Abstract
Cryotherapy is often applied after injuries of synovial joints. Although positive clinical effects on periarticular swelling and pain are well known, the effects on molecular processes of cartilage and synovial cells remained largely unknown so far. Therefore, the hypothesis was tested that hypothermia alleviates the synovial reaction and prevents chondrocyte death as well as cartilage destructive processes after blunt trauma.
Human articular cartilage and synovial tissue was obtained with informed consent from patients undergoing knee joint replacement. Cartilage explants from macroscopically intact cartilage were impacted by a drop-tower apparatus with defined energy (0.59J) and cultivated for 24h or 7d at following temperature conditions: 2h, 16h or throughout at 27°C and afterwards or throughout at 37°C. Furthermore, human fibroblast-like synoviocytes (FLS) were stimulated with conditioned medium from traumatized cartilage (t-CM) and cultivated as indicated above up to 4d. Effects of hypothermia were evaluated by live/dead assay, gene expression (RQ-PCR), and type II collagen synthesis/cleavage as well as release of MMP-2, MMP-13 and IL-6 on protein level (ELISA, gelatin zymography). Statistical analysis was performed by 2-way ANOVA. The experimental study was performed in the research laboratory of the Orthopedic Department, University Hospital Ulm, Germany.
Hypothermic treatment significantly improved chondrocyte viability 7d after blunt cartilage trauma (2h: p=0.016; 16h: p=0.036; throughout: p=0.039). 2h posttraumatic hypothermia attenuated expression of MMP-13 (m-RNA: p=0.012; protein: p=0.024). While type II collagen synthesis was significantly increased after 16h hypothermia, MMP-13 expression (mRNA: p=0.003; protein: p<0.001) and subsequent cleavage of type II collagen (p=0.049) were inhibited. Continuous hypothermia for 7d further significantly suppressed MMP release (proMMP-2, active MMP-2 and MMP-13) and type II collagen breakdown. On day 4 t-CM stimulated FLS revealed significantly suppressed gene expression of matrix-destructive enzymes (16h: ADAMTS-4; throughout: ADAMTS-4, MMP-3, MMP-13) and by trend reduced IL-6 expression in case of 16h or continuous hypothermia.
Overall, hypothermia for only 2h and/or 16h after blunt cartilage trauma exhibited significant cell- and matrix-protective effects and promoted anabolic activity of surviving chondrocytes. Expression of matrix-destructive enzymes by FLS stimulated with Danger Associated Molecular Patterns (DAMPs) released from traumatized cartilage was attenuated by more prolonged hypothermia. These findings suggest that an optimized cryotherapy management after cartilage trauma might have the potential to ameliorate early molecular processes usually associated with the pathogenesis of posttraumatic osteoarthritis.
