Osteogenic inducing medium

Multilineage differentiation capacity of hADSCs was detected as requested by the International Society for Cellular Therapy. Briefly, cells were cultured in adipogenic induction medium (Cyagen Biosciences, USA) for designated time and were stained by Oil Red‐O staining. For osteogenic differentiation, cells were cultured in osteogenic inducing medium (Cyagen Biosciences, USA) for designated time and were stained by alizarin red S. Chondrogenic differentiation was performed using the micromass culture technique maintained in chondrogenic medium (Cyagen Biosciences, USA) for up to 5 weeks, and were stained by Alcian blue.

To identify the osteogenic differentiation potential of PDLSCs, the cells (P3) were plated into 6-well plates at a density of 2 × 10⁴ cells per well at 37 °C in a humidified atmosphere of 5% CO₂. Once the cells reached 60%~70% confluence, the medium was changed to an osteogenic-inducing medium (Cyagen, USA). After induction for two to four weeks, the cells were fixed with paraformaldehyde for 30 min and stained with Alizarin Red-S (Beyotime, China) for 3 ~ 5 min.
To identify the adipogenesis differentiation potential of PDLSCs, the cells (P3) were plated into 6-well plates at a density of 2 × 10⁴ cells per well at 37℃ in a humidified atmosphere of 5% CO₂. Once the cells reached 100% confluence, the medium was changed to adipogenic inducing medium (Cyagen, USA). After induction for three to four weeks, the cells were fixed with paraformaldehyde for 30 min and stained with Oil Red O for 30 min.