Keratin-chitosan membranes as scaffold for tissue engineering of human cornea

Abstract

Purpose: To study the attachment and growth of human corneal cells on keratin-chitosan membranes. The end goal is to develop a bioengineered cornea based on this material. Methods: Keratin-chitosan membranes were prepared as previously described by Tanabe et al., 2002. Briefly, 7.15 mg/cm2 of keratin dialysate was mixed with 10wt% chitosan solution and 20 wt% glycerol. The solution was cast into a silicone mold and dried at 50°C for 36 hours. Eyes were attained from a local eye bank after penetrant-keratoplastic surgery. Human epithelial, stromal and endothelial cells were obtained of the limbal, stromal and endothelial regions. Cells were cultured on keratin-chitosan membranes, as well as on plastic dishes as controls. When cultured cells reached confluence, they were fixed, incubated with primary antibodies (E-cadherin, cytokeratin high molecular weight (CK), vimentin and Na+/K+ ATPase) and visualized by indirect immunocytochemistry. Results: Epithelial, stromal and endothelial cells were able to attach and grow on keratin-chitosan membranes. All the cells maintained their morphology and cellular markers, both in the membrane and on the culture plate. Epithelial cells stained positively for CK and Ecadherin. A positive vimentin stain was observed in all stromal cells, while endothelial cells were positive forvimentin and Na+/K+ ATPase, but negative for Ecadherin. Conclusions: Keratin-chitosan membranes have been shown to be a good scaffold for culturing epithelial, stromal and endothelial corneal cells; therefore, future applications of keratin-chitosan membranes may be developed for reconstruction of the cornea.