Circulating plastic adherent mesenchymal stem cells in aged hip fracture patients

We examined the presence of circulating plastic adherent multipotent mesenchymal stem cells (MSCs) in fracture patients. Three patient groups (n = 10–18) were evaluated, including elderly females with a femoral neck fracture treated with cemented hemiarthroplasty, an age‐ and sex‐matched group with hip osteoarthritis (OA) treated with cemented total hip arthroplasty (THA), and younger adults with surgically treated lower extremity fractures. The presence of circulating MSCs pre‐ and postoperatively was compared to bone marrow (BM) MSCs from the same subjects. Criteria for identifying MSCs included cell surface markers (CD105+, CD73+, CD90+, CD45?, CD14?), proliferation through several passages as well as osteogenic, chondrogenic, and adipogenic differentiation. Plastic adherent MSCs were found in peripheral blood (PB) from 22% of hip fracture patients, 46% of younger fracture patients, and in none of 63 pre‐ and postmenopausal women with hip OA. When detectable, circulating MSCs appeared between 39 and 101 h after fracture. PB derived MSCs did not differ from BM derived MSCs, except for a small population (<15%) of CD34+ cells among PB derived MSCs. This initial study indicates mobilization of MSCs into the circulation in response to fracture, even in very old patients, while circulating MSCs were not detectable before or after elective THA. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1634–1642, 2010     

Comparison of the osteogenic capacity of minipig and human bone marrow‐derived mesenchymal stem cells

Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM‐MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic‐adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM‐MSCs were isolated and cultured from adult fracture patients (n = 13, age 19–60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM‐MSCs had similar morphology, proliferation, and colony formation capacities as human BM‐MSCs. Unexpectedly, minipig BM‐MSCs had a significantly lower ability than human BM‐MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species‐specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1019–1025, 2012     

Estrogen reduces cellular aging in human mesenchymal stem cells and chondrocytes

Chondrocyte aging is associated with cartilage degeneration and senescence impairs the regenerative potential of mesenchymal stem cells (MSCs). Estrogen exerts profound effects on human physiology including articular cartilage and MSCs. The present study should analyze the effects of pre‐ and postmenopausal estrogen concentrations on chondrogenic cells. Physiologic premenopausal concentrations of 17β‐estradiol (E₂) significantly decelerated telomere attrition in MSCs and chondrocytes while postmenopausal E₂ concentration had no significant effects. The estrogen agonist–antagonist tamoxifen did not affect telomere biology, but inhibited the E₂‐stimulated reduction in telomere shortening. E₂ and tamoxifen did not influence cell proliferation, cell morphology, and β‐galactosidase staining in chondrogenic cells. E₂ treatment did not affect the telomere‐associated proteins TRF1 and TRF2. E₂ had no regulatory effects on the expression rates of the cell cycle regulator p21 and the DNA repair proteins SIRT1 and XRCC5. In spite of reducing telomere shortening in aging MSCs and chondrocytes, estrogen is not able to prevent somatic cells from replicative exhaustion and from finally entering senescence. The fade of telomere shortening under pre‐ to postmenopausal estrogen concentrations suggests, at least in part, a senescence‐dependent cause for the onset of osteoarthritis in women after menopause. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1563–1571, 2011     

Cartilage fragments from osteoarthritic knee promote chondrogenesis of mesenchymal stem cells without exogenous growth factor induction

Extracellular matrix (ECM) is thought to participate significantly in guiding the differentiation process of mesenchymal stem cells (MSCs). In this study, we hypothesized that cartilage fragments from osteoarthritic knee could promote chondrogenesis of MSCs. Nonworn parts of cartilage tissues were obtained during total knee arthroplasty (TKA) surgery. Cartilage fragments and MSCs were wrapped into fibrin glue; and the constructs were implanted subcutaneously into nude mice. Histological analysis showed neocartilage‐like structure with positive Alcian blue staining in the cartilage fragment–fibrin–MSC constructs. However, constructs with only MSCs in fibrin showed condensed appearance like MSCs in the pellet culture. Gene expression of type II collagen in the constructs with 60 mg cartilage fragments were significantly elevated after 4 weeks of implantation. Conversely, the constructs without cartilage fragments failed to express type II collagen, which indicated MSCs did not differentiate into a chondrogenic lineage. In conclusion, we demonstrated the effect of cartilage fragments from osteoarthritic knee in promoting chondrogenic differentiation of MSCs. This may be a favorable strategy for MSC chondrogenesis without exogenous growth factor induction. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:393–400, 2012     

Mechanical stimulation of mesenchymal stem cells: Implications for cartilage tissue engineering

Articular cartilage is a load‐bearing tissue playing a crucial mechanical role in diarthrodial joints, facilitating joint articulation, and minimizing wear. The significance of biomechanical stimuli in the development of cartilage and maintenance of chondrocyte phenotype in adult tissues has been well documented. Furthermore, dysregulated loading is associated with cartilage pathology highlighting the importance of mechanical cues in cartilage homeostasis. The repair of damaged articular cartilage resulting from trauma or degenerative joint disease poses a major challenge due to a low intrinsic capacity of cartilage for self‐renewal, attributable to its avascular nature. Bone marrow‐derived mesenchymal stem cells (MSCs) are considered a promising cell type for cartilage replacement strategies due to their chondrogenic differentiation potential. Chondrogenesis of MSCs is influenced not only by biological factors but also by the environment itself, and various efforts to date have focused on harnessing biomechanics to enhance chondrogenic differentiation of MSCs. Furthermore, recapitulating mechanical cues associated with cartilage development and homeostasis in vivo, may facilitate the development of a cellular phenotype resembling native articular cartilage. The goal of this review is to summarize current literature examining the effect of mechanical cues on cartilage homeostasis, disease, and MSC chondrogenesis. The role of biological factors produced by MSCs in response to mechanical loading will also be examined. An in‐depth understanding of the impact of mechanical stimulation on the chondrogenic differentiation of MSCs in terms of endogenous bioactive factor production and signaling pathways involved, may identify therapeutic targets and facilitate the development of more robust strategies for cartilage replacement using MSCs. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:52–63, 2018.     

Development and characterization of novel clinical grade neonatal porcine bone marrow‐derived mesenchymal stem cells

Due to recent advances in research on mesenchymal stem cells (MSCs), MSCs are expected to be used in various clinical applications. However, securing adequate cadaveric donors and safety of living donors are major issues. To solve such issues, we have examined to develop clinical grade neonatal porcine bone marrow‐derived MSCs (npBM‐MSCs). Clinical grade neonatal porcine bone marrow cells were collected, frozen, and sent to our laboratory by air. The npBM‐MSCs were isolated from thawed bone marrow cells, then frozen. The thawed npBM‐MSCs were examined for CD markers and differentiated into chondrocytes, osteocytes, and adipocytes. They were compared with human bone marrow‐derived MSCs (hBM‐MSCs) for growth rate and size. To assess the robustness of proliferation, we compared culture medium with or without gelatin. The npBM‐MSCs expressed positive MSC markers CD29, CD44, and CD90 and were differentiated into chondrocytes, osteocytes, and adipocytes. The doubling time of npBM‐MSCs was significantly shorter than that of hBM‐MSCs (17.3 ± 0.8 vs 62.0 ± 19.6 hours, P < 0.01). The size of npBM‐MSCs was also significantly smaller than that of hBM‐MSCs (13.1 ± 0.3 vs 17.5 ± 0.4 μm, P < 0.001). The npBM‐MSCs showed similar proliferation characters irrespective of with or without gelatin coating. The npBM‐MSCs secreted VEGF‐A, VEGF‐C, and TGF‐β1. We have established npBM‐MSCs which show super‐rapid growth, small size, and robust proliferation profile. The np‐MSCs might be able to solve the donor issues for MSC therapy.     

Evaluation of adult equine bone marrow‐ and adipose‐derived progenitor cell chondrogenesis in hydrogel cultures

Bone marrow mesenchymal stem cells (BM‐MSCs) and adipose‐derived progenitor cells (ADPCs) are potential alternatives to autologous chondrocytes for cartilage resurfacing strategies. In this study, the chondrogenic potentials of these cell types were compared by quantifying neo‐tissue synthesis and assaying gene expression and accumulation of extracellular matrix (ECM) components of cartilage. Adult equine progenitor cells encapsulated in agarose or self‐assembling peptide hydrogels were cultured in the presence or absence of TGFβ1 for 3 weeks. In BM‐MSCs‐seeded hydrogels, TGFβ1 stimulated ECM synthesis and accumulation 3–41‐fold relative to TGFβ1‐free culture. In ADPC cultures, TGFβ1 stimulated a significant increase in ECM synthesis and accumulation in peptide (18–29‐fold) but not agarose hydrogels. Chromatographic analysis of BM‐MSC‐seeded agarose and peptide hydrogels cultured in TGFβ1 medium showed extensive synthesis of aggrecan‐like proteoglycan monomers. ADPCs seeded in peptide hydrogel also synthesized aggrecan‐like proteoglycans, although to a lesser extent than seen in BM‐MSC hydrogels, whereas aggrecan‐like proteoglycan synthesis in ADPC‐seeded agarose was minimal. RT‐PCR analysis of TGFβ1 cultures showed detectable levels of type II collagen gene expression in BM‐MSC but not ADPC cultures. Histological analysis of TGFβ1‐cultured peptide hydrogels showed the deposition of a continuous proteoglycan‐ and type II collagen rich ECM for BM‐MSCs but not ADPCs. Therefore, this study showed both protein and gene expression evidence of superior chondrogenesis of BM‐MSCs relative to ADPCs. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:322–331, 2008     

Effect of culture duration on chondrogenic preconditioning of equine bone marrow mesenchymal stem cells in self‐assembling peptide hydrogel

Ex vivo induction of chondrogenesis is a promising approach to improve upon the use of bone marrow mesenchymal stem cells (MSCs) for cartilage tissue engineering. This study evaluated the potential to induce chondrogenesis with days of culture in chondrogenic medium for MSCs encapsulated in self‐assembling peptide hydrogel. To simulate the transition from preconditioning culture to implantation, MSCs were isolated from self‐assembling peptide hydrogel into an individual cell suspension. Commitment to chondrogenesis was evaluated by seeding preconditioned MSCs into agarose and culturing in the absence of the chondrogenic cytokine transforming growth factor beta (TGFβ). Positive controls consisted of undifferentiated MSCs seeded into agarose and cultured in medium containing TGFβ. Three days of preconditioning was sufficient to produce chondrogenic MSCs that accumulated ～75% more cartilaginous extracellular matrix than positive controls by day 17. However, gene expression of type X collagen was ～65‐fold higher than positive controls, which was attributed to the absence of TGFβ. Potential induction of immunogenicity with preconditioning culture was indicated by expression of major histocompatibility complex class II (MHCII), which was nearly absence in undifferentiated MSCs, and ～7% positive for preconditioned cells. These data demonstrate the potential to generate chondrogenic MSCs with days of self‐assembling peptide hydrogel, and the ability to readily recover an individual cell suspension that is suited for injectable therapies. However, continued exposure to TGFβ may be necessary to prevent hypertrophy indicated by type X collagen expression, while immunogenicity may be a concern for allogeneic applications. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1368–1375, 2019.     

Basement membrane molecule expression attendant to chondrogenesis by nucleus pulposus cells and mesenchymal stem cells

Bone marrow‐derived mesenchymal stem cells (MSCs) represent an autologous cell source for nucleus pulposus (NP) tissue engineering and regeneration. Although studies have demonstrated the ability of MSCs to differentiate to NP‐like chondrocytic cells, few have comparatively studied the matrix synthesis and composition of the cartilaginous tissue formed in vitro from both cell types, particularly with respect to the expression of basement membrane (BM) molecules. The objective of this study was to evaluate chondrogenesis and expression of BM molecules, laminin and type IV collagen, in monolayer and in pellet cultures of caprine NP cells and MSCs. Both cell types demonstrated comparable levels of chondrogenesis, indicated by the percentage of chondrocytic cells, and the amounts of glycosaminoglycan and type II collagen. Laminin and type IV collagen were expressed intracellularly by NP cells and MSCs cultured in monolayer. During chondrogenesis in pellet cultures, the deposition of BM molecules in NP and MSC pellets followed an orderly spatiotemporal shift in pattern from a diffuse territorial and interterritorial distribution to a defined pericellular localization, as seen in normal adult NP. These results inform the use of MSCs for NP regeneration and suggest the possible involvement of certain BM molecules in chondrogenesis and cartilage regeneration. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1136–1143, 2013     

A preliminary study comparing the use of allogenic chondrogenic pre‐differentiated and undifferentiated mesenchymal stem cells for the repair of full thickness articular cartilage defects in rabbits

Chondrogenic differentiated mesenchymal stem cells (CMSCs) have been shown to produce superior chondrogenic expression markers in vitro. However, the use of these cells in vivo has not been fully explored. In this study, in vivo assessment of cartilage repair potential between allogenic‐derived chondrogenic pre‐differentiated mesenchymal stem cells and undifferentiated MSCs (MSCs) were compared. Bilateral full thickness cartilage defects were created on the medial femoral condyles of 12 rabbits (n = 12). Rabbits were divided into two groups. In one group, the defects in the right knees were repaired using alginate encapsulated MSCs while in the second group, CMSCs were used. The animals were sacrificed and the repaired and control knees were assessed at 3 and 6 months after implantation. Quantitative analysis was performed by measuring the Glycosaminoglycans (GAGs)/total protein content. The mean Brittberg score was higher in the transplanted knees as compared to the untreated knee at 6 months (p < 0.05). Quantitative analysis of GAGs was consistent with these results. Histological and immunohistochemical analysis demonstrated hyaline‐like cartilage regeneration in the transplanted sites. Significant differences between the histological scores based on O'Driscoll histological grading were observed between contralateral knees at both 3 and 6 months (p < 0.05). No significant differences were observed between the Britberg, O'Driscoll scores, and GAGs/total protein content when comparing defect sites treated with MSC and CMSC (p > 0.05). This study demonstrates that the use of either MSC or CMSC produced superior healing when compared to cartilage defects that were untreated. However, both cells produced comparable treatment outcomes. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1336–1342, 2011     

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.     

Induction of fracture repair by mesenchymal cells derived from human embryonic stem cells or bone marrow

Development of novel therapeutic approaches to repair fracture non‐unions remains a critical clinical necessity. We evaluated the capacity of human embryonic stem cell (hESC)‐derived mesenchymal stem/stromal cells (MSCs) to induce healing in a fracture non‐union model in rats. In addition, we placed these findings in the context of parallel studies using human bone marrow MSCs (hBM‐MSCs) or a no cell control group (n = 10–12 per group). Preliminary studies demonstrated that both for hESC‐derived MSCs and hBM‐MSCs, optimal induction of fracture healing required in vitro osteogenic differentiation of these cells. Based on biomechanical testing of fractured femurs, maximum torque, and stiffness were significantly greater in the hBM‐MSC as compared to the control group that received no cells; values for these parameters in the hESC‐derived MSC group were intermediate between the hBM‐MSC and control groups, and not significantly different from the control group. However, some evidence of fracture healing was evident by X‐ray in the hESC‐derived MSC group. Our results thus indicate that while hESC‐derived MSCs may have potential to induce fracture healing in non‐unions, hBM‐MSCs function more efficiently in this process. Additional studies are needed to further modify hESCs to achieve optimal fracture healing by these cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1804–1811, 2011     

Chemotaxis of human articular chondrocytes and mesenchymal stem cells

Migration of chondrocytes and mesenchymal stem cells (MSCs) may be important in cartilage development, tissue response to injury, and in tissue engineering. This study analyzed growth factors and cytokines for their ability to induce migration of human articular chondrocytes and bone marrow‐derived mesenchymal stem cells in Boyden chamber assays.In human articular chondrocytes serum induced dose‐ and time‐dependent increases in cell migration. Among a series of growth factors and cytokines tested only PDGF induced a significant increase in cell migration. The PDGF isoforms AB and BB were more potent than AA. There was an aging‐related decline in the ability of chondrocytes to migrate in response to serum and PDGF. Human bone marrow MSC showed significant chemotaxis responses to several factors, including FBS, PDGF, VEGF, IGF‐1, IL‐8, BMP‐4, and BMP‐7. In summary, these results demonstrate that directed cell migration is inducible in human articular chondrocytes and MSC. PDGF is the most potent factor analyzed, and may be useful to promote tissue integration during cartilage repair or tissue engineering. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1407–1412, 2008     

Chondrocytes harvested from osteochondritis dissecans cartilage are able to undergo limited in vitro chondrogenesis despite having perturbations of cell phenotype in vivo

Our objective was to characterize the variation in gene expression for key genes associated with chondrogenic phenotype of osteochondrosis (OC)‐affected and normal chondrocytes, and to identify whether OC chondrocytes can redifferentiate and regain a phenotype similar to normal chondrocytes if appropriate chondrogenic signals are given. Equine articular cartilage removed at surgery to treat clinically significant OC lesions was collected (n = 10), and the gene expression evaluated and compared to aged‐matched normal samples (n = 10). Cartilage was harvested from normal (n = 4) and OC (n = 3) joints from horses at necropsy. Chondrogenic pellet cultures were established following monolayer proliferation. After 14 days in culture, the pellets were assessed by histochemical and pellet weight analysis, assay of glycosaminoglycan (GAG) content, and gene expression. Chondrocytes from OC cartilage expressed significantly more Coll‐I, ‐II, ‐III, and ‐X than chondrocytes from normal cartilage (all p < 0.0001). Furthermore, OC chondrocytes expressed significantly more MMP‐13, ADAMTS‐4 (both p < 0.0001), and TIMP‐1 (p < 0.001) and significantly less TIMP‐2 and TIMP‐3. Pellets created from OC chondrocytes contained significantly less GAG (p = 0.0069) and expressed significantly less Sox9 and significantly more superficial zone protein (SZP) (p = 0.0105) than pellets created from normal cartilage. The results suggest that chondrocytes from OC cartilage at the time of surgical treatment have perturbations in phenotype compared to cells from normal cartilage. Despite these differences, following monolayer expansion and pellet culture under chondrogenic conditions, chondrocytes derived from OC cartilage retain some ability to undergo chondrogenic differentiation and synthesize an appropriate cartilage‐like matrix. However, this chondrogenic differentiation potential is inferior to that seen in aged‐matched normal chondrocytes. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1133–1140, 2008     

Enhanced chondrogenesis from chondrocytes co-cultured on mesenchymal stromal cells: Implication for cartilage repair

Cellular therapy based on chondrocytes implantation is the most widely used procedure for inducing cartilage regeneration. However, the dedifferentiation process that these cells suffer and their limited capacity of proliferation, when they are cultured in vitro, restrict their use in cellular therapy protocols. To investigate the capacity of mesenchymal stromal cells (MSCs) to promote chondrogenesis from chondrocytes or chondrons in 2D and 3D coculture systems. Murine chondrocytes and chondrons were cocultured with MSCs at different cell ratios (100/0, 50/50, 70/30, 0/100) in two-dimensional (2D) and three-dimensional (3D) culture systems. High proliferation of cells with chondrocyte morphology, enhanced GAG production and expression of cartilage genes (aggrecan, type II collagen, and SOX-9) were observed in chondrocytes/MSCs cocultures. In contrast, fibroblastoid cells, down-regulation of cartilage gene expression and reduction of GAG production were observed in chondrons/MSCs cocultures. Chondrocytes within cartilage lacunae and surrounded by extracellular matrix were observed in chondrocytes/MSC pellets. MSCs promote the proliferation of functional chondrocytes in 2D and 3D culture systems. Transplantation of chondrogenic construct based on MSCs and chondrocytes may constitute a potential treatment for inducing cartilage repair.     

COMP‐Ang1 promotes chondrogenic and osteogenic differentiation of multipotent mesenchymal stem cells through the Ang1/Tie2 signaling pathway

Mesenchymal stem cells (MSCs) are pleiotrophic cells that differentiate to chondrocytes, osteoblasts, or adipocytes, as a result of crosstalk by specific signaling pathways including MAPK pathway. Recently cartilage oligomeric matrix protein angiopoietin1 (COMP‐Ang1), an Ang1 variant which is more potent than native Ang1 in phosphorylating Tie2 receptor was developed. The Ang1/Tie2 signaling system not only plays a pivotal role in vessel growth, remodeling, and maturation, but also protective and recruit effect on MSCs. Thus, the aim of the present study was to investigate the differentiate effect of Ang1/Tie2 signaling on MSCs in the presence of chondrogenic, osteogenic and adipogenic induction medium, and to determine the possible mechanisms. Our results clearly demonstrated that MSCs cultured in each induction medium with COMP‐Ang1 revealed strongly chondrogenic and osteogenic morphological change (3.5‐ and 2‐fold, respectively) as well as up‐regulate each gene, except for adipogenic differentiation. Accordingly, we found that phosphorylation of Tie2 expression lead to phosphorylation of p38 and AKT and then accelerating each differentiation of MSCs to chondrocytes and osteoblasts. Therefore, our findings suggest that COMP‐Ang1 present a portal to promote MSCs differentiation to chondrocytes and osteoblasts through Ang1/Tie2 signaling pathway and provide insights into novel therapies for bone diseases. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1920–1928, 2013     

miR‐483 targets SMAD4 to suppress chondrogenic differentiation of human mesenchymal stem cells

MicroRNAs (miRNAs) can regulate cellular differentiation processes by modulating multiple pathways simultaneously. Previous studies to analyze in vivo miRNA expression patterns in developing human limb cartilage tissue identified significant downregulation of miR‐483 in hypertrophic chondrocytes relative to proliferating and differentiated chondrocytes. To test the function of miR‐483 during chondrogenesis, lentiviral strategies were used to overexpress miR‐483 during in vitro chondrogenesis of human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs). While the in vivo expression patterns led us to hypothesize that miR‐483 may enhance chondrogenesis or suppress hypertrophic marker expression, surprisingly, miR‐483 overexpression reduced chondrocyte gene expression and cartilage matrix production. In addition, cell death was induced at later stages of the chondrogenesis assay. Mechanistic studies revealed that miR‐483 overexpression resulted in downregulation of the TGF‐β pathway member SMAD4, a known direct target of miR‐483‐3p. From these studies, we conclude that constitutive overexpression of miR‐483 in hBM‐MSCs inhibits chondrogenesis of these cells and does not represent an effective strategy to attempt to enhance chondrocyte differentiation and anabolism in this system in vitro. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2369–2377, 2017.

     

Effects of antithrombotic drugs fondaparinux and tinzaparin on in vitro proliferation and osteogenic and chondrogenic differentiation of bone‐derived mesenchymal stem cells

An unexpected side effect of some classes of anticoagulants has been osteoporosis which may be, at least in part, related to deranged mesenchymal stem cell (MSC) function. The aim of the present study was to compare the effect of fondaparinux (FDP), a novel antithrombotic with a traditional widely used low molecular weight heparin, tinzaparin (TZP) on MSC proliferation and differentiation. MSCs were isolated from trabecular bone of 14 trauma patients by a collagenase‐based digestion procedure and expanded in standard conditions until passage 3. Proliferation and differentiation of MSCs to chondrocytes and osteoblasts was assessed with or without the addition of FDP and TZP using standard in vitro assays and a broad range of drug concentrations. Flow cytometry was used for MSC phenotyping. In the age studied group (17–74 years old) the MSC frequency in collagenase‐released fractions was 641/10⁶ cells (range 110–2,158) and their growth characteristics were ～4 days/population doubling. Cultures had a standard MSC phenotype (CD73+, CD105+, CD146+, CD106+, and CD166+). Cell proliferation was assessed by both colony‐forming unit‐fibroblast (CFU‐F) and colorimetric tetrazolium salt XTT assays. In both assays, MSC proliferation was inhibited by the addition of TZP, particularly at high concentrations. In contrast, FDP had no effect on MSC proliferation. Osteogenic differentiation and chondrogenic differentiation were not affected by the addition of either TZP or FDP. Whilst MSC proliferation, but not differentiation, is negatively affected by TZP, there was no evidence for adverse effects of FDP in this in vitro model system which argues well for its use in the orthopedic setting. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1327–1335, 2011     

Articular cartilage repair using an intra‐articular magnet and synovium‐derived cells

The purpose of this study was to investigate the chondrogenic potential of magnetically labeled synovium‐derived cells (M‐SDCs) and examine whether M‐SDCs could repair the articular cartilage using an intra‐articular magnet after delivery to the lesion. Synovium‐derived cells (SDCs) were cultured from the synovium of a rat knee, and were magnetically labeled with ferumoxides. M‐SDCs were examined with a transmission electron microscope. A pellet culture system was used to evaluate the chondrogenic potential of M‐SDCs in a magnetic field. In a rat model, allogeneic M‐SDCs were injected into the knee after we made an osteochondral defect on the patellar groove and implanted an intra‐articular magnet at the bottom of the defect. We histologically examined the defects at 48 h, 4 weeks, 8 weeks, and 12 weeks after treatment. Electron microscopy showed the transfection of ferumoxides into SDCs. The pellet cultures revealed the chondrogenic potential of M‐SDCs in a magnetic field. M‐SDCs accumulated in the osteochondral defect at 48 h after treatment, and we confirmed the regeneration of the articular cartilage at 4 weeks, 8 weeks, and 12 weeks after treatment using an intra‐articular magnet. We demonstrated that articular cartilage defects could be repaired using an intra‐articular magnet and M‐SDCs. We believe that this system will be useful to repair human articular cartilage defects. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:531–538, 2011