Collagenase type 2

Under aseptic conditions, the cartilage tissues were repeatedly washed with phosphate-buffered saline (PBS) to remove the coagulation and appendages and cut into 1 mm³ blocks. Subsequently, the tissues were detached with 0.05% type II collagenase (Sigma-Aldrich Chemical Company, St Louis, MO, USA) for 30 minutes, centrifuged at 1000 r/min with supernatant discarded, followed by detachment with the mixture of 0.1% type II collagenase and 0.25% trypsin (Sigma-Aldrich) at 37°C for 60-100 minutes. Afterward, the cells were dispersed into cell suspension in Dulbecco’s modified Eagle’s medium (Gibco, Carlsbad, CA, USA), which was supplemented with 10% fetal bovine serum (Gibco), 100 units/mL penicillin, and 100 units/mL streptomycin. Chondrocytes (4 × 10⁵ cells/mL) were grown on culture plates and incubated at 37°C under 5% CO₂. Chondrocytes at passage 2 - 3 were used for the subsequent experiments. The primary chondrocytes were stimulated with 10 ng/mL interleukin-1 beta (IL-1β) for 12 hours to induce inflammation in vitro.

According to our previous study, primary NP cells from surgery patients were isolated and cultured as follows [34 (link)]. In brief, NP tissues of the excisional disc were gathered with tweezers and minced into flocculent pieces under sterile conditions. Then, the isolated tissue pieces were digested in 0.2% type II collagenase (Sigma, USA) for approximately 4 h, following 0.25% trypsin solution (Sigma, USA) digestion at 37°C for 20-40 min. After that, complete culture medium (DMEM/F-12 (Gibco, USA) containing 15% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin/streptomycin (Sigma, USA)) was utilized to neutralize the type II collagenase. The suspension was then centrifuged at 1500 rpm for 10 min to deposit the NP cells. Next, cells were resuspended and seeded into flasks with 5 ml complete culture medium at 37°C in an atmosphere containing 5% CO₂. And when cellular confluence grew to 80-90%, NP cells were trypsinized with 0.25% trypsin (Sigma, USA) for subculture. All experiments were carried out with NP cells at passage II.

BMMs were obtained from tibial and femoral bone of male mice. Bone marrow cells were cultured in α-MEM (Hyclone, USA) containing 10% FBS (Hyclone). After 1 day, non-adherent cells were cultured with 30 ng/mL M-CSF (R&D Systems, USA) for 3 days, as described previously.^{68 (link)} Osteoclasts were generated by culturing BMMs with 30 ng/mL M-CSF and 100 ng/mL RANKL (PeproTech, UK).
Bone marrow derived dendritic cells (BMDCs) were obtained from 6–8-week-old mice. Bone marrow cells were isolated and seeded in DMEM (Hyclone) containing 10% FBS with 20 ng/mL GM-CSF. On days 3 and 5, cells were treated with 20 ng/mL rmIL-4. Then on day 7, cells were harvested after incubation with or without 100 ng/mL LPS for 24 h.
Bone marrow stromal cells (BMSCs) were obtained from 4-week-old mice as described previously.^{69 (link)} The tibial and femoral bone were cut into small pieces and incubated for 90 min at 37 °C with shaking in a solution containing collagenase type II (C6885, Sigma-Aldrich, USA). The cells were cultured in a 100 mm culture dish.
Primary osteoblasts were harvested from calvaria as described previously.^{70 (link)} The calvariae were digested for 15 min in a solution containing collagenase type II (Sigma-Aldrich) and dispase II (Roche) at 37 °C with shaking. The cells were cultured in a 100 mm culture dish. HEK293T cells and HeLa cells were cultured in DMEM (Hyclone).