Beta glycerophosphate disodium

HBO cell lines were derived from healthy fibulas of seven pediatric subjects under appropriate Institutional Review Board approval, Segments of the fibulas were digested using Collagenase A (0.1 mg/mL) treatment for 40 minutes with replacement of Collagenase A midway at 20 minutes and then digested with 0.2 mg/ml of Collagenase A for 60 minutes at 37°C with intermittent shaking. The segments of bone were maintained in DMEM + Primocin + 10% FBS + 50 μM ascorbic acid (Sigma, 49752) + 10nM dexamethasone. Media changes were done every 5 days. Osteoblast-like cells began to grow out of the bone segments by Day 10. These cells can be passaged and frozen for storage. On addition of Osteogenic media (DMEM + Primocin + 10% FBS + 50 μM ascorbic acid + 10 nM dexamethasone + 10 mM beta-glycerophosphate disodium (Sigma, G9422), the cells form mineralized nodules indicating their osteogenic ability as seen in Supplementary Figure 1.

The osteogenic differentiation of pADSCs was induced on the scaffolds for one day (d1) and two weeks (d14) with DMEM-HG supplemented with 15% FBS, 1% penicillin/streptomycin (40 IU/ml), dexamethasone (100 nM), ascorbic acid 2-phosphate (150 µM), and beta-glycerophosphate disodium (10 mM) all from Sigma Aldrich (Munich, Germany). Cells seeded on scaffold without osteogenic induction cultured for 1 and 14 days in complete culture medium without osteogenic reagents served as the control.