Low-Intensity Pulsed Ultrasound Accelerates Tooth Movement via Activation of the BMP-2 Signaling Pathway

<div><p>The present study was designed to determine the underlying mechanism of low-intensity pulsed ultrasound (LIPUS) induced alveolar bone remodeling and the role of BMP-2 expression in a rat orthodontic tooth movement model. Orthodontic appliances were placed between the homonymy upper first molars and the upper central incisors in rats under general anesthesia, followed by daily 20-min LIPUS or sham LIPUS treatment beginning at day 0. Tooth movement distances and molecular changes were evaluated at each observation point. <i>In vitro</i> and <i>in vivo</i> studies were conducted to detect HGF (Hepatocyte growth factor)/Runx2/BMP-2 signaling pathways and receptor activator of NFκB ligand (RANKL) expression by quantitative real time PCR (qRT-PCR), Western blot and immunohistochemistry. At day 3, LIPUS had no effect on the rat orthodontic tooth movement distance and BMP-2-induced alveolar bone remodeling. However, beginning at day 5 and for the following time points, LIPUS significantly increased orthodontic tooth movement distance and BMP-2 signaling pathway and RANKL expression compared with the control group. The qRT-PCR and Western blot data <i>in vitro</i> and <i>in vivo</i> to study BMP-2 expression were consistent with the immunohistochemistry observations. The present study demonstrates that LIPUS promotes alveolar bone remodeling by stimulating the HGF/Runx2/BMP-2 signaling pathway and RANKL expression in a rat orthodontic tooth movement model, and LIPUS increased BMP-2 expression via Runx2 regulation.</p></div

LIPUS stimulation increased BMP-2 expression mediated by Runx2.

<p>(A). hPDL cells were incubated with HGF for 24 h, and BMP-2 mRNA was examined by qRT-PCR. (B and C) hPDL cells were incubated with HGF for 48 h, and BMP-2 protein amounts were detected by Western blotting. The data shows that HGF significantly increased BMP-2 expression. (D). Cells were transfected with Runx2 siRNA for 24 h followed by stimulation with LIPUS for 5 days, and BMP-2 mRNA expression was examined by qRT-PCR. (E and F) hPDL cells were transfected with Runx2 siRNA for 5 days, and BMP-2 protein expression was examined by Western blot. Transfection of cells with Runx2 siRNA reduced LIPUS-increased BMP-2 expression. *<i>P<0.05</i> (**<i>P<0.01</i>) as compared with the control group.</p

Oligodeoxynucleotide primers used for qRT-PCR.

<p>Oligodeoxynucleotide primers used for qRT-PCR.</p

LIPUS stimulation increased osteoclast number and RANKL expression on the pressure side.

<p>(A). Light micrographs of PDL tissue on the compressed side in rat on days 0, 3, and 7 after LIPUS stimulation. A few osteoclasts (arrowheads) are seen in the PDL proper on day 3 and day 7. #: PDL; &: alveolar bone, scale bar, 20 μm. (B). Osteoclast number was counted in the area indicated in Fig. 4A. The quantified data shown represent the mean ± SD for three rats, and each rat contains four random fields, and all the osteoclasts were verified by a pathologist. LIPUS stimulation 0 day vs LIPUS stimulation 3 day, <i>P<0.05;</i> 0 day vs 7 day, <i>P<0.01.</i> Rat upper first molars were stimulated with or without LIPUS for different time intervals, and RANKL mRNA (C) and protein amount (D, E) increased at 3 and 7 days after LIPUS stimulation than day 0. LIPUS stimulation 0 day vs LIPUS stimulation 3 day, <i>P<0.01;</i> 0 day vs 7 day, <i>P<0.001.</i></p

The methodology for tooth movement and a schematic diagram of LIPUS assembly for cell treatment.

<p>(A). View of rat oral cavity, showing that the upper first molar was moved mesially by a closed coil spring at 0.1N of orthodontic force. (B). The 6 well plate filled with medium was placed in the LIPUS field at a distance of 4 mm, which can generate optimized beam uniformity across the target cell region. The sterilized LIPUS transducer probe was suspended above the culture medium (using a clamp stand) partially immersed in the culture medium. The water bath was maintained at 37°C.</p

The effect of LIPUS stimulation on rat tooth movement.

<p>The amount of tooth movement in the LIPUS group was significantly greater than the control group on day 5, day 7, and day 14. *Significantly different from corresponding non-stimulation group (<i>P<0.05</i>). Values are shown as the mean ± SD, and n = 6.</p

LIPUS stimulation enhanced the BMP-2 signaling pathway gene expression <i>in vitro</i> and <i>in vivo</i>.

<p>(A). hPDL cells were cultured in the presence and absence of daily LIPUS stimulation. BMP-2 mRNA expression was determined using qRT-PCR. (B). BMP-2 protein (ROW 1; 45 kDa) can be detected in the control and LIPUS groups (1: CON; 2: LIPUS). The LIPUS groups showed higher expression of BMP-2 from day 5. (C). Quantification of BMP-2 protein expression in the LIPUS stimulation group was greater than that in the control group on days 5, 7, and 14. *indicates <i>P<0.05</i>. (D–F). Rat upper first molars were stimulated with or without LIPUS for different time intervals, and HGF, Runx2, BMP-2 were determined by qRT-PCR and Western blot, respectively. The data indicated that LIPUS increased HGF, Runx2, and BMP-2 mRNA (D: LIPUS stimulation 0 day vs LIPUS stimulation 3 day, <i>P<0.05;</i> 0 day vs 7 day, <i>P<0.01</i>) and protein expression (E, F: LIPUS stimulation 0 day vs LIPUS stimulation 3 day, <i>P<0.05;</i> 0 day vs 7 day, <i>P<0.01</i>) <i>in vivo</i>. The data are the mean ± SD of three separate experiments.</p