L ascorbic acid

Both sorted sGMSCs and unsorted primary GMSCs (pGMSCs) were seeded at a density of 1×10⁵ cells per well in a 12-well plate containing DMEM. After 72 h, the culture medium was replaced with osteogenic induction medium consisting of DMEM supplemented with 20% FBS, 1% P/S, 10 nM Dexamethasone, 100 mM L-ascorbic acid, and 10 mM β-glycerophosphate (Beyotime, Shanghai, China). The induction medium was replaced every 48 h. Following 21 days of continuous induction, the cells were stained with Alizarin Red S staining solution (Solarbio) at RT for 1 h. Osteogenic differentiation was observed under a microscope (Leica, Mannheim, Germany).

Rats were used for the animal model, and the cell line model was mouse derived. Cell culture was used as previously described
[24] (link). Briefly, the MC3T3-E1 subclone 14 cell line (BDCB, Guangzhou, China) was cultured in base medium [Eagle’s medium supplemented with 5.6 mM glucose (Biofroxx, Einhausen, Germany), 2 mM L-glutamine (Solarbio, Beijing, China), 0.5 mM β-glycerophosphate (Beyotime, Shanghai, China), 50 mg/L ascorbic acid (Beyotime), and 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany)] with 5% CO
₂ at 37°C. The (seeding) cell density was 3×10
⁵ cells/mL, and cells at passages 15 to 18 were collected. Before processing, the differentiation capacity of MC3T3-E1 cells was checked at passages 15 to 18.
For high glucose intervention, MC3T3-E1 cells were cultured in high glucose medium (base medium supplemented with 25 mM glucose) for 14 days. For metformin intervention, MC3T3-E1 cells were cultured in medium containing different concentrations of metformin (25, 50, 100 μM metformin in base medium) for 14 days. For thapsigargin intervention, MC3T3-E1 cells were cultured in base medium containing 100 nM β-thapsigargin (Beyotime) for 1 day.