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.

O surgimento das matrizes de regeneração dérmica nas duas últimas décadas permitiu um grande avanço no tratamento tanto das queimaduras agudas como das sequelas. No entanto, ainda há carência de informações sobre a relação entre os resultados clínicos e o que ocorre no tecido com cada tipo de matriz. O objetivo deste estudo foi avaliar prospectivamente os aspectos clínicos quanto à qualidade de pele, escala de Vancouver e POSAS, função e retração da área tratada e os aspectos histológicos na microscopia de luz e eletrônica, com o uso de duas matrizes de regeneração dérmica, ambas de colágeno bovino, uma de duas camadas, recoberta com lâmina de silicone e outra sem. Vinte e quatro pacientes, sorteados 12 em cada grupo, tiveram suas retrações cicatriciais secundárias à queimaduras tratadas em duas cirurgias, a primeira de liberação da retração e colocação da matriz e a segunda, colocação do auto enxerto de pele; em ambas as cirurgias foi utilizado o curativo de pressão negativa. As avaliações da escala de Vancouver e medidas da retração da área foram realizadas no pré-operatório, 1, 3, 6 e 12 meses e a escala de POSAS e avaliação funcional no pré-operatório e aos 12 meses. As biópsias foram colhidas no pré-operatório, no dia da colocação do enxerto de pele, 12 dias, 2, 6 e 12 meses após o enxerto. A avaliação clínica mostrou retração de todas as áreas tratadas, melhora da qualidade da pele e funcional em todos os pacientes. A matriz com silicone, mostrou superioridade dos resultados quanto a qualidade da pele, função e menor retração da área tratada. A análise histológica mostrou o crescimento de tecido conjuntivo denso idêntico ao tecido cicatricial original, sem diferenças entre as matrizes e que não se assemelha à derme normal. Também não foi observada diferença no diâmetro das fibrilas de colágeno do tecido neoformado, a pele normal e a cicatriz / The advent of dermal regenerate templates has fostered major advances in the treatment of acute burns and their sequelae, in the last two decades. Both data on morphological aspects of the newly-formed tissue, and clinical trials comparing different templates, are still lacking. The goal of this study was to prospectively analyze the outcome of patients treated with two of the existing templates, followed by thin skin autograft. They are both made of bovine collagen, one includes a superficial silicone layer. Surgery was performed on patients with impaired mobility resulting from burn sequelae (n = 12 per template). Negative pressure therapy was applied post-surgically; patients were monitored for 12 months. Data on scar skin quality (Vancouver and POSAS evaluation scales), rate of joint mobility recovery, and graft contraction were recorded; as well as morphological analyses at light microscopical and ultrastructural levels. Improvement in mobility and skin quality were demonstrated along with graft contraction, in all patients. The silicone-coupled template showed the best performance in all aspects. There was sub epidermal growth of dense connective tissue, indistinguishable from the original scars in both templates. The formation of tissue resembling normal dermis was not detected in any of the cases. Likewise, the ultrastructural analysis showed the same architecture of the connective tissue among the template scars and the original scar. No difference was detected when the collagen fibril diameters of the normal skin and of the scars (original and of the two templates) were compared

O surgimento das matrizes de regeneração dérmica nas duas últimas décadas permitiu um grande avanço no tratamento tanto das queimaduras agudas como das sequelas. No entanto, ainda há carência de informações sobre a relação entre os resultados clínicos e o que ocorre no tecido com cada tipo de matriz. O objetivo deste estudo foi avaliar prospectivamente os aspectos clínicos quanto à qualidade de pele, escala de Vancouver e POSAS, função e retração da área tratada e os aspectos histológicos na microscopia de luz e eletrônica, com o uso de duas matrizes de regeneração dérmica, ambas de colágeno bovino, uma de duas camadas, recoberta com lâmina de silicone e outra sem. Vinte e quatro pacientes, sorteados 12 em cada grupo, tiveram suas retrações cicatriciais secundárias à queimaduras tratadas em duas cirurgias, a primeira de liberação da retração e colocação da matriz e a segunda, colocação do auto enxerto de pele; em ambas as cirurgias foi utilizado o curativo de pressão negativa. As avaliações da escala de Vancouver e medidas da retração da área foram realizadas no pré-operatório, 1, 3, 6 e 12 meses e a escala de POSAS e avaliação funcional no pré-operatório e aos 12 meses. As biópsias foram colhidas no pré-operatório, no dia da colocação do enxerto de pele, 12 dias, 2, 6 e 12 meses após o enxerto. A avaliação clínica mostrou retração de todas as áreas tratadas, melhora da qualidade da pele e funcional em todos os pacientes. A matriz com silicone, mostrou superioridade dos resultados quanto a qualidade da pele, função e menor retração da área tratada. A análise histológica mostrou o crescimento de tecido conjuntivo denso idêntico ao tecido cicatricial original, sem diferenças entre as matrizes e que não se assemelha à derme normal. Também não foi observada diferença no diâmetro das fibrilas de colágeno do tecido neoformado, a pele normal e a cicatriz / The advent of dermal regenerate templates has fostered major advances in the treatment of acute burns and their sequelae, in the last two decades. Both data on morphological aspects of the newly-formed tissue, and clinical trials comparing different templates, are still lacking. The goal of this study was to prospectively analyze the outcome of patients treated with two of the existing templates, followed by thin skin autograft. They are both made of bovine collagen, one includes a superficial silicone layer. Surgery was performed on patients with impaired mobility resulting from burn sequelae (n = 12 per template). Negative pressure therapy was applied post-surgically; patients were monitored for 12 months. Data on scar skin quality (Vancouver and POSAS evaluation scales), rate of joint mobility recovery, and graft contraction were recorded; as well as morphological analyses at light microscopical and ultrastructural levels. Improvement in mobility and skin quality were demonstrated along with graft contraction, in all patients. The silicone-coupled template showed the best performance in all aspects. There was sub epidermal growth of dense connective tissue, indistinguishable from the original scars in both templates. The formation of tissue resembling normal dermis was not detected in any of the cases. Likewise, the ultrastructural analysis showed the same architecture of the connective tissue among the template scars and the original scar. No difference was detected when the collagen fibril diameters of the normal skin and of the scars (original and of the two templates) were compared