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Title: The characterisation of monocyte responses to mediators released from osteoarthritic and periprosthetic tissues
Author: Genasan, Krishnamurithy
ISNI:       0000 0004 7970 3808
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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BACKGROUND: Aseptic loosening is the major cause of long-term failure of an arthroplasty. Pathophysiological periprosthetic osteolysis is driven by wear debris particles (WDP) accumulating in the surrounding joint tissues. Macrophages, professional phagocytic cells, internalise submicron WDP and upregulate production of inflammatory mediators, stimulating circulating monocytes to migrate into the inflamed joint tissues. These migrated monocytes/tissue macrophages fuse, forming foreign body giant cells (FBGC) and/or osteoclasts, instigating osteolysis at the bone-implant interface. However, the activation of monocytes while in circulation, subsequent migration and differentiation in response to WDP-induced inflammatory mediators are poorly understood. Thus, the alteration in functional surface receptors, gene expression and phagocytic commitments of monocytes needs evaluation. This study details how monocytes from three cohorts of orthopaedic patients respond to mediators derived from aseptic loosening periprosthetic synovium. METHODS: Two sets of patients were recruited for this study. First set; with cohorts of end stage OA (ES-OA, N=10), stable arthroplasty (N=10) and revision arthroplasty due to aseptic loosening (N=10), to isolate, characterise and test peripheral blood mononuclear cells (PBMNCs), from blood samples. A second set with two cohorts donated tissues debrided at surgery; Hip/Knee aseptic loosening revision synovium (N=13) and knee ES-OA synovium (N=25). These tissues were used for H&E staining and preparation of tissue conditioned media (TCM). Pooled revision synovial (RTCM) or ES-OA synovial (OACM) TCM was used for proteomic analysis and PBMNCs migration assays using a 3-μm pores transwell system. Serum free media (SFM) and monocyte chemoattractant protein-1 (MCP-1) were used as spontaneous and specific chemokine directed migration controls, respectively. Migrated cells were assessed using light microscopy. Characterisation by FACs included monocyte markers (CD14 and CD16), surface receptors for chemotaxis (CCR2 and CX3CR1), inflammation (CXCR2), cell adhesion (ITGAM), FBGC/osteoclast precursor formation (DCSTAMP) and monocyte-platelet aggregation (GP1BA). Functional commitment analysis utilised gene expression profiles and 1-μm fluorescence latex beads (LB) phagocytosis assay before and after migration. Parametric and/or non-parametric statistical analyses were undertaken and results presented as mean ± standard deviation. RESULTS: FBGC were identified in sections of revision synovial tissues. Proteomic analysis revealed proteins related to FBGC formation, osteoclast activity, inflammation, cytokine secretion and phagocytosis in RTCM but not in OACM. The PBMNCs monocyte percentage count (72.74±8.69%, p=0.001), cell adhesion receptor (ITGAM, p=0.024), FBGC formation gene expression (IL13RA1, p=0.041) and LB uptake/cell (8±4, p=0.001) were significantly increased in arthroplasty patients when compared with ES-OA patients. The RTCM migrated cell count, CD14+/ classical CD14++CD16- monocyte percentage count, cell adhesion receptor (ITGAM), inflammatory (TNF-α and IL-1β), survival (MAPK1), cell adhesion (C1qR1/CD93), FBGC formation (IL13RA1) genes expression and LB uptake/cell (7±5) were significantly increased (p < 0.05) when compared with SFM. CONCLUSION: This study starts to unravel changes in monocytes in patients with an arthroplasty. Characteristic functional alterations in circulating monocytes, indicative of early commitment to the osteoclast lineage, could offer non-invasive, early warning of pathophysiological osteolysis or arthroplasty loosening. New targeted therapies could be developed to slow or prevent progression and reduce extensive revision surgery.
Supervisor: Frostick, Simon ; Roebuck, Margaret ; Barrera, valentina ; Kamarul, Tunku Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral