**2. Cells in bone engineering**

#### **2.1. MSCs as the best choice**

**1. Introduction**

224 Advanced Techniques in Bone Regeneration

artificial muscles.

alone [1].

bone structure, physiology and development.

Tissue engineering is a multidisciplinary field that applies basic concepts and techniques of life science and engineering. It is generally understood as a process of taking human or animal tissues, isolating cells from the tissues, culturing the isolated cells in a supporting material, i.e., scaffold to fabricate cell-scaffold complex, and transplanting the fabricated cell-scaffold complex into human or animal subject. It is applied to fabricate almost every human organ including artificial skin, artificial bone, artificial cartilage, artificial cornea, artificial blood vessels and

Bone is one of the few organs/tissues capable of spontaneous regeneration rather than simple repair. In other words, after disruption of its structure (fracture), its unique microanatomy and biological properties enable complete structural restoration without the creation of fibrotic scar tissue. However, in certain clinical situations where extensive injury, disease or malformation cause such large defects, it is necessary to resort to bone reconstruction, restoration and/or regeneration by a surgical procedure that replaces missing bone, i.e., by bone grafting. A bone graft is an implanted material that promotes bone healing alone or in combination with other material(s), through osteogenesis, osteoinduction and osteoconduction, in combination or

The selection of an ideal bone graft relies on several factors such as tissue viability, defect size, graft size, shape and volume, biomechanical characteristics, graft handling, cost, ethical issues, biological characteristics and associated complications. The materials used in bone grafting can be divided into several major categories, including autografts, allografts, and xenografts. Synthetic and biologically based, tissue-engineered biomaterials and combinations of these substitutes are other options. Altogether, tissue-engineered bone graft requires the optimal selection of cells that are seeded on biomaterial-based scaffolds and exposed to specific biochemical and physical signals known to induce osteogenesis. The development of the successful bone tissue-engineering protocols depends very much on our understanding of

Bone is a dynamic biological tissue consisting of metabolically active cells. The cell component of bone consists of the precursor cells (progenitors), osteoblasts, osteoclasts, osteocytes and bone marrow hematopoietic elements. Osteoblasts are metabolically active mature boneforming cells. They secrete osteoid, non-mineralized organic corpuscle that in turn undergoes mineralization process. Osteocytes are mature osteoblasts trapped within the bone matrix. Every osteocyte extends network of cytoplasmic tubules to the blood vessels and other cells. These cells are involved in the control of extracellular calcium. Osteoclasts are large multi‐ nucleated cells that degrade bone. Beside cells, bone is also composed of organic and inorganic elements. Approximately 20% of the weight of bone is water until the weight of dry inorganic bone makes calcium phosphate (65–70%) and the organic matrix of fibrous proteins and collagen (30–35%). Bone formation models in vitro are based on the fact that cell differentiation and function can be modelled according to factors that are important for embryonic develop‐ ment. Stem cells represent the building blocks of our bodies, functioning as the natural units of embryonic generation during development and adult regeneration following tissue

The important step in bone engineering is the choice of human cell sources that can efficiently produce bone grafts when attached to proper mechanical support with the addition of osteogenic supplements. Cell types that can be potentially used in bone engineering are primary osteogenic cells isolated from adult bone tissue and periosteum, embryonic stem cells (ESCs), induced PSCs (iPSCs) and ASCs.

The selection of appropriate cell source for bone tissue engineering depends on several factors such as:


Among the mentioned candidates, mesenchymal stem cells (MSCs), as a member of the ASC group, currently possess characteristics that make them the most appropriate cell source for bone tissue engineering. Unlike ESCs which are pluripotent and have unlimited potential for proliferation *in vitr*o [5], MSCs possess multilineage differentiation potential and have limited proliferation capacity [6, 7]. In bone tissue engineering, ESCs gained enormous value as a cell source for the derivation of multiple lineages present in adult bone, such as osteogenic cells, vascular cells, osteoclasts, nerve cells and others. Despite increasing interest in the application of ESCs in bone regeneration strategies, use of this cell source is limited due to political issues and ethical concerns as well as safety reasons. The primary concern is the source from which ESCs are derived. The most commonly referenced pluripotent cells are ESCs derived from the inner cell mass of blastocyst which results in destruction of the embryo [8]. It has also become clear that pluripotency is a double-edged sword; the same plasticity that permits hESCs to generate hundreds of different cell types also makes them difficult to control. Transplantation of hESCs into immune-deficient mice leads to the formation of differentiated tumors compris‐ ing all three germ layers, resembling spontaneous human teratomas [9, 10]. Karyotype abnormalities have been observed in ESCs as well as in human iPSCs [11]. Therefore, further studies are needed to ensure the stability and safety of ESC-derived progenitor populations before their potential use in clinical applications. Because these particular cells have created an ethical debate, the researchers have investigated fetal stem cells derived from voluntary interruption of pregnancy as a potential cell source for bone tissue engineering [12]. The cells that have potential medical applications, especially in organ regeneration [13–17], and importantly posess no ethical issues concerning their employment are amniotic stem cells. They are mixture of stem cells that can be obtained from the amniotic fluid [18–20] and the amniotic membrane [21]. They represent a novel class of PSCs with intermediate characteristics between embryonic and ASCs, as they are able to differentiate into lineages representative of all three germ layers but do not form tumours when injected *in vivo* [22]. They can develop into various tissue types including skin, cartilage, cardiac tissue, nerves, muscle and bone [23– 25]. In 2006, Kazutoshi Takahashi and Shinya Yamanaka established for the first time murine ES-like cell lines from mouse embryonic fibroblasts (MEFs) and skin fibroblasts by simply expressing four transcription factor genes encoding Oct4, Sox2, Klf4 and c-Myc [26]. They called these somatic cell-derived cell lines iPSCs. iPSCs exhibit similar morphology and growth properties as ESCs and express ESC-specific genes. The discovery that somatic cells can be reprogrammed into iPS cells has already had major effects on research in stem cell biology and regenerative medicine, but many obstacles remained and need to be resolved to take full advantage of this technology in research and therapy [27]. Therefore, the current clinical protocols are based on the use of autologous MSCs as the cell population that is safe and easy to obtain.

#### **2.2. Sources of human MSCs**

primary osteogenic cells isolated from adult bone tissue and periosteum, embryonic stem cells

The selection of appropriate cell source for bone tissue engineering depends on several factors

**•** Possibility of application of patient's own (autologous) cells or another person's (allogeneic)

**•** Use of cells that have both osteogenic and vasculogenic potential to support the formation

Among the mentioned candidates, mesenchymal stem cells (MSCs), as a member of the ASC group, currently possess characteristics that make them the most appropriate cell source for bone tissue engineering. Unlike ESCs which are pluripotent and have unlimited potential for proliferation *in vitr*o [5], MSCs possess multilineage differentiation potential and have limited proliferation capacity [6, 7]. In bone tissue engineering, ESCs gained enormous value as a cell source for the derivation of multiple lineages present in adult bone, such as osteogenic cells, vascular cells, osteoclasts, nerve cells and others. Despite increasing interest in the application of ESCs in bone regeneration strategies, use of this cell source is limited due to political issues and ethical concerns as well as safety reasons. The primary concern is the source from which ESCs are derived. The most commonly referenced pluripotent cells are ESCs derived from the inner cell mass of blastocyst which results in destruction of the embryo [8]. It has also become clear that pluripotency is a double-edged sword; the same plasticity that permits hESCs to generate hundreds of different cell types also makes them difficult to control. Transplantation of hESCs into immune-deficient mice leads to the formation of differentiated tumors compris‐ ing all three germ layers, resembling spontaneous human teratomas [9, 10]. Karyotype abnormalities have been observed in ESCs as well as in human iPSCs [11]. Therefore, further studies are needed to ensure the stability and safety of ESC-derived progenitor populations before their potential use in clinical applications. Because these particular cells have created an ethical debate, the researchers have investigated fetal stem cells derived from voluntary interruption of pregnancy as a potential cell source for bone tissue engineering [12]. The cells that have potential medical applications, especially in organ regeneration [13–17], and importantly posess no ethical issues concerning their employment are amniotic stem cells. They are mixture of stem cells that can be obtained from the amniotic fluid [18–20] and the

**•** Availability and ease of tissue harvesting with minimal donor site morbidity;

(ESCs), induced PSCs (iPSCs) and ASCs.

226 Advanced Techniques in Bone Regeneration

**•** Efficiency of cell isolation and cell yield;

**•** Homogeneity of the obtained cell population;

**•** Control of induction of osteogenic phenotype;

**•** Automation and good manufacturing practices production.

**•** Phenotype stability and cell safety;

**•** Potential of cell proliferation;

of vascularized bone;

such as:

cells;

Extensive research of adult MSCs started in 1970 when Freidenstein et al. discovered these cells in bone marrow tissue [28]. Later, the presence of MSC-like population was discovered in a wide range of adult tissues, including trabecular bone [29], synovium [30], adipose tissues [31], skeletal muscle [32], periosteum [33], dermis [34], blood [35, 36] deciduous teeth [37], amniotic fluid [38] and umbilical cord blood [39]. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have become one of the main cell sources for bone tissue engineering [40, 41]. Isolation of MSCs from bone marrow requires invasive procedures that can be quite painful. Bone marrow aspirate could be obtained from the iliac crest, tibia or femur. Typically, the frequency of MSCs in whole bone marrow of adults is between 5 × 10−4 and 10−5, which corresponds to yield of a hundred MSCs per milliliter of marrow. Even though BM-MSCs are rare, they are readily separated from the hematopoietic stem cells in culture by their prefer‐ ential attachment to the plastic surface [42] and can be easily expanded *ex vivo*. The presence of MSC in adipose tissue has gained considerable attention because of the ease of accessibility of adipose tissue and its abundance in the body. Adipose tissue-derived mesenchymal stem cells (AD-MSCs) were first identified in 2001 by Zuk et al. A major advantage of AD-MSCs is their relative abundance as well as their faster proliferation rate compared to BM- MSCs, which allows more rapid expansion to obtain clinically relevant cell numbers [43, 44]. AD-MSCs have similar osteogenic potential to BM- MSCs with the added advantage of being highly abundant. For example, as many as 1 × 107 AD-MSCs can routinely be isolated from 300 ml of lipoaspirate, with purity greater than 95% [45, 46]. Comparative analysis of human BM-MSCs and AD-MSCs by Li et al. revealed that AD-MSCs have biological advantages in the proliferative capacity, secreted proteins (basic fibroblast growth factor, interferon-γ and insulin-like growth factor-1) and immunomodulatory effects, but BM-MSCs have advantages in osteogenic and chondrogenic differentiation potential and secreted proteins (stem cell-derived factor-1 and hepatocyte growth factor) [47]. These biological advantages should be considered systemati‐ cally when choosing the MSC source for specific clinical application. Nevertheless, the utilization of human AD-MSCs in scaffolds for bone tissue engineering has been heralded as the alternative strategy of the twenty-first century to replace or restore the function of traumatized, damaged or lost bone.

MSC-like cells can be derived from the umbilical cord from a newborn baby which contains two arteries and a vein covered with mucus connective tissue rich in hyaluronic acid, referred to as a Wharton's jelly [48]. The blood from the umbilical cord is a rich source for pluripotent cells named as umbilical cord blood derived MSCs (UCB-MSCs). These cells are quite similar to bone marrow-derived MSCs and have osteogenic potential in an optimized culture [49]. Many investigations have thus far been conducted on bone engineering by using these cells and various scaffolds [50].

Several stem cell types in dental tissue have been reported including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), stem cells of the apical papilla (SCAP), periodontal ligament stem cells (PDLSCs) and dental follicle progenitor cells (DFPCs) [37, 51]. Since DPSCs can be easily isolated by enzymatic digestion of pulp tissue, many studies have been conducted regarding bone engineering with these cells and appro‐ priate 3D scaffolds [52, 53].

#### **2.3. Phenotypic characterization of MSCs**

Phenotypic characterization of MSCs is usually carried out using immunocytochemical detection or fluorescence-activated cell sorting (FACS) analysis of cell surface molecule expression [54, 55]. Methods of immunodepletion using such techniques as magnet-activated cell sorting (MACS) have also been used in the negative selection of MSCs [56]. However, the lack of specific markers renders the characterization of MSCs difficult and sometimes ambig‐ uous, especially because many of these epitopes are shared between hematopoietic and mesenchymal stem cells. It is interesting that MSCs from different species do not express the same markers. The use of multiple markers such as cell surface cluster of differentiation (CD) markers, ECM proteins, cell adhesion molecules, integrins, cytokines genetic and proteomic fingerprinting can help identify MSCs. The most commonly used markers to identify MSCs are CD markers. Positive MSC markers include: Stro-1, SH2 (CD105), SH3 (CD73), SH4, CD29, CD44, CD54, CD90, CD133, CD166 and p75LNGFR, whereas negative markers are CD11, Cd14, CD19, Cd31, CD34, Cd45, CD79 and HLA-DR [57]. The International Society for Cellular therapy has provided minimum criteria for defining MSCs. Acceptable MSCs meet the minimum requirements of CD73, CD90 and CD105 positive and CD14, CD34, CD45 and HLA-DR negative expression [58].

#### **2.4. Nonimmunogenic properties and immunosuppressive nature of MSCs**

Previous studies have shown that yield of MSCs is affected by age and health of a donor. The trend is that yield is decreased with donor age. Patients with degenerative diseases, such as osteoporosis and osteoarthritis, tend to have lower MSC yield although they would benefit the most from MSC-based therapies. The alternative is the use of allogeneic MSCs because they have low immunogenic potential and immunosuppressive properties. Immunologic pheno‐ types of hMSCs are: positive expression for major histocompatibility complex (MHC) class I molecules, minimal expression for MHC class II and do not express the co-stimulatory molecules CD40, CD40 ligand, CD80 and CD86 [59–62]. MSCs do not fully activate T cells owing to the absence of CD80 and CD86 in their membrane. Apart from not being recognized as alloantigens, MSCs are able to suppress the activation and proliferation of different cells of the host immune system [59, 63–66]. Interleukin-10, transforming growth factor beta (TGF-β), hematopoietic growth factor (HGF), prostaglandin E2 (PGE2), indoleamine 2,3-dioxygenase (IDO) and nitric oxide (NO) were some of the soluble molecules associated with the immu‐ nosuppressive effect of MSCs [67–69]. Another important soluble molecule involved in the immunoregulation of MSCs is HLA-G5, a non-classical human leukocyte antigen (HLA) class I protein that protects the fetus against rejection from the maternal immune system [70]. The HLA-G5 isoform released by MSCs can suppress allogeneic T cell proliferation and can also induce the expansion of CD4+ CD25highFOXP3<sup>+</sup> regulatory T cells (Tregs). With regard to innate immunity, HLA-G5 is able to inhibit the lysis of MSCs mediated by NK cells, as well as the secretion of IFN-γ by these cells [71].

#### **2.5. Tumor formation risk in MSCs application**

the alternative strategy of the twenty-first century to replace or restore the function of

MSC-like cells can be derived from the umbilical cord from a newborn baby which contains two arteries and a vein covered with mucus connective tissue rich in hyaluronic acid, referred to as a Wharton's jelly [48]. The blood from the umbilical cord is a rich source for pluripotent cells named as umbilical cord blood derived MSCs (UCB-MSCs). These cells are quite similar to bone marrow-derived MSCs and have osteogenic potential in an optimized culture [49]. Many investigations have thus far been conducted on bone engineering by using these cells

Several stem cell types in dental tissue have been reported including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), stem cells of the apical papilla (SCAP), periodontal ligament stem cells (PDLSCs) and dental follicle progenitor cells (DFPCs) [37, 51]. Since DPSCs can be easily isolated by enzymatic digestion of pulp tissue, many studies have been conducted regarding bone engineering with these cells and appro‐

Phenotypic characterization of MSCs is usually carried out using immunocytochemical detection or fluorescence-activated cell sorting (FACS) analysis of cell surface molecule expression [54, 55]. Methods of immunodepletion using such techniques as magnet-activated cell sorting (MACS) have also been used in the negative selection of MSCs [56]. However, the lack of specific markers renders the characterization of MSCs difficult and sometimes ambig‐ uous, especially because many of these epitopes are shared between hematopoietic and mesenchymal stem cells. It is interesting that MSCs from different species do not express the same markers. The use of multiple markers such as cell surface cluster of differentiation (CD) markers, ECM proteins, cell adhesion molecules, integrins, cytokines genetic and proteomic fingerprinting can help identify MSCs. The most commonly used markers to identify MSCs are CD markers. Positive MSC markers include: Stro-1, SH2 (CD105), SH3 (CD73), SH4, CD29, CD44, CD54, CD90, CD133, CD166 and p75LNGFR, whereas negative markers are CD11, Cd14, CD19, Cd31, CD34, Cd45, CD79 and HLA-DR [57]. The International Society for Cellular therapy has provided minimum criteria for defining MSCs. Acceptable MSCs meet the minimum requirements of CD73, CD90 and CD105 positive and CD14, CD34, CD45 and HLA-

**2.4. Nonimmunogenic properties and immunosuppressive nature of MSCs**

Previous studies have shown that yield of MSCs is affected by age and health of a donor. The trend is that yield is decreased with donor age. Patients with degenerative diseases, such as osteoporosis and osteoarthritis, tend to have lower MSC yield although they would benefit the most from MSC-based therapies. The alternative is the use of allogeneic MSCs because they have low immunogenic potential and immunosuppressive properties. Immunologic pheno‐ types of hMSCs are: positive expression for major histocompatibility complex (MHC) class I

traumatized, damaged or lost bone.

228 Advanced Techniques in Bone Regeneration

and various scaffolds [50].

priate 3D scaffolds [52, 53].

DR negative expression [58].

**2.3. Phenotypic characterization of MSCs**

In general, it is believed that MSCs can be safely cultured *in vitro* without risk of spontaneous malignant transformation [72], but there have been no reports of human trials demonstrating the formation of tumors with culture-expanded MSCs [73]. Concerns have been raised about the safety of MSCs for clinical use as there have been some reports of sarcoma formation by cultured murine MSCs *in vitro* and *in vivo* [74, 75]. The mechanism by which MSCs are transformed into malignant cells is known to be related to chromosomal abnormalities, including structural and numeric aberrations, and increases with higher passage numbers. Rubio et al. showed that although MSCs can be managed safely during the standard *ex vivo* expansion period (6–8 weeks), human MSCs can undergo spontaneous transformation following long-term *in vitro* culture (4–5 months), and the transformed cells lead to the formation of tumors in mice [76].

#### **2.6. Osteogenic differentiation of MSCs**

Various *in vitro* protocols have been developed to induce hMSCs to differentiate into meso‐ dermal lineages, such as osteoblasts, chondrocytes, adipocytes, as well as transdifferentiate into tissue cells derived from different germ layers, such as neuronal cells or insulin-producing cells [55, 77, 78]. Osteogenic differentiation is a highly programmed process consisting of many stages, including proliferation, differentiation, matrix deposition, mineralization and matrix maturation. The general protocol for *in vitro* bone differentiation of MSCs involves incubation of the cell monolayer in a culture medium containing DEX, β-glycerophosphate and ascorbic acid for a period of 2–3 weeks (**Figure 1**) [79]. DEX is a synthetic glucocorticoid that stimulates MSC proliferation and is essential for osteogenic differentiation [80, 81]. Although the mechanisms underlying DEX's effects are not well known, it has been speculated that DEX upregulates the beta catenin-like molecule TAZ, which results in upregulation of Runx2 related transcription factor and osteogenic differentiation [82]. The optimal concentration of this reagent for MSC bone differentiation is approximately 10 nM, which corresponds to physiologic concentrations [83]. Organic phosphate released after enzymatic hydrolysis of beta glycerol phosphate plays an important role in matrix mineralization. This free phosphate is usually applied in 5–10 mM concentrations for MSC bone differentiation. Ascorbic acid is a cofactor in the hydroxylation of prolines and lysine moiety of collagen molecules and is an abundant protein in the ECM. This reagent is used in 50–500 μM concentrations [84]. When MSCs are cultured in osteogenic media, they express the same markers as bone-forming osteoblasts that are responsible for laying down the matrix and mineral during new bone formation *in vivo*. The osteogenic differentiation of MSCs *in vitro* has been divided into three stages. The first stage (days 1–4) is the proliferation stage where a peak in the number of cells is seen. This is followed by early cell differentiation (from days 5 to 14), which is characterized by the transcription and protein expression of alkaline phosphatase (ALP). After this initial peak of ALP, its level starts to decline. Also found at an early stage is the expression of a collagen type I matrix onto which the mineral is deposited. The final stage (from days 14 to 28) results in a high expression of osteocalcin and osteopontin, followed by calcium and phosphate deposition [4, 85].

**Figure 1.** Monolayer of mesenchymal stem cells derived from human bone marrow before (A) and after 3 weeks of (B) differentiation. Arrows mark bone-forming nodules.

In addition to osteogenic supplements, there are other substances that act as biochemical signals capable of triggering cellular processes like growth, proliferation or differentiation. The most common growth factors in bone tissue engineering are listed below.

*Bone morphogenetic proteins* (*BMPs*) are a family of cytokines that stimulates the proliferation of chondrocytes and osteoblasts and increases extracellular matrix production. BMPs induce the differentiation of MSCs into osteoblasts. BMPs allow not only skeletal tissue formation during embryogenesis, growth, and adulthood but also bone healing process. In newborns' skeletons, BMPs can be found in the collagen fibers of the bone matrix and also in cells located in the periosteum and the bone marrow. After a fracture, BMPs' growth factors diffuse from bone matrix and activate osteoprogenitor cells which, in turn, produce more BMPs [86].

*Fibroblast growth factors* (*FGFs*) stimulate the proliferation of mesenchymal cells, osteoblasts and chondrocytes. FGFs enhance the growth of different tissues due to their angiogenic properties. FGF-2 or bFGF is the most studied cytokine of this family for bone regeneration applications [87].

*Insulin*-*like growth factors* (*IGFs*) promote the proliferation of osteoblasts and chondrocytes and induce matrix secretion from both cell types [87]. IGFs stimulate collagen synthesis and mineralization of bone tissue [88].

*Platelet*-*rich plasma* (*PRP*) is another known source of various growth factors, namely, plateletderived growth factor, transforming growth factor-β and vascular endothelial growth factor. The applicability of PRP for the repair of bony defects is well established [89] and several investigators have advocated the use of this product in combination with MSCs [90].
