Extracellular matrix fragmentation in young, healthy cartilaginous tissues

The composition and structure of cartilaginous tissues is complex but collagen II fibrils and the aggrecan are the most abundant assemblies in articular cartilage (AC) and the nucleus pulposus (NP) of the intervertebral disc (IVD). Whilst structural heterogeneity of intact (three globular domain) aggrecan is well characterised the extent of aggrecan fragmentation in healthy tissues is poorly defined. Using young, yet skeletally mature (18-30 months) bovine AC and NP tissue, we show that, whilst the ultrastructure of intact aggrecan is tissue dependent, most molecules (AC: 95 %; NP: 99.5 %) are fragmented (lacking one or more globular domains). Fragments are significantly smaller and more structurally heterogeneous in NP compared with AC (AC: 8543 nm2; NP: 4625 nm2; p<0.0001). In contrast, fibrillar collagen appeared structurally intact and tissue invariant. Molecular fragmentation has been considered to be indicative of pathology; however, these young skeletally mature tissues are histologically and mechanically (reduced modulus: AC: ~500 kPa; NP: ~80 kPa) comparable with healthy tissues and devoid of notable gelatinase activity (compared with murine dermis). As we also show that aggrecan fragmentation is prevalent in neonatal bovine AC (99.5 %; MAGAG: 5137 nm2), we hypothesize that targeted proteolysis may be an adaptive process which modifies aggrecan packing (as simulated computationally) and hence tissue charge density, mechanical properties and porosity. These observations provide a baseline against which pathological and/or age-related fragmentation of aggrecan can be assessed and suggest that new strategies may be required to engineer constructs which mimic the mechanical properties of native cartilaginous tissues.