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.