Silk fibroin biomaterials for skin tissue engineering applications

The limited reparative capacity of the skin and the inadequacy of conventional treatments have necessitated the development of tissue engineered skin substitutes. Several substitutes, including Integra Dermal Regeneration Template, are finding increasingly widespread application in the treatment of acute and chronic wounds. To date, these substitutes are unable to fully recreate the functionality and aesthetics of skin prior to injury. This thesis applied an integrated approach combining solution preparation, material fabrication control and biological testing to investigate electrospun silk fibroin (SF) nano-microfibrous scaffolds as potential biomimetic skin substitutes. Further to this, the improvement of the existing Integra scaffold through the incorporation of hyaluronan (HA) was assessed. Through rheological analysis of regenerated SF solutions under shear and extensional deformation a concentration regime transition at 20 wt% SF was identified. Solutions with relaxation times under 0.001 seconds were found to be unsuitable for electrospinning. The incorporation of poly(ethylene oxide) (PEO) was found to significantly increase solution relaxation times and extensional viscosity, making them much more suitable for electrospinning. Solution viscoelastic properties were shown to directly influence electrospun fibre morphology, with increases in viscosity resulting in increases in fibre diameter under stable spinning conditions. The effects of electrospinning parameters on electrospun fibre morphologies were investigated using SF-PEO blended solutions. Increased electrical field, spinneret height and decreased flow rate were found to decrease fibre diameter. In vitro assessment of the attachment, spreading, proliferation, viability and gene expression of primary human dermal fibroblasts (PHDFs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) was conducted. Both PHDFs and BM-MSCs attached and proliferated with greater rapidity on fibres of the smallest diameters (~250-300 nm) with proliferation decreasing as fibre size increased until fibre diameters reached ~1200 nm. Cells were observed to be spread, with multiple attachments between fibres in scaffolds composed of ~250-300 nm diameter fibres. Cells aligned themselves to single fibres in scaffolds composed of fibres greater than 1 micrometre. HA supplementation to Integra resulted in increased proliferation, viability and migration of PHDFs. In ex vivo cutaneous wound healing models, the invasion of Integra was enhanced when scaffolds were supplemented with HA, with increased matrix deposition observed. Optimal supplementation concentrations for in vitro and ex vivo increases in cell proliferation and migration were at 1.5 – 2 mg ml-1 HA. SF electrospun scaffolds facilitated epithelial migration in ex vivo artificial wounds, with the migratory epidermis more closely resembling the structures observed in vivo. Additional preliminary investigations into the efficacy of a paste-form of Integra, Integra Flowable Wound Matrix (IFWM) were performed ex vivo, with cell invasion comparable to the conventional scaffold format. The potential for the incorporation of viable PHDFs and BM-MSCs was also investigated and keratinocyte migration was enhanced in these scaffolds. The results in this thesis provide valuable optimisation information on the development of SF electrospun scaffolds for skin engineering. Additionally, the supplementation of Integra with HA may provide a simple and effective way to enhance the performance of the scaffold in vivo.