Long wave ultrasound may enhance bone regeneration by altering OPG/RANKL ratio in human osteoblast-like cells

Human osteoblast cell line (MG63) cells were treated with long wave (45 kHz, intensity 30 mW/cm2) continuous ultrasound (US) for 5 min and incubated for various time periods following the treatment. The reverse transcriptase polymerase chain reaction (RT-PCR) technique was used for observing the genetic expression and real-time PCR for quantitative analysis of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) along with alkaline phosphatase (ALP), an early bone marker, and osteocalcin (OCN) a late marker. ELISA was performed to estimate the amount of the cytokine released into the culture media. The osteoblasts responded to US by significantly upregulating both the OPG mRNA and protein levels. There was no RANKL mRNA expression observed in both the US and control groups and the protein levels were also very low in both groups. There was also no TNF-α expression and the TNF-α protein levels were insignificant. ALP and OCN mRNA were significantly upregulated in the US group. To our knowledge, this is the first study that shows the effect of US on OPG, RANKL and TNF-α expression. US appears to upregulate OPG and may downregulate RANKL production. From these findings, we conclude that therapeutic ultrasound may increase bone regeneration by altering the OPG/RANKL ratio in the bone microenvironment.