Magnesium promotes bone marrow mesenchymal stem cells chondrogenic differentiation by regulating macrophage polarization

Healing the tendon-bone interface requires regenerating a functional fibrocartilage zone. Beyond direct biological effects, magnesium ions (Mg 2+ ) exhibit immunomodulatory potential by polarizing macrophages toward a pro-healing phenotype. This study investigates whether Mg 2+ can achieve superior chondrogenesis indirectly through this reshaped immune microenvironment, providing a novel strategy for designing intelligent magnesium-based biomaterials. First, CCK-8 assays defined the biocompatible concentration range of Mg 2+ for BMSCs and macrophages. Immunofluorescence and RT-qPCR were used to identify the optimal concentration for inducing macrophage M2 polarization. Subsequent spreading and Transwell assays evaluated that the resulting conditioned medium enhanced stem cell migration and spreading. Finally, immunofluorescence and RT-qPCR confirmed the superior chondrogenic efficacy of this immunomodulatory microenvironment over direct Mg 2+ stimulation alone. All experiments were repeated three times. Comparisons among multiple groups were performed using one-way ANOVA or two-way ANOVA, followed by Tukey’s multiple comparison test. CCK-8 assays established 5–10 mM as the biocompatible concentration window for Mg 2+ , which effectively enhanced stem cell viability. Immunofluorescence and RT-qPCR demonstrate that Mg 2+ polarized macrophages toward an M2 phenotype at these concentrations. Conditioned medium from Mg 2+ -treated macrophages (Mg + CM) further optimized stem cell spreading and migration. Most importantly, Mg + CM induced significantly greater glycosaminoglycan/proteoglycan-rich extracellular matrix deposition, as shown by Alcian Blue/Safranin O staining, and type II collagen deposition, as evidenced by immunofluorescence. In addition, it more strongly upregulated chondrogenic gene expression (Sox9, Acan, Col2a1) than direct Mg 2+ stimulation. These results demonstrate that the immunomodulatory microenvironment orchestrated by Mg 2+ surpasses its direct effect in driving chondrogenesis. This study reveals that Mg 2+ promotes cartilage repair more effectively by polarizing macrophages to create a pro-regenerative immune microenvironment than through direct stimulation. This immunomodulatory microenvironment may support BMSC chondrogenesis, potentially involving soluble factors, informing the design of intelligent magnesium-based biomaterials guided by immunomodulation-regeneration synergy.