Ascorbic acid 2 phosphate aa

Ascorbic acid-2-phosphate (AA) and β-glycerophosphate (β-GP) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Bone morphogenetic protein-2 (BMP2) was purchased from CowellMedi Corp (Seoul, Korea). PHF20 short interfering RNA (PHF20 si-RNA) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Commercial antibodies against PHF20, Runx2 (Cell Signaling Technology, Beverly, MA, USA), Histone H3 (H3), Histone H3 Tri methyl K4 ChIP grade (H3K4me3), Sp7 (Osx) (Abcam, Cambridge, UK), β–actin (Santa Cruz Biotechnology, Dallas, TX, USA), and Myc (Thermo Fisher Scientific, Waltham, MA, USA) were used.

Undifferentiated human bone-marrow-derived mesenchymal stem cells (hBMSC, ATCC-PCS-500-012) were cultured in mesenchymal stem cell basal medium (BM, ATCC PCS500030) supplemented with 7% FBS, 100 IU/mL penicillin/streptomycin, 2.4 mM, 125 pg/mL FGF-b, and 15 ng/mL IGF-1 (ATCC, Milan, Italy), at a density of 5 × 10³ cells/cm² and incubated for 24 h at 37 °C under 5% CO₂. To induce osteogenic differentiation, hBMSC were cultured in osteogenic medium (OM), (DMEM with 10% FBS, 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine (Corning, Manassas, VA, USA), 10 mM β-glycerophosphate (BGP), 100 nM dexamethasone, 100 μM ascorbic acid 2-phosphate (AA) (Sigma Chemical Co., Milan, Italy), or cultured in BM with the presence of implant plus CGF supplemented with 7% FBS, 100 IU/mL penicillin/streptomycin, 10 mM BGP, and 100 μM AA (Device) for 21 days. The medium was replaced at a rate of 50% every 3 days. In all experiments, the implant coated with CGF was placed into a sterile transwell insert (TC-inserts, Sarstedt, Nümbrecht Germany) with a semipermeable membrane at the bottom (pores of 0.4 μm) and inserted into the 12-well culture plates (an insert in each well).