No abstract

The growth and morphology of rat fibroblasts cultured on various polymer substrates, as well as their collagen biosynthesis, were studied. A clear difference in cell growth and cell morphology was observed among the substrates. The dependence of cell growth on the water contact angle of substrate was similar to that of the adhesion. Fibroblasts could proliferate at the highest rate and showed the highest-ordered morphology when cultured on the substrate with a contact angle around 70 degrees, which was also the most favorable for cell adhesion. The amount of collagen synthesized by total cells and of adsorption of the synthesized collagen to substrates were in good correlation with the cell growth dependence on the contact angle of substrate, whereas the collagen synthesis per cell was more active on the surfaces poor for cell growth than on the good ones. Cells on surfaces promoting active collagen synthesis had a round shape and clustered upon each other. The collagen-immobilized surface had nearly the highest cell adhesion, high cell proliferation, and high collagen adsorption among the substrates studied. In addition, the highest-ordered morphology and no lag time for proliferation were observed for the collagen-immobilized surface. These results indicate that the collagen-immobilized substrate provides the most favorable surface for cell growth at the initial stage.

A new process to form fibroin spongy porous 3-D structure is reported herein. The process involves freezing and thawing fibroin aqueous solution in the presence of a small amount of an organic solvent. The process requires no freeze-drying, chemical cross-linking, or the aid of other polymeric materials. The solvent concentration, fibroin concentration, freezing temperature, and freezing duration affect the sponge formation, its porous structure, and its mechanical properties. Measurements by XRD and FTIR indicate that silk I and silk II crystalline structures exist in the fibroin sponge and that the secondary structure of fibroin is transformed to a β-sheet from a random coil during this process. The tensile strength decreased slightly, but the fibroin sponge showed no deformation after autoclaving. Therefore, the fibroin sponge was sterilized using an autoclave. For 3 weeks, MC3T3 cells proliferated in the sterilized fibroin sponge. The fibroin sponge formed by this new process is applicable as a tissue-engineering scaffold because it is formed from biocompatible pure silk fibroin and offers both porous structure and mechanical properties that are suitable for cell growth and handling.

The near-surface structure and the wettability of silk fibroin films cast from aqueous solutions on hydrophobic polystyrene substrates at various temperatures is investigated by Fourier transform infrared attenuated total reflection spectroscopy (FTIR-ATR) and measurement of contact angle. The FTIR data reveal that the near-surface region of the films is enriched in random coil conformations of the protein at the expense of a reduced fraction of α-helix and β-sheet conformations. The relative random coil/β-sheet content shows a marked dependence on the casting temperature, displaying a minimum at 50 °C. The minimum occurs concurrently with a maximum in the wettability of film surfaces by polar liquids. In the lower wettability region, the film surfaces of this hydrophilic protein are hydrophobic, whereas in the enhanced wettability range they are slightly hydrophilic. The experimental data indicate that during formation of fibron films, α-helix and β-sheet structures are rejected by the interface because of their non-surface-active character, whereas random coils are energetically favored because at the interface they convert into a surface-active conformation which effectively minimizes the interfacial free energy and renders the polymer surface hydrophobic. In the narrow range of casting temperatures centered at 50 °C, the effect of the interface is overweighed by the bulk thermodynamics favoring the β-sheet crystallization of fibroin. Though the interfacial conformation is not accessible by FTIR-ATR, its surface-active character in combination with the unique composition and amino acid sequence of fibroin allows one to conclude that the possible chain structure is one that separates the hydrophobic alanine and hydrophilic serine residues to opposite sides of the plane passing through the chain axis.

Silk of Bombyx mori can be used as various biomaterials. Especially, it is useful as a protein for coating the surface of cell culture plates since the silk possesses a biocompatibility to the cultured cells. However, the cell-adhesive ability is weaker than collagen or fibronectin, which are used for coating the plate more frequently (Yao et al. J. Biochem., 2004, 136, 643-649). To increase the biocompatibility of the silk, we constructed transgenic silkworms, inserting the modified fibroin light-chain genes for making recombinant silks that possessed partial collagen or fibronectin sequences, that is, [GERGDLGPQGIAGQRGVV(GER)3GAS]8GPPGPCCGGG or [TGRGDSPAS]8, respectively. Films were made from the recombinant silks, and the cell-adhesive activity for cultured mammalian cells was observed. The results showed that the two types of recombinant silk films possessed a much higher cell-adhesive activity as compared to the original unmodified silk. Especially, the recombinant silk with the sequence [TGRGDSPAS]8, produced by a transgenic Nd-sD mutant, gave a 6 times higher activity than the original unmodified silk.