Ascorbic acid

MSC were cultured for 21 days in αMEM with FBS (10%), ultraglutamine (1%), penicillin (100 U/mL), streptomycin (100 µg/ml) and osteogenic stimuli based on dexamethasone (1 µM; Sigma-Aldrich), β-glycerol phosphate (10 mM; Sigma-Aldrich) and ascorbic acid (0.2 mM; BAYER, Barcelona, Spain). Basal Mg²⁺ concentration in the medium was 0.8 mM. In order to increase the Mg²⁺ content in the pro-osteogenic medium, MgCl₂ (Carlo ErbaReagentiSpA, Milano, Italy) was added to achieve final Mg²⁺ concentrations of 1.2 mM and 1.8 mM during the osteogenic stimulus. Fresh medium alone or osteogenic medium supplemented with MgCl₂ was replaced every 3 days. Furthermore, 2-APB (50 µM; Tocris Bioscience, Bristol, UK) was added to the osteogenic medium containing 0.8 mM of Mg²⁺ during differentiation to determine the effects of inhibiting the Mg²⁺ channel TRPM7. To examine a direct effect of Mg²⁺ supplementation (1.2 and 1.8 m, or 2-APB) for 24 hours on undifferentiated MSC or at the different stages of differentiation (early or mature osteoblasts obtained from MSC) the NICD expression was also analyzed by western blot. All experiments were repeated at least three times.

Twenty-eight male Dunkin-Hartley guinea pigs (Harlan Laboratories, Inc., Indianapolis) with ages between 4 and 5 wk were provided with standard guinea pig chow and water supplemented with ascorbic acid (Bayer Hispania, Sant Joan Despí, Spain) ad libitum. Animals were housed in nonmetabolic cages under controlled conditions.