Upregulation of MMP-13 and TIMP-1 expression in response to mechanical strain in MC3T3-E1 osteoblastic cells

<p>Abstract</p> <p>Background</p> <p>Mechanical strain plays a significant role in the regulation of bone matrix turnover, which is mediated in part by matrix metalloproteinase (MMP)-13 and tissue inhibitors of matrix metalloproteinase (TIMP)-1. However, little is known about the correlation between mechanical strain and osteoblastic cell activities, including extracellular matrix (ECM) metabolism. Herein, we determined the effect of different magnitudes of cyclic tensile strain (0%, 6%, 12%, and 18%) on MMP-13 and TIMP-1 mRNA and protein expression in MC3T3-E1 osteoblasts. Furthermore, we employed specific inhibitors to examine the role of distinct signal transduction pathways known to mediate cellular responses to mechanical strain.</p> <p>Results</p> <p>We identified a magnitude-dependent increase in MMP-13 and TIMP-1 mRNA and protein levels in response to mechanical strains corresponding to 6%, 12%, and 18% elongation. The strain-induced increases in MMP-13 and TIMP-1 mRNA expression were inhibited by PD098059 and cycloheximide, respectively.</p> <p>Conclusions</p> <p>Our results suggest a mechanism for the regulation of bone matrix metabolism mediated by the differential expression of MMP-13 and TIMP-1 in response to increasing magnitudes of mechanical strain.</p

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 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

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

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

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