Surgery (St Vincent's) - Theses
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ItemEffect of melatonin and indomethacin on adipose-derived stem cells and fat graft survival and function( 2016)Background: Autologous fat grafting has emerged as a key technique in soft tissue reconstruction, and is currently utilized for various deformities secondary to burns, chronic wounds, trauma, irradiation injuries and post-oncological resections. Nevertheless, a significant proportion of the transplanted fat fails to survive within the host environment, which may partly be attributed to cell death of its constituents. A process known as cell-assisted transfer is now used clinically to further enhance fat graft retention by adding stromal vascular fraction (SVF), an adipose-derived stem cell (ASC) rich content to lipoaspirate. However, there are certain concerns about cell-assisted lipotransfer especially regarding its oncologic safety and feasibility. Methods: The basis of this project stems from the need for reproducible and predictable clinical outcomes following transplantation of adipose tissue. To this end, the properties of two safe, commercially available and FDA-approved drugs, melatonin and indomethacin were studied. The in-vitro effects of melatonin on human ASCs were investigated through functional and survival assays including oxidative stress and cell-death assays, MTT Assay, monolayer scratch assay, cell membrane integrity assay, a human cytokine array, and a specific TNF-alpha ELISA kit. The in-vitro effects of indomethacin and melatonin on human ASC adipogenesis were investigated through a lipid (Oil Red O) staining kit as well as reverse transcription polymerase chain reaction (RT-PCR) to determine RNA expression of key adipogenic genes. The effects of melatonin-treated autologous fat grafts injected into the dorsums were tested in-vivo with a murine model, and its weights and volumes were assessed at three time points of two, four and twelve weeks. Hematoxylin and eosin staining, perilipin and CD31 immunostaining were performed with morphometric analysis of adipose tissue and vascularization. Results: The in-vitro results indicate that melatonin significantly protects human ASCs from hydrogen peroxide induced cell-death in a dose-dependent fashion. Addition of melatonin to ASCs improved cell-viability, promoted cell migration and preserved membrane integrity as compared to controls. In addition, it induced a potent anti-inflammatory response by down-regulating acute inflammatory cytokines particularly TNF-alpha but melatonin itself did not exert an adipogenic effect on human ASCs. Indomethacin stimulates adipogenesis of human ASCs in 2 weeks cell culture and upregulates several key adipogenic genes including PPAR-gamma, lipoprotein lipase and fatty acid binding protein 4. For the first time, this work demonstrated in-vivo that melatonin enhances fat graft volume retention at 4 weeks by increasing the percentage of adipose volume within fat grafts with comparable volumes to that of cell-assisted lipotransfer. Melatonin treatment, however, had no effect on angiogenesis. Based on these findings, melatonin and indomethacin may potentially be useful pharmacological adjuncts in clinical fat grafting based on their respective cytoprotective and adipogenic properties.