Composition-function relations of cartilaginous tissues engineered from chondrocytes and mesenchymal stem cells isolated from bone marrow and infrapatellar fat pad

The objective of this study was to determine the functional properties of cartilaginous tissues generated by porcine MSCs isolated from different tissue sources, and to compare these properties to those derived from chondrocytes (CCs). MSCs were isolated from bone marrow (BM) and infrapatellar fat pad (FP), while CCs were harvested from the articular surface of the femoro-patellar joint. Culture-expanded CCs and MSCs were encapsulated in agarose hydrogels and cultured in the presence of TGFβ3. Samples were analysed biomechanically, biochemically and histologically at days 0, 21 and 42. After 42 days in free swelling culture, mean GAG content was 1.50% w/w in CC-seeded constructs, compared to 0.95% w/w in FP- and 0.43% w/w in BM-seeded constructs. Total collagen accumulation was highest in FP constructs. DNA content increased with time for all the groups. The mechanical functionality of cartilaginous tissues engineered using CCs was superior to that generated from either source of MSCs. Differences were also observed in the spatial distribution of matrix components in tissues engineered using CCs and MSCs, which appears to have a strong influence on the apparent mechanical properties of the constructs. Therefore, while functional cartilaginous tissues can be engineered using MSCs isolated from different sources, the spatial composition of these tissues is unlike that generated using chondrocytes, suggesting that MSCs and chondrocytes respond differently to the regulatory factors present within developing cartilaginous constructs.     

Notch信号通路阻断剂对人脐带间充质干细胞软骨分化的影响

背景：Notch信号通路作为一条在细胞增殖和分化过程中起重要作用的信号通路,目前它在人脐带间充质干细胞在体外向软骨细胞诱导分化过程中的作用仍然未知。目的：首次探讨Notch信号通路特异性阻断剂2,4-二氨基-5-苯噻唑（DAPT）对人脐带间充质干细胞向软骨细胞诱导分化的影响。方法：从人脐带中分离获得间充质干细胞,体外向软骨细胞诱导分化。实验分4组：①无诱导组换成含体积分数5%胎牛血清和1%双抗的DMEM高糖培养基培养。②单纯诱导组换成终浓度为6.25 mg/L胰岛素、6.25 mg/L转铁蛋白、10μg/L转化生长因子β1、0.1μmol/L地塞米松、50 mg/L维生素C、体积分数5%胎牛血清和1%双抗的软骨诱导培养基培养。③二甲基亚砜组在软骨诱导培养基中加入终浓度为0.1%的二甲基亚砜培养。④2,4-二氨基-5-苯噻唑组在软骨诱导培养基中加入终浓度为5μmol/L的2,4-二氨基-5-苯噻唑（溶于二甲基亚砜）培养,二甲基亚砜终浓度为0.1%。结果与结论：诱导人脐带间充质干细胞向软骨细胞分化后,细胞形态由长梭形变为多边形,且甲苯胺蓝染色和免疫荧光染色均呈现阳性;软骨诱导分化后Jag-1、PS-1、Notch-1、Hes-1的基因表达均明显下降（P〈0.01）;添加2,4-二氨基-5-苯噻唑后,与单纯诱导组相比,人脐带间充质干细胞中Jag-1、PS-1、Hes-1（P〈0.01）和Notch-1（P〈0.05）的基因表达显著降低;蛋白聚糖和Ⅱ型胶原蛋白含量均降低（P〈0.01）;蛋白聚糖的基因表达显著降低（P〈0.01）,Ⅱ型胶原蛋白的基因表达也出现一定程度的下降。提示Notch信号存在于人脐带间充质干细胞中,诱导人脐带间充质干细胞向软骨细胞分化后,这种信号强度迅速减弱;2,4-二氨基-5-苯噻唑可能通过Jag-1-Notch-1-Hes-1途径抑制人脐带间充质干细胞向软骨细胞分化。     

骨髓间质干细胞突变的致瘤肿瘤干细胞分离及培养

目的建立合适的骨髓间质干细胞突变的肿瘤细胞系F6(肿瘤干细胞)分离及体外长期培养方法并探讨其生物学特性。方法采用抗CD133-PE抗体经流式细胞仪分选F6肿瘤干细胞(F6 CSCs),研究不同培养体系对F6 CSCs体外生长的影响,探讨最适体外培养条件。结果一次分选获得F6 CSCs纯度达到80%。分选的F6 CSCs对Balb/c小鼠有体内致瘤能力,在含2%胎牛血清的L-DMEM营养液中可以长期稳定的培养,其CD133表面标志的表达维持在68.87%。结论低浓度胎牛血清的培养液能维持F6 CSCs的生长并保留其相应的生物学特性,这为肿瘤干细胞体外培养及研究提供了新的思路和方法。     

软骨细胞上清液诱导滑膜间充质干细胞微团培养成软骨

背景：滑膜间充质干细胞在体外具有多向分化的能力,有望成为软骨组织工程中治疗软骨缺损的种子细胞,在其向软骨细胞分化过程中,合适的生长因子起了重要作用。 目的：利用富含生长因子的软骨细胞上清液诱导滑膜间充质干细胞向软骨细胞分化,并对其鉴定。 方法：采用消化法分别获得 SD 大鼠滑膜间充质干细胞、软骨细胞。收集软骨细胞上清液离心、过滤冻存备用。培养滑膜间充质干细胞至第3代后离心成微团,并用软骨细胞上清液进行成软骨诱导分化,通过形态学观察、免疫组织化学法、RT-PCR检测进行鉴定。 结果与结论：滑膜间充质干细胞使用软骨细胞上清液成软骨诱导21 d后,微团可见似软骨样组织。免疫组化法进行Ⅱ型胶原鉴定,基质能被Ⅱ型胶原染色,细胞染色呈现棕黄色。RT-PCR 结果显示诱导后的微团表达软骨特异性基因Ⅱ型胶原和蛋白聚糖。证实软骨细胞分泌的可溶性因子可以诱导大鼠滑膜间充质干细胞向软骨方向分化。     

人骨髓间充质干细胞的培养与鉴定

目的探索体外培养和鉴定人骨髓间充质干细胞（mesenchymal stemcell,MSC）的方法,为人骨髓间充质干细胞移植于受损心脏、改善心脏功能的研究打下基础。方法采用Percoll（1．073g／ml）密度梯度离心法分离人骨髓中单个核细胞,用含10％的胎牛血清低糖DMEM培养液体外培养扩增骨髓MSC,并通过FCM对其纯度进行鉴定。结果Percoll液能成功分离出骨髓间充质干细胞。原代及传代培养显示人骨髓间充质干细胞具有活跃的增殖倍增能力。体外扩增的人骨髓间充质干细胞表达CD29、CD166等表面抗原,不表达CD34、CD45等表面抗原。结论人骨髓间充质干细胞作为一种具有多分化潜能的多能干细胞,具有强大的分裂增殖能力,体外培养无明显的分化倾向．     

自体激活富血小板血浆干预兔骨髓间充质干细胞体外成软骨分化的研究

背景：自体富血小板血浆激活后可释放多种生长因子,可以促进骨髓间充质干细胞的增殖与分化。目的：观察自体激活富血小板血浆对体外培养的兔骨髓间充质干细胞向成软骨细胞分化的影响。方法：取兔股骨骨髓,全骨髓贴壁法分离培养骨髓间充质干细胞;取第3代骨髓间充质干细胞,分别应用体积分数10%自体激活富血小板血浆和体积分数10%胎牛血清培养液进行体外培养,观察其向成软骨细胞分化情况。结果与结论：分离培养的兔骨髓间充质干细胞呈长梭形,传代后细胞生长迅速。流式细胞仪检测发现第3代细胞高表达CD29、CD44,而低表达CD45。免疫荧光细胞化学染色显示经自体激活富血小板血浆诱导的骨髓间充质干细胞表达Ⅱ型胶原;实时荧光定量PCR检测发现经自体激活富血小板血浆诱导的骨髓间充质干细胞Ⅱ型胶原α1链基因和聚集蛋白聚糖基因表达明显高于经胎牛血清诱导的骨髓间充质干细胞（P〈0.01）。可见自体激活富血小板血浆具有促进兔骨髓间充质干细胞向软骨细胞方向分化的潜能。     

大鼠骨髓间充质干细胞体外分离及对原发性骨质疏松性骨折的实验研究

目的用全骨髓贴壁分离法分离培养骨髓间充质干细胞,通过建立原发性骨质疏松性骨折模型,探讨骨髓间充质干细胞对骨质疏松性骨折愈合情况。方法无菌条件下采集双侧股骨、胫骨骨髓,用全骨髓贴壁分离法培养出原代骨髓间充质干细胞,选取11周雌性SD大鼠60只,卵巢切除术(OVX)建立骨质疏松模型后,均做双侧骨折模型,随机分成对照组(OVX骨折不处理组)、生理盐水组(OVX骨折+生理盐水组)和细胞治疗组(OVX骨折+骨髓间充质干细胞治疗组),术后于第3天、第4、8周分别进行X-光扫描。结果全骨髓贴壁法成功分离出骨髓间充质干细胞,经流式细胞仪分析,培养出来的细胞与骨髓间充质干细胞表面抗原一致。X-光结果表明,细胞治疗组相比于其它组能加快骨质疏松性骨折愈合。结论体外分离大鼠骨髓间充质干细胞对原发性骨质疏松性骨折愈合有促进作用。     

Bone differentiation of marrow-derived mesenchymal stem cells using beta-tricalcium phosphate-alginate-gelatin hybrid scaffolds

The aim of the present study was to establish a 3D culture system for bone differentiation of mesenchymal stem cells (MSCs), using a new hybrid sponge. To manufacture the scaffold, a composite of beta-tricalcium phosphate-alginate-gelatin was prepared and cast as pellets of 1 cm diameter. The sponge was then fabricated by drying in freeze-dryer for 12 h. The porosity, mean pore size, compressive modulus and strength of the composite sponge fabricated in this study were 89.7%, 325.3 microm, 1.82 and 0.196 MPa, respectively. To establish a 3D culture system, the rat bone marrow-derived MSCs were suspended in 500 microl diluted collagen gel, loaded into the porous sponge and provided with medium with or without osteogenic supplements for 3 weeks. The day after loading, the cells appeared in the scaffold's internal spaces, where later some of them from either culture survived by anchoring on the surfaces. At the end of cultivation period, individually adhered cells from both cultures were observed to be replaced by cell aggregates, in which mineralized matrix was detected by alizarin red staining. Furthermore, RT-PCR analysis indicated that the bone-specific gene osteocalcin was expressed in cultures in both the presence and absence of the osteogenic supplements. Taken together, it seems that the studied scaffolds are cell-compatible and, more importantly, possess some osteo-inductive properties.     

骨髓间充质干细胞向软骨细胞的分化

背景：以骨髓间充质干细胞构建组织工程气管尚缺乏理想的特异性表面标志物,对其鉴定主要依赖细胞形态学、细胞表型及诱导分化的功能进行分析。目的：体外分离培养、鉴定兔骨髓间充质干细胞,观察在特定条件下向气管软骨细胞分化的潜能。方法：无菌环境取兔骨髓,经全骨髓贴壁筛选法分离培养细胞至第2代,流式细胞术鉴定第1、第2代细胞表面抗原CD44、CD45的表达。无菌环境取气管,经酶消化法分离培养气管软骨细胞,甲苯胺蓝染色鉴定软骨细胞蛋白聚糖的合成。在使用转化生长因子B1的基础上,将骨髓间充质干细胞与气管软骨细胞通过Transwell小室非接触式共培养,倒置显微镜观察细胞形态,甲苯胺蓝染色鉴定蛋白聚糖的合成,荧光实时定量PCR鉴定Ⅱ型胶原和蛋白聚糖mRNA的表达。结果与结论：分离、培养的细胞呈长梭形、不规则形聚集生长,传代后细胞生长速度明显增快,呈鱼群状聚集生长。第1代有96．97％的细胞表达CD44、13.72％的细胞表达CD45,第2代有99．11％的细胞表达CD44、8．54％的细胞表达CD45。气管软骨细胞甲苯胺蓝染色阳性。在诱导后,骨髓间充质干细胞形态逐渐由长梭形变为三角形或不规则形,表达软骨细胞特异性Ⅱ型胶原和蛋白聚糖mRNA基因,甲苯胺蓝染色示阳性。结果表明全骨髓贴壁筛选法可成功分离培养骨髓间充质干细胞,第2代纯度较高,且在特定诱导条件下具有分化为气管软骨细胞的潜能。     

全骨髓法培养C57小鼠骨髓间充质干细胞的生物学特性

背景：饲养层细胞多数利用丝裂霉素C来处理,虽丝裂霉素C抑制了细胞增殖,但是细胞仍处于有分泌功能的活性状态,能够分泌不同的细胞因子。而骨髓中的非间充质骨髓细胞和血浆中的各种分泌因子,保持间充质细胞生长的微环境,提高间充质干细胞的产量。 目的：探究全骨髓培养法分离培养的小鼠骨髓间充质干细胞的生物学特性。 方法：采用全骨髓贴壁法纯化扩增C57小鼠的骨髓间充质干细胞,检测细胞增殖的动力学变化、细胞表面的免疫标志物、多向分化的潜能及细胞周期的变化。 结果与结论：全骨髓培养法获得的小鼠骨髓间充质干细胞具有黏附于塑料培养器皿的能力,流式细胞仪检测表达CD45,CD105和Sca-1,不表达CD34,CD133和C-kit等间充质干细胞的表面标记特征;细胞倍增时间（57.11＆#177;1.5） h;诱导后具有向成骨细胞、脂肪细胞和软骨细胞分化的能力;细胞周期分析表明64%的细胞处于G 0-G 1期。提示全骨髓培养法获取的C57小鼠骨髓间充质细胞具有间充质干细胞的生物学特征。     

Low‐oxygen conditions promote synergistic increases in chondrogenesis during co‐culture of human osteoarthritic stem cells and chondrocytes

There has been increased interest in co‐cultures of stem cells and chondrocytes for cartilage tissue engineering as there are the limitations associated with using either cell type alone. Drawbacks associated with the use of chondrocytes include the limited numbers of cells available for isolation from damaged or diseased joints, their dedifferentiation during in vitro expansion, and a diminished capacity to synthesise cartilage‐specific extracellular matrix components with age and disease. This has motivated the use of adult stem cells with either freshly isolated or culture‐expanded chondrocytes for cartilage repair applications; however, the ideal combination of cells and environmental conditions for promoting robust chondrogenesis remains unclear. In this study, we compared the effect of combining a small number of freshly isolated or culture‐expanded human chondrocytes with infrapatellar fat pad–derived stem cells (FPSCs) from osteoarthritic donors on chondrogenesis in altered oxygen (5% or 20%) and growth factor supplementation (TGF‐β3 only or TGF‐β3 and BMP‐7) conditions. Both co‐cultures, but particularly those including freshly isolated chondrocytes, were found to promote cell proliferation and enhanced matrix accumulation compared to the use of FPSCs alone, resulting in the development of a tissue that was compositionally more similar to that of the native articular cartilage. Local oxygen levels were found to impact chondrogenesis in co‐cultures, with more robust increases in proteoglycan and collagen deposition observed at 5% O₂. Additionally, collagen type I synthesis was suppressed in co‐cultures maintained at low‐oxygen conditions. This study demonstrates that a co‐culture of freshly isolated human chondrocytes and FPSCs promotes robust chondrogenesis and thus is a promising cell combination for cartilage tissue engineering.     

兔骨髓间充质干细胞与透明质酸钠复合修复软骨的实验研究

目的研究兔骨髓间充质干细胞(BM-MSCs)与透明质酸钠复合修复兔软骨缺损的作用。方法在麻醉下制备18只家兔关节软骨缺损模型,将其随机分成3组。实验组(A组):BM-MSCs/透明质酸钠移植入软骨缺损处;对照组(B组):透明质酸钠移植入软骨缺损处;空白组(C组):不做处理。于移植后第6、12周分别对标本行组织学检测,并对实验数据行改良Pineda法评分,最后进行相关统计学分析。结果 A组为软骨样组织修复,组织学染色为阳性;B、C组软骨缺损处是以纤维组织为主的修复,组织学染色呈弱阳性。A组的修复效果显著优于B、C组,而B、C组之间的修复效果无明显差异。结论移植的BM-MSCs/透明质酸钠复合物可有效促进兔软骨缺损修复。     

Osteoarthritic human chondrocytes proliferate in 3D co‐culture with mesenchymal stem cells in suspension bioreactors

Osteoarthritis (OA) is a painful disease, characterized by progressive surface erosion of articular cartilage. The use of human articular chondrocytes (hACs) sourced from OA patients has been proposed as a potential therapy for cartilage repair, but this approach is limited by the lack of scalable methods to produce clinically relevant quantities of cartilage‐generating cells. Previous studies in static culture have shown that hACs co‐cultured with human mesenchymal stem cells (hMSCs) as 3D pellets can upregulate proliferation and generate neocartilage with enhanced functional matrix formation relative to that produced from either cell type alone. However, because static culture flasks are not readily amenable to scale up, scalable suspension bioreactors were investigated to determine if they could support the co‐culture of hMSCs and OA hACs under serum‐free conditions to facilitate clinical translation of this approach. When hACs and hMSCs (1:3 ratio) were inoculated at 20,000 cells/ml into 125‐ml suspension bioreactors and fed weekly, they spontaneously formed 3D aggregates and proliferated, resulting in a 4.75‐fold increase over 16 days. Whereas the apparent growth rate was lower than that achieved during co‐culture as a 2D monolayer in static culture flasks, bioreactor co‐culture as 3D aggregates resulted in a significantly lower collagen I to II mRNA expression ratio and more than double the glycosaminoglycan/DNA content (5.8 vs. 2.5 μg/μg). The proliferation of hMSCs and hACs as 3D aggregates in serum‐free suspension culture demonstrates that scalable bioreactors represent an accessible platform capable of supporting the generation of clinical quantities of cells for use in cell‐based cartilage repair.     

骨髓间充质干细胞复合温敏水凝胶支架的制备

背景：壳聚糖及其衍生物制备的支架对细胞迁移和神经轴突再生有重要作用。壳聚糖及其衍生物的组织相容性好,易使干细胞在其表面附着生长,在神经组织工程具有较为广阔的应用前景。
　　目的：制备适宜骨髓间充质干细胞生长的壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性水凝胶细胞支架,观察骨髓间充质干细胞在细胞支架中的生长情况。
　　方法：将壳聚糖进行季铵盐化改性处理,通过傅里叶变换红外光谱分析谱检测确定其生成。实验以壳聚糖与壳聚糖季铵盐配比为8∶1成功制备出较为稳定的壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性温敏水凝胶细胞支架,观察成胶情况,并进行生物安全性检测。
　　结果与结论：实验在傅里叶变换红外光图谱上发现了季铵基基团的特征峰。细胞毒性实验显示,水凝胶浸提液干预的大鼠骨髓间充质干细胞无毒性。急性全身毒性实验显示,浸提液对大鼠体质量增加无明显影响,支架生物安全性较好。扫描电镜观察显示,骨髓间充质干细胞在细胞支架中能正常的生长和增殖。结果证实,实验成功制备了壳聚糖/壳聚糖季铵盐/甘油磷酸钠温敏性水凝胶细胞支架,适合骨髓间充质干细胞生长和增殖。     

人关节液促进骨髓间充质干细胞的定向分化

背景：目前的众多研究都是通过体外加入生长因子诱导骨髓间充质干细胞分化,但该方法细胞因子用量大,成本高,随着培养次数的增加,其成软骨潜能明显降低。 目的：混合培养人关节液与人骨髓间充质干细胞,观察共培养系统对人骨髓间充质干细胞定向诱导分化的影响。方法：采用全骨髓、贴壁培养法培养和分离人骨髓间充质干细胞,无菌操作下抽取健康志愿者的膝关节液,将其与P3代细胞混合用于实验,分为3组,关节液＋完全培养基,关节液＋人骨髓间充质干细胞＋完全培养基,人骨髓间充质干细胞＋完全培养基。每天在倒置显微镜下观察细胞形态变化和生长情况,分别于诱导后第7,14,21天行甲苯胺蓝染色检测和Ⅱ型胶原免疫组化染色检测。 结果与结论：人关节液与人骨髓间充质干细胞混合培养后,细胞增殖速度减慢,由长梭形变为多角形、椭圆形,细胞外基质呈甲苯胺蓝异染性阳性,Ⅱ型胶原免疫组化染色阳性。说明关节液对人骨髓间充质干细胞向软骨细胞分化有正性促进作用,关节液中可能含有促人骨髓间充质干细胞向软骨细胞方向分化的物质。     

Chondrogenesis by bone marrow‐derived mesenchymal stem cells grown in chondrocyte‐conditioned medium for auricular reconstruction

Bone marrow‐derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell‐based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte‐conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor‐beta 3 (TGF‐β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real‐time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real‐time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF‐β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF‐β3. Osteogenesis was only observed in the SM group with TGF‐β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF‐β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

山羊骨髓间充质干细胞向纤维软骨分化的形态学改变简

背景：前期初步研究发现,碱性成纤维细胞生长因子可诱导骨髓间充质干细胞向颞下颌关节盘细胞方向分化,且碱性成纤维细胞生长因子10μg/L诱导组合成胶原量明显高于5μg/L诱导组。目的：观察经不同浓度碱性成纤维细胞生长因子诱导后的骨髓间充质干细胞的超微结构变化。方法：原代分离培养山羊骨髓间充质干细胞,选P3,P4细胞,用5,10μg/L碱性成纤维细胞生长因子诱导骨髓间充质干细胞,以未加碱性成纤维细胞生长因子培养的骨髓间充质干细胞做对照。倒置相差显微镜观察细胞生长状况,用第7,14,21天的细胞爬片行蕃红O、天狼猩红和Ⅰ型胶原免疫组织化学染色,并观察第21天细胞的超微结构。结果与结论：经不同浓度碱性成纤维细胞生长因子诱导后,骨髓间充质干细胞可向颞下颌关节盘成纤维细胞样细胞形态分化,10μg/L组细胞更像关节盘成纤维细胞样细胞。提示骨髓间充质干细胞有向颞下颌关节盘细胞方向分化的形态学基础。     

人骨形态发生蛋白-2重组慢病毒载体转染羊骨髓间充质干细胞及其成骨调节作用

目的探讨人骨形态发生蛋白-2（bone morphogenetic protein-2,BMP-2）基因转染对体外培养的骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）增殖和骨向分化的影响。方法构建携带人BMP2基因的慢病毒载体,转染诱导羊第4代BMSCs为转染组,选择未转染的BMSCs为对照组。采用Western blot检测2组细胞BMP-2与成骨相关蛋白表达情况,采用实时定量PCR法检测细胞I型胶原、骨钙素、骨桥蛋白mRNA相对表达量的变化情况。结果转染组骨钙素、骨桥蛋白mRNA及蛋白表达量较对照组增高,差异有统计学意义（P〈0．05）,2组I型胶原mRNA及蛋白表达量差异无统计学意义（P〉0．05）。结论BMP-2基因转染可增强羊BMSCs增殖和骨向分化的能力,可作为组织工程骨的种子细胞。     

转化生长因子β及周期性拉伸应变条件下骨髓间充质干细胞向软骨样细胞的分化

背景：研究表明转化生长因子β对骨髓间充质干细胞的软骨方向分化具有显著的诱导作用。周期性拉伸应变可以模拟软骨细胞在体内的力学环境,对细胞的增殖和分化起着重要的调节作用。目的：探讨转化生长因子β及周期性拉伸应变在诱导骨髓间充质干细胞向软骨样细胞分化过程中是否具有协同作用。方法：取2月龄新西兰大白兔10只,骨穿针穿入股骨髓腔内,抽取骨髓3.0-4.0 mL并分离培养骨髓间充质干细胞,传至3代后随机分为4组,分别为空白组、转化生长因子β组、周期性拉伸应变组以及周期性拉伸应变＋转化生长因子β组,作用1,3,6 d后取出相应细胞,番红O染色观察大体形态,阿尔新蓝染色检测糖胺聚糖水平,ELISA检测上清液基质金属蛋白酶13及基质金属蛋白酶组织抑制剂1水平,RT-PCR检测Ⅱ型胶原、基质金属蛋白酶13及基质金属蛋白酶组织抑制剂1 mRNA相对表达量。结果与结论：番红O染色可见细胞呈长梭形或不规则三角形样改变,各实验组较空白组细胞数量及基质分泌增多。作用第3天时转化生长因子β组、周期性拉伸应变＋转化生长因子β组上清液糖胺聚糖水平较空白组均升高（P＜0.05）,周期性拉伸应变＋转化生长因子β组Ⅱ型胶原mRNA相对表达量较空白组增高（P ＜0.05）。结果提示转化生长因子β及周期性拉伸应变均可诱导骨髓间充质干细胞向软骨细胞分化,二者具有明显的协同作用。