完全脱钙骨基质与骨髓间充质干细胞体外诱导构建组织工程纤维软骨

In vitro fibrocartilage construction with bone marrow-derived mesenchymal stem cells-fully demineralized bone matrix

背景:含有骨形态发生蛋白的完全脱钙骨基质可使骨髓间充质干细胞向软骨细胞分化并维持其软骨细胞的特性,在软骨发生过程中发挥重要作用。目的:将骨髓间充质干细胞与完全脱钙骨基质在体外诱导培养成纤维软骨组织以及观察培养体系与支架材料对细胞凋亡的影响。方法:采用全骨髓培养法分离培养猪骨髓间充质干细胞,取第1代骨髓间充质干细胞均匀接种到完全脱钙骨基质上,以含有转化生长因子β1、胰岛素样生长因子、地塞米松、抗坏血酸及体积分数10%FBS的高糖DMEM诱导培养液培养作为实验组,将单层诱导培养、单层基础培养的骨髓间充质干细胞、空白完全脱钙骨基质作为对照,于培养第1,2,3,4周进行大体形态观察以及Ⅰ、Ⅱ型胶原及聚集蛋白聚糖的RT-PCR定性检测;培养4周进行细胞凋亡检测。结果与结论:实验组可检测到Ⅰ型胶原的持续表达,Ⅱ型胶原及聚集蛋白聚糖表达逐渐增加。单层诱导培养组可检测到Ⅱ型胶原、聚集蛋白聚糖的表达及Ⅰ型胶原的持续表达,但表达量远低于实验组;实验组细胞凋亡率高于单层诱导培养组、单层基础培养组。说明转化生长因子β1、胰岛素样生长因子1体外诱导培养的骨髓间充质干细胞-完全脱钙骨基质可以表达特异性软骨基质,三维环境培养细胞外基质的表达量明显高于单层诱导培养;静态培养系统对三维培养的细胞凋亡有影响。

BACKGROUND：The fully demineralized bone matrix containing bone morphogenetic protein can induce bone marrow-derived mesenchymal stem cells（BMSCs） differentiate into cartilage cells,and keep it characteristics of cartilage cells.It plays an important role in the process of chondrogenesis.OBJECTIVE：To observe the complexes of BMSCs and fully demineralized bone matrix induced into the cartilage tissue in vitro and to detect the effects on cell apoptosis of culture system and scaffold materials.METHODS：Porcine BMSCs were isolated with whole bone marrow culture method.The first passage of BMSCs were seeded onto the fully demineralized bone matrix.The BMSCs-fully demineralized bone matrix complex was incubated in inductive medium（high glucose DMEM including transforming growth factor-β1,insulin-like growth factor-Ⅰ,dexamethasone,ascorbic acid and 10% fetal bovine serum） for chondrogenic differentiation,serving as experimental group.The monolayer induced BMSCs,monolayer basic cultured BMSCs and the fully demineralized bone matrix cultured with inductive medium were the control groups.Samples were harvested at week 1,2,3 and 4 after culture respectively to observe general morphology.Collagen type Ⅰ,Ⅱ and Aggrecan could be observed by reverse-transcription polymerase chain reaction.The cell apoptosis was detected at 4 weeks.RESULTS AND CONCLUSION：The continuous expression of collagen type Ⅰ,and the increased expression of collagen type Ⅱ and Aggrecan could be detected in experimental group and monolayer induced BMSCs group.But the expression in experimental group was higher than that of monolayer induced BMSCs group.The apoptosis of experimental group was higher than monolayer induced BMSCs and monolayer basic cultured BMSCs.The complex of porcine BMSCs and fully demineralized bone matrix could be induced to express cartilage matrix in vitro with transforming growth factor-β1 and insulin-like growth factor-Ⅰ.The expression of extracellular matrix in three-dimensional culture was much higher than that in the monolayer;the static culture systems may influence the apoptosis of BMSCs cultured in three-dimensional environment.