脱细胞基质软骨支架的制备

Preparation of an acellular cartilage matrix scaffold

背景:脱细胞基质材料去除了天然材料中的细胞成分,保留了基质成分,有效降低了天然材料的免疫原性,同时能够保持材料的机械强度。目的:拟利用洗脱方法去除兔肋软骨中的细胞基质,制备天然生物支架材料。方法:取新西兰大白兔肋软骨,清除周围组织后随机分组处理,以未经处理的肋软骨作为正常对照组;48 h处理组以去污剂-酶化学消化48 h;96 h处理组以去污剂-酶化学消化96 h,3组均通过苏木精-伊红染色及电镜观察脱细胞效果。同时收集诱导第7天的兔骨髓间充质干细胞3×109 L-1,与同种异体肋软骨脱细胞基质体外复合培养,于第3,7天取复合物行电镜观察细胞在脱细胞基质表面的黏附生长情况。结果与结论:新鲜肋软骨标本每个软骨陷窝内均有排列紧密的二三个软骨细胞,去污剂-酶化学消化后软骨陷窝内的细胞逐渐脱失,至消化处理96 h后,软骨陷窝内的细胞完全脱失。共培养第3天时,脱细胞基质表面有大量骨髓间充质干细胞分布,细胞为多角形,有伪足伸出,锚定在基质表面,部分区域可见细胞在基质表面增殖分裂;第7天时,脱细胞基质表面大部分均为细胞覆盖,细胞呈扁平状,有多个足突充分伸展,细胞之间互相连接,分泌大量细胞外基质沉积在基质表面,呈冰霜样改变,表明制备的脱细胞基质具有良好的细胞相容性。

BACKGROUND： Acellular matrix which is decellularized but retains the matrix components in the nature materials can reduce the immunogenicity of natural materials effectively and keep the material mechanical strength. OBJECTIVE： To prepare the natural biological scaffold material by using elution method＇to remove the cells in the rabbit costicartilage matrix. METHODS： After removal of the surrounding tissues, the costicartilage samples from New Zealand white rabbits were randomly divided them into three groups： costicartilages untreated as normal control group, detergent-enzyme digestion for 48 hours as 48-hour treatment group, detergent-enzyme digestion for 96 hours as 96-hour treatment group. Hematoxylin-eosin staining and electron microscope were used to observe decellularized effects. At 7 days of induction, bone marrow mesenchymal stem cells, 3x 109/L, were collected and co-cultured with allogeneic acellular costicartUage matrix in vitro. At 3 and 7 days of co-culture, the composite was taken for observation of cell growth on the cell-free substrete surface under electron microscope. RESULTS AND CONCLUSION： Two or three cartilage cells were closely packed in each cartilage pit, began to depigment using the detergent-nuclease digestion method, and then completely depigmented at 96 hours after treatment. At 3 days of co-culture, there were a large amount of polygon-shaped adherent cells with pseudopodiasobserved on the acellular matrix surface in vitro, and cells could proliferate and divide in partial regions. At 7 days of co-culture, adherent cells spread mostly throughout the acellular matrix surface, these cells were fiat-shaped and extended with multiple interconnected processes. Mass of secreted excellular matrixes were deposited on the matrix surface and showed frost-like changes, indicating the prepared acellular matrix has favorable cytocompatibility.