软骨细胞与骨髓基质细胞共培养体外构建软骨的初步研究  

Preliminary study of in vitro chondrogenesis by co-culture of bone marrow stromal cells and chondrocytes

作　　者：苗春雷[1] 周广东[1] 刘天一[1] 王晓云[1] 崔磊[1] 刘伟[1] 唐胜建[2] 曹谊林[1] 

机构地区：[1]上海第二医科大学第九人民医院整复外科上海市组织工程研究与开发中心,上海200235 [2]潍坊医学院整形外科医院

出　　处：《上海第二医科大学学报》2004年第4期246-249,283,共5页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：国家高技术研究发展计划资助项目("863"计划)(2002AA205021);国家自然科学基金资助项目(30300353);上海市"重中之重"要点学科基金资助.

摘　　要：目的 探讨不同浓度软骨细胞提供的软骨微环境诱导骨髓基质干细胞(BMSC)体外构建软骨的可行性。方法 将体外分别培养扩增的猪BMSC与耳软骨细胞按不同比例混合(9:1,8:2),均以5.0×107/mL的细胞终浓度接种于聚羟基乙酸/聚乳酸(PGA/PLA)支架作为共培养组,以相同终浓度的单纯软骨细胞和单纯BMSC分别接种作为阳性及阴性对照,以20％上述浓度的单纯软骨细胞接种作为低软骨细胞浓度对照。各标本于体外培养4周时取材,通过大体观察、组织学以及免疫组化等方法对新生软骨进行初步评价。结果 各组细胞均与材料粘附良好。8:2共培养组及阳性对照组在体外培养4周时,外观已类似软骨组织并基本保持了材料的大小和形状,组织学显示有较连续的成熟软骨形成,免疫组化也均显示有大量Ⅱ型胶原分泌。9:1共培养组在培养过程中稍有缩小和变形,组织学上仅在培养物的边缘可见到连续的软骨样组织。阴性对照组明显皱缩变形,组织学未见成熟软骨陷窝。低软骨细胞浓度组复合物明显变薄,只在局部形成了不连续的软骨组织,新生软骨量明显少于共培养各组及阳性对照组。结论软骨细胞能够提供软骨微环境诱导BMSC成软骨分化并形成软骨,20％浓度的软骨细胞已能够达到良好的诱导效果。Objective This study explored the feasibility of in vitro bone marrow stromal cell (BMSC) chondro-genesis induced by the chondrogenic microenvironment that was provided by chondrocytes at various concentrations. Methods Porcine BMSCs and auricular chondrocytes were isolated and in vitro expanded respectively and then were mixed at the ratio of 9: 1 , 8:2 (BMSCs:chondrocytes). The mixed cells were seeded onto polyglycolic acid/polylac-tic acid( PGA/PLA) scaffold at the ultimate concentration of 5. 0×107/mL as co-culture group. Chondrocytes and BMSCs of the same ultimate concentration were seeded respectively onto the scaffold as positive control group ( chon-drocyte group) and negative control group (BMSC group). 20% of above concentration chondrocytes (1.0 ×107 / mL) were seeded as low chondrocyte group. The specimens were collected after in vitro culture for 4 weeks. Gross observation, histology and immunohistochemistry were used to evaluate the results. Results Cells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. In 8 : 2 co-culture group and positive control group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture. At 4 weeks, cartilage-like tissue had formed and type Ⅱ collagen could be detected for strong expression by immunohistochemistry. In 9: 1 co-culture group, the constructs shrunk slightly and histology showed cartilage-like tissue formed only at the edges of constructs. In negative control, the constructs deformed and shrunk gradually without mature cartilagelacuna in histology. The constructs in low chondrocyte group also shrunk obviously in thickness with small amount of cartilage formation. Conclusion Chondrocytes can provide chondrogenic microenvironment to promote in vitro BMSC chondrogenesis. 20% chondrocytes have fine capacity of inducing BMSC chondrogenic differentiation.

关 键 词：软骨细胞 骨髓基质细胞 细胞共培养 组织工程 微环境 胶原 软骨缺损 生物材料 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]