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Title: Repairing injured articular cartilage : investigation of potential mechanisms using mesenchymal stem cells
Author: Ahmed, Saima
ISNI:       0000 0004 6347 182X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Traumatic cartilage injuries are a known risk factor for development of osteoarthritis (OA). Classically cartilage is seen as poorly repairing, however, cartilage injuries in very young animals have been observed to heal although the mechanism of this repair is unknown. The aim of this project was to investigate if mesenchymal stem cells (MSCs) in the joint tissue can respond to cartilage injury and are involved in the potential cartilage repair mechanisms. Fluorescently-labelled synovial fluid-derived MSCs (SF MSCs) specifically adhered along the edges of the injured cartilage. SF MSCs and bone marrow-derived MSCs (BM MSCs) migrated in significantly greater numbers to injured cartilage compared to non-injured cartilage. Incubation of freshly chopped articular cartilage with serum-free media gave an injury conditioned medium (IJCM) which significantly stimulated cell migration of both SF and BM MSCs in classical 'Scratch' assays. Migration of MSCs in response to IJCM was significantly reduced by pre-treating the IJCM with immobilised heparin or using neutralising antibodies to TGFβ3, FGF2 and CXCL12, and the CXCR4 receptor inhibitor AMD3100. No SF MSC migration was observed with IJCM in cell exclusion zone migration assays (no 'wound' is created in the cell layer in this assay protocol). However, migration in response to IJCM was observed in this system when a cell injury was created (by scraping off a thin line of cells) close to the cell exclusion zone. Creating the 'scratch' wound caused release of μM amounts of ATP from the injured cells as determined by an ATP-bioluminescence assay. Supplementation of IJCM with ATP or UTP or the stable purine analogues ATP-γS or UTP-γS stimulated MSC migration in the cell exclusion zone assays, suggesting a role for a putative P2 receptor in priming of SF MSCs for migration. Furthermore, IJCM-stimulated SF MSC migration could be completely inhibited in scratch assays using the P2 purine receptor antagonist suramin. The results suggested that articular cartilage released ATP, CXCL12, TGFβ3 and FGF2 following injury and these factors together stimulated MSC migration to injured cartilage. Extracellular nucleotides such as ATP and UTP are required to prime the SF MSCs before CXCL12 and TGFβ3 stimulates cell migration to the injury site. In contrast, BM MSCs do not require any priming signal before their migration along the chemotactic gradient of CXCL12, FGF2 and TGFβ3.
Supervisor: Crawford, Aileen ; Buttle, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available