Designing Bioengineered Skin Substitutes Containing Microfabricated Basal Lamina Analogs to Enhance Skin Regeneration

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Bush, Katie Ann
Worcester Polytechnic Institute
keratinocytes; bioengineered skin substitutes; fibronectin; microenvironment
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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.