Surgery (St Vincent's) - Theses
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ItemEngineering articular cartilage from human infrapatellar fat pad stem cells for transplantation therapy( 2015)Mesenchymal stem cells (MSCs) have shown promise in cartilage tissue engineering due to their unlimited capacity for self-renewal and capability to differentiate into cartilage tissue lineage under certain physiological or experimental conditions. In this thesis, we harvested MSCs from human infrapatellar fat pad tissue (hIPFP) and further fully characterised using flow cytometry. Human IPFP-derived MSCs at passage three (P3) show good homogeneity for MSCs cluster differentiation (CD) markers including CD29, CD44, CD73, CD90, and CD105. Hyaline articular cartilage repair is a significant challenge in orthopaedics and traditional therapeutic options result in inferior outcomes. We believe traditional methods can be improved through applications based on three-dimension (3D) culture systems and tissue engineering strategies. In this thesis, we planned to investigate the chondrogenic potential of hIPFP-derived MSCs, stimulated by TGFβ3 and BMP6, over 7, 14 and 28 day in vitro in 3D pellet culture, a 3D printed chitosan scaffold and a 3D scaffold comprising methacrylated hyaluronic acid and methacrylated gelatin (called HA/GelMA). Therefore, endpoints included histology staining, immunohistochemistry, immunofluorescence, and temporal changes in expression of specific chondrogenic genes using quantitative real-time polymerase chain reaction (qPCR). In vitro 3D pellet culture maintained cells to be in close proximity to each other and promoted cell aggregation that mimics the cellular condensation process within native cartilage tissue. Furthermore, research has shown the potential of 3D biomaterial scaffolds for providing a suitable environment for chondrogenic induction and significantly enhancing the proliferation, differentiation, and chondrocytic extracellular matrix synthesis by MSCs. Collaborators at the Intelligent Polymer Research Institute (IPRI) at the Uiversity of Wollongong have developed extrusion printing for diverse bioengineering projects and this technique has developed for provision of both 3D chitosan scaffolds and 3D hyaluronic acid/biogel scaffolds for this project. The biocompatibility of chitosan and its structural similarity with glycosaminoglycan make it attractive for cartilage tissue engineering. Also, methacrylated HA and gelatin polymers were utilised to produce UV- crosslinkable HA/GelMA scaffold. A cartilage extracellular matrix component, HA, is the main non-sulphated glycosaminoglycan and offers a promise candidate for engineering of cartilage. In all three types of cultures (pellet, chitosan and HA/GelMA), over 14–28 days, clusters of encapsulated chondrocytes formed. Collagen type 2 and proteoglycan production were confirmed using immunohistochemistry and immunoflourescence. Chondrogenic lineage markers including: SRY-related transcription factor (SOX9), collagen type 2 alpha 1 (COL2A1), and aggrecan (ACAN) gene expression increased significantly over the time course. We reported that chitosan and HA/GelMA scaffolds enhance and increase the efficiency of chondrogenesis in our model. Finally, advanced microarray technique was conducted to provide novel informations about overall gene expressions during chondrogenesis across all three cultures. This is the first time that in vitro microarray has been used in the assessment of the chondrogenic differentiation of hIPFP-derived MSCs cultured in 3D pellet and seeded into chitosan and HA/GelMA scaffolds. Microarray gene analysis requires high-end programming for assessment of the test statistics that show whether a particular gene or a set of related genes are highly regulated (up- or down-regulated). Another challenge is to select a ‘ranking of expressed genes’ that may be relevant to a particular set of experimental conditions or of particular interest from a biological perspective (e.g. a particular metabolic pathway or a set of apoptotic genes). Therefore, we have successfully demonstrated in vitro production of hyaline-like cartilage from infrapatellar fat pad (IPFP)-derived MSCs in 3D culture. Microarray has provided novel informations concerning genes involved in chondrogenesis of hIPFP- derived MSCs and our approach offers a viable strategy for generating clinically relevant cartilage for therapeutic use.