Porous silk fibroin film as a transparent carrier for cultivated corneal epithelial sheets.

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 ß1 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.

Deposition of lipid, protein, and secretory phospholipase A2 on hydrophilic contact lenses.

PURPOSE: Recent studies have shown that low tear phospholipid levels are associated with tear film instability in hydrophilic contact lens wearers. The concentration of secretory phospholipase A2 (sPLA2), the enzyme that hydrolyzes phospholipids, in tears is known to exceed the levels found in serum by four orders of magnitude. This study was performed to determine the levels of sPLA2 from the deposition on two different frequent-replacement contact lens materials. METHODS: Polymacon and etafilcon A contact lenses worn for 2 weeks by 16 experienced contact lens wearers were used for the analysis. Total lipids were determined by the sulfo-phospho-vanillin reaction. Phospholipids in lipid extracts were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion. Total protein was measured by bicinchoninic acid analysis. Double-antibody sandwich enzyme-linked immunosorbent assay was used to determine sPLA2 concentrations. RESULTS: Total lipid deposition was found to be greater in the polymacon group (66.3+/-16.3 microg/lens) than in the etafilcon A group, although phospholipids were not detected in either group. The etafilcon A group had greater deposition of protein (3.7+/-0.7 mg/lens) than the polymacon group had. The etafilcon A group deposited statistically significantly more group IIa sPLA2 (1.1+/-0.3 microg/lens) than the polymacon group (0.07+/-0.04 microg/lens) did (P<0.001). CONCLUSIONS: There was a significant difference in the lipid and protein deposition profiles in the two lenses tested. A significant amount of sPLA2 in the deposition on contact lenses may play a role in tear film instability in hydrophilic contact lens wearers.