Ultrasound stimulation increases cartilage synthesis and revert dedifferentiation on human chondrocytes

Therapeutic ultrasound (US) holds promise as a potential therapy to treat articular cartilage; however, optimized and targeted US protocols are lacking. This study aimed at determining an appropriate set of US protocols for stimulating human chondrocyte activity. Human chondrocytes were exposed to various US parameters, including different central frequencies, power densities, operation modes, pulse parameters, and durations, either daily or every other day over a three-day period. Temperature was monitored during stimulation. Protocols that showed the greatest potential for increasing chondrocyte metabolic activity and proliferation were further investigated and extended to a seven-day application. Cartilage matrix synthesis was analyzed by immunocytochemistry, qPCR, and glycosaminoglycans staining. Chondrocytes exposed to 250 mW/cm² at 1.25 MHz (continuous or pulsed, every other day), 0.45 MHz (pulsed, daily), or 2.00 MHz (pulsed, every other day) exhibited significantly higher proliferation and metabolic activity. Immunocytochemistry analyses revealed that those protocols markedly increased COL II expression by up to 2.7-fold and aggrecan up to 1.7-fold compared to non-stimulated chondrocytes. Collagen type II and aggrecan mRNA levels were elevated by up to 2.0-fold and 4.5-fold, respectively. No significant effect on glycosaminoglycan production was detected. US stimulation also played an important role in counteracting chondrocyte phenotype loss, reducing collagen type I protein and mRNA expression by up to 68% and 90%, respectively, compared to untreated chondrocytes. Temperature remained stable during US stimulation. The US protocol 250 mW/cm² at 2.00 MHz (pulsed, 1 Hz, 50% duty cycle) for 20 min, every other day, appears to be the most effective protocol for eliciting cartilage components synthesis and decelerating the dedifferentiation process in chondrocytes and, thus, it should be further explored to regenerate articular cartilage.