Porous Silk Fibroin Film as a Transparent Carrier for Cultivated Corneal Epithelial Sheets

Abstract

Biological carriers, such as the amniotic membrane and serum-derived fibrin, are currently used to deliver cultivated corneal epithelial sheets to the ocular surface. Such carriers require being transparent and allowing the diffusion of metabolites in order to maintain a healthy ocular surface. However, safety issues concerning biological agents encouraged the development of safer, biocompatible materials as cell carriers. We examined the application of porous silk fibroin films with high molecular permeability prepared by mixing silk fibroin and poly(ethylene glycol) (PEG), and then removal of PEG from the silk-PEG films. Molecular permeability of porous silk fibroin film is higher than untreated silk fibroin film. Epithelial cells were isolated from rabbit limbal epithelium, and seeded onto silk fibroin coated wells and co-cultured with mitomycin C-treated 3T3 fibroblasts. Stratified epithelial sheets successfully engineered on porous silk fibroin film expressed the cornea-specific cytokeratins K3 and K12, as well as the corneal epithelial marker pax6. Basement membrane components such as type-IV collagen and integrin β1 were expressed in the stratified epithelial sheets. Further more, colony-forming efficiency of dissociated cells was similar to primary corneal epithelial cells showing that progenitor cells were preserved. The biocompatibility of fibroin films was confirmed in rabbit corneas for up to 6 months. Porous silk fibroin film is a highly transparent, biocompatible material that may be useful as a carrier of cultivated epithelial sheets in the regeneration of corneal epithelium.

Biocompatibility of helicoidal multilamellar arginine-glycine-aspartic acid-functionalized silk biomaterials in a rabbit corneal model

“…Clinical slit-lamp observations showed that the material maintained good transparency over the 90-day period of observation. Our data are in agreement with the previously published study by Vieira et al 29 and Higa et al 30 in which 2 or 5-mm diameter single-layer silk films, respectively, were used.…”

Silk fibroin in ocular tissue reconstruction

“…Results from our own preliminary studies using a rabbit model indicate that 2-mm discs cut from fibroin films are well tolerated when implanted within the central corneal stroma, with little evidence of leukocyte recruitment or fibroblast proliferation being observed over 60 days [71]. A similar result has recently been reported by Higa et al [61] using 5-mm fibroin film discs. Nevertheless, both results may well have been influenced by the avascular nature of the central cornea.…”

“…Traditionally, 3T3 fibroblast cell lines established from mouse embryos are used for this purpose, but a number of recent studies including those by our own group have shown that primary cultures of human limbal fibroblasts can also be used [31]. At time of submitting this paper, a detailed study by Higa et al has just appeared in press [61] characterising the phenotype of rabbit primary corneolimbal epithelial cells when grown on fibroin films mounted above a 3T3 feeder layer by using a porous cell culture insert. Two particularly important observations are made in this study.…”

Recent advances in the design of artificial corneas

“…While little of the native full-length protein remains following processing, the resulting peptide solutions can subsequently be used to generate a variety of stable and biocompatible materials including freestanding membranes, electro-spun fibers and porous sponges [24]. Of these three materials, the freestanding BMSF membranes have received most attention for potential use in corneal tissue reconstruction owing to their high degree of transparency and general handling properties [25][26][27][28]. Importantly, subsequent studies have revealed that BMSF-based materials support the attachment, growth and differentiation of cells isolated from all three main tissue layers of the cornea [25,[28][29][30].…”

“…Theoretically, combining these approaches could lead to creation of a full-thickness corneal tissue substitute as described in Figure 1. Preliminary studies by Higa et al [27] indicate that single BMSFmembranes are well tolerated when implanted within the corneal stroma, but safety and efficacy data for of any combination of cells grown on BMSF membranes has yet to be reported.…”