模拟微重力培养骨髓间充质干细胞定向诱导复合改良纤维蛋白原支架修复兔关节软骨缺损

Effect of bone mesenchymal stem cells and modified fibrinogen composite scaffold on repairing articular cartilage defects in the mimic microgravity environment

背景:模拟微重力培养系统通过模拟细胞生存的体内微重力环境,使细胞承受较低的剪切力,提高细胞内外、细胞间物质传递效率,从而提高细胞分化质量。目的:观察微重力培养环境在构建组织工程模块修复关节软骨缺损中的作用。设计、时间及地点:体外实验采用自身对照;体内实验采用随机分组比较,于2007-12/2008-06在广东省构建与检测实验室及南方医院动物实验中心进行。材料:3周普通级龄新西兰兔1只用于骨髓间充质干细胞培养;3月龄新西兰兔48只用于体内验证实验。方法:以纤维蛋白胶粉、凝血酶、氯化钙、抑肽酶、氨甲环酸制备纤维蛋白原支架。体外培养新西兰大白兔骨髓间充质干细胞3周,使之吸附于改良纤维蛋白原支架上在微重力环境下三维立体培养3周。48只兔做软骨钻孔制备软骨缺损动物模型,每只兔4孔。实验分为4组,每组48孔,微重力培养组在孔内植入微重力培养下的软骨细胞组织模块;普通培养组在孔内植入普通培养下的软骨细胞组织模块;单纯支架组在孔内植入空白支架;空白组孔内不植入任何材料,分别于2,6,12周处死动物,取相应标本。主要观察指标:通过生长曲线、倒置相差显微镜、组织学检测微重力培养环境在三维立体培养对软骨细胞增殖及功能的影响,以及用组织工程学评分检测微重力环境下培养的软骨模块对缺损的修复效果。结果:模拟微重力培养条件下构建的模块体外培养3周,细胞数量明显增多,并分泌较多的细胞基质,包裹在软骨细胞周围。组织工程学评分显示微重力培养组修复软骨缺损的效果明显好于其他组。结论:模拟微重力培养环境对三维立体培养软骨细胞模块有比较良好的效果。

BACKGROUND： The mimic microgravity culture system simulates an in vivo microgravity environment for cell survival to decrease shearing force of cells, increase transmission rate of intra-cellular, extra-cellular and intercellular substances, and enhance differentiation quality. OBJECTIVE： To study the effect of the mimic microgravity system on repairing articular cartilage defects. DESIGN, TIME AND SETTING： In vitro self-controlled and in vivo randomized study was performed at the Key Laboratory of Tissue Construction and Detection of Guangdong Province and Animal Experimental Center of Nanfang Hospital between December 2007 and June 2008. MATERIALS： One New Zealand rabbit of 3-week old was used for the preparation of bone mesenchymal stem cells （BMSCs）, and 48 rabbits of 3 months old were used for in vivo study. METHODS： Fibrin glue powder, thrombin, calcium chloride, aprotinin, and tranexamic acid were used to prepare fibrinogen scaffold. BMSCs were in vitro cultured for three weeks; after adhesion to modified fibrinogen scaffold, they were three-dimensionally cultured in the mimic microgravity environment for three weeks. A hole was drilled on cartilage tissue of 48 rabbits to establish defect models, with 4 holes on each rabbit. The holes were assigned into four groups, including the mimic microgravity group, normal culture group, blank scaffold group, and blank control group, with 48 wells in each group. The animals were sacrificed after 2, 6, and 12 weeks. MAIN OUTCOME MEASURES： The effects of the mimic microgravity environment on the proliferation and function of chondrocytes using growth curve, phase-contrast microscopy and histology; the effect on repairing articular cartilage defects was also detected using tissue engineering scores. RESULTS： At 3 weeks after culturing in the mimic microgravity environment, number of cells was increased, and number of cell matrixes was also increased surrounding chondrocytes. Tissue engineering scores indicated that the defect reparation in the mimic microgravity group was greater than other three groups. CONCLUSION： The mimic microgravity environment has greater effect on three-dimensionally repairing the cartilage defect.