Chondrocyte Inflammation Assay with Cytokines

To establish the inflammation in vitro model, we used human chondrocytes primary cultures treated with IL‐1β (10 ng/mL) a key pro‐inflammatory cytokine involved in the osteoarthritis (OA) pathogenesis. A slight modification of the model described by Calamia et al. [2010] was applied. In particular, 2.5 × 10⁴ cells/cm² were seeded in 24 well tissue plates, when confluence of the cells was reached the medium was changed to FBS free medium containing biotechnological chondroitin (1% w/v) and/or chondroitin sulfate (1% w/v), nothing was added in the control wells. Two hours later, IL‐1β was added at 10 ng/mL to each well, except for the negative control wells (at least three per trial) and multiwells were incubated 24 h. In order to evaluate cell response in the diverse conditions of this in vitro inflammation assay, supernatants were collected for cytokines multiplex analyses. Beside oxidative stress of cells was also evaluated through gene expression quantification (RT‐PCR) of the enzyme superoxide dismutase 2 (SOD‐2) from cell extracts. The method is described in the successive paragraph.