Type 1 collagenase

For the harvesting of SCAPs, healthy immature human third molars were collected from patients aged 16–20 years (n = 5) according to the previous studies [6 (link)]. All protocols in the present work were approved by the Ethics Committee of the School of Stomatology, Wuhan University, China. In brief, apical papilla tissues were gently separated from teeth, thoroughly rinsed with phosphate buffered saline (PBS, Hyclone, Logan, UT), minced, and digested with 3 mg/mL type I collagenase (Biosharp, Hefei, China) and 4 mg/mL dispase (Roche, Mannheim, Germany) at 37 °C for 40 min. The digested mixtures were passed through a 70 μm cell strainer to get single-cell suspensions. SCAPs were cultured in the α-MEM (Hyclone) containing 10% fetal bovine serum (FBS, Gibco, Thornton, NSW, Australia) and 100 U/mL penicillin–streptomycin (Hyclone). Cells were incubated at 37 °C in an atmosphere with 5% CO₂. SCAPs at passages 2 to 5 were used in this study.

Endometrial tissues of the control group were collected to isolate and culture endometrial stromal cells. First, the endometrial tissues were rinsed with PBS and cut into little pieces. Next, the tissues were digested with 2.5 mg/mL type I collagenase (Biosharp, Hefei, China) and 0.1 mg/mL DNase I (Biosharp, Hefei, China) for 30 min. The digested tissues were filtered through a 40 μm sieve. Then, the isolated endometrial stromal cells were resuspended in an RPMI-1640 medium with a 10% fetal bovine serum.

The study was designed according to the Declaration of Helsinki, and was approved by Ethic Committee of the Second Affiliated Hospital of Harbin Medical University (KY 2021-256). Informed consent was obtained from each donor. Synovium of 3 OA patients (age 54–70 years, Kellgren-Lawrence grade 4) that underwent total joint arthroplasty (TKA) and 3 patients (age 56–68 years) that underwent meniscectomy without OA were obtained at the time after surgery. All patients were confirmed without Rheumatoid Arthritis, acute trauma, tumor or infection of knee joint. Briefly, synovium was cut into pieces at the final size about 0.5 mm*0.5 mm, and put into 0.1% type I collagenase (Biosharp, China, BS163). α-MEM medium (Cytiva, United States, SH30265.01) was added with 10% foetal bovine serum (ExCell Bio, China, FSD500) after 2 h. Primary cells could be seen climbing out after about 3–5 days. FLS of passage 6-8 (P6-8) were used in this study. 10 ng/ml of IL-1β (PEPROTECH, United States, 200-01B) were used to stimulate FLS of OA groups for 48 h in order to imitate the environment of OA, while complete medium was added into the FLS of control group. 10 μg/ml of Etanercept and Iguratimod (MedChem Express, China, HY-108847, HY-17009) were added along with IL-1β to FLS for the following test.