Osteogenesis differentiation medium

Cell viability was assessed by a cell counting kit-8 (CCK-8). Briefly, PKH26-labelled MenSCs (PKH26-MenSCs) and unlabelled MenSCs were separately seeded at a density of 1000 cells per well in 96-well plates. Ten microliters of CCK-8 reagent (Dojindo Laboratories, Japan) was added to 6 wells of each group every 24 h, and the cells were incubated for 1 h at 37 °C in 5% CO_2. The absorbance at 450 nm was measured by a spectrophotometer (Beckman Coulter, United States), and the growth curve was drawn to compare cell proliferation ability between the two groups. To induce osteogenic differentiation, PKH26-MenSCs and unlabeled MenSCs were cultured in commercially available osteogenesis differentiation medium (Cyagen Biosciences, United States). On day 21, cells were stained with alizarin red S. To induce adipogenic differentiation, each group of cells was cultured in a commercially available adipogenesis differentiation medium purchased from Cyagen. On day 21, lipid droplets were visualized by oil red O.

To evaluate the multipotential differentiation, third-passage PO-MSCs were seeded in six-well plates and cultured at a density of 2 × 10⁵ cells per well. After the PO-MSCs reached about 80% confluence, their culture medium was changed to osteogenesis differentiation medium or adipogenic differentiation medium (all from Cyagen, Guangzhou, China) and induction 21 days according to the manufacturer’s protocol, respectively. Then, Oil Red staining and Alizarin Red S staining were subjected to verify the adipogenic differentiation and osteogenic differentiation, respectively.