Osteogenic differentiation medium

To induce adipogenic or osteogenic differentiation, MSCs were cultured in adipogenic differentiation medium (Takara Bio, Shiga, Japan) or osteogenic differentiation medium (Sigma-Aldrich), respectively, for 14 days following the manufacturers’ protocols. Oil Red O (Sigma-Aldrich) and Alizarin Red S (FUJIFILM Wako Pure Chemical, Osaka, Japan) were used to analyze adipogenic and osteogenic differentiation, respectively.

BMSCs were seeded on the hydrogel extracts with a density of 1 × 10⁶ cells/well in osteogenic differentiation medium (Sigma, Springfield, MO, USA) including α-MEM supplemented with 10% of FBS, 1% of antibiotics, 50 μM of ascorbic acid, 10 mM of β-glycerol phosphate, and 0.1 μM of dexamethasone. After the osteogenic incubation for 7 and 14 days, the cells were washed three times, fixed with 4% of paraformaldehyde for 15 min, and stained for 30 min using the ARS staining kits at room temperature. The stained BMSCs were dried, and image J 1.8.0 software (Image J2, USA) was utilized to calculate the stained areas for semi-quantitative analysis.

Human periodontal ligament stem cells (hPDLSCs) were isolated from human periodontal ligament tissue, were purchased from Procell Life Science and Technology Co., Ltd. (Cat No: CP-H234, Procell, China). These cells were both cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Keygen, China) containing 10% fetal bovine serum (FBS) (Gibco, United States) and 1% penicillin/streptomycin (HyClone, United States) at 37°C in 5% CO2. hPDLSCs between passages 3 and 6 were used for subsequent experiments. For osteogenic induction, the medium was changed to an osteogenic differentiation medium (Sigma, United States).

Synovial membrane specimens were digested with 0.2% type I collagenase (Thermo Fisher) at 37°C overnight, which were further collected by centrifugation and seeded in a high-glucose DMEM medium (Thermo Fisher, Waltham, MA, United States) supplemented with 10% FBS for 4 days to allow cell attachment. The medium was refreshed every 3 days, and at day 14, SMSCs were obtained. After blocking with human BD Fc Block™, SMSCs were stained with the following antibodies (Becton Dickinson): anti-CD34, anti-CD44, anti-CD45, anti-Sca-1, and anti-CD105 antibodies to confirm the phenotype using Guava^® easyCyte™ flow cytometer (Merck-Millipore, Billerica, MA, United States). At passage 3, SMSCs were switched to osteogenic differentiation medium (Sigma-Aldrich, St. Louis, MO, United States) for 2 weeks or StemPro Adipogenesis Differentiation Kit (Gibco) for 4 weeks. The miR-212-5p mimic or mimic-negative control (NC) (Sigma-Aldrich) were transfected with Lipofectamine^® 3,000 (Thermo Fisher) at the concentration of 100 nM according to the manufacturer’s instructions. The following sequences were used: miR-212-5p mimic (sense, 5′-ACC​UUG​GCU​CUA​GAC​UGC​UUA​CU-3'; and antisense, 5′-UAA​GCA​GUC​UAG​AGC​CAA​GGU​UU-3′), mimic-negative control miRNA (sense, 5′-UUC​UCC​GAA​CGU​GUC​ACG​UTT-3'; and antisense, 5′-ACG​UGA​CAC​GUU​CGG​AGA​ATT-3′).