Semaphorin-3A expression is up-regulated in innervated joint tissues during the development of osteoarthritis

Purpose: Osteoarthritis (OA) is a common skeletal disease affecting nearly 3.3% of the world population. The main symptoms are chronic joint pain and stiffness caused by progressive changes in the joint tissues. Abnormal nerve growth into joints was observed in OA contributing to pain. Semaphorin-3A (sema-3A) is a secreted axonal chemo-repellent which inhibits axonal growth in a concentration-dependent manner. The purpose of this study was to determine whether sema-3A plays a role in the development of joint pain and degeneration. Methods: Two mouse models of OA, the mechanical joint loading (MJL) model and STR/ort mice that spontaneously develop OA were used in this study. For the MJL model, the right knees of C57BL/6 mice (male, 12-week-old) were loaded (40 cycles of loading regimen at 9N or 11N for two weeks, 3 times/week). The knee joints and serum were collected at week 6 post loading. Male STR/ort mice were maintained for 30-40 weeks for severe OA phenotype to develop and joints collected. Joint sections were used for immunocytochemistry using sema-3A antibody (Abcam). Sema-3A levels in serum were detected by ELISA (CUSABIO). QPCR was used to analyse sema-3A mRNA expression during mouse chondrogenic cell line ATDC5 differentiation and in rat articular cartilage and dorsal root ganglia (DRGs). We used lentivirus (10ul, 5x108 TU/ml) as a tool to overexpress sema-3A-GFP in mouse knee joints by intra-articular injection using the MJL model of OA. Knee joints were collected at week 1 after loading, and then processed for cryostat sections. Results: Sema-3A is expressed in cartilage, cruciate ligaments, synovial lining and subchondral bone in healthy mouse knee joints. The overall expression of sema-3A increased in these tissues and osteophytes with the development of OA, both in the MJL and STR/ort mouse models. The expression of sema-3A in cartilage doesn’t change with OA. There was a significant increase in sema-3A expression (+25%) in mouse serum 6 weeks after MJL compared to the non-loaded mice. Sema-3A is expressed at all stages of ATDC5 chondrocyte differentiation, with a peak of expression during chondrocyte maturation. Sema-3A was also found in rat DRGs but its expression was more than 10 times higher in rat articular cartilage compared to DRGs. Intra-articular injection of lentivirus in mouse knee joints successfully expressed sema-3A-GFP in articular cartilage, meniscus, patella and tibia and fibular junction. Conclusions: Our results demonstrate that sema-3A is upregulated in the highly innervated joint tissues, suggesting that it may control joint innervation during the development of OA. Intra-articular injection of lentivirus overexpressing sema-3A is efficient and further studies will use this tool to study sema-3A function during OA development.