Hadscs

hADSCs were purchased (Lonza, Basel, Switzerland) and cultured in Dulbecco’s Modified Eagle Medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 100 units/mL of penicillin (Gibco BRL), 10% (v/v) fetal bovine serum (FBS, Gibco BRL), and 100 μg/mL of streptomycin (Gibco BRL). All experiments were performed using hADSCs within five passages. To induce hypoxic condition in vitro, hADSCs were cultured under 1% oxygen and serum-free medium, as previously described [28 (link)].

Human adipose-derived stem cells (hADSCs) were purchased from Lonza Inc. (Walkersville, MD, USA). The cells were maintained in Dulbecco’s Modification of Eagle’s Medium/Ham’s F-12 50/50 Mix (DMEM/F12; catalog number: 10-090-CV, Corning Life Sciences, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; catalog number: 35-015-CV, Corning), 1% penicillin-streptomycin (Pen/Strep; catalog number: 15140122, Thermo Fisher Scientific, Waltham, MA, USA), 10 ng/mL recombinant human HB-EGF (EGF; catalog number: 100-47, PeproTech, Rocky Hill, NJ, USA) and 2 ng/mL recombinant human FGF-acidic (bFGF; catalog number: 100-17A, PeproTech, Rocky Hill, NJ, USA) at 37 °C in a humidified atmosphere containing 5% CO₂. In order to induce osteogenic differentiation in the 2D culture condition, hADSCs suspension (1 × ${10}^5$ cells/six-well) was inoculated in a six-well plate at 1 mL/well. After 1 h, the cells were attached to the plate and incubated in an osteogenic medium composed of high glucose (DMEM; catalog number: 10-013-CV, Corning), 10% (v/v) FBS, 1% Pen/Strep, 10 mM β-glycerol-phosphate (catalog number: 50020, Sigma-Aldrich; Merck KGaA, Darmastadt, Germany), 100 ng/mL dexamethasone (catalog number: D2915, Sigma-Aldrich; Merck KGaA) and 50 μg/mL ascorbic acid (catalog number: A4403, Sigma-Aldrich; Merck KGaA). The osteogenic medium was refreshed every two days.

hADSCs (Lonza, Walkersville, MD, USA) were cultured in 150‐mm culture dishes using DMEM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% (v/v) FBS (Gibco BRL) and 1% (v/v) PS (Gibco BRL) in a 5% CO₂ incubator at 37 °C. The culture medium was replaced every other day. For experiments, hADSCs with five to seven passages were used. The passage numbers of the hADSCs used in each experiment were identical.