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Objectives and questions: In knee joints, aging is associated with progressive degradation of the extracellular matrix (ECM). Collagen II is a major component of cartilage ECM, determining its mechanical properties and undergoing age-related molecular modifications. However, other collagen types (e.g., collagen III, IX, and X) also contribute to cartilage development and function. In addition, collagen I is the main component of subchondral bone (SB), located beneath articular cartilage and subjected to sclerosis or osteoporosis during aging. The aim of the present study was to investigate age-related changes in the expression of specific collagen types and the fibril organization during aging and joint compartments.

Material and methods: 50 WT C57BL/6J mice (3-, 6-, 12-, 18- and 24-months-old, 5 males and 5 females per timepoint) were sacrificed and dissected. Paraffin-embedded knee sections were stained with Picrosirius red (PSR) and collagens I, II, III, IX and X were detected by immunohistochemistry (IHC). Staining was analyzed in menisci, different articular cartilage compartments and SB. The surface percentage of thick and thin collagen fibers was determined in PSR-stained sections, while immunostained sections were evaluated quantitatively to characterize age-related changes.

Results: The amount of thick collagen fibers decreased during aging in articular cartilage and SB (p<0,001 between 3 and 18 months of age), displaying a significant re-increase at 24 months (p<0,05, compared with 3 months old). Thick fibers were more abundant in medial compared to lateral and femoral compared to tibial cartilage compartments (max. p<0,05). Thin fibers displayed an increase between 3 and 6 months old mice in SB (p<0,05) and were more expressed in femoral compared to tibial cartilage (p<0,05). Collagen II and III IHC revealed an age-dependent decrease in articular cartilage and different expression between femoral and tibial compartments similar to the ones observed in PSR-stained thick fibers (same significances). Collagen III IHC displayed differences between lateral and medial menisci (p<0,05). A stronger expression of collagen IX and X was visually found in 24 months old mice compared to other time points. No sex-specific differences were detected.

Discussion and conclusions: A clear age-dependent change in both collagen fiber surface and expression was observed in mouse knee joints. The re-increase in thick fibers between 18 and 24 months may result from collagen molecular modifications or from reactivation of collagen synthesis and secretion by chondrocytes at later life stages. Intra-articular compartmental variations may reflect developmental differences and/or an altered mechanical loading. Differences between PSR- and IHC-derived results suggest a differential susceptibility of specific collagen types to aging-related processes. Overall, our findings reveal age-related molecular dynamics and intra-articular differences which, if confirmed in humans, could substantially advance our understanding of aging-associated osteoarthritis.