Abstract: To verify the feasibility of deep-sea fish collagen to be utilized in fabrication of the scaffold for tissue-engineered cornea, a novel three-dimensional(3D)collagen scaffold was produced using fish skin collagen for the first time and its biocompatibility was evaluated in this study. After freeze dried fish skin collagen, purified from the skin of Gadus microcephalus, was cross-linked using N-(3-Dimethylaminopropyl)-N' -ethylcarbodiimide hydrochloride crystalline(EDC)/N-Hydroxysuccinimide(NHS), a 3D-collagen scaffold was obtained. Its transparency and histological structure were detected and identified by macroscopy, photospectrometry, hematoxylin-eosin(HE)staining of frozen sections, scanning electron microscope. After DiI(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-labeled human corneal stromal(HCS)cells were inoculated onto the scaffold, the immigration and distribution of the cells were identified by HE staining of frozen sections and fluorescent microscopy. The biocompatibility of the scaffold to HCS cells was characterized by MTT assay and immunocytofluorescent microscopy to examine the cell viability and expression patterns of marker protein and functional proteins. Our results showed that the fabricated 3D-collagen scaffold had an excellent transparency, uniform organized reticular structure with pore size of 50-130 μm. After HCS cells were 山 东 大 学 学 报 (理 学 版)第51卷 - 第1期袁晓龙,等:三维鱼类胶原支架的制备及其生物相容性研究 \=-inoculated onto the scaffold, a lot of the cells could migrate into the scaffold, and the inoculated HCS cells distributed regularly in the scaffold 3 days after cultured in vitro. Moreover, the extracts of the scaffold had no obvious adverse effects on the viability of HCS cells. The inoculated HCS cells still maintained the positive expression of the marker protein of vimentin, cell junction proteins of integrin β1, E-cadherin and connexin-43, membrane transport protein of Na⁺-K⁺-ATPase, and anti-UV protein of acetaldehyde dehydrogenase. In conclusion, the 3D-collagen scaffold fabricated from G.macrocephalus skin collagen, in good transparency and histological structure, was suitable for HCS cell immigration, and has good biocompatibility to these cells. The 3D-collagen scaffold has promising potentials to be used in the in vitro construction of tissue-engineered cornea.

Key words: human corneal stromal cell, biocompatibility, fish skin collagen, Gadus macrocephalus, 3D collagen scaffold