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Title: Development of a biodegradable membrane to be used with skin explants for full thickness skin defect reconstruction
Author: Sharma, Kavita
ISNI:       0000 0004 7960 3516
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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Tissue engineered skin has advanced significantly in creating a substitute to mimic human skin. Advances in biomaterial sciences and a better understanding of wound healing have been the catalyst for development of tissue-engineered skin substitutes. We aimed to develop and evaluate a biodegradable dermal alternative composed of FDA approved polymers consisting of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) (nanofibres) and polylactide-co- glycolide (75:25) (microfibers)to be combined with skin explants as a one-step approach for full thickness skin defects reconstruction. The scaffolds consisted of an upper and under layer of PGLA 75:25 which is known to facilitate the migration of epithelial cells and a middle layer of PHBV to act as a basement membrane separating the epidermis from the underlying dermis. The undulating topography of the dermo epidermal junction was replicated by templating these undulations on the middle layer of scaffolds using metal collectors. The rete ridges are thought to create a microenvironment for stem cells to reside. To assess this, we undertook Ki67 immunohistochemistry which demonstrated that there was some activity in the trough of the undulations but this was not convincing. As part of the one step approach we used skin explants to populate the scaffolds as opposed to isolated epithelial cells which requires planning and appropriate staff and facilities. We investigated the ability for cells from skin explants to migrate from the cut edges of onto the electrospun scaffolds compared to using isolated keratinocytes and fibroblasts. It was found that cells from the explants will migrate along the fibres of the scaffold but not as much as using isolated cells in the same time period in terms of metabolic activity, collagen production and qualitative assessments using DAPI and H&E. The ability for optimization of migration of cells from the skin explants was assessed using fibrin, a biological tissue glue in current clinical use in its ability to retain the explants on the scaffold. We found that a fibrin constituent combination of thrombin 2.5 I.U and fibrinogen 18.75mg allowed manipulation of skin explants on the scaffold and migration of cells along the fibres. We also found that explants with less dermis had more outward cellular migration from their cut edges compared to those with more dermis. Finally, we assessed the ability for the constructs to have another layer of scaffold on top to aid cellular migration but found that this did not significantly influence outgrowth. Furthermore, we tested the effect of keratinocyte migration from skin explants in the presence of isolated fibroblasts pre-cultured on scaffolds as an in vitro wound bed model. This revealed significantly increased metabolic and proliferative activity of constructs in production of collagen. Although in our model for clinical translation we do not intend to use isolated fibroblasts to pre culture scaffolds, it was felt that this was the closest to a wound bed model as the scaffolds would be placed onto a prepared wound bed. Immunohistochemistry was used to demonstrate that keratinocytes were migrating from the cut edges of the skin explants and confocal imaging demonstrated that the cells penetrated the layers of the trilayer scaffold. In summary were able to design a suitable synthetic dermal scaffold and populate it with cells from skin explants. To facilitate this, we propose the use of fibrin and skin explants with less dermis to facilitate skin cell migration. Animal studies are now needed to further define parameters of the use of this in the clinical environment.
Supervisor: MacNeil, Sheila ; Ralston, David ; Giblin, Victoria Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available