Chondrogenic potential of perivascular stem cells from the infra-patellar fat pad
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Articular cartilage damage and degeneration is a siginficant clinical problem which no technique has been able to adequately and reliably repair or regenerate. Recent research has investigated the use of cell-based therapies to treat focal cartilage lesions. In clinical practice proliferated autologous chondrocytes are used and clinical trials are investigating the use of mesenchymal stem cells. The aim of this thesis was to assess aspects of current cell-based therapy and to investigate the potential of perivascular stem cells for articular cartilage repair. The phenotype of expanded matrix-applied autologous chondrocytes utilised in current cell therapies was confirmed using immunocytochemistry and polymerase chain reaction (PCR) expression of hyaluronan and proteoglycan link protein 1 (HAPLN1), transcription factor sox-9 (SOX9) and aggrecan (ACAN). Quantitative real-time PCR demonstrated that they were down-regulated for expression of COL2A1, SOX9 and ACAN but up-regulated for COL1A1 compared to unproliferated chondrocytes. Confocal laser-scanning microscopy (CLSM) demonstrated a significant decrease in cell viability and density when the membranes were subjected to levels of trauma similar to those that could be experienced during surgery. Hyperosmolar solutions did not confer a chondroprotective effect. Pericytes and adventitial cells, collectively termed perivascular stem cells (PSCs), from the infra-patellar fat pad were identified using immunohistochemistry and isolated using enzymatic digestion and fluorescence-activated cell sorting (FACS). Cell identity was ascertained using PCR, FACS and mesenchymal differentiation (osteogenesis, adipogenesis and chondrogenesis). Quantitative real-time PCR analysis of micromass cultures indicated that PSCs displayed increased chondrogenic potential compared to mesenchymal stem cells. An ovine model of perivascular stem cells was developed and a pilot study using three sheep was undertaken to confirm the viability of the model. Autologous ovine PSCs were isolated and re-implanted into articular cartilage defects. Green fluorescent protein transfected cells were identified in the cartilage defect four weeks following re-implantation using CLSM. This thesis has examined aspects of matrix-applied autologous chondrocyte implantation for cell based cartilage repair and has identified a new source of prospectively identified and purified stem cells that have demonstrated increased chondrogenic potential compared to mesenchymal stem cells, which are commonly used in clinical research. The methods to identify and purify ovine perivascular stem cells were developed to investigate the use of autologous PSCs and to track the cells following implantation.