Collagenase solution

Chondrocytes were isolated from the knee samples. The tissues were immersed in a sterilized phosphate buffer solution (pH7.4). The tissue fat and connective tissue were digested with 1% collagenase solution (Invitrogen, Thermo Fisher Scientific, Grand Isle, NY, USA), and kept at 37 °C for 50 min. The cells were isolated from tissue culture using 70 μM cell strainers. The cells were then cultured with Dulbecco Modified Eagle Medium (DMEM) (Gibco BRL, NY, USA) and antibiotics penicillin and streptomycin (100 ng/mL) (Sigma-Aldrich, Darmstadt, Germany), and incubated at 37 °C with 5% CO₂. After reaching confluence, the cells were detached with 0.1% trypsin (Sigma-Aldrich, Darmstadt, Germany) and pretreated with (10 ng/mL) IL-1β before being treated with VITD for 24 h to simulate harsh conditions. MTT assay was used to determine the toxicity of VITD (Sigma-Aldrich, Darmstadt, Germany) [36 (link),37 (link)].

All the following operations were performed under aseptic conditions. The cartilage layer and subchondral trabecular bone were carefully removed under magnifying microscope. The remaining subchondral bone plate was dissected into separate bone chips, which were then incubated in 1% trypsin (Gibco, CA, USA) in a humidified environment containing 5% CO₂ at 37 °C for 10 min. Then the trypsin solution was replaced with 0.2% collagenase solution (Invitrogen, Carlsbad, CA, USA) (16 ). After incubation for 30 min, the bone chips were transferred to a 75 cm² culture flask and cultivated in alpha-Minimal Eagle Medium (αMEM; Gibco) replenished with 20% fetal bovine serum (FBS; Gibco), 100 units/mL penicillin (HyClone, Logan, UT, USA) and 100 µg/mL streptomycin (HyClone). The medium was changed every 2 or 3 days. When cells were observed in 75 cm² culture flask, fresh medium containing 10% FBS was utilized. Primary osteoblasts were digested and passaged when cells reached 80% to 90% confluence. Only osteoblasts in passage 1 were used in our following experiments.