Comparison of low-intensity pulsed ultrasound and pulsed electromagnetic field treatments on OPG and RANKL expression in human osteoblast-like cells

Abstract

Objective: To compare two clinically applied treatments to stimulate bone healing-low-intensity pulsed ultrasound (LIPUS) and pulsed electromagnetic field (PEMF)-for their effects on RANKL and OPG expression in osteoblast-like cells in vitro.

Materials and methods: LIPUS or PEMF was applied to Saos-2 cells for 10 minutes or 3 hours. RANKL and OPG expressions were analyzed at 0, 4, 8, or 12 hours after treatment with real-time PCR. Secreted protein levels in culture supernatant were analyzed at the same posttreatment time points using specific ELISA assays.

Results: Neither LIPUS nor PEMF had an effect on RANKL protein expression. OPG protein was significantly increased by LIPUS after 0 and 4 hours (brief short-term effect) and was increased almost 2.5-fold by PEMF after 8 hours. The mRNA levels of OPG and RANKL were hardly affected by LIPUS treatment at any time point. PEMF induced a fivefold increase in RANKL mRNA expression at t = 0. A brief PEMF treatment of 10 minutes resulted in downregulation of RANKL expression after 0 and 4 hours and upregulation at 12 hours. OPG mRNA was downregulated after 8 hours.

Conclusion: The effects of LIPUS or PEMF expression on OPG and RANKL are limited. From our experiments, it seems that LIPUS treatment resulted in a quick protein response, while the response of cells to PEMF (3 hours) was delayed. The increase in OPG protein at 8 hours post PEMF treatment is indicative of reduction of osteolysis.

Figure 1

LIPUS exposure assembly. The culture plate filled with medium was positioned floating on water (37°C) with the transducers positioned 5 mm from the cells. The tank was covered with ultrasound-absorbing rubber. LIPUS was applied for 10 minutes.

Figure 2

PEMF exposure assembly. The flask filled with medium was placed inside the PEMF machine. PEMF was applied for 10 minutes or 3 hours.

Figure 3

Expression of mRNA of RANKL and OPG normalized to GAPDH and β-actin after (A) 10 minutes of LIPUS, (B) 10 minutes of PEMF, or (C) 3 hours of PEMF. (^a Significant difference from t  =  0; ^b significant difference from 4 hours; ^c significant difference from 8 hours (P < .05) * Significant difference from control (P < .05).

Figure 4

Expression of OPG protein after (A) 10 minutes LIPUS, (B) 10 minutes PEMF, and (C) 3 hours PEMF. (* Significant difference from control [P < .05]).