Bmscs

Sprague–Dawley (SD) bone marrow derived mesenchymal stem cells (BMSCs) (Cyagen Biosciences Inc., Guangzhou, China) were propagated at 37 °C under 5% CO₂ in Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Carlsbad, CA) cultured with 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (50 U mL⁻¹ penicillin and 50 ug mL⁻¹ streptomycin, Gibco). The passage 4(P4) cells were used for the in vitro experiments. SD-derived BMSCs(P3) (Cyagen Biosciences Inc., USA) that stably express GFP were propagated and the P5-GFP-BMSCs were used for in vivo experiments.

The mouse macrophage line Raw 264.7, mouse preosteoblast cell line MC3T3-E1, and BMSCs were obtained from Procell Life Science & Technology (Wuhan, China). MC3T3-E1 cells were cultured in α-MEM supplemented with 10% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin. RAW264.7 cells were cultured in DMEM supplemented with 10% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin. Mouse BMSCs were cultured in mouse MSC Medium (Cyagen) according to the manufacturer’s protocol (Nuwacell, Hefei, China). The cell cultures were maintained in an incubator at 37 °C in a humidified atmosphere with 5% CO2.
C57BL/6J female mice of specific pathogen-free (SPF) quality were purchased from the Model Animal Research Center of Tongji Medical College of Huazhong University of Science and Technology (HUST, Wuhan, China). All mice were housed under controlled conditions, which included room temperature of 25 °C, humidity maintained at 60 ± 10%, and a natural light–dark cycle throughout the study. All animals were treated according to the regulations of Chinese law and the local Ethics Committee. All animal experiments were reviewed and approved by the IACUC of HUST (IACUC Number: 3469).

Human bone mesenchymal stromal cells (BMSCs) were purchased from Cyagen Biosciences Inc. (HUXMA-01201). Cells were maintained in Dulbecco’s modified Eagle’s medium (11995, Gibco) and supplemented with 10% fetal bovine serum (FBS) (11995, Gibco) containing 1% penicillin/streptomycin, 0.2% herin, and 1% glutamax at 37°C in a humidified atmosphere containing 5% CO₂. Cells were pretreated with inhibitors for 1 h prior to stimulation.

BMSCs were from Cyagen Biosciences Inc. (Santa Clara, CA, USA; http://www.cyagen.com/) and maintained in alpha minimal essential medium (aMEM; Gibco, Invitrogen, Rockville, MD; http://www.lifetechnologies.com/), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (Gibco, Invitrogen) as described previously [16 (link)]. 293 T cells were from ATCC (Beijing, China; http://www.atcc.org/) and cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco) with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin.