site of injury by continuous synthesis of bone matrix [50]. Mesenchymal stem cells are the center of a multitude of clinical studies currently underway (http://clinicaltrials.gov) [51]. Scientific evidence shows that they are one of the best choices in cell therapy, because of their ease of access and isolation, great potential of expansion in culture, immunosuppressive properties, paracrine effect and ability to migrate to injured tissues [52]. Moreover, their great therapeutic potential has been documented in the repair and regeneration of injured tissues in nearly every organ of the body, including the heart [53], immune system [54], liver [55], kidneys [56] and bone and cartilage tissue [57].

Mesenchymal stem cells are defined as pluripotent cells capable of self-renewal and differen‐ tiation into various specialized types of mesenchymal cells, such as osteoblasts, chondrocytes, adipocytes, myocytes, fibroblasts [52, 58–61]. MSCs are a group cells that have been isolated from virtually every vascularized tissue [62]. MSCs are a group cells that have been isolated from virtually every vascularized tissue [52]; however, recent reports have documented that they can also be isolated from other sources as umbilical cord [62], peripheral blood [63], adipose tissue [64–67], hair follicle [68], periodontal ligament [69–72], gingival tissue [73] and dental pulp [74, 75], among others.

MSCs, for its ability to differentiate to multiple lineages, specifically, their osteogenic potential and their immunomodulatory, anti-inflammatory and anti-apoptotic properties, have become a major tool in cell therapy for the regenerative treatment of pathologies affecting functionally bone tissue [76–79]. In vitro analyzes show that MSCs induced by osteogenic differentiation medium increase the expression of osteogenic differentiation markers such as alkaline phosphatase, osteocalcin, osteopontin, bone sialoprotein and calcium deposits in the extrac‐ ellular matrix. The progress in the study of the biology of bone tissue and the isolation and in vitro cultivation of MSCs opened the possibility of studying the molecular and biological mechanisms of bone regeneration, making significant progress, as evidenced by the more two thousand publications of experimental reports on the application of MSCs in bone defects in animal models promoting bone regeneration, and the more than five hundred clinical trials currently registered on the NIH clinical trials website (http://clinicaltrials.gov) [51].

#### *4.1.1. MSCs mechanism of action*

**3.5. Insulin-like growth factors (IGF)**

262 Advanced Techniques in Bone Regeneration

**4. Regenerative therapies**

new bone formation [48, 49].

**4.1. Mesenchymal stem cells-based therapy**

IGF-1 and -2 play a critical role in stimulation of organogenesis and growth during the first stages of embryogenesis as well as in regulating the functions of specific tissues and organs in later stages of development [47]. The sources of IGF-1 and IGF-2 are the bone matrix, endo‐ thelial cells, osteoblasts and chondrocytes [25]. IGF-1 promotes bone matrix formation (type I collagen and non-collagenous matrix proteins) by fully differentiated osteoblasts and is more potent than IGF-2 [45]. IGF-2 acts at a later stage of endochondral bone formation and stimulates type I collagen production, cartilage matrix synthesis and cellular proliferation [25].

Clinical failure of bone tissue is defined as a discontinuity of the integrity of bone resulting from trauma, congenital malformation or surgical recession. Particularly, bone deficiency "critical size" is the bone defect that cannot regenerate spontaneously during the lifespan of

The processes that drive the biology and biomechanics of bone regeneration remain largely unknown. During regeneration of bone tissue, many highly complex interactions between multiple cell types are mediated by soluble and insoluble factors and they have not been sufficiently characterized. The challenge for tissue engineering and regenerative medicine is to rebuild the regenerative healing process of bone tissue and then join the components to produce osteoangiogenic and, therefore, osteoregenerative therapies that fulfill the biome‐

We must remember that the bone has an inherent capacity for regeneration, so it is important to not only design therapies that do not interfere with the natural regenerative processes but also complement them and work synergistically with the endogenous bone healing process.

Regenerative therapy of bone tissue should include the three essential elements of bone regeneration: osteogenesis, osteoinduction and osteoconduction. Osteogenesis refers to the ability to produce new bone by bone-forming cells. Osteoinduction is the process whereby the presence of biological mediators stimulates the recruitment of mesenchymal stem cells to the wound site and their subsequent differentiation into mature bone cells, and osteoconduction is the physical property of providing a matrix facilitating the invasion of blood vessels and the

Based on these fundamental principles, the main goal of regenerative medicine in clinical treatment is to reduce surgical morbidity by applying biological signals or cellular components that allow the reconstruction and restoration of lost tissue without autologous tissue transfer.

Bone cell-based therapies seek to create viable tissue equivalents, providing live and meta‐ bolically active cells to repair the site of injury by continuous synthesis of bone matrix [50].

the patient and, therefore, requires a surgical intervention for recovery [23].

chanical parameters for the healing of a bone defect that exceeds the critical size.

The mechanisms through which MSCs enhance the bone tissue repair process are complex, since they can participate in the three phases of bone healing: inflammation, proliferation and remodeling [80]. The in vivo identity and location of MSC have been difficult to establish. However, various reports, especially the work of Crisan et al., presented evidence of a relationship between MSCs and perivascular pericytes. Irrespective of their tissue origin, perivascular cells exhibit osteogenic, chondrogenic and adipogenic potentials and express MSC markers [81]. Based on these reports, Caplan suggests that all MSCs are pericytes, which would explain the presence of MSC in all vascularized tissues. When an injury disrupts the normal architecture of the blood vessels, pericytes are activated giving rise to MSCs that then contribute to tissue repair by secreting trophic factors that can control the endogenous inflammatory reaction, promote angiogenesis and stimulate the proliferation and differentia‐ tion of progenitor cells [82] (**Figure 2**).

**Figure 2.** Schematic model of the MSCs' paracrine effect on tissue regeneration.

As mentioned before, a growing number of recent reports in the literature have revealed that even if a therapeutic effect can be observed, the implanted MSC cells do not differentiate and do not survive for a long time. For example, in an animal model of acute myocardial infarction, it was established that the MSCs implanted do not survive, and only 4.4% of grafted MSC could be found 1–2 weeks after transplantation [17], and MSC transplantation in a model of spinal cord injury in rats revealed that MSCs implanted disappeared from the host after 1–2 weeks [18]. It has been also reported that human adipose tissue-derived MSCs effectively induce bone regeneration in rabbit jaws, but they do not differentiate and do not survive more than 12 days in the site of implantation [21]. Recent reports have demonstrated that many of the therapeutic effects of MSCs can be mediated by the secretion of trophic factors, opening the possibility that direct administration of these mediators may replace the use of the cells in some instances [57]. This implies a shift from a paradigm centered on cell differentiation to a new vision where the MSCs can have a therapeutic effect even if they are not grafted or differentiated into specific tissue cells, which significantly increases the options of MSC therapeutic applications. According to this concept, Caplan has proposed that the most important feature of the MSC which determines its therapeutic potential is not their stemness but the ability to secrete a large number of trophic factors, and he has proposed that their name to be changed to medicinal signaling cells, keeping the same MSC acronym [83].

Caplan also proposes a model whereby MSCs exert their therapeutic action at the site of the injury by two different activities: from the front of the cells, away from the area of injury, MSCs create a curtain, by the production of bioactive molecules that control local inflammation and prevent autoimmune reactions. From the back of the MSC, they produce molecules that: (1) stop scar formation, (2) inhibit cell apoptosis due to ischemia, (3) stimulate the formation and stabilization of blood vessels and (4) secrete trophic factors that induce the replication of endogenous tissue progenitors [84] (**Figure 3**).

**Figure 3.** Schematic diagram illustrating the concept of application of MSC conditioned media in bone injuries. The MSC secretome, containing chemokines and growth factors, promote the recruitment of endogenous osteogenic cells and stimulate their migration to injured sites, inducing their differentiation and bone formation.

#### **4.2. Secretome as a therapeutic strategy: conditioned media**

**Figure 2.** Schematic model of the MSCs' paracrine effect on tissue regeneration.

264 Advanced Techniques in Bone Regeneration

As mentioned before, a growing number of recent reports in the literature have revealed that even if a therapeutic effect can be observed, the implanted MSC cells do not differentiate and do not survive for a long time. For example, in an animal model of acute myocardial infarction, it was established that the MSCs implanted do not survive, and only 4.4% of grafted MSC could be found 1–2 weeks after transplantation [17], and MSC transplantation in a model of spinal cord injury in rats revealed that MSCs implanted disappeared from the host after 1–2 weeks [18]. It has been also reported that human adipose tissue-derived MSCs effectively induce bone regeneration in rabbit jaws, but they do not differentiate and do not survive more than 12 days in the site of implantation [21]. Recent reports have demonstrated that many of the therapeutic effects of MSCs can be mediated by the secretion of trophic factors, opening the possibility that direct administration of these mediators may replace the use of the cells in some instances [57]. This implies a shift from a paradigm centered on cell differentiation to a new vision where the MSCs can have a therapeutic effect even if they are not grafted or differentiated into specific tissue cells, which significantly increases the options of MSC therapeutic applications. According to this concept, Caplan has proposed that the most important feature of the MSC which determines its therapeutic potential is not their stemness

The broad spectrum of factors secreted by the different types of MSCs is generally referred as MSC secretome. Recent data demonstrate that MSC secretome factors, collected as conditioned media (CM), are sufficient to exert the MSC therapeutic effects.

Previous studies have reported many growth factors and cytokines derived from the CM of various stem cells [19–21, 85–89], which could be responsible for the paracrine protective effects of stem cells against several diseases. Consequently, the use of stem cells CM instead of direct implantation of stem cells may be a feasible approach to overcome the limitations of current cell-based therapy. In addition, because CM is not a cell, but a conjugate of many growth factors, the administration of CM has no ethics concerns related with cell therapies.

However, secretomic signatures of the various types of MSC are not completely known, and the qualitative and quantitative characterization of MSC secretomes and their functions in secretome-mediated repair will contribute to the development of new regenerative therapies that will not require cell transplants [90].

Recently, the great potential of tissue engineering and regenerative medicine strategies for bone augmentation has been demonstrated, and the feasibility of using CM from MSC as an osteoinductive agent for future clinical use is becoming more evident. CM from bone marrow-MSC increased the migration and proliferation of MSCs, vascularization and the early bone regeneration in rabbit sinus model, showing CM as a promising novel therapeutic agent to promote bone regeneration after maxillary sinus floor elevation [91]. It has been shown that CM can have stronger effects than MSCs, accelerating the mobilization of endogenous endothelial and MSC cells for bone regeneration in rat calvarial bone defect model [92]. Intravenous administration of MSC-CM provided the protection of osteoblasts and osteoclasts, induced angiogenesis, anti-apoptotic and anti-inflammatory effects in a rat bisphosphonaterelated osteonecrosis of the jaw-like model [93]. It has also been reported that the use of MSC-CM may be an alternative therapy for periodontal tissue regeneration [94]. CM from human MSC accelerates the formation of new bone callus, shortening the time period required for distraction osteogenesis treatment in a mouse model by recruiting endogenous mouse bone marrow stem cells (mBMSCs) and EC/EPCs via MCP-1/-3 and IL-3/-6 signaling [95].

We have also reported that human Ad-MSCs and their CM induce bone regeneration in a jaw rabbit model, and that morphometric, radiographic and histological analysis demonstrate that the amount and quality of neoformed bone, repaired area, bone density, arrangement of collagen fibers, maturation and inorganic matrix calcification are very similar between Ad-MSC and CM-treated groups [21] (**Figure 3**).
