AbstractCartilage damage and disease pose a critical challenge in medicine due its poor capacity to self-repair, especially with conditions such as osteoarthritis. Many cartilage repair treatments, including autologous chondrocyte implantation, involve extracting chondrocytes from their native in vivo environment and expanding them in in vitro monolayer culture. However, this change in environment can lead to rapid loss of chondrocyte phenotype, namely its rounded morphology, leaving the cells in a fibroblastic-like state. These dedifferentiated cells manufacture different levels of extracellular matrix, which are key to the mechanical properties of the in vivo cartilage, as well as an altered cytoskeletal conformation. Several techniques have been employed to attempt to redifferentiate chondrocytes to their natural phenotype. These include 3D hydrogels and scaffolds, pelleted culture, high-density culture and micropatterns. However, more research is needed to further understand the effects of cell constriction on cell behaviour, particularly on single cells. This thesis set out to improve the photolithographical techniques previously published, rendering them more repeatable and less time-consuming to produce. Subsequently, the effects of cell constraint between micropattern shapes and shape sizes were analysed. Characterisation methods (atomic force microscopy, contact angle goniometry, microscopy, X-ray photoelectron spectroscopy, cell culture and immunocytochemistry) pointed to the variability of cell attachment to glass, the difficulty of photoresist removal, and the challenges of creating a long-term cell-resistive chemistry. Following optimisation, a novel method of 2D cell culture within glass cell-adhesive micropatterns surrounded by cell-repulsive 1H,1H,2H,2H-perfluorodecyltrichlorosilane (FDTS) was developed. Using this method, cells were restricted to different shapes (square, circle, rhombus, 30Â° sector of a circle, 300Â° sector of a circle and circle) of various sizes (ranging from ~100Î¼m2 to ~40000Î¼m2). The micropattern design permitted the study of the initial attachment of isolated cells and multiple cells for 24 hours on the same substrate, providing an ideal platform for comparison. At 24 hours, a change in the cytoskeletal conformation of bovine chondrocytes was observed, more specifically in actin distribution, transforming from parallel bundles of fibres to a bulky mass surrounding the nucleus, with tubulin and vimentin forming condensed conformation around the nucleus. However, the change in ECM production, although present, was not substantial. At 24 hours, pattern size appears to be more critical for vimentin, actin, vinculin, FAK and aggrecan; however, not for tubulin. Pattern shape was shown to be essential for tubulin and did show some significance for vimentin and aggrecan.
|Date of Award||1 Aug 2019|
|Supervisor||Julie Gough (Supervisor) & Lu Shin Wong (Supervisor)|