Vit c p

Passages 3–5 of P-MSCs were categorized into six groups according to the culture media: the control group [α-MEM (HyClone), 5% FBS (Invitrogen), 10 mM of β-glycerophosphate (Sigma), and 10⁻⁷ M dexamethasone (Sigma)], Vit. C-p group (control with 100 μM Vit. C-p), 10⁻¹⁰ M calcitriol (Nang Kuang Pharmaceutical Co., Ltd) group (control with 10⁻¹⁰ M calcitriol), 10⁻⁹ M calcitriol group (control with 10⁻⁹ M calcitriol), 10⁻⁸ M calcitriol group (control with 10⁻⁸ M calcitriol), and 10⁻⁷ M calcitriol group (control with 10⁻⁷ M calcitriol).

Pre-treatment of assay microplates (Falcon^®, USA) was performed with the coating of multiple 12-well cell culture microplate wells using 50 µg/mL rat tail collagen I (Corning^®, USA). hFPTs and ASC-F cells were then seeded at a relative viable density of 1.5 × 10³ cells/cm² in the microplates in FBS- or HPL-supplemented growth medium, as described previously, respectively. Cultures were appropriately maintained in both incubation conditions (i.e., normoxia and hypoxia) until cell monolayers attained 80% confluency (i.e., 6 ± 2 days). Thereafter, the specific culture medium was replaced with a specific osteogenic induction medium, composed of high-glucose DMEM (Gibco™, USA) supplemented with 5% v/v HPL (Stemulate^®, USA), 80 µg/mL VitCp (Sigma-Aldrich^®, USA), 5 mM β-glycerophosphate (Sigma-Aldrich^®, USA), and 100 nM dexamethasone (Acros Organics™, USA). The induction medium was exchanged twice weekly for a period of 21 days for the different cell types and in both incubation conditions. At the end of the induction period, the cells were either fixed with a 4% formalin solution and stained with a classical Von Kossa staining procedure (Sigma-Aldrich^®, USA) or were fixed with 70% ethanol and stained with Alizarin Red stain (Sigma-Aldrich^®, USA) for revelation of mineralized matrix accumulation. Following staining, assay plates were photographed as described previously.