Silk Fibroin Sponge Research Articles

Spheroid Formation and Expression of Liver-Specific Functions of Human Hepatocellular Carcinoma-Derived FLC-4 Cells Cultured in Lactose−Silk Fibroin Conjugate Sponges

This study presents a hepatic tissue engineering application of three-dimensional (3D) porous sponges composed of lactose-silk fibroin (SF) conjugates (Lac-CY-SF) bearing β-galactose residues, hepatocyte-specific ligands. Lac-CY-SF sponges were prepared by freeze-drying, followed by immersion in a series of methanol aqueous solutions. Lac-CY-SF sponges showed heterogeneous pore structure with round pores about 100 μm in diameter and elongated pores 250-450 μm in length and 100-150 μm in breadth. To employ a 3D Lac-CY-SF culture system, human hepatocellular carcinoma-derived FLC-4 cells were seeded in Lac-CY-SF sponges and cultured up to 3 weeks. FLC-4 cell culture in collagen and SF sponges was also performed for comparison with the cell response to Lac-CY-SF sponges. Within 5 days of culture, FLC-4 cells cultured in Lac-CY-SF sponges, as well as the cells cultured in collagen sponges, formed multicellular spheroids with diameters from 30 to 100 μm more efficiently than did the cells cultured in SF sponges. After 3 weeks of culture, WST-1 viability assay revealed that shrinkage suppression of Lac-CY-SF sponges enabled the maintenance of viable FLC-4 cells for a long time, while the shrinkage and disintegration of collagen sponges prevented the maintenance of the cells. FLC-4 cells cultured in Lac-CY-SF sponges exhibited greater elevation of albumin secretion and sustained a higher albumin level compared with the cells cultured in collagen and SF sponges during the 3 week cultivation period. FLC-4 cells cultured in Lac-CY-SF sponges for 3 weeks expressed genes related to liver-specific functions such as transferrin and HNF-4α. On the other hand, the cells cultured in collagen and SF sponges for 3 weeks did not express these genes. These results indicated the very promising properties of Lac-CY-SF sponges as a scaffold for long-term culture of functional FLC-4 cells to study drug toxicity and hepatocyte metabolism in humans and develop a bioartificial liver model.

Chondrocyte distribution and cartilage regeneration in silk fibroin sponge

Chondrocytes distribution and cartilage formation in three types of fibroin sponges with different average pore sizes (40-80, 80-120 and 100-140 μm) was measured. The image processing was performed combining two methods to identify cells automatically: extraction of local maximum luminance and multi-threshold analysis. The results showed that initial accumulation of chondrocytes localized at surface area at 3 h in the small and medium-pore groups, however, the difference in the cell distributions become equivalent until 24 h after seeding. Cartilaginous tissue was well formed in each group at 21 days, and that in the smaller pore group tend to distribute at the surface area. Spherical tissues were located at the subsurface (200-600 μm below the surface) of the sponge in the medium- and large-pore groups at 21 days. Local cell aggregation was observed at 24 h at the same depth of the fibroin sponge as the spherical tissues observed at 21 days. These results suggest that the initial cell condensation process till 24 h after seeding play an important role in cartilage tissue formation.

Semi-interpenetrating polymer networks composed of silk fibroin and poly(ethylene glycol) for wound dressing

Semi-interpenetrating polymer networks (SIPNs) composed of silk fibroin (SF) and poly(ethylene glycol) (PEG) were prepared by photopolymerization of a PEG macromer in the presence of SF to improve the mechanical properties of SF sponge as wound dressing. The morphological structure of the SF/PEG SIPNs was observed to be composed of an interconnected microporous surface and a cross-sectional area. SF/PEG SIPNs showed non-cytotoxicity evaluated by a cell proliferation method using L929 fibroblasts. Wound contraction treated with SF/PEG SIPNs sponges was faster than that of Vaseline gauze as a control. Histological observation confirmed that the deposition of collagen in the dermis was organized by covering the wound area with SF/PEG SIPNs. The above results indicated that SF/PEG SIPNs could be used as wound dressing.

Preparation and characterization of regenerated Bombyx mori silk fibroin fiber with high strength

Regenerated Bombyx mori silk fibers were spun from hexafluoro-iso- propanol solution of silk fibroin sponge in methanol used as a coagulant solvent and then elongated in water. The stress-strain curves of the regenerated fibers changed dramatically depending on the draw ratio and the structure was studied by 13 C CP/MAS NMR and X-ray diffraction meth- ods. The patterns of 13C CP/MAS NMR spectra of two regenerated fibers with different draw ratios (1× and 3×) and native silk fiber are all β-sheet structure although the fraction of random coil/distorted β-turn decreases in the order of 1×, 3× and native fiber gradually. On the other hand, azimuthal scans of their X-ray fiber patterns changed remarkably with increasing the draw ratio. This indicates that long-range orientation of the fibroin chain changes remarkably during the drawing process, but the short-range local structure does not change significantly. Regenerated silk fiber with a draw ratio of 3× is a fiber with high strength which is comparable with that of natural silk fiber. The regenerated fiber is also more degradable than natural silk fiber in enzyme solution in vitro.

Preparation and characterization of chitin whisker-reinforced silk fibroin nanocomposite sponges

For highly porous form such as sponges or scaffolds, the induction of the β-sheet formation of silk fibroin to make the water-stable materials usually results in their high shrinkage leading to a difficulty in controlling shape and size of materials. Thus, the objective of this study was to improve dimensional stability of silk fibroin sponge by incorporating chitin whiskers as nanofiller. Chitin whiskers exhibited the average length and width of 427 and 43 nm, respectively. Nanocomposite sponges at chitin whiskers to silk fibroin weight ratio (C/S ratio) of 0, 1/8, 2/8, or 4/8 were prepared by using a freeze-drying technique. The dispersion of chitin whiskers embedded in the silk fibroin matrix was found to be homogeneous. The presence of chitin whiskers embedded into silk fibroin sponge not only improved its dimensional stability but also enhanced its compression strength. Regardless of the chitin whisker content, SEM micrographs showed that all samples possessed an interconnected pore network with an average pore size of 150 μm. To investigate the feasibility of the nanocomposites for tissue engineering applications, L929 cells were seeded onto their surfaces, the results indicated that silk fibroin sponges both with and without chitin whiskers were cytocompatible. Moreover, when compared to the neat silk fibroin sponge, the incorporation of chitin whiskers into the silk fibroin matrix was found to promote cell spreading.