Polymeric membranes for corneal epithelium engineering based on chitosan and structural proteins

Abstract Purpose To develop chitosan hydrogel scaffolds for corneal epithelium cultures. Methods The hydrogel scaffold in the form of a membrane was obtained from chitosan‐natural linear polysaccharide derived from chitin from crustacean shells. It is anti‐bacterial, biocompatible, biologically inert, stable in the natural environment and safe for human. Chitosan films containing keratin and collagen were cross‐linked with genipin, a naturally occurring and nontoxic agent compound found in the gardenia fruit extract. Corneal epithelial cells were seeded on chitosan hydrogel scaffold and were cultivated for 14 days in standard condition using DMEM/F12HAM medium with 3T3 fibroblast co‐culture. Results Mean number of cell layers was 5,2 (3‐8). Immunostaining for cytokeratin 3 and 12,protein p63, connexin 43 was performed to confirm origin of the epithelium and presence of low differentiated cells. Conclusion Genipin crosslinked chitosan hydrogel seems to be a promising material for further clinical tests directed towards the development of implantable corneal epithelial cells