Dulbecco s modified eagle medium ham s f 12 dmem f12

Human bone marrow MSCs (h-MSC) (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were identified by detecting mesenchymal stem cell surface markers through flow cytometry (Additional file 1: Figure S2A) and evaluating the potential for differentiation towards adipocytes, osteoblasts, and chondrocytes (Additional file 1: Figure S2A). The h-MSCs were cultured in Gibco Dulbecco’s modified Eagle medium/Ham’s F-12 (DMEM/F12) supplemented with 10% FBS and 100 U/mL penicillin–streptomycin (Gibco, USA) under condition of 5% CO₂, 37 °C, and 100% humidity. Upon the cells were approximately 90% confluence, 0.25% trypsin (Gibco, USA) were used to detach the cells, which then passaged at a ratio of 1:2 or 1:3. Passages between 3 and 5 were used in the following experiment. For the EMF treatment, MSCs were exposed for 8 h per day. To inhibit P2X7 or PI3K/Akt activity, cells were treated with 5 μM P2X7 blocker A740003 (Sigma–Aldrich, St. Louis, MO, USA) or 10 μM PI3K/Akt inhibitor LY294002 (Sigma–Aldrich).

The 6th passage of bone marrow mesenchymal stromal cells (MSCs) derived from C57BL/6 mice was obtained from Cyagen Biosciences (Guangzhou, China). Following culture and upon reaching the 10th passage, the purchased MSCs were characterized by evaluating their surface markers indicative of MSC identity, as well as their multipotent potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages (Figure S1A and B). The MSCs were cultured in Gibco Dulbecco’s modified Eagle medium/Ham’s F-12 (DMEM/F12) supplemented with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin (Gibco, USA), under the conditions of 5% CO₂, a temperature of 37℃, and a humidity level of 100%. Upon reaching approximately 90% confluence, trypsin at a concentration of 0.25% (Gibco, USA) was utilized to detach the cells for subsequent passaging at a ratio of 1:3. Passages ranging from 8th to 10th were employed for the following experimental procedures.