Adipogenic induction medium

The density of the third generation PDLSCs was adjusted to 2×10⁵ cells/well and inoculated into a 24-well plate. After the cells were grown to about 70%, the original culture medium was discarded, and the medium was replaced with osteogenic, chondrogenic and adipogenic induction medium (Gibco, USA). The control group was added with normal induction medium, and the other groups were added with 10 ng/mL TNF-α and/or 100 μg/mL AGEs-BSA in the induction medium according to the experimental requirements. The culture medium was changed every three days, cultured for twenty-one days, fixed with 4% paraformaldehyde for thirty minutes at room temperature. 500 ml alizarin red staining solution (osteogenic induction), toluidine blue staining solution (cartilage induction) and oil red O staining solution (fat induction) (Solarbio, China) were added to each hole and placed in incubator for twenty minutes (osteogenic and cartilage induction) and one hour respectively (fat induced), ALP staining twelve hours, discarding staining solution, PBS cleaning three times. Cartilage induction is rinsed once with absolute ethanol. Adipogenic induction wash the residual stain with 75% ethanol and 60% isopropanol. Observed under an inverted microscope and photographed. ALP staining was washed three times with PBS and photographed and compared.

Cells were cultured in adipogenic induction medium (Gibco). On day 14, cultures were stained with oil red O staining (Sigma) as an indicator of intracellular lipid accumulation.