4.3. Osteogenic Differentiation

At passage 3 to 4, cells are harvested using 0.05% Trypsin-EDTA (Gibco) and seeded on Thermanox™ coverslips (Nunc, Rochester, NY, USA) placed within 24-well plates at a density of 1.5 × 10⁴ cells/cm². For the first 24 h, cells are cultured in osteocontrol medium consisting of LG-DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1% (v/v) PEN/STREP. At this point (day 0), cells are switched into four different culture media compositions, as detailed in Table 1. Briefly, two groups are cultured with osteocontrol medium in the absence or presence of 30 µg/mL polyP-NP, respectively. The third group consists of osteocontrol medium additionally supplemented with the classic osteogenic cocktail including 10 nM Dexa, 50 µl/mL AA2P and 10 mM BGP. The fourth group is the classic osteogenic cocktail but with the BGP replaced by 30 µg/mL polyP-NP. Cells are kept under standard culture conditions for 28 days with media changed three times per week.

Media composition of the four experimental groups.

¹ LG-DMEM: low glucose (1 g/L) DMEM, PEN/STREP: Penicillin/Streptomycin, FBS: fetal bovine serum, Dexa: dexamethasone, AA2P: L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, BGP: β-Glycerolphosphate disodium salt hydrate, polyP-NP: polyphosphate nanoparticles. The “+” indicates the presence and the “-“ indicates the absence of the supplement in the medium.