Silkworm and spider silks as potential scaffold materials for tissue engineering (2008)

Silks have been described as biocompatible materials with a range of excellent mechanical properties, particularly a rare combination of strength and elasticity thought to arise from their unique molecular composition and ultrastructure. This project was aimed at studying the potential of natural silk fibres for tissue engineering. Species studied included degummed brins from the domestic silkworm Bombyx mori, the wild silkworm Antheraea pernyi and egg sac silk fibres from the spider Nephila edulis. A qualitative and quantitative description of the physical properties of the silks was carried out, where surface appearance, morphology and ultrastructure were investigated using a variety of microscopy techniques. The intrinsic fluorescence, density, linear density and cross section of silk fibres were also measured, facilitating a numerical estimation of their surface area per unit weight. Consequently, the tensile properties of the silks were measured, prior to and post cell culture preparation. The effect of autoclaving, washing and storage in growth medium was assessed through different tensile parameters. Upon culturing endothelial cells on the silk, a marked toxic effect was recorded and investigated, and different washing procedures to remove the toxic effect have been tested. Cell adhesion studies compared the ability of the different silks to support cell attachment for up to ten days. Also measured was as the effect of different treatments of the silk and scaffold design on rates of cell attachment. Finally, a new method of surface modification was developed and tested in order to functionalise natural silk fibres for endothelial guidance and angiogenesis. Results showed that all three silks shared a similar hierarchical ultrastructure of nanofibrils bundled into microfibrils, running parallel to the axis of the fibre. The silks also shared a lined, ridged topography where the fibroin monofilament was exposed or smooth topography when still coated in gum. However, tested silks differed in diameter, morphology, amino acid content and intrinsic fluorescence. Tensile tests showed that preparation for cell culture procedures, including sterilisation by autoclaving and water wetting had little or no effect on the mechanical properties, with the exception of medium incubation, which had a statistically significant effect on the mechanical properties of all tested silks. Cell growth studies showed that exposure of endothelial and myofibroblast cells to silk reduced their growth rates. The effect was mediated by iii both direct and indirect exposure to all tested silks, but most markedly by A. pernyi, which caused a severe cytostatic effect. One study showed that incubation of A.pernyi silk in medium supplemented with serum (but not non-supplemented medium) resulted in highly toxic growth medium. Among the different washing procedures devised to remove the toxicity, only lengthy enzymatic degumming was effective in reducing the toxic effect. Cell adhesion and growth studies indicated endothelial cells could attach and grow on the silk, but adhesion improved after the enzyme treatment. Scaffold design was also shown to have some effect on adhesion, with three-dimensional woven fabric proving a better scaffold than a random mesh of fibres. Finally, it was reported that the transglutaminase Factor XIII might be used to modify the surface of silk fibres with the biologically active factor L1Ig6. To conclude, studies presented in this described two poorly characterised silk species (Antheraea pernyi cocoon and Nephila edulis egg case) in term of their morphology, ultrastructure and fluorescence. They then showed that native, nonmodified silks supported cell attachment and growth provided they were treated to remove toxic coating and used in the form of a woven fabric rather then a loose mesh of fibres. Studies also presented a quantitative approach to the utilisation of silk in tissue engineering, and established wild silkworm silk Antheraea as a superior scaffold compared with egg case from the spider Nephile edulis or the domestic silkworm Bombyx mori. Finally, the novel method of silk modification using Factor XIII was reported as a potential route to further enhancing native silk fibres as cell scaffolds for specific applications. These studies present a unique approach, as they intentionally avoided harsh modifications of silk fibres before their use as scaffolds in order to preserve their excellent mechanical properties.