Rabbit Corneal Epithelial Sheets Engineered With Porous Silk Fibroin Film as a Biological Carrier

Abstract

Purpose: : Silk fibroin sutures have been used over the past decade in the clinical field. Silk fibroin can also be processed as a thin transparent film, which may be used as a transparent biological carrier. To examine the application in corneal epithelial reconstruction, we engineered rabbit epithelial sheet with silk fibroin film as a carrier.

Methods: : Porous silk fibroin films with high molecular permeability were prepared by mixing silk fibroin and polyethylene glycol (PEG), and then removal of PEG from the silk-PEG films. Epithelial cells were isolated from rabbit limbal epithelium, and then seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts in SHEM medium. The quality of cultivated rabbit epithelial were examined by immunohistochemistry and RT-PCR for cytokeratin (K)3, K12, collagen IV and Integrin β1, and colony forming assay. Transparency of cultivated sheets were compared with amniotic membrane (AM) carriers as control.

Results: : Molecular permeability of porous silk fibroin film was higher than untreated silk fibroin film. The stratification of epithelial sheets was successfully engineered by using the porous silk fibroin film, which expressed K3 and K12 as differentiation makers. Basement membrane components such as collagen IV and integrin b1 were expressed in the basal layer of stratified epithelial sheets. Colony forming efficiency of dissociated cells from fibroin sheets was similar to primary corneal epithelial cells. Epithelial sheet using porous silk fibroin were transparent compared with AM sheets.

Conclusions: : Porous silk fibroin film may be used as a transparent carrier of cultivated epithelial sheets in corneal epithelial reconstruction.

Keywords: cornea: epithelium • regeneration