Bone regeneration from mesenchymal stem cells (MSCs) and compact bone-derived MSCs as an animal model

Japanese Dental Science Review (2013) 49, 35—44

Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/jdsr

Review Article

Bone regeneration from mesenchymal stem cells (MSCs) and compact bone-derived MSCs as an animal model Eiki Yamachika a,*, Seiji Iida b a

Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama city 700-8558, Japan b Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Densitry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama city 700-8558, Japan Received 4 September 2012; accepted 11 November 2012

KEYWORDS Bone regeneration; Mesenchymal stem cells

Summary Mesenchymal stem cells (MSCs) have emerged as a promising alternative to traditional surgical techniques. The purpose of this review is to document the current state of research and identify future research directions. At present, no specific markers have been shown to specifically identify MSCs. The most commonly reported positive markers are CD105, CD90, CD44, CD73, CD29, CD13, CD34, CD146, CD106, CD54 and CD166. The most frequently reported negative markers are CD34, CD14, CD45, CD11b, CD49d, CD106, CD10 and CD31. Regarding the source of MSCs, bone marrow-derived MSCs are the most frequently studied MSCs in bone regeneration; however, no reports have demonstrated advantages of bone marrow-derived MSCs over other types of MSCs in bone regeneration. For the purpose of clinical use, serum-free media is recommended to avoid risks connected with the use of animal products. Attempts have been made to develop defined serum-free media for animal and human MSC growth; however, most products have demonstrated only limited performance. Tumorigenesis is the other major problem in MSC regeneration. It is strongly recommended to prepare MSCs for tissue regeneration at early passages to avoid potential chromosomal abnormalities. # 2012 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . Characterization of MSCs . . . . . . . . . . . . . . . . Immunophenotype of MSCs . . . . . . . . . . . . . . Sources of adult mesenchymal stem cells and

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* Corresponding author. Tel.: +81 86 235 6697; fax: +81 86 235 6699. E-mail address: [email protected] (E. Yamachika). 1882-7616/$ — see front matter # 2012 Japanese Association for Dental Science. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdsr.2012.11.003

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36 5. 6. 7. 8. 9. 10.

E. Yamachika, S. Iida Mice compact bone-derived mesenchymal stem cells. Ex vivo isolation and expansion of MSCs. . . . . . . . . . . Transformation of MSCs to bone lineage. . . . . . . . . . . Scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tumor formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The field of tissue engineering has yielded several successes in early clinical trials of therapies involving regenerative medicine and living cells. However, there has been little progress in the development of methods to regenerate bone from living cells. Therefore, autogenous bone grafting remains the gold standard for repairing damaged bone. To address this situation, many groups have focused on the use of multipotent cells, including mesenchymal stem cells (MSCs). MSCs were first recognized more than 40 years ago by Friedenstein et al. who described a population of adherent cells originating from bone marrow that were nonphagocytic, exhibited a fibroblast-like appearance and able to differentiate in vitro into bone, cartilage, adipose tissue, tendons and muscle [1]. In general, MSCs represent a minor fraction of cells in the bone marrow, and their exact frequency is difficult to calculate. However, the frequency of MSCs in human bone marrow has been estimated to be in the order of 0.001—0.01% of total nucleated cells [2]. One of the characteristics of MSCs is their multipotency, defined as the ability to differentiate into several mesenchymal lineages. Usually, trilineage differentiation into bone, adipose and cartilage is taken as the criteria for multipotentiality [2,3]. Because of their ability to repair injured tissues, MSCs are considered to be a tool for regenerative cell therapy [4]. On the other hand, multipotent cells such as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are studied aggressively, and reprogramming methods are considered to be promising tools in regenerative cell therapy. For example, murine cardiac fibroblasts have been reprogrammed into cardiomyocyte-like cells [5], and human non-neural somatic cells have been converted into neurons [6]. However, there are ethical problems associated with the use of human ES cells, and transgenic and reprogramming methods have a perpetual risk of tumorigenesis. Therefore, we believe that MSCs have many advantages over ES and iPS cells for bone regeneration in clinical practice. To succeed in regenerating bone with MSCs, the cells should be extracted, expanded, transformed and implanted successfully. There are many studies of MSCs; however, the information is very confusing. For the extraction of MSCs from the adult body, there are no clear answers regarding which tissues and methods are best for isolating MSCs. In order to expand MSCs, the cells must be cultured; however, there are no clear answers regarding which media and conditions are best. The purpose of this review is to document the available evidence on the use of MSCs for bone regeneration.

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All experiments shown here were performed in accordance with the guidelines of Okayama University regarding the use of laboratory animals.

2. Characterization of MSCs Many scientists believe that putative mesenchymal stem or progenitor cells exist in adult organisms that are founders of fibroblasts, osteoblasts, chondrocytes, adipocytes and smooth muscle cells in vivo. The definitive evidence that bone marrow includes cells that can generate connective tissue-forming cells was originally provided by the pivotal work of Friedenstein et al. [7]. First, these authors, using heterotopic transplantation, demonstrated the existence of a minor population of cells in human bone marrow that are precursors of osteoblasts. The cells were distinguishable from the majority of hematopoietic cells by their rapid adherence to plastic and by their elongated fibroblast-like appearance in culture. Therefore, the authors showed that seeding bone marrow cells at the clonal level results in the formation of colonies initiated by single cells, named colonyforming unit fibroblasts (CFU-Fs). CFU-Fs have since been used as the hallmark for measuring the quality and growth potential of human MSC isolates in vitro. However, in the murine system, CFU-Fs are highly contaminated with hematopoietic cells, at least in early cultures initiated with unfractionated bone marrow [8], making this assay inappropriate as a predictive factor for the quality and growth potential of murine stromal cells. Others have since extended observations supporting the finding that the cells identified by Friedenstein are multipotent. In particular, Pittenger et al. showed that trilineage potential (osteoblast, chondrocyte and adipocyte lineages) clones are present in human bone marrow and provided a substantial description of the cell surface phenotype of these cells [2].

3. Immunophenotype of MSCs Most of the information available on the phenotypic and functional properties of MSCs is derived from studies performed on cells cultured in vitro [3,9,10]. However, at present, no specific markers have been shown to specifically identify MSCs, and little is known about the characteristics of primary precursor cells in vivo since it is not currently possible to isolate the most primitive mesenchymal cells from bulk cultures. One of the hurdles has been the inability to isolate MSCs due to their low frequency and the lack of specific markers. In fact, to date, MSC identification has primarily relied on the adherent properties, immunophenotype (determined with flow cytometry) and differentiation potential of the cells.

Summary of positive cell surface markers of mesenchymal stem cells. CD13

Dominici et al. (1) Wongchuensoontorn et al. (2) Gronthos et al. (3) Tsai et al. (4) Igura et al. (5) Zuk et al. (6) Hasebe et al. (7) Yu et al. (8) Kyurkchiev et al. (9) Royer et al. (10) Orciani et al. (11) Ohgushi et al. (12) Iwata et al. (13) Latif et al. (14) Buhring et al. (15) Zvaifler et al. (16) Simmons et al. (17) Simmons et al. (18) Miura et al. (19) Pittenger et al. (20)

CD29

CD34

CD44

CD54

CD44 CD13 CD13 CD13

CD29 CD29 CD29 CD29

CD13

CD34

CD34

CD29 CD29 CD29

CD44 CD44 CD44 CD44 CD44 CD44

CD44

CD73

CD90

CD105

CD73 CD73

CD90

CD105 CD105

CD54 CD73 CD54 CD73 CD73 CD73 CD73

CD44 CD44

CD13

CD90 CD90 CD90 CD90 CD90 CD90 CD90 CD90 CD90 CD90

CD106

CD166 Human bone marrow-derived, expanded Human bone marrow-derived, primary

CD105 CD105 CD105 CD105 CD105 CD105 CD105 CD105 CD105

CD146

CD73 CD34 CD34 CD34

CD146

CD146

CD106 CD146 CD106

CD166

Human adipose tissue-derived, expanded Human amniotic fluid-derived, expanded Human placenta-derived, expanded Human adipose tissue-derived, expanded Mouse dermis-derived, expanded Human adipose tissue-derived, expanded Human endometrium-derided, primary, expanded Human bone marrow-derived, expanded Human skin-derived, expanded Human bone marrow-derived, expanded Human periodontal ligament-derived, expanded Human bone marrow-derived, expanded Human bone marrow-derived, expanded Human blood-derived, expanded Human bone marrow-derived, expanded Human bone marrow-derived, expanded Human deciduous tooth-derived, expanded Human bone marrow-derived, expanded

Bone regeneration from mesenchymal stem cells

Table 1

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38 Table 2

E. Yamachika, S. Iida Summary of negative cell surface markers of mesenchymal stem cells. CD10

Dominici et al. (1) Wongchuensoontorn et al. (2) Tsai et al. (4) Igura et al. (5) Zuk et al. (6) Orciani et al. (11) Ohgushi et al. (12) Zvaifler et al. (16) Pittenger et al. (20) Conget et al. (21)

CD10

CD10

CD11b

CD14

CD11b

CD14

CD11b

CD14

CD11b

CD14 CD14 CD14 CD14

CD31

CD31 CD31

CD34

CD45

CD34 CD34

CD45 CD45

CD34 CD34 CD34 CD34 CD34

CD45 CD45

CD49d

Human bone marrow-derived, expanded Human bone marrow-derived, primary

CD49d

CD45 CD34 CD34

The most commonly reported positive markers are CD105 [11— 21], CD90 [11,14—23], CD44 [12—18,21,23,24], CD73 [11,12, 15,18—21,25], CD29 [13,14,16,18,21—23], CD13 [13,15,16, 22,25], CD34 [13,18,26—28], CD146 [13,25,29], CD106 [2,28], CD54 [13,17] and CD166 [13] (Table 1). The most frequently reported negative markers are CD34 [2,11,12, 14—16,21,22,30], CD14 [11,14,16,21,22,26], CD45 [11,12, 15,16,26], CD11b [11,14,21], CD49d [2,16,30], CD106

CD106

CD106

CD106 CD49d CD49d

Human Human Human Human Human Human Human Human

amniotic fluid-derived, expanded placenta-derived, expanded adipose tissue-derived, expanded skin-derived, expanded bone marrow-derived, expanded blood-derived, expanded bone marrow-derived, expanded bone marrow-derived, expanded

[16,26], CD10 [14,21] and CD31 [15,16] (Table 2). A number of other cell surface markers, including STRO-1 [13,16,29], SH2 [14,16], SH3 [14,16], SH4 [14], HLA-A [14,21], HLA-B [14,21], HLA-C [14,21], HLA-DR [14,15,21], HLA-I [14], DP [14], EMA [14], DQ [14] (MHC Class II), Oct 4 [31], Oct 4A [31], Nanog [31], Sox-2 [31], TERT [31], Stat-3 [31], fibroblast surface antigen [24], smooth muscle alpha-actin [24], vimentin [24], integrin subunits alpha4 [30], alpha5 [30], beta1 [30],

Figure 1 A flow cytometry analysis of Sca1, CD29, CD34, CD44, CD45 and CD90 in murine compact bone-derived MSCs cultured in bFGF-conditioned medium at 19 passages. The open histogram represents reactivity with the indicated antibodies and the shaded histogram shows the background signal. The incidence of Sca1-positive cells significantly increased over time, and more than 90% of the cells expressed Sca1 at 19 passages. These MSCs were 30% positive for CD29 and 100% positive for CD44. The expressions of CD34, 45 and 90 were not evident.

Bone regeneration from mesenchymal stem cells integrins alphavbeta3 [30] and alphavbeta5 [30] and ICAM-1 [30], have also been reported. In detail, ex vivo expanded MSCs have been phenotypically characterized on the basis of the expression of nonspecific markers, including CD105, CD73, CD90, CD166, CD44 and CD29. In addition, culture-expanded cells lack the expression of some hematopoietic and endothelial markers such as CD34, CD14 and CD45. In our own experiment, we isolated Sca1(+), CD29(+), CD34( ), CD44(+), CD45( ) and CD90( ) MSCs from mice (Fig. 1) and confirmed the trilineage potential of the cells. In our observations, the cultured cells usually showed contamination with hematopoietic cells at an early passage stage. Additionally, one of the most reliable selection methods for MSCs is to rule out hematopoietic lineage cells. In this point, the immunophenotype of CD34( ), CD14( ) and CD45( ) for MSCs was acceptable.

4. Sources of adult mesenchymal stem cells and their applicability for bone applications MSCs exist in almost all postnatal tissues. There are increasing reports that MSCs can be isolated from various adult mesenchymal tissues such as the synovium [32], the periosteum [33], skeletal muscle [34] and adipose tissue [16] in addition to bone marrow [35]. These MSCs have been assumed to be similar, irrespective of their original tissue source, since they all have the potential for self-renewal and multidifferentiation with common surface epitopes [36]. However, the properties of MSCs can be affected by preparation [37—39], which is not properly controlled for in some studies. In general, MSCs are a minor fraction of the cells in bone marrow and other tissues; the exact frequency is difficult to calculate due to differences in methods of harvest and separation. However, the frequency in human bone marrow has been estimated to be on the order of 0.001—0.01% of total nucleated cells, approximately 10-fold less abundant than HSCs [2]. It is important to note that there is much evidence demonstrating that MSCs derived from various sources exhibit different characteristics in gene expression profiles, proliferation and differentiation potential and functional properties, although most of the cells satisfy the minimal criteria for defining MSCs and, thus, are considered to be MSCs as a whole [40—43]. For example, studies have shown that adipose tissuederived MSCs exhibit in vitro immunomodulatory properties at higher efficiencies compared to their bone marrow-derived counterparts [40]. Another example can be found in a study comparing the differentiation potential of MSCs derived from bone marrow and the pancreas to become insulin-producing endocrine cells [41]. That study revealed that MSCs derived from the pancreas are committed to an endocrine fate and thus have a greater propensity to generate insulin-producing cells compared to bone marrow-derived MSCs. Therefore, to select an ideal source of MSCs for therapeutic use, the functional properties of the cells (e.g. differentiation potential, immunomodulation, secretion of bioactive factors) should be critically evaluated in comparison with the properties of cells from other potential sources. Although bone marrow-derived MSCs are among the most frequently used types in bone regeneration studies [44—47],

39 several investigators have suggested the use of other sources of MSCs, namely, peripheral blood-derived MSCs, fetal MSCs and adipose tissue-derived MSCs [48—52]. However, there are no clear guidelines indicating which sources are the most suitable for bone regeneration.

5. Mice compact bone-derived mesenchymal stem cells To develop MSC-based methods for bone regeneration, mice are suitable experimental animals; however, standard culture conditions, including plastic culture flasks and standard culture media do not support the passage of pure MSCs derived from murine bone marrow. Several groups have reported independent methods for purifying MSCs obtained from murine bone marrow, including plastic adherent selection [53], retroviral infection [54] and unique culture systems [55—59]. However, the long time passage of MSCs has not been successfully achieved. Moreover, the fact that murine bone marrow harbors very few MSCs and contains hematopoietic cells [60] shows that murine bone marrow may not be a suitable source of murine MSCs. To overcome the difficulties in culturing MSCs obtained from mice, Sun et al. [61] established murine MSC cultures by adding fragments of murine bone to murine bone marrow cells. Short et al. [62], using a CFU-Fs assay, found that the femoral bone itself is a richer source of murine MSCs than the marrow within the bone. Additionally, we [63] succeeded in maintaining murine MSCs for more than 120 days in culture in the presence of basic fibroblast growth factor (bFGF). Based on this information, we confirmed that cells derived from compact bone and propagated in bFGF-conditioned medium are murine MSCs and that bFGF-conditioned medium supports the selfrenewal of murine MSCs and maintains the potential of these MSCs to differentiate along multiple lineages, including chondrocyte, osteocyte and adipocyte lineages (Fig. 2).

6. Ex vivo isolation and expansion of MSCs To date, MSC isolation has primarily relied on the adherent properties, immunophenotype (determined with flow cytometry) and differentiation potential of the cells. Additionally, due to the low frequency of mesenchymal progenitors in adult tissues, in vivo use of MSCs requires that the cells be extensively manipulated ex vivo to achieve the numbers necessary for clinical application [64,65]. MSCs can be expanded in vitro; however, the cell yield after expansion varies with the age and condition of the donor and the harvesting technique. Therefore, differences in isolation methods, culture conditions and media additives greatly affect the cell yield and possibly also the phenotype of the expanded cell products [37,66,67]. In this regard, MSCs are primarily cultured under either experimental or clinicalgrade conditions in the presence of 10% fetal calf serum (FCS) [2—4,9,64,65]. In addition, serum batches are routinely pre-screened to guarantee both the optimal growth of the MSCs and the biosafety of the cellular products. Nonetheless, the use of FCS in clinical-grade preparations raises concerns because FCS theoretically might be responsible for the transmission of prions and still unidentified zoonoses or cause immune reactions in the host, especially

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E. Yamachika, S. Iida

Figure 2 (A) Murine compact bone-derived MSCs were cultured in normal medium (a-MEM and 20% FBS) and b-FGF-conditioned medium (basic FGF (5 ng/ml) added to normal medium), and differences between the cultures were assessed. Initially, cells from each group were plated at a density of 3  106 cells per 25 cm2 and the cultures were incubated continuously. The data are expressed as the calculated mean live cell numbers of three replicate flasks at 0, 20, 40, 60, 80, 100 and 120 days. (B) Alizarin red S staining of murine compact bone-derived MSCs grown in 12-well plates with osteogenic medium. MSCs subcultured in bFGF-conditioned medium for 19 passages were seeded in the two wells. The control groups were cultured in bFGF-conditioned medium, and the cells in the left well were cultured in osteogenic medium. This staining showed that when murine compact bone-derived MSCs are subcultured in bFGFconditioned medium and treated with osteogenic medium, they produce a well-mineralized matrix. (C) Cell nodules cultured under chondrogenic differentiation micromass conditions for two weeks. Murine compact bone-derived MSCs subcultured in bFGF-conditioned medium for 19 passages formed cell nodules, which had a cartilage-like structure (left panel, HE staining) and were associated with a collagen type II-positive extra cellular matrix (right panel, Collagen II staining) (scale bar = 500 mm). (D) Oil Red O staining of cells grown in 12-well plates with adipogenic medium. Murine compact bone-derived MSCs subcultured in bFGF-conditioned medium had small lipid droplets (scale bar = 50 mm).

if repeated infusions are needed, with the consequent rejection of the transplanted cells [68—71]. In view of these considerations, the use of serum-free media appropriate for extensive expansion and devoid of the risks connected with the use of animal products has been investigated, and several serum-free media, developed based on the use of cytokines and growth/attachment factors such as b-FGF and transforming growth factor beta (TGF-b), have been tested under experimental conditions [72,73]. Attempts have been made to develop defined serum-free media for animal or human MSC growth; however, most products have demonstrated only limited performance [74,75]. These media formulations have been shown to support only cell expansion for single-passage cultures or multiple-passage cultures at slow rates. Moreover, all of these studies used cells that had been previously exposed to serum during the initial isolation/expansion phase. Serum-derived contaminants are most likely carried over with the cells when placed under serum-free conditions after exposure to serum. Therefore, exposure to serum may ultimately limit their therapeutic use.

7. Transformation of MSCs to bone lineage Osteogenic differentiation is a highly programmed process that consists 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 dexamethasone, beta glycerol phosphate and ascorbic acid for a period of two to three weeks [76]. Dexamethasone is a synthetic glucocorticoid that stimulates MSC proliferation and is essential for osteogenic differentiation [77,78]. Although the mechanisms underlying dexamethasone’s effects are not well known, it has been speculated that this reagent upregulates the beta cateninlike molecule TAZ, which results in upregulation of Runx2related transcription factor and osteogenic differentiation [79]. The optimal concentration of this reagent for MSC bone differentiation is approximately 10 nM, which corresponds to physiologic concentrations [80]. Organic phosphate released after enzymatic hydrolysis of beta glycerol phosphate plays an important role in matrix mineralization. This free

Bone regeneration from mesenchymal stem cells

41

phosphate is usually applied in 5—10 mM concentrations for MSC bone differentiation [81]. Ascorbic acid is a cofactor in the hydroxylation of prolins and lysine moiety of collagen molecules and is an abundant protein in the ECM. This reagent is used in 50—500 mM concentrations [82]. In addition to these osteogenic supplements, there are other osteogenic factors, including BMP-2 and bFGF. Bone formation up to 84% has been reported with the application of human bone marrow-derived MSCs with hydrogel and 10 mg/mL of BMP2 in a rat calvarial defect model [83]. Another investigation reported significantly greater bone formation with BMP-2and bFGF-treated human bone marrow-derived MSCs [84]. Platelet rich plasma (PRP) is another known source of various growth factors, namely, platelet-derived growth factor, transforming growth factor-b and vascular endothelial growth factor. The applicability of PRP for the repair of bony defects is well established [85], and several investigators have advocated the use of this product in combination with MSCs [86,87].

8. Scaffold Many scaffolds have been used in different MSC-based bone augmentation procedures. At present, no perfect scaffold/ carrier for MSC transfer has been developed, and evidence regarding the subject is sparse. Hydroxy apatite (HA), btricalcium phosphate (b-TCP) or a mixture of the two are often used as MSC transfer scaffolds [88,89]. Several studies have suggested the application of fibrin glue for cell delivery because fibrin glue is a biocompatible tissue adhesive that stabilizes seeded cells and provides an equally distributed population of cells throughout the carrier [87,90].

9. Tumor formation In general, it is believed that MSCs can be safely cultured in vitro without risk of spontaneous malignant transformation [91]. Stenderup and colleagues cultured several strains of hMSCs from bone marrow at various ages (i.e. aged 18—81 years) until the cells reached their maximal life span without any evidence of transformation [92]. Furthermore, there have been no reports of human trials demonstrating the formation of tumors with culture-expanded hMSCs [93]. On the other hand, concerns have been raised about the safety of MSCs for clinical use, with studies reporting the potential risk of in vitro expanded MSCs developing into tumors on transplantation. In mice, there have been some reports of sarcoma formation by cultured murine MSCs in vitro and in vivo [94—96]. 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. Miura et al. intentionally induced spontaneous immortalization after numerous passages (P29 to P54) and demonstrated the contribution of the transformed cells to fibrosarcoma formation in vivo [95]. We have also confirmed sarcoma formation by cultured murine compact bonederived MSCs (Fig. 3). Rubio et al. showed that, although MSCs can be managed safely during the standard ex vivo expansion period (six to eight weeks), human MSCs can undergo spontaneous transformation following long-term in

Figure 3 The tumorigenic capacity of murine compact bonederived MSCs subcultured in bFGF-conditioned medium was investigated in vivo using ectopic implantation into immunedeficient SCID mice. Implanted cells were collected four weeks after implantation of MSCs. Tumor formation was confirmed when late passage MSCs (40 passages) were implanted (A). HE staining of the tumor showing a sarcoma-like structure (B) (scale bar = 100 mm).

vitro culture (four to five months), and the transformed cells lead to the formation of tumors in mice [97]. This information indicates that unmodified MSCs should be used at early passages to avoid potential chromosomal abnormalities that result from long-term culture. Additionally, it has been recommended by the Food and Drug Administration (FDA) that ‘‘minimally manipulated’’ cells be used for human clinical trials. In this regard, attempts are being made to develop an efficient production system to produce clinically relevant numbers of hMSCs in relatively shorter periods of time with lower passage numbers [98].

10. Conclusion As adult stem cells, MSCs, are free from ethical concerns, residents of multiple tissues, able to efficiently differentiate along an osteogenic lineage and can be used as vehicles for bone gene therapy. These characteristics make MSCs promising candidates for use in bone engineering and regeneration. Unfortunately, to date, no markers have yet been identified that specifically identify MSCs and it is difficult to culture MSCs in chemically-defined media. Additionally, while the characteristics of multipotency and autonomous growth are

42 suitable for producing qualified cells for tissue regeneration, at the same time, these characteristics are very close to those of tumorigenesis. Therefore, regarding clinical use, this review did not reach any significant conclusions as to the most promising model for MSC reconstruction. However, this review does show the need for additional collaborative studies using similar designs and data analysis in advancing the science of bone reconstruction using MSCs.

Conflict of interest statement The authors have no conflicts of interest to declare.

Acknowledgement This work was supported in part by a Grant-in-Aid for funding scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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