与软骨细胞共培养和条件培养液诱导骨髓间充质干细胞向软骨细胞分化的比较

背景：骨髓间充质干细胞体外转化很大程度上依赖于合适的培养条件。 目的：比较与软骨细胞共培养和条件培养液2种不同的诱导方案诱导骨髓间充质干细胞向软骨细胞分化的特点。 方法：分离培养大鼠骨髓间充质干细胞和耳软骨细胞,采用骨髓间充质干细胞与软骨细胞共培养及条件培养液诱导成软骨的方法,诱导骨髓间充质干细胞向软骨细胞分化。以MTT法及流式细胞仪检测细胞活性及周期,糖胺多糖、甲苯胺蓝以及免疫组化染色检测细胞生物学特性,以RT-PCR法检测诱导后的软骨细胞Ⅱ型胶原RNA表达情况。 结果与结论：采用共培养方式诱导的软骨细胞,其生物学特性与采用条件培养液诱导的软骨细胞相比,前者优于后者,如分泌糖胺多糖的能力以及基质分泌量均较高。提示共培养方式诱导的软骨细胞更接近正常软骨细胞,更有利于作为组织工程软骨的种子细胞。     

同种异体脱钙骨与骨髓间充质干细胞关节腔内共培养:与同腔软骨性状的对比

背景:临床发现膝关节内游离体能长期存在于关节腔内并能保持一定的软骨组织学特性和生理学特性,因此大胆提出假设:关节腔环境可能是软骨细胞生长、发育的较佳环境并提出"腔内培养,腔内移植"的理念。目的:观察兔骨髓间充质干细胞复合同种异体脱钙骨基质体外培养或关节腔内培养组织工程软骨与同腔软骨的性状差异。方法:实验分3组进行,体外培养组将经成软骨诱导的乳兔骨髓间充质干细胞与成年兔脱钙骨基质体外复合培养;腔内培养组将经成软骨诱导的乳兔骨髓间充质干细胞与成年兔脱钙骨基质以筋膜包裹,复合培养于成年新西兰兔膝关节腔内,以同腔内正常软骨为对照。结果与结论:培养12周后:①体外培养组苏木精-伊红染色见软骨细胞少量增生,胞核蓝染;甲苯胺蓝染色见软骨细胞排列无序,少量周围基质包绕;Masson染色阳性区域小,细胞排列无序;Ⅱ型胶原免疫组织化学见软骨细胞胞浆及胞外基质少量黄色颗粒。②腔内培养组苏木精-伊红染色见软骨细胞增生,胞核蓝染;甲苯胺蓝染色见软骨细胞成串排列,软骨陷窝形成,周围基质包绕;Masson染色阳性,软骨细胞多,基质蓝染,按一定应力方向排列;Ⅱ型胶原免疫组织化学见细胞外基质中出现较多棕黄色颗粒,Ⅱ型胶原染色阳性。说明骨髓间充质干细胞与同种异体脱钙骨基质复合物可在体外及膝关节腔内培养出组织工程软骨,关节腔内培养的软骨比体外培养的软骨更接近正常软骨。     

骨髓间充质干细胞与同种异体脱钙骨基质组合生长因子诱导向软骨细胞分化

背景：用组织工程学方法高质量修复关节软骨缺损并达到很好的远期疗效目前尚无定论。鉴于此,课题组提出“同种异体骨髓间充质干细胞关节腔内定向培养组织工程软骨”的实验设技。 目的：同种异体脱钙骨基质组合转化生长因子β1和胰岛素样生长因子Ⅰ诱导骨髓间充质干细胞向软骨分化的能力,同时探索其在关节腔内培养促进向关节软骨定向分化的方法。 方法：分离兔骨髓间充质干细胞并行体外培养,分为两组：实验组DMEM培养液中加入转化生长因子β1和胰岛素样生长因子Ⅰ,对照组中未加入诱导因子。比较两组细胞的增殖情况和成软骨分化情况。制备同种异体脱钙骨基质支架材料,实验组骨髓间充质干细胞负载到同种异体脱钙骨基质中,构建组织工程软骨复合体,取另2只兔的腰背筋膜包裹后缝合固定到30只兔膝关节腔内行腔内培养。分别于植入后4,8,12周各取10只标本进行组织学切片观察及Ⅱ型胶原免疫组织化学观测,并对结果进行分析。 结果与结论：实验组中骨髓间充质干细胞集落形成效率明显高于对照组(u=3.326,P < 0.01)。实验组细胞爬片做Ⅱ型胶原免疫组化检测呈阳性,对照组未见阳性细胞。组织工程复合体在腔内培养12周后,苏木精-伊红染色见大量软骨细胞增生,胞核染色呈蓝色;甲苯胺蓝染色见软骨细胞成串排列,大量软骨陷窝形成,周围大量基质包绕;Ⅱ型胶原免疫组织化学反应见细胞外基质中出现大量棕黄色颗粒,Ⅱ型胶原染色强阳性。提示转化生长因子β1和胰岛素样生长因子Ⅰ可显著促进骨髓间充质干细胞增殖和成软骨分化,骨髓间充质干细胞与同种异体脱钙骨基质结合后可在关节腔内成功培养出组织工程软骨。同种异体脱钙骨基质符合组织工程软骨支架材料的基本要求。     

壳聚糖-胶原凝胶复合骨髓间充质干细胞修复兔关节软骨缺损的组织学变化

背景:大块软骨损伤目前临床上尚无方法治疗,干细胞技术出现后为解决这一难题提供了理论支持。采用新型的支架材料"壳聚糖-胶原蛋白"结合干细胞诱导分化移植给动物模型是一种较新的尝试。目的:观察壳聚糖-胶原凝胶复合骨髓间充质干细胞修复兔关节软骨缺损的组织学变化。方法:体外培养扩增兔骨髓间充质干细胞。采用软骨诱导分化培养基对P2代细胞进行成软骨诱导。同时制备"壳聚糖-胶原蛋白"支架和兔关节软骨缺损模型。缺损用含有骨髓间充质干细胞的壳聚糖-胶原凝胶填充,另一条关节用没有细胞的支架或不做处理。其中12个关节作为实验组(接受骨髓间充质干细胞+支架),8个关节作为空白对照组(不做处理),8个关节作为单纯支架组(未植入细胞)。造模后于2,4,8,16周进行组织学评分并处死动物行组织学染色。结果与结论:造模后16周移植的细胞一致分化为软骨细胞,大部分软骨缺损区被新生软骨修复,组织学评分实验组关节修复良好。提示应用骨髓间充质干细胞结合"壳聚糖-胶原蛋白"复合物可以修复关节软骨缺损。     

三维培养诱导人脂肪间充质干细胞微球的成软骨分化能力

背景：关节软骨损伤后修复结果不满意,需要新的手段,而脂肪间充质干细胞较适宜做种子细胞诱导软骨,然而怎么能够使诱导的软骨具有功能需要研究。 目的：采用三维培养体系诱导人脂肪间充质干细胞微球向软骨分化。 方法：无菌切取吸脂术后脂肪组织,分离培养人脂肪间充质干细胞,传至第3代进行流式细胞术分析,成骨成脂肪诱导等鉴定,同时也给予合适的培养条件用三维培养的方式向软骨细胞诱导,并行阿利辛蓝染色鉴定糖胺多糖的合成,苏木精-伊红染色进行组织学分析,免疫荧光检测Ⅱ型胶原表达,称质量测量软骨硬度。 结果与结论：分离的人脂肪间充质干细胞CD105,CD44,CD29均高表达,而 CD45,CD34低表达,并且成骨成脂诱导后细胞茜素红染色和油红O染色均为阳性。三维培养法诱导的软骨细胞可表达大量糖胺多糖及Ⅱ型胶原。结果证实,三维培养法诱导人脂肪间充质细胞向软骨分化后,具有软骨细胞的特性。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

胶原支架复合兔骨髓间充质干细胞体外模拟的微重力培养

背景:骨髓间充质干细胞向软骨细胞诱导的方法包括体外高密度微团培养、体外单层细胞培养、体外三维支架环境诱导、与软骨细胞体外共培养诱导和基因转染诱导培养等。目的:验证模拟微重力条件下诱导后的兔骨髓间充质干细胞在Ⅰ、Ⅱ型胶原支架上黏附、伸展和增殖情况。方法:对兔骨髓间充质干细胞体外进行原代和传代培养,按加入诱导条件不同分为2组:实验组(转化生长因子β1+胰岛素样生长因子1诱导)组、空白对照组。3周后分别作MTT比色试验、糖胺聚糖检测和免疫组织化学染色。将诱导后的实验组细胞分别种植于Ⅰ、Ⅱ型胶原支架,分为4组培养:复合Ⅱ型胶原支架静置培养、复合Ⅱ型胶原支架模拟微重力培养组、复合Ⅰ型胶原支架静置培养、复合Ⅰ型胶原支架模拟微重力培养组,1周后作苏木精-伊红、甲苯胺蓝染色。结果与结论:实验组MTT吸光度值和糖胺聚糖水平检测结果均大于空白对照组,且Ⅱ型胶原免疫组织化学检测阳性。苏木精-伊红、甲苯胺蓝染色显示,Ⅱ型胶原支架复合细胞的数量明显高于Ⅰ型胶原支架;模拟微重力培养条件下胶原支架复合细胞的数量明显高于静置培养组。结果说明微重力培养环境有利于高密度细胞的黏附、增殖,有利于细胞之间的信号传递,为维系细胞的生长和代谢提供适宜的微环境;作为诱导后骨髓间充质干细胞的支架材料Ⅱ型胶原支架优于Ⅰ型胶原。     

骨髓间充质干细胞向软骨细胞分化过程中miR130α的表达

目的诱导骨髓间充质干细胞向软骨细胞分化,初步探索miR130a在骨髓间充质干细胞向软骨细胞分化过程中的调控作用。方法体外TGF-β1诱导骨髓间充质干细胞向软骨细胞分化,免疫荧光法和免疫组化染色鉴定Ⅱ型胶原,阿辛兰染色鉴定氨基葡聚糖。用Real-time PCR法检测细胞miR130a的表达。结果骨髓间充质干细胞在TGF-β1诱导下可分化为软骨细胞。培养7 d后,诱导培养组细胞miR130a表达的水平显著低于培养前的水平(P<0.05)。结论骨髓间充质干细胞体外诱导可以分化为软骨细胞,在向软骨细胞分化的早期,miR130a表达降低伴随着软骨细胞的分化,提示与软骨形成的过程有关。     

全骨髓贴壁法获取组织工程髓核种子细胞的相关研究*

目的明确兔骨髓间充质干细胞的分化能力及分化条件,以TGF-β3和BMP-7诱导兔骨髓间充质干细胞向类髓核细胞分化,为组织工程髓核的构建提供良好的种子细胞。方法利用全骨髓贴壁法分离获取兔骨髓间充质干细胞,并对细胞进行培养和传代,以特定的环境及细胞诱导液进行诱导,促使其向特定的中胚层细胞分化。再将细胞分为四组,A:空白对照组,B:BMP-7诱导组,C:TGF-β3诱导组,D:TGF-β3与BMP-7共同诱导组,诱导21天后对蛋白聚糖、Ⅰ型胶原、Ⅱ型胶原、Ⅹ型胶原和SOX9基因进行realtime-PCR检测。结果全骨髓贴壁法分离获得的原代兔骨髓间充质干细胞生长良好,2周后显微镜下观察细胞间形态较为一致,成纺锤形。细胞传代后生长良好。细胞表面表达CD29、CD105、CD166表面标记物。在成骨诱导培养液、成软骨诱导培养液和脂肪诱导培养液的诱导下,培养21天后,通过特定染色发现兔骨髓间充质干细胞向预定方向分化生长。以TGF-β3和BMP-7生长因子诱导21天后,蛋白聚糖、Ⅱ型胶原和SOX9基因表达C组和D组明显高于A组和B组(P0.05),X型胶原表达A组和B组明显高于C组和D组(P0.05),Ⅰ型胶原表达四组之间无明显差异。结论全骨髓贴壁法可以成功分离获得状态良好的兔骨髓间充质干细胞,在成骨诱导培养液、成软骨诱导培养液和脂肪诱导培养液的诱导下,兔骨髓间充质干细胞可向预定的方向分化且生长良好。TGF-β3和BMP-7诱导兔骨髓间充质干细胞后可明显提高其类髓核细胞具有的下游基因表达水平。     

转化生长因子β1诱导骨髓间充质干细胞向软骨细胞分化

背景:转化生长因子β1是关节软骨组织工程研究的首选生长因子,适当浓度能刺激关节软骨细胞增殖、分裂和分化。目的:建立含有转化生长因子β1的特殊诱导体系培养条件下骨髓间充质干细胞分化为软骨细胞的能力,观察诱导后的细胞形态及表型变化。方法:于兔胫骨结节内侧抽取骨髓,采用贴壁培养法分离纯化兔骨髓间充质干细胞,取第3代骨髓间充质干细胞行流式细胞仪检测鉴定其表面抗原,以含有转化生长因子β1的特殊软骨诱导体系的培养条件下对第3代骨髓间充质干细胞诱导培养21d,诱导后与人鼻中隔软骨细胞进行比较。采用免疫组织化学法对Ⅱ型胶原进行定性检测。结果与结论:贴壁培养法可分离并纯化兔骨髓间充质干细胞,所得第3代骨髓间充质干细胞表面抗原CD44阳性,CD34、CD45阴性。经诱导培养21d后细胞形态变为不规则,Ⅱ型胶原免疫组织化学染色显示可见阳性细胞。提示含有转化生长因子β1的特殊软骨诱导体系的培养条件下,骨髓间充质干细胞可以转化为软骨细胞,且与正常软骨细胞无明显差异。     

外源性透明质酸对兔骨髓间充质干细胞定向分化为软骨细胞的影响

背景：透明质酸是关节腔滑液最主要的成分,对细胞的形态发生起着非常重要的作用,但其用于软骨缺损修复时对骨髓间充质干细胞的影响如何呢？ 目的：通过分析外源性透明质酸对兔骨髓间充质干细胞体外增殖及定向分化为软骨细胞的影响,探讨关节腔内环境对骨髓间充质干细胞的作用。 方法：全骨髓法+贴壁培养法分离培养兔骨髓间充质干细胞,取第4代细胞用于实验,实验组细胞加入透明质酸诱导液,以转化生长因子β3诱导组作为阳性对照,阴性对照组加入常规培养液。分别于诱导后第7,14,21 d行甲苯胺蓝染色检测蛋白聚糖表达,免疫组化染色及RT-PCR检测细胞Ⅱ型胶原表达。 结果与结论：经透明质酸诱导后,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和Ⅱ型胶原免疫组化阳性,RT-PCR检测示Ⅱ型胶原mRNA表达阳性,表现出软骨细胞的分化特点,但表达均比阳性对照组弱。结果提示,外源性透明质酸具有诱导兔骨髓间充质干细胞向软骨细胞分化的能力,但比转化生长因子β3的诱导能力弱,关节腔内环境对骨髓间充质干细胞向软骨细胞分化有正性促进作用,支持透明质酸作为软骨组织工程基质使用。     

Conditioned Medium from Chondrocyte/Scaffold Constructs Induced Chondrogenic Differentiation of Bone Marrow Stromal Cells

For the application of bone marrow stromal cells (BMSCs) in cartilage tissue engineering, it is imperative to develop efficient strategies for their chondrogenic differentiation. In this study, the conditioned media derived from chondrocyte/scaffold constructs were used to direct chondrogenic differentiation of BMSCs. The porcine articular chondrocytes were seeded on the PGA/PLA scaffolds to form chondrocyte/scaffold constructs and were cultured to form engineered cartilage in vitro. The culture media were collected as conditioned media and used for chondrogenic induction of BMSC pellets (experimental group, Exp.). The chondrocyte pellets and BMSC pellets were cultured routinely as positive control (PC) and negative control (NC), respectively. After 4 weeks, the wet weight and GAG content in Exp. group and PC group were significantly higher than that in NC group. Histological and immunohistochemical analysis showed that cartilaginous tissue was formed with typical cartilage lacuna structure and positive staining of collagen Type II (Col II) in the peripheral area of the BMSC pellets in Exp. group. Gene expression of Sox9, Col II, and COMP in Exp. group and PC group were significantly higher than that in NC group. The growth factors in the conditioned media derived from human costal chondrocytes‐scaffold constructs were tested by protein microassay. The conditioned media contained low levels of TGF‐β1,2,3, IGF‐1 and high levels of IGF‐2, FGF‐4, and IGFBP4,6, and so forth. The soluble factors derived from the engineered cartilage can induce chondrogenic differentiation of BMSCs independently. Many cytokines may function in chondrogenesis in a coordinated way. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

Effect of nicotine on chondrogenic differentiation of rat bone marrow mesenchymal stem cells in alginate bead culture

This study was carried out to explore environmental compound such as nicotine can cause adverse effect on chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were capsulated in alginate beads incubated with a chondrogenic differentiation medium and while chondrogenic differentiation of rat BMSCs were cultured for 4 weeks treated with nicotine at concentrations of 25, 50 and 100 μM. The effect of nicotine on BMSCs viability was tested using MTT assay. After chondrogenic differentiation, alginate beads sections were stained for glycosaminoglycan (GAG) with alcian blue and safranin-O. The mRNA expression of chondrogenesis related genes, including collagen type 2 alpha 1 (Col2A1), aggrecan, insulin-like growth factor-1 (IGF-1) were determined by RT-PCR. Nicotine did not affect viability of BMSCs at any indicated concentration. Continuous exposure to nicotine for 4 weeks resulted in significant decrease of the area stained with alcian blue and safranin-O in a concentration-dependent manner compared with the control (P<0.05). After 4 weeks in chondrogenic medium, nicotine dose-dependently decreased the expression of aggrecan, Col2A1 and IGF-1 genes in rat BMSCs chondrogenesis compared with the control (P<0.05). It turned out that nicotine suppresses chondrogenic differentiation potential of BMSCs, leading to a poorly differentiated cartilage.     

Differentiation of a fibrin gel encapsulated chondrogenic cell line

Hyaline cartilage has very limited regenerative capacity following damage. Therefore engineered tissue substitutes have been the focus of much research. Our objective was to develop a fibrin-based scaffold as a cell delivery vehicle and template for hyaline cartilage regeneration, and compare its cellular properties against monolayer and pellet culture for chondrogenic cells. The chondrogenic precursor cell line, RCJ 3.1C5.18 (C5.18), was chosen as a test system for evaluating the effect of various culture conditions, including cell encapsulation, on articular chondrogenic cell differentiation. The C5.18 cells in monolayer showed elevated expression of collagen II, an articular chondrogenic marker, but also markers for fibrocartilage differentiation (collagen I and versican) when cultured with chondrogenic medium as compared to basic maintenance medium. Pellets of C5.18 cells cultured in chondrogenic medium were histologically more organized in structure than pellets cultured in control maintenance medium. The chondrogenic medium cultured pellets also secreted an extracellular matrix that was comprised of type II with very little type I collagen, indicating a trend towards a more hyaline-like cartilage. Moreover, when cultured in chondrogenic medium, fibrin-encapsulated C5.18 cells elaborated an extracellular matrix containing type II collagen, as well as aggrecan, which are both components of hyaline cartilage. This indicated a more articular-like chondrogenic differentiation for fibrin encapsulated C5.18 cells. The results of these experiments provide evidence that the C5.18 cell line can be used as a tool to evaluate potential scaffolds for articular cartilage tissue engineering.     

同种异体和异种来源骨髓间充质干细胞诱导成软骨修复喉软骨缺损

BACKGROUND: Because chondrocytes have no regeneration ability, to select suitable seed cells is the primary problem to repair cartilage defects. OBJECTIVE: To investigate the effect of allogeneic versus heterologous bone marrow mesenchymal stem cells (BMSCs) in repairing laryngeal cartilage defects after chondrogenic induction. METHODS: BMSCs from human and rabbits were isolated and cultured. Passage 3 cells were cultured in chondrogenic induction medium containing transforming transforming growth factor beta 1 and bone morphogenetic protein, and then were dropped onto a poly(lactic-co-glycolic acid) (PLGA) scaffold. Thirty New Zealand rabbits were randomly assigned into three groups: blank control group, human BMSCs group, rabbit BMSCs group. Animal models of laryngeal cartilage defects were made in the three groups. After modeling, saline-soaked PLGA scaffold, PLAG scaffold with human BMSCs or with rabbit BMSCs were implanted respectively into the rabbits in the normal blank, human BMSCs and rabbit BMSCs groups. The expression of type II collagen in the larynx and its surrounding tissues was detected by immunohistochemistry at 4 and 8 weeks postoperatively. RESULTS AND CONCLUSION: The animals in each group breathed normally with no presence of wheezing, and their eating and activity were good. Moreover, there was no purulency or infection in the three groups. At 4 and 8 weeks after operation, the positive rates of type II collagen in the two BMSCs groups were significantly higher than that in the blank control group (P < 0.05). There was no significant difference between two BMSCs groups (P > 0.05). These results show that both allogeneic and heterologous BMSCs have good therapeutic effects on the repair of laryngeal cartilage defects in rabbits.      

成年比格犬骨髓间质干细胞体外软骨方向分化的实验研究

目的：体外诱导成年比格犬骨髓间质干细胞（BMSCs）定向分化为软骨细胞，探讨体外诱导成软骨的方法和条件。方法:比格犬股骨取骨髓10 mL，体外行原代和传代培养扩增，加入转化生长因子（TGF-β1），以高密度细胞团块培养，诱导BMSCs分化为软骨细胞。甲苯胺蓝染色检测软骨基质的分泌，免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。结果:诱导的软骨样组织甲苯胺蓝染色阳性;Ⅱ型胶原免疫组织化学检测阳性。结论： 应用含TGF-β1的诱导液在体外可以诱导比格犬BMSC分化为软骨细胞，诱导的软骨细胞可作为软骨组织工程较理想的种子细胞。     

小鼠间充质干细胞三系分化中SOX9与Ⅱ型胶原mRNA的定量

背景：研究表明,软骨中的主要成分Ⅱ型胶原的基因-Col2a1在软骨细胞中的表达与SOX9 的浓度呈剂量依赖正相关关系。 目的：通过成骨、成软骨、成脂肪诱导干细胞分化,分析3种分化过程及不同时期的SOX9与Ⅱ型胶原 mRNA含量的变化,探讨SOX9在不同时空分布的表达规律及与Ⅱ型胶原的相关关系。 方法：取4周龄昆明小鼠骨髓间充质细胞,体外培养得到间充质干细胞并传达至第3代,对间充质干细胞进行流式细胞仪鉴定细胞表型,共分3组每组设3个时间段,通过成骨、成软骨、成脂肪3种诱导培养液对3组细胞进行诱导,另设不进行诱导的细胞作为对照组。分别在诱导3,7,14 d后收集提取细胞的总RNA,通过RT-PCR进行SOX9与Ⅱ型胶原的mRNA定量检测,同时对诱导后的细胞进行染色、免疫荧光染色,观察其分化状态及相关统计分析。 结果与结论：第3代骨髓间充质干细胞生长良好,流式细胞仪鉴定细胞表型证实为干细胞,对诱导后细胞进行染色、免疫荧光染色结果证实细胞分化为骨、软骨、脂肪细胞。经RT-PCR检测,在3组诱导分化细胞中SOX9 mRNA含量由高到低分别是成软骨、成骨、成脂肪,Ⅱ型胶原 mRNA含量由高到低分别是成软骨、成脂肪、成骨。在成软骨分化中SOX9在3,7 d表达不断升高,14 d呈下降趋势。Ⅱ型胶原在3,7,14 d均逐渐升高。在成骨分化中SOX9 mRNA含量随着时间推移而增加,而Ⅱ型胶原则随着时间推移而不断降低。在成脂肪分化中SOX9 mRNA表达与对照组比较差异无显著性意义(P > 0.05);而Ⅱ型胶原的表达没有规律可循,时间点的延伸及检测未观察到。结果提示,SOX9在软骨分化中作用优于成骨、成脂肪组,且软骨分化中SOX9与Ⅱ型胶原存在相关性,可能在软骨分化的早期Ⅱ型胶原随着SOX9的变化而变化;且软骨分化和成骨分化过程中SOX9可能起到了一个互相协调促进平衡的关键作用。     

脂肪基质干细胞小肠黏膜下层双面复合的体外成软骨诱导

背景：使用体外构建的组织工程软骨治疗软骨损伤是目前的研究热点,材料与支架材料的选择仍有较多问题尚待解决。 目的：观察脂肪基质干细胞-小肠黏膜下层复合物在体外经成软骨诱导培养基诱导分化的效果。 方法：将第3代脂肪基质干细胞接种于复水后的小肠黏膜下层双面,加入成软骨诱导培养基,于体外诱导培养7 d和14 d后,使用实时荧光定量RT-PCR检测Ⅱ型胶原mRNA,免疫组织化学染色检测Ⅱ型胶原蛋白,甲苯胺蓝染色观察细胞外基质,扫描电镜观察体外成软骨诱导14 d后细胞在支架材料上的生长状况。 结果与结论：Ⅱ型胶原mRNA实时荧光定量RT-PCR检测提示,体外成软骨诱导后7,14 d的脂肪基质干细胞-小肠黏膜下层复合物Ⅱ型胶原mRNA的标化值与未诱导的脂肪基质干细胞-小肠黏膜下层复合物差异有显著性意义(P  < 0.05),诱导  14 d与诱导7 d相比,差异亦有显著性意义(P < 0.05)。脂肪基质干细胞-小肠黏膜下层复合物成软骨诱导后14 d,Ⅱ型胶原免疫组织化学染色为阳性,甲苯胺蓝染色可见基质异染,未诱导的复合物Ⅱ型胶原免疫组织化学染色为阴性。扫描电镜检测显示诱导14 d时,细胞长满支架材料的双面。提示脂肪基质干细胞复合至小肠黏膜下层后,在体外经成软骨诱导培养基成软骨诱导后,能够向成软骨细胞分化。     

BMP7 induces the differentiation of bone marrow-derived mesenchymal cells into chondrocytes

Injured articular cartilage has a poor capacity for spontaneous healing. So far, a satisfactory solution to repair the injured cartilage has not been found, but transgenic therapy might be a promising treatment. This study aims to evaluate the potential of transfecting bone morphogenetic protein-7 (BMP-7), a secretory protein, into bone marrow-derived mesenchymal stem cells (BMSCs), in inducing the differentiation of bone marrow stromal cells into chondrocytes in vitro. The phenotypes of the cells were observed by alcian blue staining and H&E staining with an inverted microscope. The glycosaminoglycan (GAG) content of BMSCs transfected with pcDNA3.1-BMP7 or induced by inducing medium was examined after 7, 14, or 21 days of incubation. A standard curve as reference for BMSCs’ GAG content was plotted using galacturonic acid. The content of type II collagen in culture medium was detected by ELISA. Our results demonstrated that BMP7-transfected BMSCs or BMSCs incubated with inducing medium possess the ability to differentiate into chondrocytes. BMP7-induced BMSCs secrete type II collagen and GAG. There was no significant difference between BMP7-induced BMSCs in their secreted protein content when compared with the positive control group (TGF-β1 and dexamethasone) (P > 0.05), but there was significant difference in the secreted protein profile when compared with the negative control group (P < 0.05).     

Chondrogenic differentiation of human mesenchymal stem cells in collagen type I hydrogels

The chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (MSCs) in a collagen type I hydrogel, which is in clinical use for matrix-based autologous chondrocyte transplantation (ACT), was investigated. Collagen hydrogels with 2.5 x 10(5) MSCs/mL were fabricated and cultured for 3 weeks in a serum-free, defined, chondrogenic differentiation medium containing 10 ng/mL TGF-beta1 or 100 ng/mL BMP-2. Histochemistry revealed morphologically distinct, chondrocyte-like cells, surrounded by a sulfated proteoglycan-rich extracellular matrix in the TGF-beta1 and BMP-2 treated group, with more elongated cells seen in the BMP-2 treated group. Immunohistochemistry detected collagen type II (Col II) in the TGF-beta1 and BMP-2 treated group. Collagen type X (Col X) staining was positive in the TGF-beta1 but only very weak in the BMP-2 treated group. RT-PCR analyses revealed a specific chondrogenic differentiation with the expression of the cartilage specific marker genes Col II, Col X, and aggrecan (AGN) in the TGF-beta1 and the BMP-2 treated group, with earlier expression of these marker genes in the TGF-beta1 treated group. Interestingly, MSC-gels cultured in DMEM with 10% FBS (control) indicated few isolated chondrocyte-like cells but no expression of Col II or Col X could be detected. The results show, that MSCs cultured in a collagen type I hydrogel are able to undergo a distinct chondrogenic differentiation pathway, similar to that described for MSCs cultured in high-density pellet cultures. These findings are valuable in terms of ex vivo predifferentiation or in situ differentiation of MSCs in collagen hydrogels for articular cartilage repair.     

兔脂肪干细胞在含有转化因子和转铁蛋白培养基中向软骨细胞的分化

背景：成软骨的种子细胞的选择是软骨组织工程研究中的关键因素。 目的：观察脂肪干细胞在含有转化因子和转铁蛋白诱导条件下向软骨细胞分化的能力。 方法：取新西兰大白兔颈背部脂肪组织,机械分离及酶消化法获得脂肪干细胞,显微镜下观察细胞黏附及生长情况;加入含有转化因子β1和转铁蛋白的诱导培养基培养2周后以免疫组化方法检测Ⅱ型胶原的表达。 结果与结论：从兔脂肪组织中分离出的干细胞原代培养时24 h贴壁,96 h后达80%融合;于软骨诱导培养基内向软骨诱导7 d后形成软骨结节,14 d后Ⅱ型胶原免疫组化阳性。结果初步表明兔脂肪干细胞经诱导后可以向软骨细胞分化。