Cell culture and identification
4-5 weeks old male Wistar rats were obtained from the Animal Experimental center of Shandong University (Jinan, china). The present study was approved by the Ethics committee of the School of Stomatology, Shandong University. Rat BMSCs were isolated and cultured as previously described (Li et al. , 2018). Briefly, after rats were euthanized, the femur and tibia were removed and the bone marrow cavity was rinsed with α-minimum essential medium (α-MEM) supplemented with 20% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. Bone marrow fluid was incubated at 37˚C in an atmosphere containing 5% CO2. The medium was changed every 3 days, non-adherent cells were discarded. When cells reached 80-90% confluence, they were sub-cultured in α-MEM supplemented with 10% FBS (control medium). Rat BMSCs of passage 3 were used in the following experiments.
For osteogenic induction, cells were cultured in the osteogenic medium (osteogenic group). Cells in control group were cultured in the control medium. Following 21 days culture, cells were fixed with 4% paraformaldehyde at 37˚C for 30 min, then incubated with 0.1% (pH 4.2) alizarin red S at 37˚C for 10 min. After washing with phosphate buffered saline (PBS), samples were observed using the phase-contrast microscope to verify the presence of mineralized nodules.
For adipogenic induction, cells were cultured in the adipogenic medium as previously described (Li et al. , 2018). Cells in control group were cultured in the control medium. Following 21 days culture, cells were fixed as mentioned above. Then cells were incubated with oil red O at 37˚C for 30 min and observed using the phase-contrast microscopy.
For clonogenesis experiment, rat BMSCs were seeded at a density of 3 cells/cm2 and cultured in control medium. Following 10 days culture, cells were fixed with 4% paraformaldehyde for 30 min, then stained with 0.1% crystal violet for 10 min at 37˚C. After washing with the tri-distilled water, cell clone was observed using the phase-contrast microscope. More than 50 cells were regarded as one cell clone. The clone formation rate was calculated as the number of cell clone / the total number of seeded cells × 100%, and the average value of six samples was obtained.
For subsequent CORM-3-induced osteogenic differentiation experiments, cells were cultured in the osteogenic medium containing 200 μΜ CORM-3 (CORM-3 group).