Primary chondrocyte isolation and culture

Primary chondrocytes were extracted from the knee joints of five 6-week-old normal Sprague Dawley rats. All experiments were executed following animal approved protocols and guidelines. Cartilage was separated from the joints and washed with 1× phosphate-buffered saline (PBS) three times. After cutting the cartilage into small pieces, 0.25% trypsin was added for 30 min, followed by incubation with 2 mg/mL collagenase for another 4 h at 37 °C. The cells were then centrifuged at 1000 rpm for 5 min and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (HyClone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (HyClone, USA) in 5% CO₂ in a humidified atmosphere in an incubator at 37 °C. The culture medium was replaced every 2 days. The chondrocytes in the third and fourth passages were used for further experiments.