Growth factor release upon cartilage injury is due to aggrecan-dependent sodium flux that is lost in osteoarthritis

Purpose: Growth factors sequestered in the pericellular matrix (PCM) of cartilage are released upon tissue injury and are thought to confer largely chondroprotective effects in vivo. Of the four that we have identified, all of them are heparan sulfate binding and are liberated from the matrix within seconds of injury suggesting that they share a common release mechanism. Here we hypothesise that injury causes a flux of sodium (from the glycosaminoglycan (GAG) rich territorial matrix), and this is responsible for displacing growth factors in the PCM. Furthermore, we test whether this response is lost in OA when cartilage proteoglycan is depleted. Methods: Full-depth porcine articular cartilage explants (4mm diameter) were obtained from the metacarpophalangeal joint. Explants were rested in serum-free DMEM medium for 48 h before re-cutting (injury). This was performed on explants that were dead (freeze-thawed), in the presence of exogenously added NaCl (137-822 mM), or treated with interleukin-1 (50 ng/ml) for 7 days (aggrecan depletion). Interleukin-1 treated explants were vacuum sealed and placed in a custom-made compression cell. Imaging and spectroscopy were conducted for both proton and sodium nuclei on a Bruker 9.4T Ultra-high field microimaging system. Full-depth human explants from joint arthroplasty of osteoarthritic patients, stratified by disease severity using a modified Outerbridge classification, were cut from the joint (injury) into serum-free DMEM. Some explants were digested in proteinase K to measure GAG content. Others were treated with 2.5 M NaCl followed by lysis buffer. Injury conditioned medium was assessed for growth factors using Western blot and ELISA. Results: Heparan sulfate binding and release upon cartilage injury was validated for three growth factors (FGF2, CTGF and HDGF). Addition of high concentrations of NaCl to rested cartilage caused release of these growth factors. Upon injury, exogenous NaCl increased the amount of growth factor release in a dose-dependent manner. This was irrespective of whether the explant was alive or dead (freeze-thawed). Interleukin-1 treated cartilage over 7 days caused near complete proteoglycan depletion, and led to reduced growth factor release upon injury. Pilot data showed a change in 23Na dynamics during compression of interleukin-1 treated cartilage. Modified Outerbridge score correlated with cartilage GAG content. Severely osteoarthritic regions of cartilage with less GAG released less growth factor upon injury despite having growth factor that was readily extractable by exogenous NaCl. Conclusions: We have uncovered the principal mechanism by which heparan sulfate-bound growth factors are released from the PCM of cartilage upon injury. Our results suggest that a major unappreciated role for aggrecan is its ability to trap a pool of matrix sodium that can be mobilised to alter growth factor bioavailability. Loss of aggrecan in OA prevents this normal injury response and impedes activation of pathways normally associated with tissue repair.