Chondrogenic differentiation media

To evaluate their ability to differentiate into other cell types, DPSCs were seeded into six-well plates at a density of 3 × 10⁵ cells per well. The cells were cultured in different media upon reaching 90% confluence. For the osteogenic differentiation assay, the cell culture medium was supplemented with 15% FBS, 10 mmol·L⁻¹ 10% β-glycerophosphate, 5 mg·mL⁻¹ ascorbic acid 2-phosphate, and 10⁻⁵ mol·L⁻¹ 0.1% dexamethasone for 4 weeks of induction. For adipogenic differentiation, a mixture including 0.5 mmol·L⁻¹ isobutylmethylxanthine, 0.5 mmol·L⁻¹ hydrocortisone, 60 μmol·L⁻¹ indomethacin, and 15% FBS was added before culturing for 4 weeks. To induce chondrogenic differentiation, cell pellets (of 2.5 × 10⁵ cells per pellet) were cultured in chondrogenic differentiation media (Gibco, Carlsbad, CA, USA) for 4 weeks. After the induction periods, cells or cell pellets were fixed in 4% paraformaldehyde for 20 min. The degrees of osteogenic, adipogenic, and chondrogenic differentiation were determined by Alizarin red staining, the formation of lipid vacuoles (visualized by Oil red O staining according to standard protocols), and Alcian blue staining, respectively. Each test was repeated at least three times.

For cellular differentiation, cells were treated with or without 5-aza-CN for 24 h, then washed with growth media and replaced in a proper differentiation medium. For osteoblastic induction, we replaced them into DMEM supplemented 2% FBS containing 170 μM ascorbic acid 2-phosphate (AA; Sigma-Aldrich), 5 mM β-Glycerophosphate (β-GP; Sigma-Aldrich) and 200 ng/ml BMP2 (R&D Systems, Minneapolis, MN, USA). For adipogenic differentiation, the media were changed to DMEM supplemented 2% FBS with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich), 1 μM dexamethasone (DEX) (Sigma-Aldrich), 100 μM Indomethacin (Sigma-Aldrich), and 10 μg/ml Insulin (Sigma-Aldrich). For chondrogenic differentiation, the media were changed to chondrogenic differentiation media (Gibco BRL). For neurogenic differentiation, the cells were induced by culturing in DMEM supplemented 2% FBS, 2% dimethyl sulfoxide (Sigma-Aldrich), 100 μM butylated hydroxyanisole (Sigma-Aldrich). All cell differentiation media were changed every other day.