一种新型脱细胞猪角膜基质载体支架的制备及其鉴定研究

Preparation and characterization of a novel acellular porcine cornea stromata carrier scaffold

为了获得基于异种角膜材料的理想组织工程角膜载体支架,首次建立了脱氧胆酸钠(SD)和原钒酸钠(SO)联合处理的技术方法,利用新鲜猪角膜进行了脱细胞角膜基质(aPCS)的制备及其鉴定研究。用角膜板层刀从新鲜猪角膜中切下厚度450μm的角膜片,分别选用SD联合SO、十二烷基磺酸钠(SDS)和Triton X-100的去细胞处理方法制备出SD-aPCS、SDS-aPCS和Triton-aPCS共3种支架,利用外观照相、分光光度计、石蜡切片苏木紫-伊红(HE)染色、冰冻切片DAPI和阿利新蓝染色检测了其理化性质和组织结构;对SD-aPCS进行扫描和透射电镜鉴定后,进而利用噻唑蓝(MTT)、石蜡切片HE染色、冰冻切片DiI荧光观察以及免疫荧光细胞化学染色评估了其对非转染人角膜基质(ntH CS)细胞的毒性与生物相容性。检测结果发现,3种aPCS的细胞脱除干净且在干重和含水量上没有显著差异;其中,SD-aPCS的透明性与糖胺聚糖(GAG)含量最高、SDS-aPCS次之、Triton-aPCS最低;除Triton-aPCS的组织结构出现了明显的紊乱外,SD-aPCS和SDS-aPCS的组织结构均排列规则。在电镜下,SD-aPCS的前弹力层表面平整、无裂痕,板层结构和胶原纤维超微结构正常;此外,SD-aPCS浸提液对ntH CS细胞没有毒性作用,注射接种到SD-aPCS支架内的ntH CS细胞与支架嵌合紧密,随体外培养时间的延长而逐渐伸展和迁移,且细胞仍保持有其固有标志蛋白—波形蛋白,细胞连接蛋白—间隙连接蛋白-43和整联蛋白,以及膜运输蛋白—钠钾泵的阳性表达。由此可见,利用SD联合SO的方法所制备SD-aPCS具有理想的理化性质、组织结构和生物相容性,可作为一种理想的载体支架用于组织工程角膜的体外构建及其相关应用研究。

To obtain a satisfied carder scaffold from xenogeneic corneal stroma for corneal tissue engineering, a novel technique using detergents of sodium deoxycholate （SD） and sodium orthovanadate （SO） was established to prepare an acellular porcine cornea stromata （aPCS） scaffold from fresh porcine cornea, and characterize its essential property for the first time in this study. The anterior lamella, 450 mm in thickness and sliced off from each cornea of fresh porcine eyeballs using a microkeratome, was decellularized with SD combined with SO, sodium dodecyl sulfate （ SDS）, and Triton X-100, and three kinds of aPCS scaffolds, ie SD-aPCS, SDS-aPCS and Triton-aPCS, were obtained, respective： ly. The physichemical property and histological structure of them was evaluated by light microscopy, spectrophotometer, paraffin section with hematoxylin-eosin （HE） staining, and frozen section with DAPI staining, and alcian blue staining, respectively. The ultrastructure of the SD-aPCS scaffold was verified by scanning electronic microscopy （SEM） and transmission electron microscopy （TEM）, and their cytotoxicity and biocompatibility to non-transfected human corneal stromal （ntHCS） cells was evaluated by methyl thiazolyl tetrazolium （MTT）, HE staining, DiI-fluorescence observation, and immunocytofluorescent staining. The results showed that the dry weight and water content of the three kinds of aPCS scaffolds had no significant difference, their innate cells were removed, and all of them were completely decellularized. Among them, the SD-aPCS scaffold had the highest transparency and glycosaminoglycan （GAG） content, followed by the SDS-aPCS and Triton-aPCS scaffold successively. In addition to the obvious disorder of the Triton-aPCS scaffold, the histological structure of SD-aPCS and SDS-aPCS scaffolds was very regular. Furthermore, the surface of Bowmen＇s membrane of the SD-aPCS scaffold was smooth and no cracks were found, and the orientation and arrangement of collagen fiber were normal in ultrastructure. SD-aPCS scaffold had no cytotoxicity to ntHCS cells, and the cells injected into the scaffold could adhere tightly to collagen fibers, and spread and immigrated within scaffold lamella with the time of in vitro culture. These cells still maintained the positive expression pattern of the marker protein of vimentin, the cell junction proteins of integrin 131 and connextin-43, and the membrane transport protein of Na ＋ -K＋ pump. In conclusion, the SD-aPCS scaffold prepared by SD and SO detergents, with ideal physichemical property, histological structure and biocompatibility, could be used as an ideal scaffold in the in vitro construction of tissue engineered cornea and other related application researches.