双相组织工程椎间盘纤维环构建与生物学功能研究

项目名称： 双相组织工程椎间盘纤维环构建与生物学功能研究

项目编号： No.30872614

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 冯刚

作者单位： 川北医学院

项目金额： 31万元

中文摘要： 前期研究中我们合成了一种高分子材料PPCLM，并和骨基质明胶BMG结合形成BMG/PPCLM双相支架。初步实验结果表明BMG/PPCLM双相支架同时具有较好的机械强度和伸缩性，以及良好的组织细胞相容性，可以用于纤维环组织工程构建。本研究利用最新的"诱导的多能干细胞"技术，将小鼠成纤维细胞诱导为具有多向分化潜能的干细胞，并通过RT-PCR、碱性磷酸酶染色、染色体核型分析和成瘤性分析等手段进行验证。随后在体外将这些干细胞诱导分化为"内纤维环细胞样细胞"和"外纤维环细胞样细胞"，通过番红染色、CollagenⅡ#21644;Aggrecan染色对其进行验证。随后分别将这两种细胞种植到BMG和PPCLM支架上继续生长，随后组装成双相组织工程纤维环。在经过体外培养，通过HE染色、番红染色和免疫组化染色，证明细胞在支架上生长良好，植入体内以后，可以最终可以形成能真实模拟纤维环组织结构和细胞分布特点的，具有生物学功能的组织工程纤维环。

中文关键词： 椎间盘退行性变疾病；纤维环；组织工程；干细胞

英文摘要： In preliminary reseach, a biomaterial poly(polycaprolactone triol malate) (PPCLM) was synthesised, and assembled into BMG/PPCLM biophasic scaffold with bone matrix gelatin (BMG).And the BMG/PPCLM scaffold has excellent performances in mechanical strength, flexibility, and biocompatibility, which is suitable for annulus fibrosus tissue engineering scaffold. In this study, mouse fibroblasts were induced into "induced pluripotent stem cells" (iPS cells) with multiple differentiation potention, as confirmed by RT-PCR, alkaline phosphatase staining, chromosome karyotype analysis and tumorigenicity analysis. Then these iPS cells were induced into inner annulus fibrosus cell and outer annulus fibrosus cell in vitro, and the results were proved by H&E staining,Collagen II and Aggrecan immunostaining. Subsequently the two kinds of cells were implanted on the PPCLM scaffold and cultured 7 days, then the cells-scaffold was assembled into BMG/PPCLM biophasic scaffold with BMG. After cultured in vitro, the cells could growth well and secrete matrix on the BMG/PPCLM scaffold, according to HE staining, Safranin staining and immunohistochemical staining. Finally after the biophasic scaffold was cultured in vivo, the artificial annulus fibrosus with biological function was formed, which could simulate the structure of annulus fibrosus and the distribution of annulus fibrosus cells.

英文关键词： Disc degeneration disease;Annulus fibrosus;Tissue engineering;stem cells