Cell cultures and osteogenic induction

Primary human adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). All cell-based in-vitro studies were repeated in triplicate using MSCs from three donors.

Bone marrow tissues were collected from Sham and ovariectomized (OVX) mice through flushing the femurs and tibias with DMEM alpha modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA). The bone marrow suspension was concentrated, washed twice in DMEM alpha modified Eagle’s medium containing 10% fetal bovine serum (FBS; Invitrogen), 2 mmol/L glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen), and then cultured at 37 °C with 5% CO₂. Cells were collected after about 2 weeks when they had expanded. MSCs were grown in a humidified incubator under 5% CO₂ at 37 °C in DMEM alpha modified Eagle’s medium (Invitrogen) supplemented with 10% FBS (Invitrogen), 2 mmol/L glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For osteogenic differentiation, cells were cultured in osteogenic medium (OM), which consisted of DMEM alpha modified Eagle’s medium with 10% (v/v) FBS, 1% (v/v) antibiotics, 100 nM dexamethasone, 10 mM β-glycerophosphate, and 0.2 mM l-ascorbic acid. Human embryonic kidney 293 T cells were maintained in complete DMEM medium with 10% FBS (Invitrogen), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). For TNF-α (R&D Systems, Minneapolis, MN, USA) or BAY117082 (Selleck, Houston, TX, USA) treatment, MSCs were starved for 24 h to synchronize the cells in the DMEM alpha modified Eagle’s medium without serum, and then changed to routine culture medium and treated with TNF-α or BAY117082.