Silk Fibroin as a Biomaterial Substrate for Corneal Epithelial Cell Sheet Generation

Abstract

To evaluate a silk fibroin (SF) biomaterial as a substrate for corneal epithelial cell proliferation, differentiation, and stratification in vitro compared with denuded human amniotic membrane (AM). Primary human and rabbit corneal epithelial cells and immortalized human corneal limbal epithelial cells were cultured on the SF and denuded AM, respectively. The biological cell behavior, including the morphology, proliferation, differentiation, and stratification, on the two substrates was compared and analyzed. Corneal epithelial cells can adhere and proliferate on the SF and denuded AM with a cobblestone appearance, abundant microvilli on the surface, and wide connection with the adjacent cells. MTT assay showed that cell proliferation on denuded AM was statistically higher than that on SF at 24 and 72 hours after plating (P = 0.001 and 0.0005, respectively). Expression of ΔNp63a and keratin 3/12 was detected in primary cell cultures on the two substrates with no statistical difference. When cultured at the air-liquid interface for 7 days, cells on SF could form a comparable stratified graft with a 2- to 3-cell layering, which compared similarly to AM cultures. SF, a novel biomaterial, could support corneal epithelial cells to proliferate, differentiate, and stratify, retaining the normal characteristic epithelium phenotype. Compared with AM, its unique features, including the transparency, ease of handling, and transfer, and inherent freedom from disease transmission, make it a promising substrate for corneal wound repair and tissue-engineering purposes.