The effects of superoxide dismutase on osteoarticular cells and structural analysis of ochronosis, in a disease model of alkaptonuria

Kammath, Vishnu (2017) The effects of superoxide dismutase on osteoarticular cells and structural analysis of ochronosis, in a disease model of alkaptonuria. Masters thesis, UNSPECIFIED.

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Introduction: Alkaptonuria (AKU) is a rare autosomal recessive condition resulting from deficiency of homogentisate 1,2 dioxygenase, causing inability to metabolise homogentisic acid (HGA). Over time HGA polymerises and deposits as a dark pigment within collagenous tissues; this process is ochronosis. Ochronosis causes rapid and early onset joint arthropathy, mimicking osteoarthritis in presentation. Ochronosis causes damage to the extracellular matrix and chondrocyte death. It is hypothesised that superoxide dismutase (SOD) may have a beneficial effect on chondrocytes and limit this polymerisation process. This research aims to observe the effects of SOD in an in vitro model of ochronosis, and identify structural changes in this model via Raman spectroscopy. Methods: Immortalised human chondrocytes (C20) and osteosarcoma (MG63) cell lines were cultured in DMEM containing HGA, SOD or a combination of both for up to 14 days. Cell viability was assessed using trypan blue assay. Pigmentation was quantified using Schmorl’s staining technique. A dimethylmethylene blue assay was performed to assess damage to proteoglycans. Structural analysis was then performed on an in vitro model of ochronosis via Raman spectroscopy. Results: Addition of SOD to the culture medium indicates there is little effect on percentage viability of osteoarticular cells. All C20 cultures suggest a decrease in pigmentation upon addition of SOD by day 14. Pigmentation analysis of MG63 cultures indicate contrasting results at different magnification settings. In C20 and MG63 cells, there does not appear to be a difference in levels of sulphated glycosaminoglycan release between these treatment groups. Results from Raman spectroscopy identify structural differences between control and HGA treated cultures. Conclusion: The results indicate that SOD does have a role in the prevention of ochronosis. This suggests that SOD has the potential to be used as a therapeutic approach in the prevention of ochronosis in articular cartilage. Raman spectroscopy is able to highlight structural differences caused by ochronosis. This has implications for the use of Raman spectroscopy as a potential diagnostic tool in AKU, in the future.

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Thesis (Masters)
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06 Oct 2017 20:18
Last Modified:
19 Oct 2023 23:47