Effect of Microcurrent Stimulation on Cartilage Regeneration in Engineered Chondrocyte Spheroids

Microcurrent stimulation has emerged as a promising biophysical cue for cartilage regeneration, but its dose-dependent effects in 3-dimensional engineered cartilage constructs remain unclear. Here, we investigated the effects of continuous direct microcurrent stimulation on auricular chondrocytes and engineered cartilage spheroids using 0-, 10-, 25-, 50-, 100-, and 200-μA stimulation for 30 min daily. In 2-dimensional culture, microcurrent stimulation did not markedly increase the DNA content or Cell Counting Kit-8 value, indicating that it did not markedly promote cell expansion or overall metabolic activity. 5-Ethynyl-2'-deoxyuridine and Ki67 assays showed that low to moderate (<50 μA) stimulation had limited effects on proliferation, whereas 100 to 200 μA attenuated proliferative activity. Nevertheless, microcurrent stimulation enhanced chondrogenic marker expression in a biphasic manner, with type II collagen, SRY-box transcription factor 9, aggrecan, and cartilage oligomeric matrix protein increasing from 0 to 50 μA and declining from 50 to 200 μA. In 3-dimensional spheroids, 50 μA produced the most favorable morphology, cartilage-specific extracellular matrix deposition, mechanical properties, and hyaline cartilage-like phenotype. Excessive stimulation increased catabolic, inflammatory, and apoptosis-associated molecular responses without causing obvious cytotoxicity, apoptosis, reactive oxygen species production, temperature change, or pH alteration. Mechanistically, 50 μA induced coordinated Ca²⁺ signaling and promoted nuclear factor of activated T cells (NFAT) nuclear translocation, whereas 200 μA generated dysregulated Ca²⁺ responses and inefficient NFAT activation. Verapamil, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, and cyclosporin A attenuated 50-μA-induced NFAT activation, supporting the involvement of the Ca²⁺/calcineurin/NFAT pathway. Furthermore, 50-μA-preconditioned spheroids showed superior cartilage-forming capacity after subcutaneous implantation. These findings identify 50 μA as a favorable bioelectrical stimulation condition within the tested intensity range and provide a practical strategy for enhancing cartilage spheroid maturation in cartilage tissue engineering.