Oil red o staining solution

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.

Osteogenic differentiation: The ADMSCs at passage 4 were cultured in a 12-well plate with ADMSCs medium. When the cells growth reached 60% confluence, the ADMSCs were replaced with osteogenic differentiation medium (MEM medium containing 10% FBS, 100 nM dexamethasone (Selleck), 30 μg/ml ascorbic acid (sigma) and 10 mM sodium ß-glycerophosphate (Solarbio)) and differentiated for another 14 days. After that, the cells were fixed with 4% paraformaldehyde, washed with PBS, and incubated with alizarin red staining solution for 30 min. Finally, cells are used for photography after removing alizarin red staining solution and washing with PBS for three times.
Adipogenic differentiation: The ADMSCs were cultured similarly as above, then replaced with adipogenic differentiation medium (MEM medium containing 10% FBS, 5% Insulin-Transferrin-Selenium-Ethanolamine (ITS-X) (Selleck), 1 μM dexamethasone (Selleck) and 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX) (Selleck)). After 14 days’ differentiation, the cells were fixed with 4% paraformaldehyde, washed with PBS, and incubated with oil red O staining solution (Solarbio) for 60 min. Finally, cells are used for photography after removing staining solution and washing with PBS for three times.

For the osteogenic differentiation, the induction medium (IM) included DMEM supplemented with 10% FBS (Gibco, USA), 50 µM vitamin C (Sigma-Aldrich, USA), 10 mM sodium β-glycerophosphate (Sigma-Aldrich, USA), and 0.1 µM dexamethasone (Sigma-Aldrich, USA). iPSC-MSCs were fixed with 95% ethanol after 14 days of differentiation and stained with Alizarin red staining solution (Solarbio, China).
For adipogenic differentiation, cells were cultured in DMEM with 10% FBS, 0.5 µM IBMX (Sigma-Aldrich, USA), 200 µM indomethacin (Sigma-Aldrich, USA), 10 µM insulin (Sigma-Aldrich, USA), and 1  µM dexamethasone. Cells were stained with Oil Red O staining solution (Solarbio, China).
For chondrogenic differentiation, cells were cultured in DMEM with 10% FBS, 10 ng/ml TGF-β1 (R&D Systems, USA), 40 µg/ml proline (Sigma-Aldrich, USA), 100 µg/ml sodium pyruvate (Sigma-Aldrich, USA), 50 µg/ml vitamin C, and 0.1 µM dexamethasone. Cells were stained with Alcian blue staining solution (Solarbio, China).