Human myogenic endothelial cells exhibit chondrogenic and osteogenic potentials at the clonal level

We have previously reported the high regenerative potential of murine muscle‐derived stem cells (mMDSCs) that are capable of differentiating into multiple mesodermal cell lineages, including myogenic, endothelial, chondrocytic, and osteoblastic cells. Recently, we described a putative human counterpart of mMDSCs, the myogenic endothelial cells (MECs), in adult human skeletal muscle, which efficiently repair/regenerate the injured and dystrophic skeletal muscle as well as the ischemic heart in animal disease models. Nevertheless it remained unclear whether human MECs, at the clonal level, preserve mMDSC‐like chondrogenic and osteogenic potentials and classic stem cell characteristics including high proliferation and resistance to stress. Herein, we demonstrated that MECs, sorted from fresh postnatal human skeletal muscle biopsies, can be grown clonally and exhibit robust resistance to oxidative stress with no tumorigeneity. MEC clones were capable of differentiating into chondrocytes and osteoblasts under inductive conditions in vitro and participated in cartilage and bone formation in vivo. Additionally, adipogenic and angiogenic potentials of clonal MECs (cMECs) were observed. Overall, our study showed that cMECs not only display typical properties of adult stem cells but also exhibit chondrogenic and osteogenic capacities in vitro and in vivo, suggesting their potential applications in articular cartilage and bone repair/regeneration. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1089–1095, 2013     

Pyrvinium,a potent small molecule Wnt inhibitor,increases engraftment and inhibits lineage commitment of mesenchymal stem cells (MSCs)

We and others have found that Wnt signaling inhibition is important in mesenchymal stem cell (MSC) self‐renewal. Pyrvinium was identified as a potent Wnt inhibitor in a chemical screen for small molecules. In the present study, we hypothesized that pyrvinium will enhance MSC self‐renewal to improve the clinical efficacy of MSC therapy. Pyrvinium increased MSC proliferation in vitro while inhibiting their osteogenic and chondrogenic lineage commitment by reducing cytoplasmic β‐catenin. Although MSCs are a promising target for cell therapy, strategies to enhance their survival and maintain their stemness in the wounded area are essential. Using an in vivo model of granulation tissue formation, we demonstrated that pyrvinium enhanced long‐term MSC engraftment. Pyrvinium‐treated MSC‐generated granulation tissue also demonstrated less ectopic differentiation into bone or cartilage. This study highlights the potential of using a therapeutic Wnt inhibitor to enhance MSC‐driven regenerative therapy.     

Osteogenic stem cells and the stromal system of bone and marrow

According to current hypothesis, cells of the osteogenic lineage, which includes both osteoblasts and chondroblasts, are derived from a stromal stem cell in the postnatal organism. That there exist osteogenic precursors in association with the soft, fibrous tissue of the marrow stroma is well established. An osteogenic tissue comprised of cartilage and bone is formed when marrow or marrow cell suspensions are cultured in vivo within diffusion chambers. Bone with a functional marrow organ is formed when marrow or marrow cell suspensions are transplanted heterotopically, e.g., under the renal capsule. Cultures of marrow stromal fibroblasts are readily established in vitro from single-cell bone marrow suspensions. Such cultures do not demonstrate overt differentiation in an osteogenic direction in vitro. When transplanted in vivo, however, they differentiate to form cartilage and bone in diffusion chambers and bone with a functional marrow organ when transplanted heterotopically. Single-cell bone marrow suspensions can be cultured in vitro under conditions that facilitate the formation of stromal fibroblast colonies. Circumstantial evidence supports the conclusion that each colony is derived from a single initiating cell termed a colony-forming unit-fibroblastic (CFU-F). A proportion of CFU-F demonstrates extensive proliferative potential both in vitro and in vivo. In vitro the extensive proliferative potential of a subset of CFU-F has been shown to be associated with a capacity for extensive self-renewal. On transplantation in vivo, the progeny of a proportion of CFU-F has been shown to be capable of proliferating and differentiating into all the stromal cell lines necessary for the formation of bone and reconstitution of the hematopoietic inductive microenvironment. These findings provide strong circumstantial evidence to support the hypothesis that there are stem cells present within the marrow stroma that are capable of giving rise to cells of a number of different lineages, including those of the osteogenic lineage (chondroblasts and osteoblasts).     

Adipose tissue is an ideal stem cell source for tissue reconstruction of bone，cartilage，and soft tissue defects

目的 验证人脂肪基质细胞是否具有向成骨细胞、软骨细胞、脂肪细胞分化的能力,从而为骨、软骨、软组织再建寻找一种理想的干细胞来源.方法 分别用成骨向分化培养基（DMEM＋10?S＋地塞米松＋维生素C＋β-甘油磷酸）、软骨向分化培养基（DMEM＋1?S＋胰岛素＋维生素C＋转化生长因子β1）及脂肪向分化培养基（DMEM＋10?S＋地塞米松＋胰岛素＋吲哚美辛＋异丁基甲基黄嘌呤）诱导人脂肪基质细胞向成骨细胞、软骨细胞及脂肪细胞分化.用von Kossa和碱性磷酸酶染色鉴定成骨细胞分化,而软骨细胞分化和脂肪细胞分化分别用Alcian blue染色和油红O染色显示.成骨细胞、软骨细胞以及脂肪细胞特异相关或标志基因的表达用RT-PCR检测.结果 体外实验表明,人脂肪基质细胞在定向分化诱导剂的作用下可分别向成骨细胞、软骨细胞及脂肪细胞分化.结论 人脂肪基质细胞中包含有多向分化能力的干细胞,可用于今后骨、软骨、软组织的组织工程再建.     

Isolation and characterization of turkey bone marrow‐derived mesenchymal stem cells

Flexor tendon injury is often associated with suboptimal outcomes and results in substantial digit dysfunction. Stem cells have been isolated from several experimental animals for the growing interest and needs of utilizing cell‐based therapies. Recently, turkey has been developed as a new large animal model for flexor tendon research. In the present study, we reported the isolation and characterization of bone marrow‐derived mesenchymal stem cells (BMSCs) from 8‐ to 12‐month‐old heritage‐breed turkeys. The isolated cells demonstrated fibroblast‐like morphology, clonogenic capacity, and high proliferation rate. These cells were positive for surface antigens CD90, CD105, and CD44, but were negative for CD45. The multipotency of turkey BMSCs was determined by differentiating cells into osteogenic, adipogenic, chondrogenic, and tenogenic lineages. There was upregulated gene expression of tenogenic markers, including mohawk, tenomodulin, and EGR1 as well as increased collagen synthesis in BMP12 induced cells. The successful isolation and verification of bone marrow‐derived MSCs from turkey would provide opportunities of studying cell‐based therapies and developing new treatments for tendon injuries using this novel preclinical large animal model. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1419–1428, 2019.     

Distinguishing characteristics of stem cells derived from different anatomical regions of human degenerated intervertebral discs

Introduction

Several types of stem cells have been successfully demonstrated to exist in the human degenerated intervertebral disc (IVD), which is composed of annulus fibrosus (AF), nucleus pulposus (NP) and cartilage endplate (CEP). However, the differences in the biological characteristics among these and bone marrow derived mesenchymal stem cells (BM-MSCs) remain unclear.

Materials and methods

To investigate this issue, cells were harvested from human AF, NP, CEP, and bone marrow, respectively; passage 2 cells were selected using the agarose suspension culture system to obtain stem cell clones. Following expansion in vitro, stem cells from different anatomical regions were compared regarding the morphology, proliferation ability, immunophenotypic expression, and multi-lineage differentiation capacity. In addition, stem cell-alginate bead compositions were constructed for the comparison of DNA and sGAG content.

Results

There were subtle differences regarding cell morphology, but no significant differences in proliferation ability among the four types of stem cells. For the immunophenotypic analysis, all stem cells basically fulfilled the criteria for mesenchymal stem cells (MSCs), which have been published by the International Society for Cellular Therapy (ISCT), with a significant difference in CD105 expression. A comparison of the osteogenic capacities indicated: cartilage endplate-derived stem cells (CESCs) > annulus fibrosus-derived stem cells (AFSCs) > BM-MSCs > nucleus pulposus-derived stem cells (NPSCs). The chondrogenesis difference was similar to osteogenesis. For adipogenesis: BM-MSCs >NPSCs >CESCs >AFSCs. In the stem cell/alginate composition, the CESCs consistently showed the superior chondrogenic potential among all those cell types.

Conclusions

Our data indicated that all the four types of stem cells shared some similar biological properties (regarding shape, proliferation ability and immunophenotypic expression). CESCs, which had the strongest osteogenic and chondrogenic potentials, may serve as excellent seed cells for NP/cartilage or bone tissue engineering.

     

MT1‐MMP and Type II Collagen Specify Skeletal Stem Cells and Their Bone and Cartilage Progeny

Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic activity associated with the membrane‐type 1 matrix metalloproteinase (MT1‐MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1‐MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1‐MMP activity in the type II collagen‐expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1‐MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1‐MMP under the control of the type II collagen promoter in an MT1‐MMP‐deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived from nontransgenic MT1‐MMP‐deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic and osteogenic lineage progeny where collagenolytic activity is a requisite for proper cell and tissue function.     

The effect of two- and three-dimensional cell culture on the chondrogenic potential of human adipose-derived mesenchymal stem cells after subcutaneous transplantation with an injectable hydrogel

Articular cartilage is an avascular tissue composed of chondrocytes, a unique cell type responsible for abundant matrix synthesis and maintenance. When damaged, it never heals spontaneously under physiological circumstances. Therefore, the delivery of mesenchymal stem cells using hydrogel has been considered for cartilage repair. This study aims at investigating the influence of in vitro chondrogenic differentiation of human adipose tissue-derived stem cells (hATSCs) on in vivo cartilage formation when associated with a cellulose-based self-setting hydrogel (Si-HPMC). hATSCs were characterized for their proliferation, surface marker expression, and multipotency. The in vitro chondrogenic potential of hATSCs cultured within Si-HPMC in control or chondrogenic medium was evaluated by measuring COL2A1, ACAN, SOX9, and COMP expression by real-time PCR. Alcian blue and type II collagen staining were also performed. To determine whether in vitro chondrogenically differentiated hATSCs may give rise to cartilage in vivo, cells differentiated as a monolayer or in pellets were finally associated with Si-HPMC and implanted subcutaneously into nude mice. Cartilage formation was assessed histologically by alcian blue and type II collagen staining. Our data demonstrate that hATSCs exhibited proliferation and self-renewal. hATSCs also expressed typical stem cell surface markers and were able to differentiate towards the adipogenic, osteogenic, and chondrogenic lineages. Real-time PCR and histological analysis indicated that Si-HPMC enabled chondrogenic differentiation of hATSCs in inductive medium, as demonstrated by increased expression of chondrogenic markers. In addition, histological analysis of implants showed that chondrogenically differentiated hATSCs (monolayers or pellets) have the ability to form cartilaginous tissue, as indicated by the presence of sulphated glycosaminoglycans and type II collagen. This study therefore suggests that an in vitro induction of hATSCs in 2D was sufficient to obtain cartilaginous tissue formation in vivo. Si-HPMC associated with autologous hATSCs could thus be a significant tool for regenerative medicine in the context of cartilage damage.     

Osteogenic potential of postnatal skeletal muscle-derived stem cells is influenced by donor sex.

This study compared the osteogenic differentiation of F-MDSCs and M-MDSCs. Interestingly, M-MDSCs expressed osteogenic markers and underwent mineralization more readily than F-MDSCs; a characteristic likely caused by more osteoprogenitor cells within the M-MDSCs than the F-MDSCs and/or an accelerated osteogenic differentiation of M-MDSCs. INTRODUCTION: Although therapies involving stem cells will require both female and male cells, few studies have investigated whether sex-related differences exist in their osteogenic potential. Here, we compared the osteogenic differentiation of female and male mouse skeletal muscle-derived stem cells (F- and M-MDSCs, respectively), a potential cell source for orthopedic tissue engineering. MATERIALS AND METHODS: F- and M-MDSCs were stimulated with bone morphogenetic protein (BMP)4, followed by quantification of alkaline phosphatase (ALP) activity and expression of osteogenic genes. F- and M-MDSCs were also cultured as pellets in osteogenic medium to evaluate mineralization. Single cell-derived colonies of F- and M-MDSCs were stimulated with BMP4, stained for ALP, and scored as either Low ALP+ or High ALP+ to detect the presence of osteoprogenitor cells. F- and M-MDSCs were transduced with a BMP4 retrovirus (MDSC-BMP4 cells) and used for the pellet culture and single cell-derived colony formation assays. As well, F- and M-MDSC-BMP4 cells were implanted in the intramuscular pocket of sex-matched and sex-mismatched hosts, and bone formation was monitored radiographically. RESULTS AND CONCLUSIONS: When stimulated with BMP4, both F- and M-MDSCs underwent osteogenic differentiation, although M-MDSCs had a significantly greater ALP activity and a larger increase in the expression of osteogenic genes than F-MDSCs. In the pellet culture assay, M-MDSCs showed greater mineralization than F-MDSCs. BMP4 stimulation of single cell-derived colonies from M-MDSCs showed higher levels of ALP than those from F-MDSCs. Similar results were obtained with the MDSC-BMP4 cells. In vivo, F-MDSC-BMP4 cells displayed variability in bone area and density, whereas M-MDSC-BMP4 cells showed a more consistent and denser ectopic bone formation. More bone formation was also seen in male hosts compared with female hosts, regardless of the sex of the implanted cells. These results suggest that M-MDSCs may contain more osteoprogenitor cells than F-MDSCs, which may have implications in the development of cellular therapies for bone healing.     

Tissue-engineered cartilage and bone using stem cells from human infrapatellar fat pads

Multipotential processed lipoaspirate (PLA) cells extracted from five human infrapatellar fat pads and embedded into fibrin glue nodules, were induced into the chondrogenic phenotype using chondrogenic media. The remaining cells were placed in osteogenic media and were transfected with an adenovirus carrying the cDNA for bone morphogenetic protein-2 (BMP-2). We evaluated the tissue-engineered cartilage and bone using in vitro techniques and by placing cells into the hind legs of five severe combined immunodeficient mice. After six weeks, radiological and histological analysis indicated that the PLA cells induced into the chondrogenic phenotype had the histological appearance of hyaline cartilage. Cells transfected with the BMP-2 gene media produced abundant bone, which was beginning to establish a marrow cavity. Tissue-engineered cartilage and bone from infrapatellar fat pads may prove to be useful for the treatment of osteochondral defects.     

Biological alchemy: engineering bone and fat from fat-derived stem cells

Adipose tissue contains a population of pluripotent stem cells capable of differentiating along multiple mesenchymal cell lineages. In this study the authors isolated these fat-derived stem cells successfully from Lewis rats and induced differentiation along adipogenic and osteogenic lineages in vitro and in vivo. Induction was stimulated by exposing stem cells to lineage-specific induction factors. Adipocyte-inducing media contained dexamethasone, insulin, and isobutyl-methylxanthine. Osteoblast inducing media contained dexamethasone, beta-glycerophosphate, and ascorbic acid. Undifferentiated stem cells were maintained in minimal essential media alpha and fetal bovine serum. At 10 days, cells cultured in adipogenic media differentiated into adipocytes in vitro, as evidenced by positive Oil red O staining of lipid vacuoles. At 21 days, cells cultured in osteogenic media differentiated into osteoblasts in vitro as demonstrated by Alizarin red staining of a calcified extracellular matrix and immunohistochemical staining for osteocalcin. Differentiated cells were seeded at a density of 5 x 106 cells onto 15 x 15-mm polyglycolic acid grafts and implanted subcutaneously into three groups of Lewis rats: Group I contained undifferentiated stem cell grafts, group II contained adipocyte grafts, and group III contained osteoblast grafts. At weeks 4 and 8, in vivo fat formation was demonstrated in group II rats, as confirmed by Oil red O staining. At 8 weeks, group III rats demonstrated in vivo bone formation, as confirmed by the presence of osteocalcin on immunohistochemistry and the characteristic morphology of bone on hematoxylin-eosin staining. Group I rats demonstrated no in vivo bone or fat formation at either time interval. These results demonstrate the ability to isolate pluripotent stem cells from adipose tissue, to induce their differentiation into osteoblasts and adipocytes in vitro, and to form bone and fat subsequently in vivo. This is the first published report of in vivo bone formation from fat-derived stem cells. These cells may eventually serve as a readily available source of autologous stem cells for the engineering of bone and fat.     

Skeletal Cell Fate Decisions Within Periosteum and Bone Marrow During Bone Regeneration

Bone repair requires the mobilization of adult skeletal stem cells/progenitors to allow deposition of cartilage and bone at the injury site. These stem cells/progenitors are believed to come from multiple sources including the bone marrow and the periosteum. The goal of this study was to establish the cellular contributions of bone marrow and periosteum to bone healing in vivo and to assess the effect of the tissue environment on cell differentiation within bone marrow and periosteum. Results show that periosteal injuries heal by endochondral ossification, whereas bone marrow injuries heal by intramembranous ossification, indicating that distinct cellular responses occur within these tissues during repair. Next, lineage analyses were used to track the fate of cells derived from periosteum, bone marrow, and endosteum, a subcompartment of the bone marrow. Skeletal progenitor cells were found to be recruited locally and concurrently from periosteum and/or bone marrow/endosteum during bone repair. Periosteum and bone marrow/endosteum both gave rise to osteoblasts, whereas the periosteum was the major source of chondrocytes. Finally, results show that intrinsic and environmental signals modulate cell fate decisions within these tissues. In conclusion, this study sheds light into the origins of skeletal stem cells/progenitors during bone regeneration and indicates that periosteum, endosteum, and bone marrow contain pools of stem cells/progenitors with distinct osteogenic and chondrogenic potentials that vary with the tissue environment.     

Recruitment of mesenchymal stem cells and expression of TGF-beta 1 and VEGF in the early stage of shock wave-promoted bone regeneration of segmental defect in rats.

Extracorporeal shock wave (ESW) treatment has recently been established as a method to enhance bone repair. Here, we reported that ESW-promoted healing of segmental defect via stimulation of mesenchymal stem cell recruitment and differentiation into bone forming cells. Rats with a segmental femoral defect were exposed to a single ESW treatment (0.16 mJ/mm(2), 1 Hz, 500 impulses). Cell morphology and histological changes in the defect region were assessed 3, 7, 14, and 28 days post-treatment. Presence of mesenchymal stem cell was assayed by immuno-staining for RP59, a recently discovered marker, and also production of TGF-beta 1 and VEGF was monitored. ESW treatment increased total cell density and the proportion of RP59 positive cells in the defect region. High numbers of round- and cuboidal-shaped cells strongly expressing RP59 were initially found. Later, the predominant cell type was spindle-shaped fibroblastic cells, subsequently, aggregates of osteogenic and chondrogenic cells were observed. Histological observation suggested that bone marrow stem cells were progressively differentiated into osteoblasts and chondrocytes. RP59 staining was initially intense and decreased with the appearance of expression depended on the differentiation states of osteogenic and chondrogenic cells during the regeneration phase. Mature chondrocytes and osteoblasts exhibited only slight RP59 immuno-reactivity. Expression of TGF-beta 1 and VEGF-A mRNA in the defect tissues was also significantly increased (P<0.05) after ESW treatment as determined by RT-PCR. Intensive TGF-beta 1 immuno-reactivity was induced immediately, whereas a lag period was observed for VEGF-A. Chondrocytes and osteoblasts at the junction of ossified cartilage clearly exhibited VEGF-A expression. Our findings suggest that recruitment of meseoblasts at the junction of ossified cartilage clearly exhibited mesenchymal stem cells is a critical step in bone reparation that is enhanced by ESW treatment. TGF-beta 1 and VEGF-A are proposed to play a chemotactic and mitogenic role in recruitment and differentiation of mesenchymal stem cells.     

Distinct roles of bone morphogenetic proteins in osteogenic differentiation of mesenchymal stem cells.

Efficacious bone regeneration could revolutionize the clinical management of many bone and musculoskeletal disorders. Bone morphogenetic proteins (BMPs) can regulate the differentiation of mesenchymal stem cells into cartilage, bone, tendon/ligament, and fat lineages. Early data documented the osteogenic potential of rhBMP2 and rhBMP7/OP-1. However, prior to this work that summarized several of our recent studies, no comprehensive analysis had been undertaken to characterize relative osteogenic activity of all BMPs. Using recombinant adenoviruses expressing 14 BMPs, we have demonstrated that, besides BMP2 and BMP7, BMP6 and BMP9 exhibit the highest osteogenic activity both in vitro and in vivo. We further demonstrated that several BMPs may exert synergistic effect on osteogenic differentiation, and that osteogenic BMPs produce a distinct set of molecular fingerprints during osteogenic differentiation. The reported work should expand our current understanding of BMP functions during osteogenic differentiation. It is conceivable that osteogenic BMPs (i.e., BMP2, 4, 6, 7, and 9) may be used to formulate synergistic pairs among themselves and/or with other less osteogenic BMPs for efficacious bone regeneration in clinical settings.     

成软骨诱导的骨髓间充质干细胞膜片修复肋软骨供区缺损的实验研究

目的采用兔胸廓损伤动物模型,观察成软骨诱导的骨髓间充质干细胞膜片对肋软骨供区再生修复的影响。方法将16只家兔随机分为4组,每组4只,分别为健康对照组,实验1、2、3组。健康对照组家兔无任何处理,对实验组的每组双侧第4—6肋软骨均采用不同的2种方法处理,同侧3根肋软骨采用同一种方法处理,3种方法在每组中两两配对。3种方法分别为：①直接缝合软骨膜;②骨髓间充质干细胞膜片折叠数层成圆筒状填塞人肋软骨缺损处缝合;③成软骨诱导的骨髓间充质干细胞膜片同法折叠数层成圆筒状填塞入肋软骨缺损处,缝合封闭缺损。3种方法在各实验组兔两侧肋软骨中两两配对,健康对照组不做处理。术后16周,处死家兔取材进行大体观察,常规HE染色,并行生物力学检测,测定所有肋软骨的抗压强度及弯曲强度。结果各实验组家兔的胸廓整体形态均较良好,各组及各处理方法间无明显差别。生物力学检测显示,3种处理方法之间均存在差异（P〈0．01）,方法3处理的修复组织的抗压、弯曲强度与健康对照组比较,差异无统计学意义（P〉0．05）;方法1、2处理的修复组织的抗压、弯曲强度明显低于健康对照组（P〈0．01）;方法2处理的修复组织的抗压、弯曲强度优于方法1。组织切片HE染色病理观察,可见方法1、2处理的修复组织主要为纤维组织,方法3处理的修复组织内,可见新生的软骨细胞和大量的软骨细胞外基质。结论成软骨诱导的骨髓间充质干细胞膜片可以促进肋软骨供区软骨细胞的再生,修复肋软骨供区缺损,维持胸廓的正常形态和稳定性,从而降低术后胸廓畸形的发生率。     

Comparison of Multipotent Differentiation Potentials of Murine Primary Bone Marrow Stromal Cells and Mesenchymal Stem Cell Line C3H10T1/2

Murine C3H10T1/2 cells have many features of mesenchymal stem cells (MSCs). Whether or not the multipotent differentiation capability of C3H10T1/2 cells is comparable to that of primary bone marrow–derived MSCs (BM-MSCs) was investigated in this study. For in vitro osteogenic differentiation, both BM-MSCs and C3H10T1/2 cells differentiated to osteoblastic cell lineage and showed positive staining for alkaline phosphatase (ALP) and increased mRNA expression of Runx2, Col1αI, and osteocalcin. C3H10T1/2 cells and BM-MSCs induced similar amounts of bone formation in the biomaterials. Under chondrogenic induction in the presence of TGF-β1, cell pellets of both BM-MSCs and C3H10T1/2 cells formed cartilage-like tissues with cartilage matrix components including proteoglycan, type II collagen, and aggrecan. However, C3H10T1/2 cells presented lower adipogenic differentiation potential, with only about 10% C3H10T1/2 cells (but about 70% of BM-MSCs) being committed to adipogenesis. In this study we confirmed that C3H10T1/2 cells coimplanted with osteoconductive scaffolds can form bone spontaneously in vivo and that C3H10T1/2 cells have a basal level of osteocalcin expression, suggesting that they may be a good alternative source of primary BM-MSCs for investigating osteogenic and chondrogenic differentiation in bone or cartilage tissue engineering studies. Caution is needed when using C3H10T1/2 cells for adipogenic studies as they appear to have lower adipogenic potential than BM-MSCs.     

Noggin resistance contributes to the potent osteogenic capability of BMP9 in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors and can differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bone morphogenetic proteins (BMPs) play important roles in stem cell proliferation and differentiation. We recently demonstrated that BMP9 is a potent but less understood osteogenic factor. We previously found that BMP9‐induced ectopic bone formation is not inhibited by BMP3. Here, we investigate the effect of BMP antagonist noggin on BMP9‐induced osteogenic differentiation. BMP antagonists noggin, chording, gremlin, follistatin, and BMP3 are highly expressed in MSCs, while noggin and follistatin are lowly expressed in more differentiated pre‐osteoblast C2C12 cells. BMP9‐induced osteogenic markers and matrix mineralization are not inhibited by noggin, while noggin blunts BMP2, BMP4, BMP6, and BMP7‐induced osteogenic markers and mineralization. Likewise, ectopic bone formation by MSCs transduced with BMP9, but not the other four BMPs, is resistant to noggin inhibition. BMP9‐induced nuclear translocation of Smad1/5/8 is not affected by noggin, while noggin blocks BMP2‐induced activation of Smad1/5/8 in MSCs. Noggin fails to inhibit BMP9‐induced expression of downstream targets in MSCs. Thus, our results strongly suggest that BMP9 may effectively overcome noggin inhibition, which should at least in part contribute to BMP9's potent osteogenic capability in MSCs. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1796–1803, 2013     

Transient chondrogenic phase in the intramembranous pathway during normal skeletal development.

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.     

The enhancement of osteogenesis through the use of dental pulp pluripotent stem cells in 3D

The potential for osteogenic differentiation of dental pulp mesenchymal stem cells (DPMSCs) in vitro and in vivo has been well documented in a variety of studies. Previously, we obtained a population of cells from human dental pulp called dental pulp pluripotent stem cells (DPPSCs) that could differentiate into mesodermal, ectodermal and endodermal progenies. We compared the osteogenic capacity of DPPSCs and DPMSCs that had been isolated from the same donors (N=5) and cultivated in the same osteogenic medium in 3D (three dimensions) Cell Carrier glass scaffolds. We also compared the architecture of bone-like tissue obtained from DPPSCs and human maxillary bone tissue. Differentiation was evaluated by scanning electron microscopy, whereas the expression of bone markers such as ALP, Osteocalcin, COLL1 and Osteonectin was investigated by quantitative real time polymerase chain reaction (qRT-PCR). We also used calcium quantification, Alizarin red staining and alkaline phosphatase (ALP) activity to compare the two cell types. New bone tissue formed by DPPSCs was in perfect continuity with the trabecular host bone structure, and the restored bone network demonstrated high interconnectivity. Significant differences between DPPSCs and DPMSCs were observed for the expression of bone markers, calcium deposition and ALP activity during osteogenic differentiation; these criteria were higher for DPPSCs than DPMSCs. Both DPPSCs and differentiated tissue showed normal chromosomal dosage after being cultured in vitro and analysed using short-chromosome genomic hybridisation (short-CGH). This study demonstrates the stability and potential for the use of DPPSCs in bone tissue engineering applications.