"Background Osteoarthritis (OA) is a common degenerative joint disease, that affects the whole joint. Knee joint distraction (KJD) has been proposed as an alternative joint-preserving treatment strategy for relative young patients with end stage OA. Although there is evidence for the clinical and structural benefits of KJD, the regenerative mechanisms behind KJD remain unclear. This study explores the role of the synovial membrane (SM), an important, but to date limited researched part of the distracted joint, by studying the SM secretome and its effect on chondrocytes.
Methods Osteoarthritis was bilaterally induced using the groove OA model and allowed to develop for 10 weeks in 12 dogs. Subsequently, KJD was applied unilateral to the right hindlimb for 8 weeks. To determine the role of the SM during KJD, the SM and conditioned medium of the synovial membrane of OA (OA-CM) and OA+KJD joints (KJD-CM) were investigated directly after KJD treatment (n=4), and after 10 weeks of follow-up after KJD treatment (n=8) using RT-qPCR analysis and a canine-specific multiplex ELISA. Furthermore, clinically normal articular chondrocytes (n=3), synovial membrane (n=6) and CM (normal-CM) from healthy dogs were collected for comparison. The effect of OA-CM and KJD-CM on isolated normal articular chondrocytes was investigated using RT-qPCR and Nano luciferase response element reporter assays for targeted signalling analysis of pathways involved in anabolic, catabolic and inflammatory processes during OA.
Results In the synovial membrane and conditioned media the expression of pro-inflammatory cytokines (CCL2, IL-6, IL-7, IL-15, and IL-18) was increased directly after KJD compared to the normal SM. After 10 weeks of follow-up, these cytokines were still elevated, except for IL-6, compared to directly after KJD. Directly after KJD, the OA and the KJD-CM had a clear catabolic effect on articular chondrocytes, as established by decreased expression of the cartilage matrix genes ACAN and COL2A1 and increased expression of the catabolic genes MMP13 and ADAMTS5. After 10 weeks of follow-up, this effect shifted towards a less catabolic response. The reporter assays showed an upregulation of CRE and SIE signalling after stimulation with OA-CM and KJD-CM compared to normal-CM. SRE and SRF signalling was downregulated in the presence of the KJD-CM at follow-up compared to the KJD-CM from directly after KJD.
Conclusion OA and KJD result in inflammation of the SM, characterized by increased levels of pro-inflammatory cytokines and chemokines. The SM secretome directly influences cartilage matrix metabolism and cellular response, corresponding with the catabolic environment in the cartilage that is found directly after KJD. After follow-up the catabolic influence of the SM decreases. Therefore, the SM may contribute to the effect that joint distraction exerts on the OA joint. However, as the effect is small, in order to gain further insights, the sample size should be increased. Furthermore, more cartilage protective pathways should be investigated to investigate the role of the SM in the regenerative mechanisms behind joint distraction."