ABSTRACT
Background Biomaterials for corneal tissue engineering must demonstrate several critical features for potentialutility invivo, includingtransparency, mechanicalintegrity, biocompatibilityand slow biodegradation. Silk film biomaterial had been characterized to meet these functional requirements. ObjectiveThis study was to investigate the feasibility of physico-crosslink regenerated silk fibroin film as tissue engineered corneal scaffold. MethodsHuman corneal epithelial cells(CECs) links were cultured by regular method and CECs in logarithmic phase were than incubated on physico-crosslink regenerated silk fibroin film membrane. The shape of cultured human CECs was observed after 24,48 and 72 hours under the inverted microscope and scanning electron microscope( SEM ) ,and the CECs were cultured on culture plates as controls. The growth state of CECs on regenerated silk fibroin film was observed daily for 7 days by MTT, and cell cycle analysis and the presence of apoptosis of human CECs were examined by flow cytometry after incubation on regenerated silk fibroin film. Regenerated silk fibroin filmCECs (4 mm×3 mm) were implanted into the corneal stroma of the right eyes of New Zealand white rabbits. At the end of 4 and 8 weeks after implantation, the appearance of the ocular surface was examined using slit lamp and corneal neovascular area was measured. Corneal histopathological examination was carried out to assess the degradation of graft materials and immunohistochemistry was performed to detect the expression of CD34 in the corneal tissue after operation. ResultsThe morphology and structure of CECs were identical using the two cultured Methods when observed under the inverted microscope and SEM after 24,48 and 72 hours. No significant difference was found in the A490 value 1,2,3,4,5,6 or 7 days after incubation on regenerated silk membrane and in culture plates ( Fmethod =0. 641 ,P＞0.05 ). The apoptosis rates of CECs on regenerated silk membrane or culture plates were 1.8％ and 2.0％ and the amount of cells in G2/G1 phase was 1. 956 and 1. 945, respectively. Histopathological examination showed that the regenerated silk membrane material degraded and was replaced by regular collagen tissue 2 months after implantation,and the presence of neovascular area and inflammatory cells were less prominent in 2 months than 1 month post-implantation. The expression level of CD34 in corneal tissue was evidently lower 1 and 2 months after operation than the Ad-VEGF165-induced positive control group (P＜0. 05), and no significant differences were seen when compared with normal CECs(P＞0.05). ConclusionsPhysico- crosslink regenerated silk fibroin film is an excellent biomaterial for tissue engineered corneal scaffold with good biocompatibility.