Free Radical Generation by Ultrasound Application

Objectives:
Previous studies have shown that Low Intensity Pulsed Ultrasound (LIPUS) induces stimulatory effect on the bone formation and other tissue regeneration. However, the exact mechanism of action is still unclear. The aim of this study was to evaluate the generation of reactive oxygen species (ROS) in the mesenchymal stem cells after LIPUS application.
Methods: Bone marrow stem cells were harvested from rat femur and were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics at 37°C in 5% CO₂. At P4 stage the cells were trypsinized and were suspended in HBSS in concentration of 1X10⁶ cells/ml. Electron Paramagnetic Resonance (EPR) spectrometry was used for the detection of ROS using 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) spin trap. LIPUS parameters used in the experiments were:1.5 MHz sine wave repeating at 1 kHz with temporal averaged intensity of 30 mW/ cm². Groups were divided according to treatment timings in: 5 min, 15 min and 20 min of LIPUS application. Readings were taken at one time period only i.e. immediately after LIPUS application. The cells without LIPUS application were used as the control group.
Results:
EPR spectra showed formation of a DMPO^•/OH adduct after LIPUS treatment; and the height of signal produced increased with increased LIPUS duration, i.e., a more intense signal was observed using 20 min LIPUS as compared to 5 min LIPUS. Experiments carried out in media alone did not show formation of DMPO^•/OH, indicating that cells contributed to the spectrum, which is possibly derived from the trapping of hydroxylradical (OH^•).
Conclusions: EPR assay showed that LIPUS led to generation of ROS, particularly OH^•. Further research is required to confirm these findings and to detect relevant gene expression and protein synthesis after LIPUS application.