Hbmscs

Primary human bone marrow stem cells (hBMSCs) were purchased from ATCC (Manassas, Virginia, USA). The hBMSCs were cultured in alpha-MEM containing 15% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, Missouri, USA), 1% penicillin/streptomycin (PS, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37°C, 5% CO₂, and 99% humidity until 80% confluence. Cells at passage P3 were used in the experiments.
For osteogenic differentiation, hBMSCs (5 × 10³ cells) were seeded onto the electroactive surfaces (1 cm²) and incubated for 24 h to allow cell adhesion. The cells were then maintained in the normal culture medium supplemented with 10 mM β-glycerophosphate (β-GP, Sigma-Aldrich), 100 µM L-ascorbic acid phosphate (Sigma-Aldrich), and dexamethasone sodium phosphate (10⁻⁸ M), for 14 days [40 (link)].

Human bone marrow mesenchymal stem cells (hBMSCs) were obtained from Sciencell (Carlsbad, CA, USA) and cultured in Mesenchymal Stem Cell Medium (MSCM, Sciencell, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution at 37 °C under humid conditions with 5% CO₂. For osteogenesis differentiation, hBMSCs were seeded into six-well plate at a density of 2 × 10⁴ cells per well under normal culture condition. After 2 days, the cells were cultured with medium with 100 ng/mL of BMP2 (Invitrogen, Carlsbad, CA, USA) for osteogenic differentiation in vitro. And also, Osteogenic differentiation of hBMSCs was carried out using osteogenic medium containing 50 mg/ml ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), 5 mM β-glycerophosphate (Sigma-Aldrich) and 10 nM dexamethasone (Sigma-Aldrich).

Human bone mesenchymal stem cells (hBMSCs) were bought from the BeNa Culture Collection (BNCC, Beijing, China). hBMSCs were cultured in minimum essential medium Alpha Medium (α‐MEM) supplemented containing 10% FBS, 2Mm L‐Glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin and stored in a humidified atmosphere of 5% CO₂ at 37°C. Osteogenic (OS) differentiation of hBMSCs was induced according to a previously published protocol.15 The OS medium was supplemented with osteogenic inducers: including 10‐8 mol L⁻¹ dexamethasone (Dex); ascorbic acid 2‐phosphate (AsAP) with a concentration of 50 μg/mL; and 10 m mol L⁻¹ Glycerol 2‐phosphate (Gly) (Sigma, USA). hBMSCs were seeded at density of 5 x 10⁵ cells/well in a 12‐well plate, and the culture medium was replaced with an OS medium when the cells reached 80% confluence. Moreover, OS medium were changed every 3 days, and then induced cells were harvested and analysed at 0, 7, 14, and 21 days.

The primary culture of hBMSCs were obtained from ScienCell Research Laboratory (Carlsbad, CA, USA) and cells at passages 3–6 were used for this study. Cells were cultured in α-modified Eagle’s medium (α-MEM; Gibco, Grand island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco) as the proliferation medium (PM) as previously described [17 (link)]. Osteoinduction began when hBMSCs reached 70–80% confluence, and the osteogenic medium (OM) contained 10 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), 100 nM dexamethasone (Sigma), and 200 μM L-ascorbic acid (Sigma). Human embryonic kidney (293 T) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). All cells were cultured in a humid atmosphere with 95% air, 5% CO₂ at 37 °C.

hBMSCs were obtained from American Culture Collection Company (ATCC, USA) and cultured in DMEM-F12 (Gibco, USA) with 10% fetal bovine serum (Takara, Japan) and 1% penicillin–streptomycin. Cells were cultured at 37 °C in an incubator containing 5% CO₂. To stimulate osteogenic differentiation, hBMSCs were cultured in an osteogenic differentiation medium containing 10 mM β-glycerophosphate (Sigma-Aldrich, USA) and 50 ng/ml L-ascorbic acid (Sigma-Aldrich, USA). The medium is renewed every 3 days.