Complete medium

Adipose and osteogenic differentiation of CD133⁺ OSCC cells was induced according to manufacturer instructions (Cyagen, Santa Clara, CA, USA). CD133⁺ cells were cultured in a 6-well plate at a density of 2 × 10⁴ cells in 2 mL of complete medium (Cyagen) per well. The medium was aspirated when cell confluence reached 60% to 70%, and 2 mL of adult bone marrow mesenchyme (Cyagen) was added to each well. Stem cell osteogenic differentiation medium (Cyagen) was changed every 3 days. After induction for 2 to 4 weeks, calcified nodules were fixed with formaldehyde for 40 min and then stained with Alizarin Red. Adipose differentiation was performed in a similar way, with adipose tissue formed by CD133⁺ OSCC cells stained with Oil Red O (Cyagen).

BMSCs were obtained from Cyagen (Guangzhou, China) and cultured in a complete medium (Cyagen, Guangzhou, China). BMSCs were cultivated separately in either the medium alone or with various USPIO concentrations, with or without EMF exposure. Mouse primary chondrocytes were acquired from the Procell Cell Resource Center (Wuhan, China) and cultured at the Roswell Park Memorial Institute-1640 (Procell, Wuhan, China). Normal chondrocytes were cultivated in a basal medium supplemented with 10% FBS (SORFA Life Science, China). Chondrocytes in the OA conditions were treated with IL-1β (10 ng/mL) (R&D Systems, Abingdon, UK) for 24 h.

The vertical migration of mMSCs was detected using the Transwell migration assay. Transwell inserts (6.5-mm diameter and 8-mm pore size, Corning Inc., Corning, NY, USA) were loaded with 2 × 10⁴ BM-MSCs in 200 μl of complete medium (Cyagen, Suzhou, China), and 600 μl of serum-free DMEM/F12 (Corning Inc., Corning, NY, USA) with or without 1.5 × 10⁶ cells in the lower chambers. The complete medium containing different drugs (10 μg/ml aristolochic acid I sodium salt (AA-I), Sigma, USA; 10 ng/ml TNF-α, Peprotech, USA; 5 ng/ml transforming growth factor-β1 (Tgroup). The sham control (sham+salineGF-β1), Sino Biological China) was placed in the lower well to induce cell migration. The cells were allowed to migrate at 37 °C in a humidified CO₂ incubator for 36 h. The nonmigrated cells on the upper side of the membranes were removed using cotton swabs. Migrated cells on the lower side of the membranes were fixed with 4% paraformaldehyde for 30 min at 4 °C and stained with 0.1% crystal violet for 20 min. Five different visual fields (× 200 magnification) were randomly selected for observation and photographed under a fluorescence microscope (Olympus Optical Co., Ltd.). Quantification was performed by calculating the percentage of stained area to the total area using FIJI software.