**3. Stem cells as source**

applications. Therefore, MSCs presently seem to be the most promising candidates for bone regeneration, due to their excellent osteogenic differentiation capacity. They can be isolated from a number of adult mesenchymal tissues, among others, umbilical cord blood, peripheral blood, placenta, synovial fluid, adipose tissue, skeletal muscle or BM, as mentioned, where they contribute to normal tissue turnover and repair. Recently, the multitude of cell surface markers used in various studies has been limited to a marker panel representing, in addition to plastic adherence and differentiation capacity, the minimal criteria for the identification of MSCs. The molecular mechanisms of human MSCs regulation and the importance of specific GFs during the different stages of osteogenic differentiation, as well as the secreted signaling proteins known as *Wnts,* implicated in various differentiation programs including osteogen‐ esis, are subjects of intensive research right now. Several studies have demonstrated improved results of MSCs therapy with genetically modified cells which produce osteogenic and angiogenic GFs in a local delivery of therapy strategy for bone healing. Also, there is recent information about the use of endothelial progenitor cells (EPCs) that improves the treatment

Besides their unique ability to differentiate into different cell types, MSCs secrete a variety of cytokines, showing anti-inflammatory activity and create an anabolic microenvironment. Furthermore, direct cell-cell contact immunomodulation has also been shown. On one hand, they indirectly influence tissue regeneration by secretion of soluble factors. On the other hand, they are able to modulate the inflammatory response. The differentiation potential of MSCs in bone engineering has been extensively studied *in vitro* and *in vivo.* By first time, Urist (1965), and Reddi and Huggins (1972) showed the capacity of a molecule, called bone morphogenetic protein (BMP), with potent osteoinductive properties in healing fractures and bone regenera‐ tion. Their experiments demonstrated the presence of osteoinductive cytokines in bone matrix that have abilities to induce MSCs differentiation into osteoblasts. The GFs, also including transforming growth factor-beta (TGF-β), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF) or insulin-like growth factor (IGF), among others, are delivered of paracrine or autocrine manner, generating a chemotaxis

Extracellular matrix (ECM) is the native scaffold in most tissues. Besides the direct injection in the surrounding tissue, biomaterials are frequently used as carriers for cells, bioactive mole‐ cules and drugs. These materials have to be immune-compatible and nontoxic, whereas the bio-degradation process must neither release toxic substances nor tissue-toxic concentrations of degradation products. Scaffolds must be of three-dimensional structures, with great influence on cell growth and differentiation, and must be highly porous with interconnected pores of a diameter of at least 100 μm to allow ingrowth of cells and vessels. Despite the tissue engineering of bone, for which various inorganic materials, such as HA, calcium phosphate, calcium carbonate (due to their similarity to bone mineral, as well as their inherent biocom‐ patibility and osteoconductivity), or glasses was tested, mainly organic biomaterials have been investigated for scaffold production. These are either naturally derived, for example, collagen, fibrin, agarose, alginate, gelatin, silk or hyaluronic acid; or produced synthetically, such as polyhydroxyacids. Since natural bone consists of an ECM with nanosized apatitic minerals

process toward MSCs by recruitment, which induce their differentiation.

of fracture and bone regeneration.

620 Regenerative Medicine and Tissue Engineering

The popularity of SCs in the clinical arena has significantly increases, given the rapid im‐ provement in our understanding of their biology. Classically, SCs are defined by their capacity to retain an undifferentiated state for a prolonged period while retaining the potential to differentiate along one lineage (unipotent), multiple lineages (multipotent), or into all three germ layers (pluripotent) (Young, 2003). These cells can be broadly categorized into two major classes: embryonic and adult SCs.

Embrionic stem cells (ESCs), isolated from the inner cell mass of the blastocyst, are pluripotent cells with the potential of differentiating into tissues from all three germ layers (Fig. 4) (Buehr et al., 2008).

While ESCs have significant regeneration capacity, their clinical application has been limited as a result of multiple factors including: 1) a propensity to form teratomas, 2) ethical concerns with isolation, 3) rejection by the host immune system after transplantation, and 4) the use of a feeder layer to retain an undifferentiated state *in vitro* (Cho et al., 2010). Recently discovered, another source of pluripotent SCs are induced pluripotent stem (iPS) cells, derived from somatic cells treated with few defined factors (Hamilton et al., 2009). While iPS cell-based therapy has the potential to revolutionize the field of Regenerative Medicine, many obstacles must be overcome before their clinical application can be realized (Lengner et al., 2010).

#### **3.1. Mesenchymal stem cells as candidates**

Furthermore, naturally occurring adult SCs have also been identified and categorized into their hematopoietic stem cells (HSCs), a source of various hematopoietic cell lineages, and nonhe‐ matopietic SCs, which can give rise to cells of mesenchymal origin (de Barros et al., 2010). Many reports have suggested that these nonhematopoietic SCs, also known as mesenchymal stem cells (MSCs), can be isolated from a wide variety of adult tissues such as blood, adipose, skin, mandibule, trabecular bone, fetal blood, liver, lung and even the umbilical cord and placenta

**Figure 4.** Origin of the different types of stem cells available. Derivation of embryonic stem cells (ESCs), embryonic germ cells (EGCs) and adult stem cells (SCs). *Taken from M.E. Arias and R. Felmer 2009, with permission*

(Steinhardt et al., 2008). The wide range of sources, methods, and acronyms are standardized by the International Society for Cellular Therapy in 2005.

Upon harvest, these cells can be expanded *in vitro* with high efficiency without sacrificing differentiation capacity (Kassem, et al., 2004). While these multipotent progenitor cells share many similar characteristics, they can be differentiated based on their expression profile and differentiation propensity along various lineages (Wagner et al., 2005). Amongst the various sources, MSCs isolated from the BM are considered to have the greatest potential for multili‐ neage differentiation and have been the most characterized (Kuznetsov et al., 2009).

MSCs were initially described by Friedenstein and colleagues more than 40 years ago as adherent cells, with a fibroblast-like appearance capable of differentiating *in vitro* into osteoblasts, chondroblasts, adipocytes, and tenocytes (Friedenstein et al., 1968; Alonso et al., 2008; Prockop et al., 2009; Andrades et al., in press (a). Unlike ESCs, MSCs provide the flexibility of autologous transplantation, circumventing ethical concerns or immunological rejection (Igura et al., 2004). These cells also play a sentinel role in proliferation and differen‐ tiation of hematopoietic cells (Briquet et al., 2010). Mankani et al. illustrated that the formation of both hematopoiesis and mature bone organ is correlated with the high local density of MSCs (Mankani et al., 2007). Additionally, MSCs are considered to be immune privileged and have the capacity for allogenic transplantation a property has been used in the clinical setting for the treatment of various autoimmune diseases (Le Blanc et al., 2008). While many studies have suggested that MSCs are immunoprivileged and do not undergo rejection, others have cast doubt on this notion, showing that in certain scenarios, MSCs induce immune rejection (Nauta et al., 2006) (Fig. 5). More investigations should be conducted to provide further insight into the specific interaction between these progenitor cells and the host immune system.

(Steinhardt et al., 2008). The wide range of sources, methods, and acronyms are standardized

**Figure 4.** Origin of the different types of stem cells available. Derivation of embryonic stem cells (ESCs), embryonic

germ cells (EGCs) and adult stem cells (SCs). *Taken from M.E. Arias and R. Felmer 2009, with permission*

Upon harvest, these cells can be expanded *in vitro* with high efficiency without sacrificing differentiation capacity (Kassem, et al., 2004). While these multipotent progenitor cells share many similar characteristics, they can be differentiated based on their expression profile and differentiation propensity along various lineages (Wagner et al., 2005). Amongst the various sources, MSCs isolated from the BM are considered to have the greatest potential for multili‐

MSCs were initially described by Friedenstein and colleagues more than 40 years ago as adherent cells, with a fibroblast-like appearance capable of differentiating *in vitro* into osteoblasts, chondroblasts, adipocytes, and tenocytes (Friedenstein et al., 1968; Alonso et al., 2008; Prockop et al., 2009; Andrades et al., in press (a). Unlike ESCs, MSCs provide the flexibility of autologous transplantation, circumventing ethical concerns or immunological rejection (Igura et al., 2004). These cells also play a sentinel role in proliferation and differen‐ tiation of hematopoietic cells (Briquet et al., 2010). Mankani et al. illustrated that the formation of both hematopoiesis and mature bone organ is correlated with the high local density of MSCs (Mankani et al., 2007). Additionally, MSCs are considered to be immune privileged and have the capacity for allogenic transplantation a property has been used in the clinical setting for the treatment of various autoimmune diseases (Le Blanc et al., 2008). While many studies have suggested that MSCs are immunoprivileged and do not undergo rejection, others have cast doubt on this notion, showing that in certain scenarios, MSCs induce immune rejection (Nauta

neage differentiation and have been the most characterized (Kuznetsov et al., 2009).

by the International Society for Cellular Therapy in 2005.

622 Regenerative Medicine and Tissue Engineering

**Figure 5.** Stem cells participate in tissue regeneration in different ways. They directly differentiate into tissue-specific cells and thus substitute damaged or lost cells (A). They indirectly influence tissue regeneration by secretion of soluble factors. Here they promote vascularization, cell proliferation, differentiation within the tissue (B) and modulate inflam‐ matory processes (C). *Taken from A. Schmitt et al. 2012, with permission*

Considerable effort has been put forth to identify specific surface markers that characterize MSCs, yet disagreement within the literature has prevented the creation of definitive stand‐ ards. The minimal criteria identified by the International Society for Cellular Therapy for identifying MSCs requires the cells: 1) to be plastic adherent while maintained in cell culture; 2) to express CD73, CD90, and CD105, and lack expression of CD11b, CD14, CD19, CD34, CD45, CD79-alpha, and HLA-DR; and 3) to differentiate into osteoblasts, adipocytes, and chondroblasts *in vitro* (Dominici et al., 2006; Claros et al., 2008; 2012). Additional studies have also suggested that CD146 is considered an important marker of BM progenitor cells (Sorrentino et al., 2008). These guidelines were set in place to enable a unified approach for comparison amongst different studies.

BM is generally considered milieu plentiful for various cell types, collectively referred to as stromal cells. Amongst these, the multipotent subset of MSCs comprises a small fraction (<0.01) (Dazzi et al., 2006), yet despite their small numbers, the relative ease with which MSCs can be harvested has propelled their experimental use. Researchers have pioneered the creation of stable animal models aimed at mimicking human conditions to study the therapeutic capacity of these BM-derived cells (Kadiyala et al., 1997). Because of their ubiquity, tolerance of expansion, paracrine capabilities, and multipotency, the potential for clinical applications of MSCs in the orthopaedic realm is countless (Becerra et al., 2011).

The first problem that arises when Cell Therapy methods are used to rebuild bone tissue is how to obtain a sufficiently large number of osteocompetent cells for the intervention to be successful. Hence, the idea of using SCs, which are self-renewing and differentiate into various tissues, surfaced.
