Frequency-tuned microcurrent stimulation directs ADSC spheroid chondrogenesis through Ca²⁺-calcineurin-NFAT signaling

Adipose-derived stem cells (ADSCs) are promising for cartilage regeneration, but efficient, safe strategies to enhance chondrogenesis remain limited. This study examined frequency-dependent effects of microcurrent stimulation (MS) on rabbit ADSCs cultured as monolayers and three-dimensional spheroids. Cells were exposed to 0 Hz, 10 Hz, 100 Hz, 1 kHz, 10 kHz, or 60 kHz MS for 20 min daily. Chondrogenesis, extracellular matrix deposition, spheroid morphology, mechanical properties, inflammatory/catabolic responses, apoptosis, and Ca²⁺-calcineurin-NFAT signaling were evaluated. Among tested frequencies, 1 kHz MS most effectively enhanced chondrogenic differentiation, increasing SOX9, COL2A1, ACAN, and COMP expression and promoting collagen II and glycosaminoglycan deposition. It also improved spheroid morphology and mechanical properties while suppressing fibrocartilaginous, hypertrophic, inflammatory, and catabolic markers. MS did not alter medium pH, temperature, or reactive oxygen species levels and did not impair viability or increase apoptosis. Mechanistically, 1 kHz MS promoted Ca²⁺ influx, NFAT nuclear translocation, and NFATc1 expression; inhibition of L-type Ca²⁺ channels, intracellular Ca²⁺, or calcineurin attenuated this response. These findings identify frequency-tuned MS as a noninvasive strategy for promoting ADSC chondrogenesis and cartilage-like matrix formation.