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.     

生长分化因子5促进软骨细胞肥大成熟的实验研究

[目的]探讨生长分化因子5(GDF-5)在诱导成人骨髓间充质干细胞(hBMSCs)成软骨分化的同时,促进其肥大成熟的作用.[方法]体外分离培养hBMSCs,取第四代细胞实验.将细胞消化、悬浮,以5×106/ml的细胞密度进行高密度培养,分别用含0.500ng/ml GDF-5的软骨诱导液(CM)诱导培养3、4周.免疫组化、甲苯胺蓝染色检测Ⅱ型胶原、Ⅹ型胶原和蛋白多糖的表达情况.荧光实时定量RT-PCR检测两组微团Ⅱ型胶原、Ⅹ型胶原和蛋白多糖mRNA的表达情况.[结果]实验组微团中的细胞体积较对照组稍大,细胞密度稍低.甲苯胺蓝和免疫组化染色结果显示各组微团均有Ⅱ型胶原、Ⅹ型胶原和蛋白多糖的表达和分泌,且4周实验组的表达最为强烈.荧光实时定量RT-PCR检测示,干预后3周实验组Ⅱ型胶原、蛋白多糖基因表达量明显高于3周对照组(P＜0.05),但Ⅹ型胶原基因表达与对照组相比无显著性差异(P＞0.05);干预后4周实验组Ⅹ型胶原基因表达高于其余各组(P＜0.05),Ⅱ型胶原、蛋白多糖基因表达明显高于两对照组(P＜0.05),但与3周实验组相比较,差异无统计学意义(P＞0.05).[结论]GDF-5在诱导细胞成软骨分化的同时,还可促进其成熟肥大.     

Low-intensity ultrasound stimulation enhances chondrogenic differentiation in alginate culture of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-beta) treatment under the 3-dimensional (3-D) culture condition. However, more efficient and versatile methods for chondrogenic differentiation of MSCs are still in demand for its clinical application. Recently, low-intensity ultrasound (LIUS) was shown to enhance fracture healing in vitro and induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of LIUS on the chondrogenesis of rabbit MSCs (rMSCs) in a 3-D alginate culture and on the maintenance of chondrogenic phenotypes after replating them on a monolayer culture. The LIUS treatment of rMSCs increased: (i) the matrix formation; (ii) the expression of chondrogenic markers such as collagen type II, aggrecan, and Sox-9; (iii) the expression of tissue inhibitor of metalloprotease-2 implicated in the integrity of cartilage matrix; and (iv) the capacity to maintain the chondrogenic phenotypes in a monolayer culture. Notably, LIUS effects were clearly shown even without TGF-beta treatment. These results suggest that LIUS treatment could be an efficient and cost-effective method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering.     

Induction of bone marrow mesenchymal stem cell chondrogenesis following short‐term suspension culture

The objective of this study was to evaluate mesenchymal stem cell (MSC) chondrogenesis following incubation in chondrogenic suspension cultures from which single cells were obtained. MSCs were maintained in suspension over a nonadherent surface for 3 days, dissociated into a suspension, and then evaluated for chondrogenesis in agarose in the presence or absence of transforming growth factor beta (TGFβ). In a second experiment, MSCs from suspension culture were returned to monolayer expansion for 2 days prior to testing for chondrogenesis. In both cases, undifferentiated MSCs were evaluated as controls. Suspension culture alone did not stimulate chondrogenesis. Suspension followed by expansion stimulated a four‐ to ninefold increase in extracellular matrix (ECM) synthesis in TGFβ‐free cultures, a finding that was attributed to an increase in viable MSCs that secreted a proteoglycan‐rich ECM. Gene expression of aggrecan and type II collagen increased with suspension culture, but decreased with postsuspension expansion. Therefore, stimulation of ECM synthesis without additional TGFβ exposure could not be attributed to an enhancement of chondrogenesis with monolayer culture. ECM synthesis of suspension/expansion‐conditioned MSCs without additional TGFβ exposure was less than samples maintained in TGFβ throughout the differentiation culture. Based on these findings, a better understanding of factors associated with early‐stage chondrogenesis and MSC differentiation to a highly active phenotype may lead to improved methods for stimulating chondrogenesis during short‐term culture. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:26–32, 2011     

The use of mesenchymal stem cells for chondrogenesis

The application of autologous chondrocytes in cartilage repair procedures is associated with several disadvantages, including injury of healthy cartilage in a preceding surgery frequently resulting in formation of inferior fibrocartilage at defect sites. In order to improve the quality of regeneration, adult mesenchymal stem cells (MSC) are regarded as a promising alternative. The great challenge, when considering MSC for articular cartilage repair, is to generate cells with features of stable chondrocytes which are resistant to hypertrophy and terminal differentiation, as found in hyaline articular cartilage. Common in vitro protocols for chondrogenic differentiation of MSC successfully induce expression of multiple cartilage-specific molecules, including collagen type II and aggrecan, and result in a chondrocyte-like phenotype. However, in vitro chondrogenesis of MSC additionally promotes induction of fibrocartilage-like features such as expression of collagen type I, and hypertrophy, as demonstrated by up-regulation of collagen type X, MMP13 and ALP-activity. As a consequence, differentiated MSC pellets undergo mineralisation and vascularisation after ectopic transplantation in a process similar to endochondral ossification. This review discusses the complexity and entailed challenges when considering MSC from various sources for clinical application and the necessity to optimise chondrogenesis by repressing hypertrophy to obtain functional and suitable cells for cartilage repair.     

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     

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     

骨髓间充质干细胞在β3转化生长因子联合胰岛素样生长因子-1定向诱导下的软骨组织工程

目的探讨β3转化生长因子（TGF-β3）在诱导骨髓间充质干细胞（mesenchymal stem cells,MSCs）向软骨细胞分化中胰岛素样生长因子-1（IGF-1）的作用以及在软骨组织工程中的应用。方法在体外用TGF—β3或（和）IGF-1诱导藻酸钠微球中的MSCs向软骨细胞定向分化,免疫组织化学、逆转录-聚合酶链反应（RT—PCR）检测Ⅱ型胶原和聚集蛋白聚糖（aggrecan）的表达,Western印迹法检测Sox9蛋白的表达,激光共聚焦显微镜和扫描电镜观察该软骨细胞在壳聚糖支架上的生长。结果TGF-β3。能诱导藻酸钠微球中的MSCs表达软骨特异性的Ⅱ型胶原、聚集蛋白聚糖和Sox9,IGF-1能显著性地增强这种作用（P〈0．05）。Ⅱ型胶原、聚集蛋白聚糖和Sox9之间的相关系数分别为0．95和0．91。诱导的软骨细胞能在壳聚糖支架上黏附、迁徙、增殖。结论在TGF-β3诱导MSCs分化成软骨细胞地过程中,IGF-1可能通过促进Sox9的表达起到协同作用。诱导分化后的软骨细胞与壳聚糖复合支架表现出良好的组织相容性。     

Physiological testosterone levels enhance chondrogenic extracellular matrix synthesis by male intervertebral disc cells in vitro,but not by mesenchymal stem cells

Background contextTestosterone (T) is a hormone and regulator involved in the processes of development of the organism (ie, promoting development of bone and muscle mass). Although T effects on the mesenchyme-derived muscle, bone, and adipose tissues are well studied, T effects on intervertebral disc (IVD) have not been reported.PurposeThe aim was to test the following hypothesis: if a physiological concentration of T (～30 nM) can improve in vitro chondrogenesis of human IVD cells and mesenchymal stem cells (MSCs).Study design/settingHuman IVD cells and MSCs were differentiated to chondrogenic lineage on gelatin scaffolds for 4 weeks, in the presence or absence of T.MethodsChondrogenesis was assessed by cell viability, by measuring gene expression with quantitative polymerase chain reaction and extracellular matrix (ECM) accumulation with immunoblotting, immunohistochemical, and biochemical methods.ResultsSupplementation of T to chondrogenic culture did not affect viability. In male IVD cells, T had a beneficial impact on chondrogenesis, particularly in nucleus pulposus cells, demonstrated by an increased expression of aggrecan, collagen type I, and especially collagen type II. Conversely, T had no effects on chondrogenesis of female IVD cells or MSCs from both genders. A gene expression array of transforming growth factor β/bone morphogenetic protein signaling cascade showed that in male IVD cells, T promoted a stable general but nonsignificant increase in gene expression. Furthermore, aromatase inhibitor anastrazole repressed the effect of T on ECM expression by IVD cells. The results suggest that T increased ECM accumulation in male IVD cells in combination with its conversion to estradiol by the enzyme aromatase.ConclusionsWe demonstrated that T effectively enhances in vitro chondrogenesis in male IVD cells, rising the interest in the possible role of sex hormones in IVD degeneration. Nevertheless, T does not affect chondrogenic differentiation of female IVD cells and MSCs from both genders.     

Runx1/AML1/Cbfa2 mediates onset of mesenchymal cell differentiation toward chondrogenesis.

Runx proteins mediate skeletal development. We studied the regulation of Runx1 during chondrocyte differentiation by real-time RT-PCR and its function during chondrogenesis using overexpression and RNA interference. Runx1 induces mesenchymal stem cell commitment to the early stages of chondrogenesis. INTRODUCTION: Runx1 and Runx2 are co-expressed in limb bud cell condensations that undergo both cartilage and bone differentiation during murine development. However, the cooperative and/or compensatory effects these factors exert on skeletal formation have yet to be elucidated. MATERIALS AND METHODS: Runx1/Cbfa2 and Runx2/Cbfa1 were examined at different stages of embryonic development by immunohistochemistry. In vitro studies used mouse embryonic limb bud cells and assessed Runx expressions by immunohistochemistry and real-time RT-PCR in the presence and absence of TGFbeta and BMP2. Runx1 was overexpressed in mesenchymal cell progenitors using retroviral infection. RESULTS: Immunohistochemistry showed that Runx1 and Runx2 are co-expressed in undifferentiated mesenchyme, had similar levels in chondrocytes undergoing transition from proliferation to hypertrophy, and that there was primarily Runx2 expression in hypertrophic chondrocytes. Overall, the expression of Runx1 remained significantly higher than Runx2 mRNA levels during early limb bud cell maturation. Treatment of limb bud micromass cultures with BMP2 resulted in early induction of both Runx1 and Runx2. However, upregulation of Runx2 by BMP2 was sustained, whereas Runx1 decreased in later time-points when type X collagen was induced. Although TGFbeta potently inhibits Runx2 and type X collagen, it induces type II collagen mRNA and mildly but significantly inhibits Runx1 isoforms in the early stages of chondrogenesis. Virus-mediated overexpression of Runx1 in mouse embryonic mesenchymal cells resulted in a potent induction of the early chondrocyte differentiation markers but not the hypertrophy marker, type X collagen. Knockdown or Runx1 potently inhibits type II collagen, alkaline phosphatase, and Runx2 and has a late inhibitory effect on type X collagen. CONCLUSION: These findings show a distinct and sustained role for Runx proteins in chondrogenesis and subsequent chondrocyte maturation. Runx1 is highly expressed during chondrogenesis in comparison with Runx2, and Runx1 gain of functions stimulated this process. Thus, the Runx genes are uniquely expressed and have distinct roles during skeletal development.     

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     

Cross‐linking affects cellular condensation and chondrogenesis in type II collagen‐GAG scaffolds seeded with bone marrow‐derived mesenchymal stem cells

The formation of cartilaginous tissue by chondroprogenitor cells, whether in vivo or in vitro, appears to require a critical initial stage of “condensation” in which intercellular space is reduced through an aggregation of cells, leading to development of cell‐to‐cell junctions followed by chondrocytic differentiation. The objective of this study was to investigate the association of aggregation (condensation) of mesenchymal stem cell (MSCs) and chondrogenesis in vitro. Previous work with chondrocytes indicated that the cross‐link density and related cell‐mediated contraction of collagen scaffolds significantly affects cartilaginous tissue formation within the cell‐seeded construct. Based on this finding, we hypothesized that the cell‐aggregating effect of the contraction of MSC‐seeded collagen scaffolds of lower cross‐link density favors chondrogenesis; scaffolds of higher cross‐link density, which resist cell‐mediated contraction, would demonstrate a lower cell number density (i.e., subcritical packing density) and less cartilage formation. Type II collagen–GAG scaffolds, chemically cross‐linked to achieve a range of cross‐link densities, were seeded with caprine MSCs and cultured for 4 weeks. Constructs with low cross‐link densities experienced cell‐mediated contraction, increased cell number densities, and a greater degree of chondrogenesis (indicated by the chondrocytic morphology of cells, and synthesis of GAG and type II collagen) compared to more highly cross‐linked scaffolds that resisted cellular contraction. These results provide a foundation for further investigation of the mechanisms by which condensation of mesenchymal cells induces chondrogenesis in this in vitro model, and may inform cross‐linking protocols for collagen scaffolds for use in cartilage tissue engineering. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1184–1192, 2010     

Hypoxic conditions during expansion culture prime human mesenchymal stromal precursor cells for chondrogenic differentiation in three-dimensional cultures

Cell-based approaches using mesenchymal stromal precursor cells (MSCs) for the regeneration of intervertebral discs are attracting increased interest, even though the intervertebral disc is a very demanding environment. Implanted cells eventually face acidic pH, hypoxia, and a lack of nutrients. While the regenerative potential of MSCs for skeletal tissues has been well described, it is still questionable whether human MSCs can be prepared for prolonged survival and proper functioning and whether they can differentiate under the adverse conditions encountered in the disc. Here we examined the influence of hypoxia during expansion and differentiation on the chondrogenesis of MSCs. Chondrogenic differentiation was performed in in situ solidifying gelatin hydrogels, which represent a suitable matrix for delivering and anchoring cells within the disc tissue. To consider limitations in nutrition in the intervertebral disc, differentiation was performed at low cell concentrations in the gelatin hydrogels. Standard high-density micromass cultures served as reference controls. To determine the quality of chondrogenesis we analyzed typical marker molecules such as collagen types I, II, X, Sox-9, MIA, and aggrecan mRNA using RT-qPCR and determined protein deposition by histological stainings and biochemical methods. We could demonstrate that in gelatin-based hydrogels chondrogenic differentiation of human MSCs is possible at low cell concentrations. The quality of chondrogenic differentiation could be improved by hypoxia. Best results were obtained when the entire in vitro process, including MSC expansion and subsequent differentiation, was done under hypoxic conditions. MSCs that were expanded under reduced oxygen tension were primed for a chondrogenic differentiation.     

诱导骨髓间充质干细胞向软骨细胞分化的体外研究

目的 探讨转化生长因子β1（transforming growth factor β1，TGF—β1）、胰岛素样生长因子1（insulinlike growth factor1，IGF-1）在诱导骨髓间充质干细胞（marrow mesenchymal stem ceils，MSCs）向软骨细胞分化过程中的相互作用，并研究细胞密度对MSCs向软骨细胞分化的影响。方法 取健康昆明种小白鼠骨髓，用全骨髓贴壁法筛选获得MSCs，体外培养传代。采用特定的诱导培养使MSCs向软骨细胞分化，按培养基内添加生长因子的不同分成3个实验组和对照组。实验组分别为：TGF—β1＋IGF-1联合应用组（TGF—β1 10ng／ml、IGF-1 50ng／m1）；TGF—β1单独应用组（TGF—β1 10ng／m1）；IGF-1单独应用组（IGF-1 50ng／m1）；对照组不添加任何生长因子。TGF—β1＋IGF-1联合应用组于诱导14d和21d，分别进行甲苯胺蓝染色及免疫荧光双染法鉴定；于诱导7、14和21d各组分别提取诱导细胞总RNA，进行RT—PCR扩增，检测TGF—β1、IGF-1对诱导细胞Ⅱ型胶原表达量的影响；比较MSCs在平板培养及细胞团培养时，Ⅱ型胶原表达量的差异。结果TGF—β1＋IGF-1联合应用组诱导培养14d，诱导软骨细胞甲苯胺蓝染色呈阳性，免疫荧光染色可见诱导软骨细胞的细胞外基质含有Ⅱ型胶原。各组基因扩增产物的凝胶电泳可见，TGF—β1＋IGF-1联合应用组和TGF—β1单独应用组Ⅱ型胶原扩增片段呈阳性；IGF-1单独应用组和对照组，未见Ⅱ型胶原扩增条带；凝胶成像系统灰度扫描示Ⅱ型胶原表达量TGF—β1＋IGF-1联合应用组各时间点均比TGF—β1单独应用组明显增加（P〈0．05）。细胞团培养模式下，诱导细胞表达Ⅱ型胶原比平板培养模式更加显著。结论 MSCs向软骨细胞诱导分化时，IGF-1对TGF—β1有明显的促进作用；细胞培养密度提高有利于MSCs成软骨细胞表型。     

Synthetic triterpenoids, CDDO-Imidazolide and CDDO-Ethyl amide, induce chondrogenesis

Novel methods for inducing chondrogenesis are critical for cartilage tissue engineering and regeneration. Here we show that the synthetic oleanane triterpenoids, CDDO-Imidazolide (CDDO-Im) and CDDO-Ethyl amide (CDDO-EA), at concentrations as low as 200 nM, induce chondrogenesis in organ cultures of newborn mouse calvaria. The cartilage phenotype was measured histologically with metachromatic toluidine blue staining for proteoglycans and by immunohistochemical staining for type II collagen. Furthermore, real-time polymerase chain reaction (PCR) analysis using mRNA from calvaria after 7-day treatment with CDDO-Im and CDDO-EA showed up-regulation of the chondrocyte markers SOX9 and type II collagen (alpha1). In addition, TGF-β; BMPs 2 and 4; Smads 3, 4, 6, and 7; and TIMPs-1 and -2 were increased. In contrast, MMP-9 was strongly down-regulated. Treatment of human bone marrow-derived mesenchymal stem cells with CDDO-Im and CDDO-EA (100 nM) induced expression of SOX9, collagen IIα1, and aggrecan, as well as BMP-2 and phospho-Smad5, confirming that the above triterpenoids induce chondrogenic differentiation. This is the first report of the use of these drugs for induction of chondrogenesis.     

Gene and protein expressions of bone marrow mesenchymal stem cells in a bone tunnel for tendon-bone healing

Purpose

Bone marrow mesenchymal stem cells (BMSCs) injected around tendon grafts can enhance tendon-bone healing and promote fibrocartilage formation. To understand gene and protein expressions of cells during tendon-bone healing, auto-BMSCs were implanted into a bone tunnel in anterior cruciate ligament reconstruction in a rabbit model.

Methods

BMSCs were harvested from New Zealand white rabbits. By an anterior cruciate ligament reconstruction model, 1 × 10⁶ BMSCs in 0.35 mL of fibrin glue was injected into bone tunnel as Fibrin-BMSC group. Only fibrin glue (Fibrin group) was injected as control. Three chondrogenesis genes and proteins, including Sox 9, collagen Type II (COII), aggrecan, and three osteogenesis genes and proteins, including Runx2, collagen type I (COI), and osteocalcin, between Fibrin-BMSC and Fibrin group were compared by real-time polymerase chain reaction assay and immunohistochemical assay postoperation.

Results

In real-time polymerase chain reaction assay, Sox9, COII, aggrecan, COI, and osteocalcin expressions upregulated and Runx2 downregulated were determined in Fibrin-BMSC group at 1 week. COII, aggrecan upregulated, and Runx2 and osteocalcin downregulated were determined at 4 weeks. In immunohistochemical assay, only Sox9, COII, and aggrecan proteins in only Fibrin-BMSC group were observed at 4 weeks. The protein expression as same as gene expression was obtained in a bone tunnel.

Conclusion

Auto-BMSCs promoted COII and aggrecan expression and reduced Runx2 and osteocalcin expression in a bone tunnel. It demonstrated that these cells could enhance fibrocartilage formation because of higher chondrogenesis expression during tendon-bone healing.