Investigation of the chondrocyte response to varying mechanical load - assessment of impact stress

Abstract

Affecting over 250 million people worldwide, Osteoarthritis (OA) is the most prevalent musculoskeletal disease and the leading cause of disability among elderly people. An accelerated form of OA, termed post-traumatic OA (PTOA), is triggered by one or more injurious, high impact loading events which result in chondrocyte cell death and a 'phenotypic shift' in residual cells toward a more catabolic state. Conversely, cyclic loading within the physiological range has been shown to have beneficial effects on joint health, with longitudinal patient studies demonstrating a lack of rhythmic mechanical stimulation results in cartilage atrophy. Here, the differences in cartilage tissue response to physiological cyclic and pathological impact loads were examined, to elucidate the mechanosensitive pathways responsible for destabilising chondrocyte homeostasis, driving the initiation of PTOA. Assembly and optimisation of a novel ex vivo porcine loading model, incorporating an ElectroForce 5110 mechanical testing instrument, delivered compressive strains comparable to that seen in the joint, providing physiological cyclic (10% stain) and pathological impact (50% strain) loading regimes for further investigation. Biological readouts including cell death, sGAG release and structural damage demonstrated the catabolic and anti-catabolic effects of these two loading programmes. Subsequent development of a protein extraction protocol and employment of a data-driven discovery proteomics study permitted unbiased examination of the two aforementioned loading regimes and their short-term (< 72 h) global effects on cartilage health. Similarities were observed through a sub-population of indiscriminately mechanosensitive proteins associated with cytoskeletal remodelling, whilst divergence was detected in IL-1, integrin and mTOR signalling pathways, among others. Most notably, this work elucidated the induction of a number of small ubiquitin related modifier (SUMO)-associated proteins via mechanical stress ex vivo, chiefly following impact loading. Further investigation of this stress response pathway was conducted in a human chondrocyte cell line in vitro via co-immunoprecipitation mass spectrometry. A global shift towards deSUMOylation was observed following PIEZO1-mediated calcium influx, with the greatest alteration in protein SUMOylation state associated with RNA processing and the cytoskeleton. Overall, this study demonstrated the effective use of a novel ex vivo mechanical loading model and protein extraction methodology for the proteomic examination of the chondrocyte response to variable mechanical load. These techniques and the identification of a unique mechanosensitive pathway (SUMOylation) aim to further the field of orthopaedic research by progressing our understanding of PTOA disease progression in the hope of identifying new therapeutic targets for the future.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Stephen Richardson (Supervisor) & Paul Townsend (Supervisor)