Preparation and characterization of a novel acellular porcine cornea stromata carrier scaffold
- FAN Ting-jun, SHAN Ming, PANG Xin
JOURNAL OF SHANDONG UNIVERSITY(NATURAL SCIENCE). 2017, 52(5):
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To obtain a satisfied carrier 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, respectively. 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) 山 东 大 学 学 报 (理 学 版)第52卷 - 第5期樊廷俊,等: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 \=-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 Bowmens 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 β1 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.