Bioengineered skin substitutes have been developed to treat burn and non-healing wounds; however limitations still hinder their clinical success rates. Optimizing these current design strategies requires an understanding of how biochemical and topographical features of the native tissue modulate keratinocyte processes involved in tissue functionality. In this thesis, a novel bioengineered skin substitute was developed that contains a microfabricated basal lamina analog that recapitulates the native microenvironment found at the dermal-epidermal junction (DEJ). In native skin, this microenvironment consists of both biochemical and topographical cues which play critical roles in maintaining tissue architecture and overall homeostasis with the external environment. Therefore, we hypothesize that microfabricated basal lamina analogs with extracellular matrix cues and three-dimensional features that mimics the cellular microenvironment of the DEJ will promote enhanced epithelialization and increase epidermal stem cell clustering on the surface of bioengineered skin substitutes. We determined that the extracellular matrix protein fibronectin (FN) found in the cellular microenvironment of the DEJ enhanced keratinocyte attachment, proliferation, and epithelialization of a collagen based basal lamina analog. It was also found that the collagen material used to create the basal lamina analog as well as the FN conjugation strategy to this material significantly influenced the bioactivity of FN and its ability to modulate keratinocyte functions through integrin based mechanism. To investigate spatial tissue organization and the role it plays in the cellular microenvironment of the DEJ on epithelialization and epidermal stem cell localization, we used photolithography coupled with materials processing techniques to create microfabricated basal lamina analogs. It was determined that epidermal thicknesses found in narrow channels of microfabricated basal lamina analogs (50 µm and 100 µm widths with 200 µm depths) were similar to cultures on de-epithelialized acellular dermis and native foreskin tissues after 7 days of in vitro culture. We also determined that the microfabricated basal lamina analogs created an epidermal stem cell niche that promoted epidermal stem cell clustering in the channels which is critical for longevity of the tissue. Overall, we developed a platform technology that was specifically used to produce a highly functional bioengineered skin substitute with regenerative capacity that mimics native skin. We anticipate through the use of this technology, we can further improve bioengineered skin substitutes by incorporating epidermal structures of native skin including hair follicles and sweat glands as well as improve overall cosmetic appearance. Additionally, this novel bioengineered skin substitute can serve as a model system to further our understanding of pathological conditions and diseases of the skin as well as facilitate robust preclinical screenings of epidermal responses to new therapeutic agents as well as to cosmetic and chemical products.

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Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-08-08T13:00:42Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Diogo do nascimento Gomes.pdf: 2107017 bytes, checksum: c3c9abeaebac392329936bc5b1b8fb97 (MD5) / Made available in DSpace on 2017-08-08T13:00:42Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Diogo do nascimento Gomes.pdf: 2107017 bytes, checksum: c3c9abeaebac392329936bc5b1b8fb97 (MD5) Previous issue date: 2016-07-29 / A pele funciona como uma barreira contra agentes físicos e patógenos, sem ela, diversos tipos de doenças causadas por agentes patógenos podem levar o indivíduo ao óbito. Substitutos temporários de pele auxiliam na regeneração cutânea, prevenindo a perda de líquidos e eletrólitos e o aparecimento de infecções. Alginatos são biomateriais biocompatíveis que podem ser utilizados para a confecção de substitutos temporários de pele. Adicionalmente, extratos e tinturas da Anadenanthera colubrina (Vell.). Brenan são amplamente utilizados na medicina tradicional, por apresentarem ações antimicrobianas e cicatrizantes, podem ser úteis na produção tais substitutos. Assim, este trabalho teve por objetivo produzir filmes baseados em alginato com ou sem adição de cloreto de cálcio, além da incorporação de extratos de A. colubrina. Foi avaliada a liberação de extratos de A. colubrina incorporados aos filmes em meio aquoso, além de ensaios físicos para verificar transparência, espessura, teores de umidade de todos os filmes de alginato e aqueles com incorporação de extratos. Adicionalmente foi verificada a distribuição do extrato no filme. Os filmes foram caracterizados por espectroscopia na região do Ultravioleta/Visível (UV/VIS) e do Infravermelho com transformada de Fourier (FT-IR). Adicionalmente foi avaliada a ação antimicrobiana qualitativa dos filmes incorporados com extratos etanólicos (50% e 80%) com ou sem a presença do Ca++, frente a linhagens multirresistentes Staphylococcus aureus. Os filmes sem incorporação de extratos apresentaram-se finos e mais transparentes (0,16 mm; 12,63) quando comparados com Cálcio (0,3 mm; não transparentes). Os filmes contendo extratos vegetais apresentaram diferenças apenas na transparência, de acordo com o extrato utilizado: 50% (0,61 mm; 3,04), a 80% (0,62 mm; 1,18) ou com CaCl2 e extrato (a 50% 0,62 mm e 2,17 a 80 % 0,75 mm; 2,03). As análises por UV/VIS evidenciaram homogeneidade na incorporação dos extratos nos filmes quando observados a 282 nm. Observouse uma pronunciada atividade bactericida destes filmes frente ao S. aureus, inclusive para as linhagens multirresistentes. O filme de Alginato com incorporação do extrato de A. colubrina 50% (FA50) apresentou maior inibição para S. aureus UFPEDA 700 (73.10-3 UFC), já o filme de Alginato reticulado com cálcio e com incorporação do extrato de A. colubrina 80% (FACa80) apresentou maior inibição do crescimento das cepas UFPEDA 711 (6.10-1) e 731 (261.10-1). Nos ensaios realizados (físico-químicos, espectroscópicos e microbiológicos) ficou evidenciada a diferença da liberação controlada dos extratos de A. colubrina (Vell.) Brenan em filmes com Ca++. / The skin acts as a barrier against physical and pathological agents. without it, all kinds of diseases caused by pathogens can cause the individual to death. Skin temporary replacements help in skin regeneration, preventing the loss of fluids and electrolytes and the onset of infections. Alginate is biocompatible biomaterials, which can be used for making skin substitutes. Additionally extracts and tinctures of Anadenanthera colubrina (Vell.) Brenan are widely used in traditional medicine, because they have antimicrobial and healing actions that can be useful in skin substitutive. This study aimed to produce films based on alginate with or without calcium chloride addition besides we tested an incorporation of extracts A. colubrina (Vell.) Brenan. Release of A. colubrina (Vell.) Brenan extracts, which was incorporated into the films, was performed in aqueous solution. Physical tests were performed to verify transparency, thickness, moisture content of all these films and alginate embedding extracts. Additionally it was verified the distribution of the extract into the film. In addition, the films were characterized by spectroscopy in the UV region / Visible (UV / VIS) and infrared Fourier transform (FT-IR). Additionally it was evaluated the qualitative antimicrobial action of the films incorporated with extracts (extracted with 50% and 80% ethanol) and with Ca ++ against Staphylococcus aureus, but also for multiresistant strains of this bacterium. The film without addition of extracts present is thinner and more transparent (0.16 mm; 12,63) than those crosslinked with calcium (0.3 mm; not transparent). The films containing plant extracts showed differences only in transparency in accordance with the extract used: 50% (0.61 mm; 3.04), 80% (0,62mm; 1.18) or CaCl2 and extract (the 50% 0.62 mm 0.75 mm 2.17 to 80%; 2.03). Analysis of UV / VIS showed homogeneous incorporation of the extracts in the movies when observed at 282 nm. There was a pronounced bactericidal activity of these films opposite to S. aureus, including multidrug-resistant strains. Alginate film with incorporation of A. colubrina extract 50% (FA50) showed greater inhibition for the strain S. aureus UFPEDA 700 (73.10-3 UFC), since the alginate film crosslinked with calcium and incorporation of A. colubrina extract 80% (FACa80) showed greater inhibition of growth of the strains UFPEDA 711 (6.10-1) and UFPEDA 731 (261.10-1). In tests (physico-chemical, spectroscopic and microbiological) was evident the difference of controlled release of A. colubrina extracts in films with Ca ++.

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.

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