Multilineage Differentiation of DPSCs

The in vitro differentiation potential of DPSCs (into adipocytes, osteocytes, and chondrocytes) was evaluated using a previously published protocol with minor modifications (Jang et al. 2018 (link)). Briefly, adipogenesis was carried out in the adipogenic medium with 10 µM insulin, 1 µM dexamethasone, 100 µM indomethacin, and 500 µM isobutylmethylxanthine (IBMX). Adipogenesis was confirmed by the accumulation of oil droplets upon staining of the differentiated cells with Oil red O solution. Osteogenesis was performed in the osteogenic medium consisting of 50 µM ascorbate-2-phosphate, 0.1 µM dexamethasone, and 10 mM glycerol-2-phosphate. Osteogenesis was then confirmed by the accumulation of calcium nodules upon staining of the differentiated cells with alizarin red and von Kossa. Chondrogenesis was carried out using the commercial chondrogenic medium (StemPro® Osteocyte/Chondrocyte Differentiation Basal Medium; StemPro® Chondrogenesis supplement, Gibco, Life technologies). Chondrogenesis was confirmed by staining the differentiated cells with Safranin O and Alcian blue.