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.

Lamellar structure in poly(ala-gly) determined by solid-state NMR and statistical mechanical calculations.

Lamellar structure of poly(Ala-Gly) or (AG)n in the solid was examined using 13C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1-13C]Gly14[1-13C]Ala15- and [1-13C]Gly18[1-13C]Ala19 of (AG)15 were examined by two-dimensional (2D) 13C spin diffusion NMR in the solid state. In addition five doubly labeled [15N,13C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3-13C] in each of the successive Ala residues were utilized for REDOR and 13C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted beta-turns with a large distribution of the torsion angles and antiparallel beta-sheets. The relative proportion of the distorted beta-turn form was evaluated by examination of 13C CP/MAS NMR spectra of [3-13C]Ala-(AG)15. In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13C and 15N nuclei which were also interpreted in terms of a combination of beta-sheets and beta-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cbeta 13C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.