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Title: In-vitro modelling of stromal and immune cell interactions following surgical bone marrow stimulation
Author: Frankham-Wells, Sophie Louise
ISNI:       0000 0004 8501 1058
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Osteoarthritis (OA) results in degeneration of cartilage and bone within a joint, leading to pain and loss-of-function. While effective treatments exist for end-stage OA, earlier disease stages lack treatment options. Micro-fracture and micro-drilling, often termed bone marrow stimulation techniques, offer an early-stage repair and regenerative strategy. These techniques result in the formation of a haematoma containing Bone Marrow Stromal Cells (BMSC) and immune cells at the joint surface, leading to repair. However, the quality of repair after marrow stimulation is, at present, sub-optimal. BMSC are known to interact with immune subpopulations during repair, although the consequences of these interactions for repair outcomes are not fully understood. This thesis aims to further the current understanding of how interactions between immune subpopulations and BMSCs in the environment of a haematoma may influence repair outcomes following surgical bone marrow stimulation techniques. In order to understand the influence of hypoxia on BMSC, strains of human BMSCs were isolated and cultured under normoxic (18.9% oxygen) and hypoxic (3.0% oxygen) conditions. Characterisation demonstrated that hypoxic culture had functional consequences on BMSC phenotype and behaviour. To explore the effect of BMSC-immune cell interactions on BMSC migration, immuno-regulation, and cell phenotypes, parallel experiments were performed under normoxic and hypoxic conditions. A unique approach was taken to identify an immune subpopulation resulting in BMSC migration, which was used as an indicator of potential co-localisation and interaction. BMSC were exposed to paired enriched and depleted populations of peripheral blood mononuclear cells, which had been sequentially fractionated using specific markers. The migration stimulated in the BMSC by the enriched and depleted fractions was compared. A Natural Killer (NK) cell population was found to have induced the greatest BMSC migration relative to its paired fraction. Co-cultures indicated that the influence of NK cells on BMSC, particularly NK cell-mediated cytotoxicity, were heavily dependent on culture conditions, including NK cell number and oxygen levels. In a further study, co-cultures of BMSC and monocytes, which interact with BMSC and have a potential role in fibrosis, were co-cultured under normoxic and hypoxic conditions. A pro-angiogenic, immunomodulatory phenotype developed in normoxic co-cultures, which was significantly reduced in hypoxic co-cultures. In conclusion, these data suggest that BMSC and immune subpopulations have diverse interactions which are strongly influenced by the local oxygen tension and cellular environment. These findings demonstrate the importance of the microenvironment formed by bone marrow stimulation techniques on cellular interactions, with potential consequences for the outcome of the repair. Furthermore, this work indicates that manipulation of the haematoma and surrounding environment following bone marrow stimulation could improve repair and regenerative outcomes.
Supervisor: McCaskie, Andrew ; Birch, Mark Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Tissue engineering ; osteoarthritis ; hypoxic ; bone marrow stromal cell ; mesenchymal stem cell ; co-culture ; immune cell ; surgical bone marrow stimulation ; Regenerative medicine