Surgery (St Vincent's) - Theses
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ItemOsteochondral repair using structured biological scaffold and stem cell technologies( 2015)Introduction: Articular cartilage damage can result in pain and loss of function for many patients. The traditional management of moderate to severe defects has been difficult due to the lack of intrinsic capacity for cartilage to regenerate. Current methods of cartilage repair include microfracture, osteochondral grafting and autologous chondrocyte implantation. Whilst some individual studies comparing these techniques have shown improvements in long-term clinical outcomes in some patient groups compared to microfracture, major randomised control trials have failed to show consistent long-term differences in clinical outcomes between microfracture, osteochondral grafting, and autologous chondrocyte implantation. Hence, there is a clinical need to explore novel methods of cartilage repair and regeneration using biological techniques such as tissue engineering, stem cells, biomaterials, and growth/differentiation factors to improve cartilage regeneration. The aim of this project was to develop a technique using human infrapatellar fat pad adipose stem cells (IPFP-ASCs) in 3D cultures for chondrogenesis; this required extensive characterisation of the cartilage formed in the 3D cultures/scaffolds (3D pellet culture, chitosan and acellular dermal matrix) for in vitro chondrogenesis. In vivo testing and characterisation of osteochondral defect repair was achieved using a small animal rabbit model for preliminary testing of the ADM-engineered structures. This preliminary testing in the small animal model may then lead to pre-clinical trials in larger animals and human pilot studies in the future. Materials and methods: In vitro IPFPs were harvested from total knee replacements and digested to release adipose stem cells (IPFP-ASCs) which were expanded in vitro. Pellet cultures were developed using TGF-β3 and BMP-6 for chondrogenesis. IPFP-ASCs were seeded onto 3D printed chitosan scaffolds and acellular dermal matrix (ADM) material under the same chondrogenic conditions as the pellet cultures. Four-week cultures were analysed using histology, immunohistochemistry, and gene expression analysis using qPCR. In vivo Osteochondral defects were drilled into distal femoral condyles of adult New Zealand White rabbits. The defects were repaired using either (1) ADM alone (2) autologous IPFP-ASC (3) ADM with autologous IPFP-ASC) or (4) left empty as control. The animals were euthanised at 12 weeks. Repairs were analysed using histology and immunohistochemistry for collagen Type II and Type I. The modified O’Driscoll score was for histological scoring. Further image analysis was conducted to assess quality and quantity of repair. Results: IPFP-ASCs were capable of undergoing chondrogenesis in vitro using pellet cultures and when cultured directly on 3D chitosan and ADM scaffolds using the growth factor combination of TGF-β3 and BMP-6. The method of chondrogenesis was robust and was replicated across both human and rabbit IPFP-ASCs. A one-step single site surgical process was developed for the in vivo modelling of osteochondral defect repair and autologous IPFP-ASC implantation. In rabbits, the rabbit ADM only group achieved the highest ratio of collagen Type II to Type I (77.3%) on image analysis using area measures based from protein expression by immunohistochemistry. This indicated a higher quality of cartilage repair resembling hyaline or hyaline-like cartilage (p<0.05). Conclusion: IPFP-ASCs exhibited robust chondrogenesis under in vitro conditions used in this study; the combination of TGF-β3 and BMP-6 for generation of hyaline cartilage has demonstrated the potential for improving cartilage repair in vitro. ADM, as a support matrix, promoted ASC ingrowth in vitro and proved to be an excellent substrate to promote formation of hyaline-like cartilage in vitro. In the small animal in vivo experiments, it was clear that ADM exhibited positive outcomes when used as a substrate for osteochondral defect repair. These experiments need to be performed in a larger animal model to consolidate these findings prior to consideration of translational to pre-clinical studies.
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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.