Intensity-dependent microcurrent stimulation enhances BMSC-derived cartilage microtissue formation under defined culture conditions

Engineering phenotypically favorable cartilage-like tissue from bone marrow mesenchymal stem cells (BMSCs) remains challenging because chondrogenic differentiation can be accompanied by insufficient cartilage-associated extracellular matrix accumulation and unfavorable hypertrophic and catabolic responses. Here, we established a microcurrent-assisted culture system for scaffold-free BMSC-derived cartilage microtissues (CMTs) and investigated the intensity-dependent effects of continuous unidirectional direct-current stimulation at 0, 50, 100, 150, 250, or 500 μA under a defined electrical and culture configuration. Among the tested conditions, 150 μA (nominal electrode current density, 3 μA/mm 2 ) showed the most consistently favorable overall response, with enhanced cartilage-associated gene and protein expression, greater type II collagen- and glycosaminoglycan-rich matrix accumulation, improved CMT morphology, and higher apparent mechanical properties. In contrast, 250 and 500 μA were associated with reduced cartilage-associated matrix formation and increased fibrotic, hypertrophic, catabolic, inflammatory, and apoptosis-related markers. The 150 μA condition was also associated with a distinct intracellular Ca 2+ response pattern and increased NFATc1 nuclear localization; verapamil, BAPTA-AM, and cyclosporin A attenuated NFATc1 nuclear localization, supporting involvement of Ca 2+ /calcineurin/NFAT-related signaling without establishing a unique causal mechanism. After 2 weeks of xenogeneic subcutaneous implantation in nude mice, CMTs preconditioned at 150 μA exhibited greater cartilage-like matrix accumulation and higher apparent compressive properties than those preconditioned at 0 or 500 μA. These findings identify 150 μA as the best-performing tested condition under the present electrical configuration and provide short-term ectopic proof-of-concept evidence, while longer-term orthotopic studies are required to evaluate phenotypic stability, tissue integration, and functional outcomes in cartilage-defect models.