Reactive Oxygen Species Mediate Therapeutic Ultrasound-Induced, Mitogen-Activated Protein Kinase Activation in C28/I2 Chondrocytes

Low-intensity pulsed ultrasound (LIPUS) has been used for the treatment of non-healing fractures because of its therapeutic properties of stimulating enhancing endochondral bone formation. However, its mechanism of action remains unclear. In this study, we hypothesized that LIPUS activates mitogen-activated protein kinases through generation of reactive oxygen species. C28/I2 cells were stimulated with LIPUS for 10 and 20 min, while the control group was treated using a sham LIPUS transducer. Through quantitative reverse transcription polymerase chain reaction and immunoblot analyses, we determined that LIPUS application increased reactive oxygen species generation and cell viability in C28/I2 cells. There were increases in the phosphorylation level of ERK1/2 and in expression of SOX9, COL2 A1 and ACAN genes. These effects were reversed when cells were treated with diphenylene iodonium, which is known to inhibit NADPH oxidase. It was concluded that exposure of chondrocytes to LIPUS led to reactive oxygen species generation, which activated MAPK signaling and further increased chondrocyte-specific gene markers involved in chondrocyte differentiation and extracellular matrix formation.