脱细胞异种角膜基质的制备及细胞相容性

Preparation of Xenogenic Corneal Stroma and Its Cytoconsistency

目的用牛角膜制备脱细胞基质,作为组织工程角膜的载体,为细胞提供贴附生长的支持物。方法选取新鲜牛角膜9个,每个分层剥离为2片组织。用0.25%胰蛋白酶,37℃下分为20、40、60min消化,漂洗。每组取样本分别做扫描电镜观察和HE染色观察。其余冻干处理后消毒备用。将消毒后冻干牛角膜基质常规培养基浸泡24h,取浸提液原液,稀释101、102、103倍培养人成纤维细胞,观察细胞生长情况。将冻干牛角膜基质复水,常规培养基浸泡24h,以2×105个/cm2接种兔角膜缘细胞,培养7d后,标本扫描电镜检查、HE染色观察。结果电镜观察各组牛角膜片表层细胞均被脱去,60min组基质胶原间细胞碎片残留较少,20min组较多,胶原纤维有序排列以60min组破坏最大。浸提液的各稀释倍数及原液所培养人成纤维细胞生长良好,各组间差异无显著性。电镜及HE染色观察复合材料培养的兔角膜缘干细胞,均见细胞在材料表面贴附生长,材料部分表面呈多层生长。结论以酶消化法和冻干处理的脱细胞牛角膜基质可以作为细胞的载体在体外与细胞复合培养。

Objective To explore the possibility that using the bovine corneal stroma to provide a suitable carrier on which the cells can grow for tissue engineering cornea. Methods Nine fresh bovine corneas were selected. Each cornea was cut into 2 pieces, and exposed to 0.25 % trypsinase for various lengths of time （20 minutes, 40 minutes, and 60 minutes） to get the stroma part with least cells and maintaining the collagen fibers arrangement. Samples obtained from each group were examined with scanning electron microscopy and HE staining. The left ones were freeze-dried and sterilized. The various concentrations of extraction were used to cultivate human fibroblasts, and a 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyhetrazolium bromide （MTT）-based colorimetric assay was taken to evaluate the exhistance of cytotoxinic effects. Then the proper corneal stroma was used as a carrier to cultivated the rabbit corneal limbal cells which were planted on it in a concentration of 2 ×10^5/cm^2 in vitro. The cell-cartier samples were sent for scanning electron microscopy and HE staining. Results The corneal stroma had the least cells in the group acted with typsin for 60 minutes, while the collagen fibers arrangement was not so orderly as before. The extractions showed no significant difference in cell culture, and no obviously harmful effect on the cell growth. The rabbit corneal limbal cells presented a stratified on the bovine corneal stroma. Conclusion The bovine corneal stroma without cells prepared using the typsin and lyophilization can be a suitable cartier for cell culture in vitro.