Osteogenesis

Cells were differentiated as previously described⁶⁴. In short, once they reached 90% confluence cells were placed in a mix of high glucose and low glucose DMEM (50:50 v/v; Sigma-Aldrich) supplemented with 10% FBS, 1% Penicillin/Streptomycin, 100 nM dexamethasone (Sigma-Aldrich), 10 mM sodium β-glycerophosphate (Sigma-Aldrich) and 0.1 mM stabilized ascorbic acid (Sigma-Aldrich). After 3 days, cells were switched to DMEM low glucose supplemented as above and cultured for up to 17 days with media change every 3 days. Control cells were maintained in Hams F10 nutrient mix, 10% FBS, and 1% Penicillin/Streptomycin. After diifferentiation cells were fixed in paraformaldehyde (4%) for 15 min and stained with Alizarin Red (2%; pH 4.2) for 30–45 min. Samples were imaged in a Zeiss Axiovert 25 Inverted Phase microscope using Zen Blue software (Advanced Micro Devices).