L ascorbic acid 2 phosphate

The osteogenic differentiation protocol was described in our previous studies [17 (link), 18 (link)]. Briefly, the BMSCs were cultured in growth medium in 24-well plates at a density of 1 × 10⁴ cells/cm². When over 80% confluence was reached, the medium was replaced with osteogenic induction medium (OIM), which consisted of growth medium supplemented with 1 nM dexamethasone, 50 μM l-ascorbic acid-2-phosphate and 20 mM β-glycerophosphate (Cyagen Biosciences, Guangzhou, China). Catalpol (10, 50 or 250 μM) was added to OIM in the experimental group, while the control group was cultured in OIM without catalpol. In accordance with a previous study [21 (link)], to examine the involvement of WNT/β-catenin signalling, OIM was supplemented with 50 μM catalpol in the presence or absence of 0.1 μg/ml Dickkopf-related protein 1 (DKK1; Peprotech, CT, USA), an antagonist of the WNT/β-catenin pathway.

The protocol for BMSC osteogenic differentiation was described in our previous study.²⁵ Briefly, HG‐treated BMSCs were seeded into 24‐well or 6‐well plates at a density of 1 × 10⁴ cells/cm² and then cultured in HG‐DMEM (25 mmol/L), and BMSCs cultured in LG‐DMEM (5.5 mmol/L) were considered the control group. After the cells reached 80% confluence, the medium was replaced with osteogenic induction medium (OIM), and 1 nmol/L dexamethasone, 50 μmol/L L‐ascorbic acid‐2‐phosphate and 20 mmol/L β‐glycerophosphate (Cyagen Biosciences, Guangzhou, China) were added to the corresponding culture medium with different glucose concentrations. Morroniside, FPS‐ZM1 and BBGCP₂ were added to the OIM with the concentrations mentioned above.