Hunt, Nicola Claire (2010)
Ph.D. thesis, University of Birmingham.
There is a clinical need for effective alternative skin replacements to autografts, allografts and xenografts. In this thesis a bi-layer skin graft was developed by encapsulation of fibroblasts in calcium-alginate hydrogel and culture of keratinocytes on the surface. Initially, the use of 5% and 2% w/v alginate hydrogels were investigated. Both scaffolds maintained fibroblast viability for at least 150 days encapsulation and caused reversible mitotic and catabolic inhibition, as assessed by fluorescent staining, immunochemistry and the thiazolyl blue assay. Sustained expression of angiogenic factors such as vascular endothelial growth factor, interleukin 6 and nerve growth factor were seen by fibroblasts encapsulated in both scaffolds, by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. Histological staining demonstrated that following degradation of the scaffolds, fibroblasts secreted ECM to facilitate dermal repair. Comparison of degradation of the scaffolds over time by measuring release of calcium, and changes in rheological properties, morphology and mass, indicated that 5% w/v alginate hydrogel degraded more slowly and was preferable to 2% w/v alginate hydrogel. Fibroblasts encapsulated in 5% w/v alginate hydrogel were shown to express keratinocyte growth factor by RT-PCR, to support keratinocyte proliferation and differentiation, and keratinocytes cultured on the 5% w/v alginate hydrogel surface were seen to form multi-layered epidermal structures by histology, immunostaining and RT-PCR.
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