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.

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.

13 C high-resolution solid-state NMR coupled with selective 13 C isotope-labeling of different Ala one methyl carbons was used to clarify the structure of (AG) 15 peptide in the silk II structure as a model for the crystalline domain of Bombyx mori silk fiber. At the inner part of the peptide, the fraction of the peak at 16.6 ppm of the Ala Cb resonance assigned to b-turn structure increased at 11th and 19th positions. These data indicate the appearance of the most probable lamellar structure having a turn structure at these two positions, although the position of turn was distributed along the chain.

In an attempt to produce mineralized composite materials with potential use as biomaterials or scaffolds for tissue engineering, we designed silklike peptides based on Ala-Gly repeated sequences with a lamellar structure and Asp as a Ca binding site in the turn part as in Tirrell's work (for example: Macromolecules 1996, 29, 1540-1553). We further modified the design of the lamella structure by introducing a Ser residue between (GlyAla) 3 and (AlaGly) 3 sequences. At first, we synthesized three labeled versions of 41SDSDS, (GlyAla) 3 Ser-(AlaGly) 3 Asp(GlyAla) 3 Ser(AlaGly) 3 Asp(GlyAla) 3 Ser(AlaGly) 3 , with 13 C labeling in different positions to characterize the lamellar structure using 13 C CP/MAS and spin-diffusion solid-state NMR. The β-sheet fraction in Ala residues increased with increased distance from the Asp residue in the turn part. The introduced Ser residue took almost 100% β-sheet structure probably because it forms an extra hydrogen bond stabilizing the stem part of (AlaGly) n . Thus, position-selective and sensitive information useful to characterize the detailed lamella structure with heterogeneous local conformations, can be obtained by 13 C selective labeling of the peptide and determining 13 C conformation-dependent NMR chemical shifts. We then produced an analogous recombinant protein, 14DS16, ThrSer[(AlaGly) 3 Asp(GlyAla) 3 Ser] 16 in Escherichia coli as a possible biomaterial. Films of this protein treated with simulated body fluid were rapidly mineralized with hydroxyapatite.