Osteoarthritis (OA) is a musculoskeletal disease for which no cure currently exists. Its main hallmark is the progressive degeneration of the extracellular matrix (ECM) in the articular cartilage (AC). Ageing and female sex are considered important predisposing factors for OA development, nonetheless, the molecular mechanisms directly linking them to OA remain elusive. The purpose of this study is to determine how age and sex can affect the composition of the AC proteome and link them to the onset of OA.

In this thesis, we initially focused on evaluating age and sex differences in the ECM proteome of the AC, with the optimisation processes for decellularisation (DC) and proteomics spatial imaging, detailed in Chapters 3 and 4, respectively. Upon, unsuccessful attempts to isolate the ECM, we performed a Tandem Mass Tag Mass Spectrometry (TMT-MS) analysis to compare the proteomic profile of the AC removed from the hip joints of male and female healthy young (11- or 12-weeks old) and old (17- or 18-month-old) mice. This proteomic dataset was interrogated, and proteins were categorised accordingly into three subcellular locations: (Chapter 5) ECM-, intracellular and other-, and mitochondrial-related (Chapter 6) proteins. Across these chapters, we quantitatively explored age and sex differences in the proteomes of the AC. Additionally, the ECM-related proteins (Chapter 5) underwent peptide-location fingerprinting (PLF) analysis to delve further into identifying any protein structural modifications specific to the sex- and/or age of the AC samples. Finally, Chapter 7 evaluated the role of one of the many interesting proteins identified in our TMT-MS analysis, Collagen type V (Col5a2; ColV) in cartilage homeostasis during ageing. This marker has been previously associated with cartilage ‘maturity’. For this study, in vitro assessments were conducted using two-dimensional (gelatin/ColV) and three-dimensional (GelMA-ColV hydrogel) environments. ColV levels were varied to mimic age-related changes, allowing for the evaluation of their phenotypic impact on cultured adult human articular chondrocytes (AHACs).

This research demonstrated distinct proteomic profiles of young, old, male and female AC tissue, identifying multiple novel proteins associated with age and sex differences. With further investigations on a biochemical level and assessments in human AC, our findings could help with identifying suitable biomarkers for the early diagnosis of OA.