L ascorbic acid 2 phosphate

Cells were cultured in osteogenic medium containing 10 mM β-glycerophosphate (Sigma-Aldrich), 100 μM L-ascorbic acid 2-phosphate (Wako), and 100 nM dexamethasone (Sigma) for indicated time periods. For alkaline phosphatase staining, after 4 weeks of induction, cells were fixed with 4% paraformaldehyde for 1 min at room temperature and incubated with a solution of 0.25% naphthol AS-BI phosphate and 0.75% Fast Blue BB (Sigma-Aldrich, St Louis, MO, USA) dissolved in 0.1 M Tris buffer (pH 9.3). For Alizarin Red staining to detect mineralized nodule formation, cells were fixed cells for 30 min at room temperature and incubated with 2% Alizarin Red (Sigma-Aldrich, St Louis, MO, USA).

Osteoblast differentiation was performed in cells plated at 10,000/cm² in osteoblast-induction media (OIM) containing DMEM supplemented with 10% FBS 10 mM beta glycerophosphate (Calbiochem-Merck, Germany), 50 μg/mL L-ascorbic acid-2-phosphate (Wako Chemicals GmbH, Germany) and 10nM dexamethasone (Sigma-Aldrich, Denmark). The medium was changed every three days during induction period.

To generate EBs, sees2 and sees5 (5 × 10³/well) were dissociated into single cells with 0.5 mM EDTA (Life Technologies) after exposure to the rock inhibitor (Y-27632: A11105-01, Wako, Japan) and cultivated in 96-well plates (Thermo Fisher Scientific) in the EB medium (76% Knockout DMEM, 20% 35 kGy irradiated Xeno-free Knockout Serum Replacement (XF-KSR, Life Technologies, CA, USA), 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, and 50 μg/ml l-ascorbic acid 2-phosphate (Sigma-Aldrich, St. Louis, MO, USA)) for 4 days. The EBs were transferred to T25 flasks coated with NMP collagen PS (Nippon Meat Packers Inc.) and cultivated in the XF32 medium (85% Knockout DMEM, 15% 35 kGy-irradiated XF-KSR, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 50 μg/ml l-ascorbic acid 2-phosphate, 10 ng/ml heregulin-1β (recombinant human NRG-beta 1/HRG-beta 1 EGF domain; Wako, Japan), 200 ng/ml recombinant human IGF-1 (LONG R3-IGF-1; Sigma-Aldrich), and 20 ng/ml human bFGF (Kaken Pharmaceutical Co. Ltd.)) for 60 to 70 days. The flasks were gently shaken to detach the cells. The detached cells were aggregated and could thus be easily removed by a pipette. The remaining adherent cells in the flasks were used for a resource of mesenchymal stromal cells. The adherent cells were then propagated in α-MEM medium supplemented with 10% FBS (Gibco or HyClone) and 1% Pen-Strep for further in vitro analysis.

Cells were seeded at 5 × 10⁴ cells/cm² and cultured with or without a sealer extract in an osteogenic medium containing 5 mM beta-glycerol phosphate (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 0.2 mM L-ascorbic acid-2-phosphate (FUJIFILM Wako Pure Chemical, Osaka, Japan). Mineralized nodules were fixed with methanol and stained with Alizarin red S (FUJIFILM Wako Pure Chemical, Osaka, Japan). The density of mineralized nodules was measured with ImageJ software (ver 1.53e, U. S. National Institutes of Health, Bethesda, MD, USA).

Isolation and culture of SCAP were according to the previous study [1 (link)]. SCAP were isolated from the apical papilla tissue of the developing tooth root apex of extracted human impacted lower third molars of healthy donors as described in Additional file 2: Supplementary Methods. Attached colony-forming cells on plastic culture flasks were expanded. The growth medium consisted of 15% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX), 100 μM l-ascorbic acid 2-phosphate (Wako Pure Chemicals, Osaka, Japan), 2 mM l-glutamine (Nacalai Tesque, Kyoto, Japan), and premixed antibiotics containing 100 U/ml penicillin and 100 μg/ml streptomycin (Nacalai Tesque) in minimum essential medium Eagle alpha modification (αMEM; Thermo Fisher Scientific, Waltham, MA). The passage 3 (P3) cells were analyzed for determining the characterization as MSCs and SCAP according to previous reports [1 (link), 24 (link)] and were used for further experiments.