Effects of low magnitude high frequency mechanical vibration combined with compressive force on human periodontal ligament cells in vitro

Effects of low magnitude high frequency mechanical vibration combined with compressive force on... Summary Objective Vibration can be used to accelerate tooth movement, though the exact mechanisms remain unclear. This study aimed to investigate the effects of low magnitude high frequency (LMHF) vibration combined with compressive force on periodontal ligament (PDL) cells in vitro. Materials and methods Human PDL cells were isolated from extracted premolar teeth of four individuals. To determine the optimal frequency for later used in combination with compressive force, three cycles of low-magnitude (0.3 g) vibrations at various frequencies (30, 60, or 90 Hz) were applied to PDL cells for 20 min every 24 h. To investigate the effects of vibration combined with compressive force, PDL cells were subjected to three cycles of optimal vibration frequency (V) or 1.5 g/cm2 compressive force for 48 h (C) or vibration combined with compressive force (VC). Cell viability was assessed using MTT assay. PGE2, soluble RANKL (sRANKL), and OPG production were quantified by ELISA. RANKL, OPG, and Runx2 expression were determined using real-time PCR. Results Cell viability was decreased in groups C and VC. PGE2 and RANKL, but not OPG, were increased in groups V, C, and VC, thus increasing the RANKL/OPG ratio. The highest level was observed in group VC. sRANKL was increased in groups V, C, and VC; however, no significant different between the experimental groups. Runx2 expression was reduced in groups C and VC. Conclusions Vibration increased PGE2, RANKL, and sRANKL, but not OPG and Runx2. Vibration had the additive effects on PGE2 and RANKL, but not sRANKL in compressed PDL cells. Introduction Orthodontic treatment usually takes a long time to complete, which can lead to several complications (1). Many attempts have been made to accelerate tooth movement, including physical (2), pharmacological (3), and surgical approaches (4). However, complications such as local pain, severe root resorption (5), and drug-induced side effects can occur. Low magnitude high frequency (LMHF) mechanical vibration is a non-invasive method that can be applied in conjunction with orthodontic treatment to increase the rate of tooth movement (6). A number of vibratory devices are commercially available, such as AcceleDent (OrthoAccel Technologies, Inc., Houston, Texas, USA). Several in vivo studies have investigated the effects of vibration during the acceleration of tooth movement in animal models (7) and humans (6, 8), but have reported conflicting results (9). Moreover, the mechanisms of action of vibration on the surrounding tissues and cells, either periodontal ligament (PDL) cells or bone cells, have not been determined. Therefore, it would be interesting to investigate the mechanisms of action and cellular responses to vibration during application of orthodontic force in vitro. Periodontal ligament cells play a major role in initiation of the remodelling process during orthodontic tooth movement (10). Compression of PDL is a prerequisite for tooth movement. The balance between RANKL and OPG in PDL cells regulates bone remodelling during tooth movement (11). Compressive force upregulates RANKL via a PGE2-dependent mechanism in PDL cells (12). PGE2 is an inflammatory mediator produced by PDL cells in response to mechanical stress that acts in autocrine and paracrine manners to stimulate RANKL expression and promote bone resorption (12). In addition, the direct action of prostaglandins on an increasing of osteoclast function and bone resorption has been reported (13). The transcription factor Runx2 also plays important role in osteoblastic differentiation and bone deposition (14). LMHF vibration has been reported to induce Runx2 in PDL stem cells (PDLSCs) (15). Although the effects of compressive force on PGE2, RANKL, and OPG in PDL cells have been determined (11,12), the effects of vibration combined with compressive force on these osteogenic factors in PDL cells have not yet been assessed. This study aimed to investigate whether vibration enhances or inhibits the osteogenic factors PGE2, RANKL, OPG, and Runx2 in compressed PDL cells. Materials and methods Cell culture This study was approved by the Institutional Ethics Committee Board of the Prince of Songkla University (EC5803-06-P-LR). Human PDL cells were scraped from healthy, non-carious premolar roots extracted for orthodontic treatments from four individuals (2 males and 2 females; 17–20 years of age). PDL cells were grown in normal culture medium (NCM) at 37°C in humidified incubator with 5 per cent CO2. NCM consisted of Dulbecco’s modified essential medium (DMEM; Gibco BRL, Grand Island, New York, USA), supplemented with 10 per cent foetal bovine serum (FBS; Gibco BRL), 1 per cent penicillin (10 000 U/ml)–streptomycin (10 000 µg/ml; Gibco BRL), and 1 per cent fungizone (250 µg/ml AmphotericinB; Gibco BRL). Cell preparations were established from each individual donor. All experiments were carried out using cell cultures at third to fifth passages and performed in triplicate using the four independently isolated cell preparations. Morphological analysis and characterization of PDL cells Periodontal ligament cells were identified by spindle-shaped cell morphology, the expression of Scleraxis mRNA which is the ligament-specific marker (16), the expression of Fibromodulin and Periostin mRNA which specifically expressed in PDL cells (17,18), and the ability to initiate an in vitro calcification after culture in osteogenic condition medium. To induce osteogenic differentiation, PDL cells were cultured in NCM supplemented with 50 µg/ml ascorbic acid, 10 mM β-glycerophosphate, and 0.1 µM dexamethasone (Sigma-Aldrich, St Louis, Missouri, USA) for 21 days. To observe calcium deposition, cells were stained with 2 per cent Alizarin Red stain solution (Sigma-Aldrich) and examined by phase contrast microscopy (Nikon Eclipse Ti-S; Nikon Instruments Inc., Melville, New York, USA). Determination of optimal LMHF vibration Periodontal ligament cells (1 × 105 cells) were seeded in 35 mm culture dishes and cultured in NCM to 70–80 per cent confluence, then the medium was changed to DMEM with 2 per cent FBS for 24 h to synchronize the cell cycle. Prior to application of mechanical stimulus, the culture medium was changed to NCM. Culture dishes were mounted onto the platform of a GJX-5 vibration calibrator (Beijing Sending Technology, Beijing, China) that generates perpendicular mechanical vibration when the platform is parallel with the ground (15), as illustrated in Figure 1A. Three cycles of low-magnitude (0.3 g) vibrations at various frequencies (30, 60, or 90 Hz) were applied to PDL cells for 20 min every 24 h. The first cycle was applied at time zero, so the total experimental time was 48 h. Non-vibrated control cells were cultured in a similar manner, but placed on a stationary plate for the same periods of time. The magnitude and frequencies of vibration were based on studies that reported positive bone remodelling (19, 20). Immediately after the end of mechanical vibration, cell viability was assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay. PGE2 levels were quantified using an enzyme-linked immunosorbent assay (ELISA). The expression of RANKL and OPG was quantified by quantitative real-time PCR (qPCR). The lowest frequency that led to a significant difference in the RANKL/OPG ratio compared to the control was selected as the optimal frequency. Application of LMHF vibration and compressive force Periodontal ligament cells were cultured and the cell cycle was synchronized as described above prior to the application of mechanical stimulus. Cells were randomly divided into four groups: control without mechanical stimulation (Con), selected optimal vibration frequency (V), compressive force (C), and vibration combined with compressive force (VC). Vibration was generated using the selected optimal vibration frequency as described above. Compressive force was performed at 1.5 g/cm2 for 48 h using a modified version of the method described by Kanzaki et al. (12). A glass cylinder containing acrylic mass was placed over the 70–80 per cent confluent monolayer in each 35 mm culture dish, as illustrated in Figure 1B. Vibration combined with compressive force group was done by mounted the compressed cell onto the platform of a GJX-5 vibration calibrator and vibrated at the selected optimal vibration frequency. Figure 1. View largeDownload slide Model used to generate vibration and compressive force in vitro. (A) GJX-5 vibration calibrator generates vibration perpendicular to the bottom of the culture dish. (B) PDL cells were continuously compressed using a glass cylinder containing acrylic mass with a total force of 1.5 g/cm2. Figure 1. View largeDownload slide Model used to generate vibration and compressive force in vitro. (A) GJX-5 vibration calibrator generates vibration perpendicular to the bottom of the culture dish. (B) PDL cells were continuously compressed using a glass cylinder containing acrylic mass with a total force of 1.5 g/cm2. Immediately after the end of mechanical stimulation, cell viability was assessed by the MTT assay. PGE2, soluble RANKL (sRANKL), and OPG levels were quantified using ELISA. The expression of RANKL, OPG, and Runx2 was quantified by qPCR. Cell viability assay The cell viability was determined with the MTT assay (Sigma-Aldrich) using a microplate spectrophotometer (Multiskan GO; Thermo Scientific, Waltham, Massachusetts, USA) at a wavelength of 570 nm. Percentage cell viability was calculated relative to the control. Quantification of PGE2, sRANKL, and OPG The protein levels of PGE2, sRANKL, and OPG in the cell culture media were determined using a commercially available kit (DuoSet® ELISA Development kit; R&D Systems, Minneapolis, Minnesota, USA) in accordance with the manufacturer’s instructions. Absorbance was determined using a Multiskan GO microplate spectrophotometer at 450 nm with wavelength correction at 540 nm. The protein levels were calculated by comparison with the standard curve. Values were normalized to total protein content, measured using Pierce™ BCA Protein Assay Kit (Thermo Scientific). Changes in experimental groups were expressed as fold changes relative to the control. RNA isolation and quantitative real-time PCR Total RNA was isolated from cultured cells using innuPREP DNA/RNA mini kits (Analytic-Jena, Konrad-Zuse-Strasse 1, Jena, Germany) according to the manufacturer’s protocol. The concentration and purity of isolated RNA were assessed using a spectrophotometer at 260 nm. Aliquots containing amounts 300 ng of total RNA were reverse transcribed to cDNA using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s protocol. Aliquots containing equal amounts of cDNA template were subjected to qPCR amplification on a Rotor-Gene® Q (Qiagen, Qiagen Str. 1, Hilden, Germany) using SensiFASTTM SYBR No-ROX Kit (Bioline Inc, Taunton, Massachusetts, USA) according to the manufacturer’s protocol. The primers for Scleraxis (16), Fibromodulin (16), Periostin (21), RANKL (22), OPG (16), Runx2 (15), and GAPDH (23) are listed in Table 1. Appropriate intron spanning primers of all genes were chosen in order to avoid co-amplification of genomic DNA. The polymerase activation started the PCR at 95°C for 2 min, then denaturing at 95°C for 5 s, following by annealing at a temperature optimized for each primer pair (Table 1) for 10 s, and an extension at 72°C for 20 s for 35 cycles. The fluorescence data were analysed using Rotor-Gene Q software version 2.0.2 (Build 3) to determine Ct values. The Ct values of interested gene were calculated in relation to GAPDH that served as an internal control. The internal control gene was validated to demonstrate that its expression was unaffected by the experiment. Gene expression levels were calculated using the 2−ΔΔCt method. Changes in experimental groups were expressed as fold changes relative to the control. All PCR efficiencies were comparable. To ensure the presence of single amplification products, melting curves analysis, and 1.5 per cent agarose gel electrophoresis of the PCR amplification products were performed. Table 1. Primers used for real-time PCR Gene and sequences Accession number Exon–exon junction spanning Product (BP) Annealing temperature (°C) Scleraxis F: 5ʹ-ACACCCAGCCCAAACAGAT-3ʹ R: 5ʹ-TCTTTCTGTCGCGGTCCTT-3ʹ NM_001080514.2 1, 2 75 60 Fibromodulin F: 5ʹ-GGGACGTGGTCACTCTCTG-3ʹ R: 5ʹ-CTGGGAGAGGGAGAAGAGC-3ʹ NM_002023.4 1, 2 93 60 Periostin F: 5ʹ-TGTTGCCCTGGTTATATGAG-3ʹ R: 5ʹ-ACTCGGTGCAAAGTAAGTGA-3ʹ NM_006475.2 3, 4 180 60 RANKL F: 5ʹ-TCCCATCTGGTTCCCATAAA-3ʹ R: 5ʹ-GGTGCTTCCTCCTTTCATCA-3ʹ NM_033012.3 6, 7 260 60 OPG F: 5ʹ-GAAGGGCGCTACCTTGAGAT-3ʹ R: 5ʹ-GCAAACTGTATTTCGCTCTGG-3ʹ NM_002546.3 2, 3 102 62 Runx2 F: 5ʹ-CAGATGGGACTGTGGTTACTGT-3ʹ R: 5ʹ-GTGAAGACGGTTATGGTCAAGG-3ʹ NM_001024630.3 4, 5 169 60 GAPDH F: 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ R: 5ʹ-ATGGTGGTGAAGACGCCAGT-3ʹ NM_002046.5 4, 5 142 62 Gene and sequences Accession number Exon–exon junction spanning Product (BP) Annealing temperature (°C) Scleraxis F: 5ʹ-ACACCCAGCCCAAACAGAT-3ʹ R: 5ʹ-TCTTTCTGTCGCGGTCCTT-3ʹ NM_001080514.2 1, 2 75 60 Fibromodulin F: 5ʹ-GGGACGTGGTCACTCTCTG-3ʹ R: 5ʹ-CTGGGAGAGGGAGAAGAGC-3ʹ NM_002023.4 1, 2 93 60 Periostin F: 5ʹ-TGTTGCCCTGGTTATATGAG-3ʹ R: 5ʹ-ACTCGGTGCAAAGTAAGTGA-3ʹ NM_006475.2 3, 4 180 60 RANKL F: 5ʹ-TCCCATCTGGTTCCCATAAA-3ʹ R: 5ʹ-GGTGCTTCCTCCTTTCATCA-3ʹ NM_033012.3 6, 7 260 60 OPG F: 5ʹ-GAAGGGCGCTACCTTGAGAT-3ʹ R: 5ʹ-GCAAACTGTATTTCGCTCTGG-3ʹ NM_002546.3 2, 3 102 62 Runx2 F: 5ʹ-CAGATGGGACTGTGGTTACTGT-3ʹ R: 5ʹ-GTGAAGACGGTTATGGTCAAGG-3ʹ NM_001024630.3 4, 5 169 60 GAPDH F: 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ R: 5ʹ-ATGGTGGTGAAGACGCCAGT-3ʹ NM_002046.5 4, 5 142 62 F, forward primer; R, reverse primer. View Large Table 1. Primers used for real-time PCR Gene and sequences Accession number Exon–exon junction spanning Product (BP) Annealing temperature (°C) Scleraxis F: 5ʹ-ACACCCAGCCCAAACAGAT-3ʹ R: 5ʹ-TCTTTCTGTCGCGGTCCTT-3ʹ NM_001080514.2 1, 2 75 60 Fibromodulin F: 5ʹ-GGGACGTGGTCACTCTCTG-3ʹ R: 5ʹ-CTGGGAGAGGGAGAAGAGC-3ʹ NM_002023.4 1, 2 93 60 Periostin F: 5ʹ-TGTTGCCCTGGTTATATGAG-3ʹ R: 5ʹ-ACTCGGTGCAAAGTAAGTGA-3ʹ NM_006475.2 3, 4 180 60 RANKL F: 5ʹ-TCCCATCTGGTTCCCATAAA-3ʹ R: 5ʹ-GGTGCTTCCTCCTTTCATCA-3ʹ NM_033012.3 6, 7 260 60 OPG F: 5ʹ-GAAGGGCGCTACCTTGAGAT-3ʹ R: 5ʹ-GCAAACTGTATTTCGCTCTGG-3ʹ NM_002546.3 2, 3 102 62 Runx2 F: 5ʹ-CAGATGGGACTGTGGTTACTGT-3ʹ R: 5ʹ-GTGAAGACGGTTATGGTCAAGG-3ʹ NM_001024630.3 4, 5 169 60 GAPDH F: 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ R: 5ʹ-ATGGTGGTGAAGACGCCAGT-3ʹ NM_002046.5 4, 5 142 62 Gene and sequences Accession number Exon–exon junction spanning Product (BP) Annealing temperature (°C) Scleraxis F: 5ʹ-ACACCCAGCCCAAACAGAT-3ʹ R: 5ʹ-TCTTTCTGTCGCGGTCCTT-3ʹ NM_001080514.2 1, 2 75 60 Fibromodulin F: 5ʹ-GGGACGTGGTCACTCTCTG-3ʹ R: 5ʹ-CTGGGAGAGGGAGAAGAGC-3ʹ NM_002023.4 1, 2 93 60 Periostin F: 5ʹ-TGTTGCCCTGGTTATATGAG-3ʹ R: 5ʹ-ACTCGGTGCAAAGTAAGTGA-3ʹ NM_006475.2 3, 4 180 60 RANKL F: 5ʹ-TCCCATCTGGTTCCCATAAA-3ʹ R: 5ʹ-GGTGCTTCCTCCTTTCATCA-3ʹ NM_033012.3 6, 7 260 60 OPG F: 5ʹ-GAAGGGCGCTACCTTGAGAT-3ʹ R: 5ʹ-GCAAACTGTATTTCGCTCTGG-3ʹ NM_002546.3 2, 3 102 62 Runx2 F: 5ʹ-CAGATGGGACTGTGGTTACTGT-3ʹ R: 5ʹ-GTGAAGACGGTTATGGTCAAGG-3ʹ NM_001024630.3 4, 5 169 60 GAPDH F: 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ R: 5ʹ-ATGGTGGTGAAGACGCCAGT-3ʹ NM_002046.5 4, 5 142 62 F, forward primer; R, reverse primer. View Large Statistical analysis All data are presented as the mean ± SD for the four independently isolated cell preparations assessed in triplicate. The Kruskal–Wallis test and Mann–Whitney U-test were performed using SPSS software version 17.0 (SPSS Inc., Chicago, Illinois, USA); P < 0.05 was defined as statistically significant. Results Morphological analysis and characterization of PDL cells The isolated cells exhibited a spindle-shaped morphology, expressed Scleraxis, Fibromodulin, and Periostin mRNA and had the ability to undergo calcification in vitro, confirming they were PDL cells (Figure 2). Figure 2. View largeDownload slide Characterization of the isolated PDL cells. (A) The cells exhibited spindle-shaped morphology. (B) Expression of Scleraxis (SCX), Fibromodulin (FMOD), and Periostin (POSTN) mRNA on 1.5 per cent agarose gel electrophoresis. (C) Alizarin Red staining after culture in osteogenic medium for 21 days. Figure 2. View largeDownload slide Characterization of the isolated PDL cells. (A) The cells exhibited spindle-shaped morphology. (B) Expression of Scleraxis (SCX), Fibromodulin (FMOD), and Periostin (POSTN) mRNA on 1.5 per cent agarose gel electrophoresis. (C) Alizarin Red staining after culture in osteogenic medium for 21 days. Effects of different vibration frequency and determination of optimal vibration frequency The vibration at all frequencies did not affect the viability of PDL cells (Figure 3A). PDL cells exposed to vibration at 30, 60, or 90 Hz had significantly higher PGE2 and RANKL than control cells (P = 0.014 and P = 0.021, respectively); however, no significant difference was found between the groups of different vibration frequency (Figure 3B and C). Vibration did not significantly affect the expression of OPG (Figure 3D). Therefore, the RANKL/OPG ratio significantly increased at all vibration frequencies (P = 0.021; Figure 3E). The frequency at 30 Hz was the lowest frequency that led to a significant difference in the RANKL/OPG ratio compared to the control, which was designated as the optimal vibration frequency for combination with compressive force. Figure 3. View largeDownload slide The cell viability, relative PGE2, and relative mRNA expression levels of RANKL, OPG, and the RANKL/OPG ratio in human PDL cells between the control group (Con) and the cells after exposed to three cycles of vibration at 30, 60, or 90 Hz, 0.3 g for 20 min every 24 h in vitro. (A) Vibration at all frequencies did not affect the viability of PDL cells. (B) PGE2 increased in all experimental groups. Values shown are expressed as fold changes relative to control levels. Absolute values of control group range from 24.48 ± 4.33 to 151.68 ± 1.16 pg/mg. (C) RANKL increased in all experimental groups. (D) OPG was not changed. (E) The RANKL/OPG ratio increased in all experimental groups. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Figure 3. View largeDownload slide The cell viability, relative PGE2, and relative mRNA expression levels of RANKL, OPG, and the RANKL/OPG ratio in human PDL cells between the control group (Con) and the cells after exposed to three cycles of vibration at 30, 60, or 90 Hz, 0.3 g for 20 min every 24 h in vitro. (A) Vibration at all frequencies did not affect the viability of PDL cells. (B) PGE2 increased in all experimental groups. Values shown are expressed as fold changes relative to control levels. Absolute values of control group range from 24.48 ± 4.33 to 151.68 ± 1.16 pg/mg. (C) RANKL increased in all experimental groups. (D) OPG was not changed. (E) The RANKL/OPG ratio increased in all experimental groups. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Effects of LMHF vibration and compressive force Cell viability was significantly decreased in groups C and VC (P = 0.014 and P = 0.014, respectively; Figure 4A). However, mechanical stimuli did not result in obvious morphologic changes in any treatment groups (Figure 4B). Figure 4. View largeDownload slide The viability and morphology of PDL cells between the control group (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. (A) Cell viability was decreased in groups C and VC. (B) Cell morphology observed with phase contrast microscopy found no obvious morphologic changes in all groups. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Figure 4. View largeDownload slide The viability and morphology of PDL cells between the control group (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. (A) Cell viability was decreased in groups C and VC. (B) Cell morphology observed with phase contrast microscopy found no obvious morphologic changes in all groups. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Cells in groups C and VC expressed significantly higher RANKL compared to the control (P = 0.021 and P = 0.021, respectively; Figure 5A), while OPG expression was not affected (Figure 5B). Therefore, cells in groups C and VC had significantly higher RANKL/OPG ratios than the control (P = 0.021 and P = 0.021, respectively; Figure 5C). In addition, the RANKL/OPG ratio in group VC was significantly higher than group C (P = 0.021; Figure 5C). Figure 5. View largeDownload slide The relative mRNA expression levels of RANKL, OPG, the RANKL/OPG ratio, and Runx2 in PDL cells between the control (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. (A) RANKL increased in all experimental groups. (B) OPG was not changed in all groups. (C) The RANKL/OPG ratio significantly increased in all experimental groups; the highest level was observed in group VC. (D) Runx2 was not changed in group V, while it significantly decreased in groups C and VC. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Figure 5. View largeDownload slide The relative mRNA expression levels of RANKL, OPG, the RANKL/OPG ratio, and Runx2 in PDL cells between the control (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. (A) RANKL increased in all experimental groups. (B) OPG was not changed in all groups. (C) The RANKL/OPG ratio significantly increased in all experimental groups; the highest level was observed in group VC. (D) Runx2 was not changed in group V, while it significantly decreased in groups C and VC. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Vibration alone (30 Hz) did not affect the expression of Runx2. In contrast, cells in groups C and VC significantly downregulated Runx2 expression compared to the control (P = 0.021 and P = 0.021, respectively; Figure 5D). PGE2 was significantly increased in PDL cells exposed to mechanical stimuli; the highest level was observed in group VC (Figure 6A). Cells in groups V, C, and VC had significantly higher levels of sRANKL compared to the control (P = 0.014, P = 0.014, and P = 0.014, respectively; Figure 6B); however, no significant difference was found between the experimental groups (Figure 6B). Mechanical stimuli did not significantly affect the production of OPG (Figure 6C). Figure 6. View largeDownload slide The relative PGE2, sRANKL, and OPG secretion from PDL cells between the control group (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. Values shown are expressed as fold changes relative to control. (A) PGE2 increased in all experimental groups, absolute values of control group range from 42.23 ± 9.29 to 81.55 ± 2.59 pg/mg. (B) sRANKL increased in all experimental groups, absolute values of control group range from 0.87 ± 0.11 to 3.33 ± 0.13 pg/mg. (C) OPG was not changed in all groups, absolute values of control group range from 14 055.26 ± 2772.11 to 18 378.01 ± 538.79 pg/mg. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Figure 6. View largeDownload slide The relative PGE2, sRANKL, and OPG secretion from PDL cells between the control group (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. Values shown are expressed as fold changes relative to control. (A) PGE2 increased in all experimental groups, absolute values of control group range from 42.23 ± 9.29 to 81.55 ± 2.59 pg/mg. (B) sRANKL increased in all experimental groups, absolute values of control group range from 0.87 ± 0.11 to 3.33 ± 0.13 pg/mg. (C) OPG was not changed in all groups, absolute values of control group range from 14 055.26 ± 2772.11 to 18 378.01 ± 538.79 pg/mg. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Discussion To examine the mechanism by which vibration accelerates tooth movement, we applied vibration in combination with compressive force to human PDL cells, mimicking the application of vibration on the compression side of the tooth during orthodontic treatment. To the best of our knowledge, there are no reports on the effects of vibration in combination with compressive force on PGE2, the RANKL/OPG ratio, and Runx2 in human PDL cells. Based on the study of Kanzaki et al. (12), RANKL expression increased in a force-dependent manner, with the peak response observed at 2 g/cm2 compressive force. We used a lower force (1.5 g/cm2) to investigate whether vibration enhanced or inhibited the effects of compressive force on the RANKL/OPG ratio. In addition, the effects of vibration depend on the frequency (24). We selected the frequency at 30 Hz as the optimal vibration frequency for combination with compressive force, which was the lowest frequency that led to a significant difference in the RANKL/OPG ratio compared to control cells. This study showed that vibration had the additive effects on PGE2 secretion, RANKL, and the RANKL/OPG ratio in compressed PDL cells, but had no effect on Runx2. When determined RANKL protein expression, mechanical stimuli were able to significantly stimulated sRANKL secretion from the PDL cells comparing to the control, but the protein level was not significantly different between experimental groups, which caused the discrepancy between the results of mRNA and protein levels. It is possible that the protein level of RANKL determined in this experiment was only soluble RANKL secreted into the culture media. There is also a membrane-bound form which needs to be determined (25). Further investigation is required to analyse both sRANKL and membrane-bound form of RANKL when PDL cells were exposed to the mechanical stimuli. Periodontal ligament cells may respond directly to vibration by increasing RANKL expression or indirectly upregulate RANKL in response to increased release of PGE2. A previous study reported that vibration in combination with orthodontic force accelerated the tooth movement with an increasing of IL-1β (8), which is an inflammatory mediator that can induce RANKL expression and osteoclast activities (26). In addition, Nishimura et al. (7) reported that vibration increased RANKL and the rate of tooth movement in a rat model. Collectively, it is possible that effects of vibration observed in our and previous studies (7,8) may correlated with the increasing of the inflammatory mediators by PDL cells. Further in vivo study is needed to confirm the effects of vibration to accelerate tooth movement on these mechanisms. We found that cell viability was significantly decreased in groups C and VC, however the cell morphology was unaffected; which was similar to previous reports (27,28). Indeed, compressive force can be increased up to 2 g/cm2 with no any damage to the cells (29). Our and previous studies indicated that mechanical stimuli affected cell proliferation but did not damage PDL cells. However, we found slightly decreased in viable cell number than the previous reports (27,28), which may be due to cell loss during removal of a glass cylinder used to generate compressive force. Moreover, the application of vibration in combination with compressive force did not increase the reduction in cell proliferation observed under compressive force alone. Compressive force increased PGE2 and RANKL in PDL cells; which was similar to previous reports (11, 12, 16, 27). The effects of compressive force on OPG were still controversial. In our study, compressive force had no significant effect on OPG expression, in agreement with previous studies (12, 29). However, one study reported compressive force (0.5–4.0 g/cm2) upregulated OPG (30), while another reported exposure to compressive force downregulated OPG (11). Overall, it appears that compressive force increases PGE2 and upregulates RANKL in PDL cells. However, it is possible that the expression of OPG responses to compressive force depends on several factors, including force magnitude, duration, and inter-individual variations. Further investigation using a larger number of samples and/or different compressive force protocols is needed to establish the mode of OPG production in compressed PDL cells. It is possible that PDL cells response to the mechanical stimuli on the expression of RANKL and OPG in different signal transduction pathways (12). A previous study reported that static compressive force significantly downregulated Runx2 in osteoblast-like cells (31,32). In our study, Runx2 was downregulated in the groups under compression, C and VC. It is possible that the application of compressive force can downregulate Runx2 in PDL cells in the similar manner as in the osteoblasts. Vibration with various frequencies had no effects on the viability of PDL cells, in agreement with previous report in mouse osteoblast-like cells (33). In contrast, Zhang et al. (15) reported that exposure of PDLSCs to vibration periodically over 3 days reduced the cell proliferation. This discrepancy may be due to the differences in the cell types, culture conditions, and vibration protocols used. All vibration frequencies tested significantly increased PGE2 and RANKL, but not OPG expression. PDL cells exposed to 30 Hz vibration significantly increased the protein levels of sRANKL, while had no effect on OPG. Lau et al. (19) showed that application of vibration with the same magnitude and frequency to osteocytes for 1 h significantly decreased PGE2 and RANKL, with had no effect on OPG. These inconsistent results may be due to cell types and/or different durations of vibration. The vibration-induced increases in PGE2 and the RANKL/OPG ratio were similar for all frequencies tested. Further studies at a wider range of frequencies are necessary to evaluate if the response of PDL cells to vibration is frequency-dependent. PGE2 and RANKL are known to stimulate osteoclast and bone resorption; therefore, this study indicates application of vibration may tend to promote toward bone resorption in PDL cells. In contrast, previous studies reported vibration enhanced bone formation in human PDLSCs (15), mouse osteoblast-like cells (33), and rat bone marrow-derived mesenchymal stromal cells (34); these differences may reflect the use of different research models. PDL cells may respond to mechanical stimuli in a different manner to bone cells or different vibration protocols may induce varied responses. Indeed, the response of cells to vibration may depend on several other factors, such as the magnitude (33), frequency (15), duration (34), and schedule of mechanical stimuli (35). As the cellular response depends on several factors, further studies with larger sample sizes and using different vibration protocols are necessary to confirm our findings and to define the ideal vibration regimen. In addition, the in vivo responses to mechanical stimulation are likely to be more complex than the in vitro. Further in vivo studies are needed to examine the effects of different force regimens on the gene expression, protein production, and osteoclast function. Additional molecular studies are required to investigate the mechanisms underlying the cellular responses to mechanical stimulation. This research sheds light on the mechanisms by which PDL cells respond to vibration and vibration combined with compressive force. This study establishes a range of parameters for further in vitro and in vivo analyses. Moreover, LMHF vibration may indirectly induce RANKL expression via a signalling pathway related to PGE2 in PDL cells. We aim to investigate the effects of PGE2 on the expression of RANKL and characterize this transduction pathway in future work. Conclusions LMHF vibration had no effect on the viability of PDL cells in vitro. PDL cells respond to 30, 60, and 90 Hz vibration by increasing PGE2, and upregulating RANKL leading to a higher RANKL/OPG ratio. LMHF vibration had the additive effects on PGE2, RANKL, and the RANKL/OPG ratio in compressed PDL cells, but had no effect on OPG and Runx2. Funding This work was supported by grant from Graduate School and Faculty of Dentistry, Prince of Songkla University. Conflict of interest None to declare. Acknowledgements The authors gratefully acknowledge Prof. Dr. Prasit Pavasant for his helpful suggestions. We thank Research facilitation and development unit, Faculty of Dentistry, Prince of Songkla University for kind assistance. References 1. Sundararaj , D. , Venkatachalapathy , S. , Tandon , A. and Pereira , A . ( 2015 ) Critical evaluation of incidence and prevalence of white spot lesions during fixed orthodontic appliance treatment: a meta-analysis . Journal of International Society of Preventive and Community Dentistry , 5 , 433 – 439 . Google Scholar CrossRef Search ADS PubMed 2. Kau , C.H. , Kantarci , A. , Shaughnessy , T. , Vachiramon , A. , Santiwong , P. , de la Fuente , A. , Skrenes , D. , Ma , D. and Brawn , P . ( 2013 ) Photobiomodulation accelerates orthodontic alignment in the early phase of treatment . Progress in Orthodontics , 14 , 30 . Google Scholar CrossRef Search ADS PubMed 3. Leiker , B.J. , Nanda , R.S. , Currier , G.F. , Howes , R.I. and Sinha , P.K . ( 1995 ) The effects of exogenous prostaglandins on orthodontic tooth movement in rats . American Journal of Orthodontics and Dentofacial Orthopedics , 108 , 380 – 388 . Google Scholar CrossRef Search ADS PubMed 4. Leethanakul , C. , Kanokkulchai , S. , Pongpanich , S. , Leepong , N. and Charoemratrote , C . ( 2014 ) Interseptal bone reduction on the rate of maxillary canine retraction . The Angle Orthodontist , 84 , 839 – 845 . Google Scholar CrossRef Search ADS PubMed 5. Brudvik , P. and Rygh , P . ( 1991 ) Root resorption after local injection of prostaglandin E2 during experimental tooth movement . European Journal of Orthodontics , 13 , 255 – 263 . Google Scholar CrossRef Search ADS PubMed 6. Kau , C.H. , Nguyen , J.T. and Jeryl , D . ( 2010 ) The clinical evaluation of a novel cyclical force generating device in orthodontics . Orthodontic Practice , 1 , 43 – 44 . 7. Nishimura , M. , Chiba , M. , Ohashi , T. , Sato , M. , Shimizu , Y. , Igarashi , K. and Mitani , H . ( 2008 ) Periodontal tissue activation by vibration: intermittent stimulation by resonance vibration accelerates experimental tooth movement in rats . American Journal of Orthodontics and Dentofacial Orthopedics , 133 , 572 – 583 . Google Scholar CrossRef Search ADS PubMed 8. Leethanakul , C. , Suamphan , S. , Jitpukdeebodintra , S. , Thongudomporn , U. and Charoemratrote , C . ( 2016 ) Vibratory stimulation increases interleukin-1 beta secretion during orthodontic tooth movement . The Angle Orthodontist , 86 , 74 – 80 . Google Scholar CrossRef Search ADS PubMed 9. Woodhouse , N.R. , DiBiase , A.T. , Johnson , N. , Slipper , C. , Grant , J. , Alsaleh , M. , Donaldson , A.N. and Cobourne , M.T . ( 2015 ) Supplemental vibrational force during orthodontic alignment: a randomized trial . Journal of Dental Research , 94 , 682 – 689 . Google Scholar CrossRef Search ADS PubMed 10. Middleton , J. , Jones , M. and Wilson , A . ( 1996 ) The role of the periodontal ligament in bone modeling: the initial development of a time-dependent finite element model . American Journal of Orthodontics and Dentofacial Orthopedics , 109 , 155 – 162 . Google Scholar CrossRef Search ADS PubMed 11. Nishijima , Y. , Yamaguchi , M. , Kojima , T. , Aihara , N. , Nakajima , R. and Kasai , K . ( 2006 ) Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro . Orthodontics and Craniofacial Research , 9 , 63 – 70 . Google Scholar CrossRef Search ADS PubMed 12. Kanzaki , H. , Chiba , M. , Shimizu , Y. and Mitani , H . ( 2002 ) Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis . Journal of Bone and Mineral Research , 17 , 210 – 220 . Google Scholar CrossRef Search ADS PubMed 13. Yamasaki , K. , Shibata , Y. , Imai , S. , Tani , Y. , Shibasaki , Y. and Fukuhara , T . ( 1984 ) Clinical application of prostaglandin E1 (PGE1) upon orthodontic tooth movement . American Journal of Orthodontics , 85 , 508 – 518 . Google Scholar CrossRef Search ADS PubMed 14. Marie , P.J . ( 2008 ) Transcription factors controlling osteoblastogenesis . Archives of Biochemistry and Biophysics , 473 , 98 – 105 . Google Scholar CrossRef Search ADS PubMed 15. Zhang , C. , Li , J. , Zhang , L. , Zhou , Y. , Hou , W. , Quan , H. , Li , X. , Chen , Y. and Yu , H . ( 2012 ) Effects of mechanical vibration on proliferation and osteogenic differentiation of human periodontal ligament stem cells . Archives of Oral Biology , 57 , 1395 – 1407 . Google Scholar CrossRef Search ADS PubMed 16. Römer , P. , Köstler , J. , Koretsi , V. and Proff , P . ( 2013 ) Endotoxins potentiate COX-2 and RANKL expression in compressed PDL cells . Clinical Oral Investigations , 17 , 2041 – 2048 . Google Scholar CrossRef Search ADS PubMed 17. Lallier , T.E. , Spencer , A. and Fowler , M.M . ( 2005 ) Transcript profiling of periodontal fibroblasts and osteoblasts . Journal of Periodontology , 76 , 1044 – 1055 . Google Scholar CrossRef Search ADS PubMed 18. Han , X. and Amar , S . ( 2002 ) Identification of genes differentially expressed in cultured human periodontal ligament fibroblasts vs. human gingival fibroblasts by DNA microarray analysis . Journal of Dental Research , 81 , 399 – 405 . Google Scholar CrossRef Search ADS PubMed 19. Lau , E. , Al-Dujaili , S. , Guenther , A. , Liu , D. , Wang , L. and You , L . ( 2010 ) Effect of low-magnitude, high-frequency vibration on osteocytes in the regulation of osteoclasts . Bone , 46 , 1508 – 1515 . Google Scholar CrossRef Search ADS PubMed 20. Rubin , C. , Judex , S. and Qin , Y.X . ( 2006 ) Low-level mechanical signals and their potential as a non-pharmacological intervention for osteoporosis . Age and Ageing , 35 Suppl 2 , ii32 – ii36 . Google Scholar CrossRef Search ADS PubMed 21. Manokawinchoke , J. , Limjeerajarus , N. , Limjeerajarus , C. , Sastravaha , P. , Everts , V. and Pavasant , P . ( 2015 ) Mechanical Force-induced TGFB1 Increases Expression of SOST/POSTN by hPDL Cells . Journal of Dental Research , 94 , 983 – 989 . Google Scholar CrossRef Search ADS PubMed 22. Hayata , K. , Weissbach , L. , Kawashima , M. , Rubah , H. and Shanbhag , A . ( 2005 ) Bisphosphonates modulate RANKL and OPG expression in human osteoblasts . The Orthopaedic Journal at Harvard Medical School , 7 , 81 – 83 . 23. Jiang , S.Y. , Shu , R. , Song , Z.C. and Xie , Y.F . ( 2011 ) Effects of enamel matrix proteins on proliferation, differentiation and attachment of human alveolar osteoblasts . Cell Proliferation , 44 , 372 – 379 . Google Scholar CrossRef Search ADS PubMed 24. Judex , S. , Lei , X. , Han , D. and Rubin , C . ( 2007 ) Low-magnitude mechanical signals that stimulate bone formation in the ovariectomized rat are dependent on the applied frequency but not on the strain magnitude . Journal of Biomechanics , 40 , 1333 – 1339 . Google Scholar CrossRef Search ADS PubMed 25. Nakashima , T. , Kobayashi , Y. , Yamasaki , S. , Kawakami , A. , Eguchi , K. , Sasaki , H. and Sakai , H . ( 2000 ) Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines . Biochemical and Biophysical Research Communications , 275 , 768 – 775 . Google Scholar CrossRef Search ADS PubMed 26. Fukushima , H. , Jimi , E. , Okamoto , F. , Motokawa , W. and Okabe , K . ( 2005 ) IL-1-induced receptor activator of NF-kappa B ligand in human periodontal ligament cells involves ERK-dependent PGE2 production . Bone , 36 , 267 – 275 . Google Scholar CrossRef Search ADS PubMed 27. Nettelhoff , L. , Grimm , S. , Jacobs , C. , Walter , C. , Pabst , A.M. , Goldschmitt , J. and Wehrbein , H . ( 2016 ) Influence of mechanical compression on human periodontal ligament fibroblasts and osteoblasts . Clinical Oral Investigations , 20 , 621 – 629 . Google Scholar CrossRef Search ADS PubMed 28. Kang , Y.G. , Nam , J.H. , Kim , K.H. and Lee , K.S . ( 2010 ) FAK pathway regulates PGE₂ production in compressed periodontal ligament cells . Journal of Dental Research , 89 , 1444 – 1449 . Google Scholar CrossRef Search ADS PubMed 29. Kim , J.W. , Lee , K.S. , Nahm , J.H. and Kang , Y.G . ( 2009 ) Effects of compressive stress on the expression of M-CSF, IL-1B, RANKL and OPG mRNA in periodontal ligament cells . The Korean Journal of Orthodontics , 39 , 248 – 256 . Google Scholar CrossRef Search ADS 30. Nakajima , R. , Yamaguchi , M. , Kojima , T. , Takano , M. and Kasai , K . ( 2008 ) Effects of compression force on fibroblast growth factor-2 and receptor activator of nuclear factor kappa B ligand production by periodontal ligament cells in vitro . Journal of Periodontal Research , 43 , 168 – 173 . Google Scholar CrossRef Search ADS PubMed 31. Tripuwabhrut , P. , Mustafa , M. , Gjerde , C.G. , Brudvik , P. and Mustafa , K . ( 2013 ) Effect of compressive force on human osteoblast-like cells and bone remodelling: an in vitro study . Archives of Oral Biology , 58 , 826 – 836 . Google Scholar CrossRef Search ADS PubMed 32. Zhou , S. , Zhang , J. , Zheng , H. , Zhou , Y. , Chen , F. and Lin , J . ( 2013 ) Inhibition of mechanical stress-induced NF-κB promotes bone formation . Oral Diseases , 19 , 59 – 64 . Google Scholar CrossRef Search ADS PubMed 33. Ota , T. , Chiba , M. and Hayashi , H . ( 2016 ) Vibrational stimulation induces osteoblast differentiation and the upregulation of osteogenic gene expression in vitro . Cytotechnology , 68 , 2287 – 2299 . Google Scholar CrossRef Search ADS PubMed 34. Zhou , Y. , Guan , X. , Zhu , Z. , Gao , S. , Zhang , C. , Li , C. , Zhou , K. , Hou , W. and Yu , H . ( 2011 ) Osteogenic differentiation of bone marrow-derived mesenchymal stromal cells on bone-derived scaffolds: effect of microvibration and role of ERK1/2 activation . European Cells and Materials , 22 , 12 – 25 . Google Scholar CrossRef Search ADS PubMed 35. Sen , B. , Xie , Z. , Case , N. , Styner , M. , Rubin , C.T. and Rubin , J . ( 2011 ) Mechanical signal influence on mesenchymal stem cell fate is enhanced by incorporation of refractory periods into the loading regimen . Journal of Biomechanics , 44 , 593 – 599 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The European Journal of Orthodontics Oxford University Press

Effects of low magnitude high frequency mechanical vibration combined with compressive force on human periodontal ligament cells in vitro

Loading next page...
 
/lp/ou_press/effects-of-low-magnitude-high-frequency-mechanical-vibration-combined-f2SAtDqepW
Publisher
Oxford University Press
Copyright
© The Author(s) 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com
ISSN
0141-5387
eISSN
1460-2210
D.O.I.
10.1093/ejo/cjx062
Publisher site
See Article on Publisher Site

Abstract

Summary Objective Vibration can be used to accelerate tooth movement, though the exact mechanisms remain unclear. This study aimed to investigate the effects of low magnitude high frequency (LMHF) vibration combined with compressive force on periodontal ligament (PDL) cells in vitro. Materials and methods Human PDL cells were isolated from extracted premolar teeth of four individuals. To determine the optimal frequency for later used in combination with compressive force, three cycles of low-magnitude (0.3 g) vibrations at various frequencies (30, 60, or 90 Hz) were applied to PDL cells for 20 min every 24 h. To investigate the effects of vibration combined with compressive force, PDL cells were subjected to three cycles of optimal vibration frequency (V) or 1.5 g/cm2 compressive force for 48 h (C) or vibration combined with compressive force (VC). Cell viability was assessed using MTT assay. PGE2, soluble RANKL (sRANKL), and OPG production were quantified by ELISA. RANKL, OPG, and Runx2 expression were determined using real-time PCR. Results Cell viability was decreased in groups C and VC. PGE2 and RANKL, but not OPG, were increased in groups V, C, and VC, thus increasing the RANKL/OPG ratio. The highest level was observed in group VC. sRANKL was increased in groups V, C, and VC; however, no significant different between the experimental groups. Runx2 expression was reduced in groups C and VC. Conclusions Vibration increased PGE2, RANKL, and sRANKL, but not OPG and Runx2. Vibration had the additive effects on PGE2 and RANKL, but not sRANKL in compressed PDL cells. Introduction Orthodontic treatment usually takes a long time to complete, which can lead to several complications (1). Many attempts have been made to accelerate tooth movement, including physical (2), pharmacological (3), and surgical approaches (4). However, complications such as local pain, severe root resorption (5), and drug-induced side effects can occur. Low magnitude high frequency (LMHF) mechanical vibration is a non-invasive method that can be applied in conjunction with orthodontic treatment to increase the rate of tooth movement (6). A number of vibratory devices are commercially available, such as AcceleDent (OrthoAccel Technologies, Inc., Houston, Texas, USA). Several in vivo studies have investigated the effects of vibration during the acceleration of tooth movement in animal models (7) and humans (6, 8), but have reported conflicting results (9). Moreover, the mechanisms of action of vibration on the surrounding tissues and cells, either periodontal ligament (PDL) cells or bone cells, have not been determined. Therefore, it would be interesting to investigate the mechanisms of action and cellular responses to vibration during application of orthodontic force in vitro. Periodontal ligament cells play a major role in initiation of the remodelling process during orthodontic tooth movement (10). Compression of PDL is a prerequisite for tooth movement. The balance between RANKL and OPG in PDL cells regulates bone remodelling during tooth movement (11). Compressive force upregulates RANKL via a PGE2-dependent mechanism in PDL cells (12). PGE2 is an inflammatory mediator produced by PDL cells in response to mechanical stress that acts in autocrine and paracrine manners to stimulate RANKL expression and promote bone resorption (12). In addition, the direct action of prostaglandins on an increasing of osteoclast function and bone resorption has been reported (13). The transcription factor Runx2 also plays important role in osteoblastic differentiation and bone deposition (14). LMHF vibration has been reported to induce Runx2 in PDL stem cells (PDLSCs) (15). Although the effects of compressive force on PGE2, RANKL, and OPG in PDL cells have been determined (11,12), the effects of vibration combined with compressive force on these osteogenic factors in PDL cells have not yet been assessed. This study aimed to investigate whether vibration enhances or inhibits the osteogenic factors PGE2, RANKL, OPG, and Runx2 in compressed PDL cells. Materials and methods Cell culture This study was approved by the Institutional Ethics Committee Board of the Prince of Songkla University (EC5803-06-P-LR). Human PDL cells were scraped from healthy, non-carious premolar roots extracted for orthodontic treatments from four individuals (2 males and 2 females; 17–20 years of age). PDL cells were grown in normal culture medium (NCM) at 37°C in humidified incubator with 5 per cent CO2. NCM consisted of Dulbecco’s modified essential medium (DMEM; Gibco BRL, Grand Island, New York, USA), supplemented with 10 per cent foetal bovine serum (FBS; Gibco BRL), 1 per cent penicillin (10 000 U/ml)–streptomycin (10 000 µg/ml; Gibco BRL), and 1 per cent fungizone (250 µg/ml AmphotericinB; Gibco BRL). Cell preparations were established from each individual donor. All experiments were carried out using cell cultures at third to fifth passages and performed in triplicate using the four independently isolated cell preparations. Morphological analysis and characterization of PDL cells Periodontal ligament cells were identified by spindle-shaped cell morphology, the expression of Scleraxis mRNA which is the ligament-specific marker (16), the expression of Fibromodulin and Periostin mRNA which specifically expressed in PDL cells (17,18), and the ability to initiate an in vitro calcification after culture in osteogenic condition medium. To induce osteogenic differentiation, PDL cells were cultured in NCM supplemented with 50 µg/ml ascorbic acid, 10 mM β-glycerophosphate, and 0.1 µM dexamethasone (Sigma-Aldrich, St Louis, Missouri, USA) for 21 days. To observe calcium deposition, cells were stained with 2 per cent Alizarin Red stain solution (Sigma-Aldrich) and examined by phase contrast microscopy (Nikon Eclipse Ti-S; Nikon Instruments Inc., Melville, New York, USA). Determination of optimal LMHF vibration Periodontal ligament cells (1 × 105 cells) were seeded in 35 mm culture dishes and cultured in NCM to 70–80 per cent confluence, then the medium was changed to DMEM with 2 per cent FBS for 24 h to synchronize the cell cycle. Prior to application of mechanical stimulus, the culture medium was changed to NCM. Culture dishes were mounted onto the platform of a GJX-5 vibration calibrator (Beijing Sending Technology, Beijing, China) that generates perpendicular mechanical vibration when the platform is parallel with the ground (15), as illustrated in Figure 1A. Three cycles of low-magnitude (0.3 g) vibrations at various frequencies (30, 60, or 90 Hz) were applied to PDL cells for 20 min every 24 h. The first cycle was applied at time zero, so the total experimental time was 48 h. Non-vibrated control cells were cultured in a similar manner, but placed on a stationary plate for the same periods of time. The magnitude and frequencies of vibration were based on studies that reported positive bone remodelling (19, 20). Immediately after the end of mechanical vibration, cell viability was assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay. PGE2 levels were quantified using an enzyme-linked immunosorbent assay (ELISA). The expression of RANKL and OPG was quantified by quantitative real-time PCR (qPCR). The lowest frequency that led to a significant difference in the RANKL/OPG ratio compared to the control was selected as the optimal frequency. Application of LMHF vibration and compressive force Periodontal ligament cells were cultured and the cell cycle was synchronized as described above prior to the application of mechanical stimulus. Cells were randomly divided into four groups: control without mechanical stimulation (Con), selected optimal vibration frequency (V), compressive force (C), and vibration combined with compressive force (VC). Vibration was generated using the selected optimal vibration frequency as described above. Compressive force was performed at 1.5 g/cm2 for 48 h using a modified version of the method described by Kanzaki et al. (12). A glass cylinder containing acrylic mass was placed over the 70–80 per cent confluent monolayer in each 35 mm culture dish, as illustrated in Figure 1B. Vibration combined with compressive force group was done by mounted the compressed cell onto the platform of a GJX-5 vibration calibrator and vibrated at the selected optimal vibration frequency. Figure 1. View largeDownload slide Model used to generate vibration and compressive force in vitro. (A) GJX-5 vibration calibrator generates vibration perpendicular to the bottom of the culture dish. (B) PDL cells were continuously compressed using a glass cylinder containing acrylic mass with a total force of 1.5 g/cm2. Figure 1. View largeDownload slide Model used to generate vibration and compressive force in vitro. (A) GJX-5 vibration calibrator generates vibration perpendicular to the bottom of the culture dish. (B) PDL cells were continuously compressed using a glass cylinder containing acrylic mass with a total force of 1.5 g/cm2. Immediately after the end of mechanical stimulation, cell viability was assessed by the MTT assay. PGE2, soluble RANKL (sRANKL), and OPG levels were quantified using ELISA. The expression of RANKL, OPG, and Runx2 was quantified by qPCR. Cell viability assay The cell viability was determined with the MTT assay (Sigma-Aldrich) using a microplate spectrophotometer (Multiskan GO; Thermo Scientific, Waltham, Massachusetts, USA) at a wavelength of 570 nm. Percentage cell viability was calculated relative to the control. Quantification of PGE2, sRANKL, and OPG The protein levels of PGE2, sRANKL, and OPG in the cell culture media were determined using a commercially available kit (DuoSet® ELISA Development kit; R&D Systems, Minneapolis, Minnesota, USA) in accordance with the manufacturer’s instructions. Absorbance was determined using a Multiskan GO microplate spectrophotometer at 450 nm with wavelength correction at 540 nm. The protein levels were calculated by comparison with the standard curve. Values were normalized to total protein content, measured using Pierce™ BCA Protein Assay Kit (Thermo Scientific). Changes in experimental groups were expressed as fold changes relative to the control. RNA isolation and quantitative real-time PCR Total RNA was isolated from cultured cells using innuPREP DNA/RNA mini kits (Analytic-Jena, Konrad-Zuse-Strasse 1, Jena, Germany) according to the manufacturer’s protocol. The concentration and purity of isolated RNA were assessed using a spectrophotometer at 260 nm. Aliquots containing amounts 300 ng of total RNA were reverse transcribed to cDNA using the SuperScript® III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s protocol. Aliquots containing equal amounts of cDNA template were subjected to qPCR amplification on a Rotor-Gene® Q (Qiagen, Qiagen Str. 1, Hilden, Germany) using SensiFASTTM SYBR No-ROX Kit (Bioline Inc, Taunton, Massachusetts, USA) according to the manufacturer’s protocol. The primers for Scleraxis (16), Fibromodulin (16), Periostin (21), RANKL (22), OPG (16), Runx2 (15), and GAPDH (23) are listed in Table 1. Appropriate intron spanning primers of all genes were chosen in order to avoid co-amplification of genomic DNA. The polymerase activation started the PCR at 95°C for 2 min, then denaturing at 95°C for 5 s, following by annealing at a temperature optimized for each primer pair (Table 1) for 10 s, and an extension at 72°C for 20 s for 35 cycles. The fluorescence data were analysed using Rotor-Gene Q software version 2.0.2 (Build 3) to determine Ct values. The Ct values of interested gene were calculated in relation to GAPDH that served as an internal control. The internal control gene was validated to demonstrate that its expression was unaffected by the experiment. Gene expression levels were calculated using the 2−ΔΔCt method. Changes in experimental groups were expressed as fold changes relative to the control. All PCR efficiencies were comparable. To ensure the presence of single amplification products, melting curves analysis, and 1.5 per cent agarose gel electrophoresis of the PCR amplification products were performed. Table 1. Primers used for real-time PCR Gene and sequences Accession number Exon–exon junction spanning Product (BP) Annealing temperature (°C) Scleraxis F: 5ʹ-ACACCCAGCCCAAACAGAT-3ʹ R: 5ʹ-TCTTTCTGTCGCGGTCCTT-3ʹ NM_001080514.2 1, 2 75 60 Fibromodulin F: 5ʹ-GGGACGTGGTCACTCTCTG-3ʹ R: 5ʹ-CTGGGAGAGGGAGAAGAGC-3ʹ NM_002023.4 1, 2 93 60 Periostin F: 5ʹ-TGTTGCCCTGGTTATATGAG-3ʹ R: 5ʹ-ACTCGGTGCAAAGTAAGTGA-3ʹ NM_006475.2 3, 4 180 60 RANKL F: 5ʹ-TCCCATCTGGTTCCCATAAA-3ʹ R: 5ʹ-GGTGCTTCCTCCTTTCATCA-3ʹ NM_033012.3 6, 7 260 60 OPG F: 5ʹ-GAAGGGCGCTACCTTGAGAT-3ʹ R: 5ʹ-GCAAACTGTATTTCGCTCTGG-3ʹ NM_002546.3 2, 3 102 62 Runx2 F: 5ʹ-CAGATGGGACTGTGGTTACTGT-3ʹ R: 5ʹ-GTGAAGACGGTTATGGTCAAGG-3ʹ NM_001024630.3 4, 5 169 60 GAPDH F: 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ R: 5ʹ-ATGGTGGTGAAGACGCCAGT-3ʹ NM_002046.5 4, 5 142 62 Gene and sequences Accession number Exon–exon junction spanning Product (BP) Annealing temperature (°C) Scleraxis F: 5ʹ-ACACCCAGCCCAAACAGAT-3ʹ R: 5ʹ-TCTTTCTGTCGCGGTCCTT-3ʹ NM_001080514.2 1, 2 75 60 Fibromodulin F: 5ʹ-GGGACGTGGTCACTCTCTG-3ʹ R: 5ʹ-CTGGGAGAGGGAGAAGAGC-3ʹ NM_002023.4 1, 2 93 60 Periostin F: 5ʹ-TGTTGCCCTGGTTATATGAG-3ʹ R: 5ʹ-ACTCGGTGCAAAGTAAGTGA-3ʹ NM_006475.2 3, 4 180 60 RANKL F: 5ʹ-TCCCATCTGGTTCCCATAAA-3ʹ R: 5ʹ-GGTGCTTCCTCCTTTCATCA-3ʹ NM_033012.3 6, 7 260 60 OPG F: 5ʹ-GAAGGGCGCTACCTTGAGAT-3ʹ R: 5ʹ-GCAAACTGTATTTCGCTCTGG-3ʹ NM_002546.3 2, 3 102 62 Runx2 F: 5ʹ-CAGATGGGACTGTGGTTACTGT-3ʹ R: 5ʹ-GTGAAGACGGTTATGGTCAAGG-3ʹ NM_001024630.3 4, 5 169 60 GAPDH F: 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ R: 5ʹ-ATGGTGGTGAAGACGCCAGT-3ʹ NM_002046.5 4, 5 142 62 F, forward primer; R, reverse primer. View Large Table 1. Primers used for real-time PCR Gene and sequences Accession number Exon–exon junction spanning Product (BP) Annealing temperature (°C) Scleraxis F: 5ʹ-ACACCCAGCCCAAACAGAT-3ʹ R: 5ʹ-TCTTTCTGTCGCGGTCCTT-3ʹ NM_001080514.2 1, 2 75 60 Fibromodulin F: 5ʹ-GGGACGTGGTCACTCTCTG-3ʹ R: 5ʹ-CTGGGAGAGGGAGAAGAGC-3ʹ NM_002023.4 1, 2 93 60 Periostin F: 5ʹ-TGTTGCCCTGGTTATATGAG-3ʹ R: 5ʹ-ACTCGGTGCAAAGTAAGTGA-3ʹ NM_006475.2 3, 4 180 60 RANKL F: 5ʹ-TCCCATCTGGTTCCCATAAA-3ʹ R: 5ʹ-GGTGCTTCCTCCTTTCATCA-3ʹ NM_033012.3 6, 7 260 60 OPG F: 5ʹ-GAAGGGCGCTACCTTGAGAT-3ʹ R: 5ʹ-GCAAACTGTATTTCGCTCTGG-3ʹ NM_002546.3 2, 3 102 62 Runx2 F: 5ʹ-CAGATGGGACTGTGGTTACTGT-3ʹ R: 5ʹ-GTGAAGACGGTTATGGTCAAGG-3ʹ NM_001024630.3 4, 5 169 60 GAPDH F: 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ R: 5ʹ-ATGGTGGTGAAGACGCCAGT-3ʹ NM_002046.5 4, 5 142 62 Gene and sequences Accession number Exon–exon junction spanning Product (BP) Annealing temperature (°C) Scleraxis F: 5ʹ-ACACCCAGCCCAAACAGAT-3ʹ R: 5ʹ-TCTTTCTGTCGCGGTCCTT-3ʹ NM_001080514.2 1, 2 75 60 Fibromodulin F: 5ʹ-GGGACGTGGTCACTCTCTG-3ʹ R: 5ʹ-CTGGGAGAGGGAGAAGAGC-3ʹ NM_002023.4 1, 2 93 60 Periostin F: 5ʹ-TGTTGCCCTGGTTATATGAG-3ʹ R: 5ʹ-ACTCGGTGCAAAGTAAGTGA-3ʹ NM_006475.2 3, 4 180 60 RANKL F: 5ʹ-TCCCATCTGGTTCCCATAAA-3ʹ R: 5ʹ-GGTGCTTCCTCCTTTCATCA-3ʹ NM_033012.3 6, 7 260 60 OPG F: 5ʹ-GAAGGGCGCTACCTTGAGAT-3ʹ R: 5ʹ-GCAAACTGTATTTCGCTCTGG-3ʹ NM_002546.3 2, 3 102 62 Runx2 F: 5ʹ-CAGATGGGACTGTGGTTACTGT-3ʹ R: 5ʹ-GTGAAGACGGTTATGGTCAAGG-3ʹ NM_001024630.3 4, 5 169 60 GAPDH F: 5ʹ-GCACCGTCAAGGCTGAGAAC-3ʹ R: 5ʹ-ATGGTGGTGAAGACGCCAGT-3ʹ NM_002046.5 4, 5 142 62 F, forward primer; R, reverse primer. View Large Statistical analysis All data are presented as the mean ± SD for the four independently isolated cell preparations assessed in triplicate. The Kruskal–Wallis test and Mann–Whitney U-test were performed using SPSS software version 17.0 (SPSS Inc., Chicago, Illinois, USA); P < 0.05 was defined as statistically significant. Results Morphological analysis and characterization of PDL cells The isolated cells exhibited a spindle-shaped morphology, expressed Scleraxis, Fibromodulin, and Periostin mRNA and had the ability to undergo calcification in vitro, confirming they were PDL cells (Figure 2). Figure 2. View largeDownload slide Characterization of the isolated PDL cells. (A) The cells exhibited spindle-shaped morphology. (B) Expression of Scleraxis (SCX), Fibromodulin (FMOD), and Periostin (POSTN) mRNA on 1.5 per cent agarose gel electrophoresis. (C) Alizarin Red staining after culture in osteogenic medium for 21 days. Figure 2. View largeDownload slide Characterization of the isolated PDL cells. (A) The cells exhibited spindle-shaped morphology. (B) Expression of Scleraxis (SCX), Fibromodulin (FMOD), and Periostin (POSTN) mRNA on 1.5 per cent agarose gel electrophoresis. (C) Alizarin Red staining after culture in osteogenic medium for 21 days. Effects of different vibration frequency and determination of optimal vibration frequency The vibration at all frequencies did not affect the viability of PDL cells (Figure 3A). PDL cells exposed to vibration at 30, 60, or 90 Hz had significantly higher PGE2 and RANKL than control cells (P = 0.014 and P = 0.021, respectively); however, no significant difference was found between the groups of different vibration frequency (Figure 3B and C). Vibration did not significantly affect the expression of OPG (Figure 3D). Therefore, the RANKL/OPG ratio significantly increased at all vibration frequencies (P = 0.021; Figure 3E). The frequency at 30 Hz was the lowest frequency that led to a significant difference in the RANKL/OPG ratio compared to the control, which was designated as the optimal vibration frequency for combination with compressive force. Figure 3. View largeDownload slide The cell viability, relative PGE2, and relative mRNA expression levels of RANKL, OPG, and the RANKL/OPG ratio in human PDL cells between the control group (Con) and the cells after exposed to three cycles of vibration at 30, 60, or 90 Hz, 0.3 g for 20 min every 24 h in vitro. (A) Vibration at all frequencies did not affect the viability of PDL cells. (B) PGE2 increased in all experimental groups. Values shown are expressed as fold changes relative to control levels. Absolute values of control group range from 24.48 ± 4.33 to 151.68 ± 1.16 pg/mg. (C) RANKL increased in all experimental groups. (D) OPG was not changed. (E) The RANKL/OPG ratio increased in all experimental groups. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Figure 3. View largeDownload slide The cell viability, relative PGE2, and relative mRNA expression levels of RANKL, OPG, and the RANKL/OPG ratio in human PDL cells between the control group (Con) and the cells after exposed to three cycles of vibration at 30, 60, or 90 Hz, 0.3 g for 20 min every 24 h in vitro. (A) Vibration at all frequencies did not affect the viability of PDL cells. (B) PGE2 increased in all experimental groups. Values shown are expressed as fold changes relative to control levels. Absolute values of control group range from 24.48 ± 4.33 to 151.68 ± 1.16 pg/mg. (C) RANKL increased in all experimental groups. (D) OPG was not changed. (E) The RANKL/OPG ratio increased in all experimental groups. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Effects of LMHF vibration and compressive force Cell viability was significantly decreased in groups C and VC (P = 0.014 and P = 0.014, respectively; Figure 4A). However, mechanical stimuli did not result in obvious morphologic changes in any treatment groups (Figure 4B). Figure 4. View largeDownload slide The viability and morphology of PDL cells between the control group (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. (A) Cell viability was decreased in groups C and VC. (B) Cell morphology observed with phase contrast microscopy found no obvious morphologic changes in all groups. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Figure 4. View largeDownload slide The viability and morphology of PDL cells between the control group (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. (A) Cell viability was decreased in groups C and VC. (B) Cell morphology observed with phase contrast microscopy found no obvious morphologic changes in all groups. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Cells in groups C and VC expressed significantly higher RANKL compared to the control (P = 0.021 and P = 0.021, respectively; Figure 5A), while OPG expression was not affected (Figure 5B). Therefore, cells in groups C and VC had significantly higher RANKL/OPG ratios than the control (P = 0.021 and P = 0.021, respectively; Figure 5C). In addition, the RANKL/OPG ratio in group VC was significantly higher than group C (P = 0.021; Figure 5C). Figure 5. View largeDownload slide The relative mRNA expression levels of RANKL, OPG, the RANKL/OPG ratio, and Runx2 in PDL cells between the control (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. (A) RANKL increased in all experimental groups. (B) OPG was not changed in all groups. (C) The RANKL/OPG ratio significantly increased in all experimental groups; the highest level was observed in group VC. (D) Runx2 was not changed in group V, while it significantly decreased in groups C and VC. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Figure 5. View largeDownload slide The relative mRNA expression levels of RANKL, OPG, the RANKL/OPG ratio, and Runx2 in PDL cells between the control (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. (A) RANKL increased in all experimental groups. (B) OPG was not changed in all groups. (C) The RANKL/OPG ratio significantly increased in all experimental groups; the highest level was observed in group VC. (D) Runx2 was not changed in group V, while it significantly decreased in groups C and VC. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Vibration alone (30 Hz) did not affect the expression of Runx2. In contrast, cells in groups C and VC significantly downregulated Runx2 expression compared to the control (P = 0.021 and P = 0.021, respectively; Figure 5D). PGE2 was significantly increased in PDL cells exposed to mechanical stimuli; the highest level was observed in group VC (Figure 6A). Cells in groups V, C, and VC had significantly higher levels of sRANKL compared to the control (P = 0.014, P = 0.014, and P = 0.014, respectively; Figure 6B); however, no significant difference was found between the experimental groups (Figure 6B). Mechanical stimuli did not significantly affect the production of OPG (Figure 6C). Figure 6. View largeDownload slide The relative PGE2, sRANKL, and OPG secretion from PDL cells between the control group (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. Values shown are expressed as fold changes relative to control. (A) PGE2 increased in all experimental groups, absolute values of control group range from 42.23 ± 9.29 to 81.55 ± 2.59 pg/mg. (B) sRANKL increased in all experimental groups, absolute values of control group range from 0.87 ± 0.11 to 3.33 ± 0.13 pg/mg. (C) OPG was not changed in all groups, absolute values of control group range from 14 055.26 ± 2772.11 to 18 378.01 ± 538.79 pg/mg. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Figure 6. View largeDownload slide The relative PGE2, sRANKL, and OPG secretion from PDL cells between the control group (Con) and the cells after exposed to three cycles of 30 Hz vibration at 0.3 g for 20 min every 24 h (V), 1.5 g/cm2 compressive force for 48 h (C), or vibration combined with compressive force (VC) in vitro. Values shown are expressed as fold changes relative to control. (A) PGE2 increased in all experimental groups, absolute values of control group range from 42.23 ± 9.29 to 81.55 ± 2.59 pg/mg. (B) sRANKL increased in all experimental groups, absolute values of control group range from 0.87 ± 0.11 to 3.33 ± 0.13 pg/mg. (C) OPG was not changed in all groups, absolute values of control group range from 14 055.26 ± 2772.11 to 18 378.01 ± 538.79 pg/mg. Values are mean ± SD of the four different cell lines, each assessed in triplicate (n = 4) (*P < 0.05, Mann–Whitney U-test). Discussion To examine the mechanism by which vibration accelerates tooth movement, we applied vibration in combination with compressive force to human PDL cells, mimicking the application of vibration on the compression side of the tooth during orthodontic treatment. To the best of our knowledge, there are no reports on the effects of vibration in combination with compressive force on PGE2, the RANKL/OPG ratio, and Runx2 in human PDL cells. Based on the study of Kanzaki et al. (12), RANKL expression increased in a force-dependent manner, with the peak response observed at 2 g/cm2 compressive force. We used a lower force (1.5 g/cm2) to investigate whether vibration enhanced or inhibited the effects of compressive force on the RANKL/OPG ratio. In addition, the effects of vibration depend on the frequency (24). We selected the frequency at 30 Hz as the optimal vibration frequency for combination with compressive force, which was the lowest frequency that led to a significant difference in the RANKL/OPG ratio compared to control cells. This study showed that vibration had the additive effects on PGE2 secretion, RANKL, and the RANKL/OPG ratio in compressed PDL cells, but had no effect on Runx2. When determined RANKL protein expression, mechanical stimuli were able to significantly stimulated sRANKL secretion from the PDL cells comparing to the control, but the protein level was not significantly different between experimental groups, which caused the discrepancy between the results of mRNA and protein levels. It is possible that the protein level of RANKL determined in this experiment was only soluble RANKL secreted into the culture media. There is also a membrane-bound form which needs to be determined (25). Further investigation is required to analyse both sRANKL and membrane-bound form of RANKL when PDL cells were exposed to the mechanical stimuli. Periodontal ligament cells may respond directly to vibration by increasing RANKL expression or indirectly upregulate RANKL in response to increased release of PGE2. A previous study reported that vibration in combination with orthodontic force accelerated the tooth movement with an increasing of IL-1β (8), which is an inflammatory mediator that can induce RANKL expression and osteoclast activities (26). In addition, Nishimura et al. (7) reported that vibration increased RANKL and the rate of tooth movement in a rat model. Collectively, it is possible that effects of vibration observed in our and previous studies (7,8) may correlated with the increasing of the inflammatory mediators by PDL cells. Further in vivo study is needed to confirm the effects of vibration to accelerate tooth movement on these mechanisms. We found that cell viability was significantly decreased in groups C and VC, however the cell morphology was unaffected; which was similar to previous reports (27,28). Indeed, compressive force can be increased up to 2 g/cm2 with no any damage to the cells (29). Our and previous studies indicated that mechanical stimuli affected cell proliferation but did not damage PDL cells. However, we found slightly decreased in viable cell number than the previous reports (27,28), which may be due to cell loss during removal of a glass cylinder used to generate compressive force. Moreover, the application of vibration in combination with compressive force did not increase the reduction in cell proliferation observed under compressive force alone. Compressive force increased PGE2 and RANKL in PDL cells; which was similar to previous reports (11, 12, 16, 27). The effects of compressive force on OPG were still controversial. In our study, compressive force had no significant effect on OPG expression, in agreement with previous studies (12, 29). However, one study reported compressive force (0.5–4.0 g/cm2) upregulated OPG (30), while another reported exposure to compressive force downregulated OPG (11). Overall, it appears that compressive force increases PGE2 and upregulates RANKL in PDL cells. However, it is possible that the expression of OPG responses to compressive force depends on several factors, including force magnitude, duration, and inter-individual variations. Further investigation using a larger number of samples and/or different compressive force protocols is needed to establish the mode of OPG production in compressed PDL cells. It is possible that PDL cells response to the mechanical stimuli on the expression of RANKL and OPG in different signal transduction pathways (12). A previous study reported that static compressive force significantly downregulated Runx2 in osteoblast-like cells (31,32). In our study, Runx2 was downregulated in the groups under compression, C and VC. It is possible that the application of compressive force can downregulate Runx2 in PDL cells in the similar manner as in the osteoblasts. Vibration with various frequencies had no effects on the viability of PDL cells, in agreement with previous report in mouse osteoblast-like cells (33). In contrast, Zhang et al. (15) reported that exposure of PDLSCs to vibration periodically over 3 days reduced the cell proliferation. This discrepancy may be due to the differences in the cell types, culture conditions, and vibration protocols used. All vibration frequencies tested significantly increased PGE2 and RANKL, but not OPG expression. PDL cells exposed to 30 Hz vibration significantly increased the protein levels of sRANKL, while had no effect on OPG. Lau et al. (19) showed that application of vibration with the same magnitude and frequency to osteocytes for 1 h significantly decreased PGE2 and RANKL, with had no effect on OPG. These inconsistent results may be due to cell types and/or different durations of vibration. The vibration-induced increases in PGE2 and the RANKL/OPG ratio were similar for all frequencies tested. Further studies at a wider range of frequencies are necessary to evaluate if the response of PDL cells to vibration is frequency-dependent. PGE2 and RANKL are known to stimulate osteoclast and bone resorption; therefore, this study indicates application of vibration may tend to promote toward bone resorption in PDL cells. In contrast, previous studies reported vibration enhanced bone formation in human PDLSCs (15), mouse osteoblast-like cells (33), and rat bone marrow-derived mesenchymal stromal cells (34); these differences may reflect the use of different research models. PDL cells may respond to mechanical stimuli in a different manner to bone cells or different vibration protocols may induce varied responses. Indeed, the response of cells to vibration may depend on several other factors, such as the magnitude (33), frequency (15), duration (34), and schedule of mechanical stimuli (35). As the cellular response depends on several factors, further studies with larger sample sizes and using different vibration protocols are necessary to confirm our findings and to define the ideal vibration regimen. In addition, the in vivo responses to mechanical stimulation are likely to be more complex than the in vitro. Further in vivo studies are needed to examine the effects of different force regimens on the gene expression, protein production, and osteoclast function. Additional molecular studies are required to investigate the mechanisms underlying the cellular responses to mechanical stimulation. This research sheds light on the mechanisms by which PDL cells respond to vibration and vibration combined with compressive force. This study establishes a range of parameters for further in vitro and in vivo analyses. Moreover, LMHF vibration may indirectly induce RANKL expression via a signalling pathway related to PGE2 in PDL cells. We aim to investigate the effects of PGE2 on the expression of RANKL and characterize this transduction pathway in future work. Conclusions LMHF vibration had no effect on the viability of PDL cells in vitro. PDL cells respond to 30, 60, and 90 Hz vibration by increasing PGE2, and upregulating RANKL leading to a higher RANKL/OPG ratio. LMHF vibration had the additive effects on PGE2, RANKL, and the RANKL/OPG ratio in compressed PDL cells, but had no effect on OPG and Runx2. Funding This work was supported by grant from Graduate School and Faculty of Dentistry, Prince of Songkla University. Conflict of interest None to declare. Acknowledgements The authors gratefully acknowledge Prof. Dr. Prasit Pavasant for his helpful suggestions. We thank Research facilitation and development unit, Faculty of Dentistry, Prince of Songkla University for kind assistance. References 1. Sundararaj , D. , Venkatachalapathy , S. , Tandon , A. and Pereira , A . ( 2015 ) Critical evaluation of incidence and prevalence of white spot lesions during fixed orthodontic appliance treatment: a meta-analysis . Journal of International Society of Preventive and Community Dentistry , 5 , 433 – 439 . Google Scholar CrossRef Search ADS PubMed 2. Kau , C.H. , Kantarci , A. , Shaughnessy , T. , Vachiramon , A. , Santiwong , P. , de la Fuente , A. , Skrenes , D. , Ma , D. and Brawn , P . ( 2013 ) Photobiomodulation accelerates orthodontic alignment in the early phase of treatment . Progress in Orthodontics , 14 , 30 . Google Scholar CrossRef Search ADS PubMed 3. Leiker , B.J. , Nanda , R.S. , Currier , G.F. , Howes , R.I. and Sinha , P.K . ( 1995 ) The effects of exogenous prostaglandins on orthodontic tooth movement in rats . American Journal of Orthodontics and Dentofacial Orthopedics , 108 , 380 – 388 . Google Scholar CrossRef Search ADS PubMed 4. Leethanakul , C. , Kanokkulchai , S. , Pongpanich , S. , Leepong , N. and Charoemratrote , C . ( 2014 ) Interseptal bone reduction on the rate of maxillary canine retraction . The Angle Orthodontist , 84 , 839 – 845 . Google Scholar CrossRef Search ADS PubMed 5. Brudvik , P. and Rygh , P . ( 1991 ) Root resorption after local injection of prostaglandin E2 during experimental tooth movement . European Journal of Orthodontics , 13 , 255 – 263 . Google Scholar CrossRef Search ADS PubMed 6. Kau , C.H. , Nguyen , J.T. and Jeryl , D . ( 2010 ) The clinical evaluation of a novel cyclical force generating device in orthodontics . Orthodontic Practice , 1 , 43 – 44 . 7. Nishimura , M. , Chiba , M. , Ohashi , T. , Sato , M. , Shimizu , Y. , Igarashi , K. and Mitani , H . ( 2008 ) Periodontal tissue activation by vibration: intermittent stimulation by resonance vibration accelerates experimental tooth movement in rats . American Journal of Orthodontics and Dentofacial Orthopedics , 133 , 572 – 583 . Google Scholar CrossRef Search ADS PubMed 8. Leethanakul , C. , Suamphan , S. , Jitpukdeebodintra , S. , Thongudomporn , U. and Charoemratrote , C . ( 2016 ) Vibratory stimulation increases interleukin-1 beta secretion during orthodontic tooth movement . The Angle Orthodontist , 86 , 74 – 80 . Google Scholar CrossRef Search ADS PubMed 9. Woodhouse , N.R. , DiBiase , A.T. , Johnson , N. , Slipper , C. , Grant , J. , Alsaleh , M. , Donaldson , A.N. and Cobourne , M.T . ( 2015 ) Supplemental vibrational force during orthodontic alignment: a randomized trial . Journal of Dental Research , 94 , 682 – 689 . Google Scholar CrossRef Search ADS PubMed 10. Middleton , J. , Jones , M. and Wilson , A . ( 1996 ) The role of the periodontal ligament in bone modeling: the initial development of a time-dependent finite element model . American Journal of Orthodontics and Dentofacial Orthopedics , 109 , 155 – 162 . Google Scholar CrossRef Search ADS PubMed 11. Nishijima , Y. , Yamaguchi , M. , Kojima , T. , Aihara , N. , Nakajima , R. and Kasai , K . ( 2006 ) Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro . Orthodontics and Craniofacial Research , 9 , 63 – 70 . Google Scholar CrossRef Search ADS PubMed 12. Kanzaki , H. , Chiba , M. , Shimizu , Y. and Mitani , H . ( 2002 ) Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis . Journal of Bone and Mineral Research , 17 , 210 – 220 . Google Scholar CrossRef Search ADS PubMed 13. Yamasaki , K. , Shibata , Y. , Imai , S. , Tani , Y. , Shibasaki , Y. and Fukuhara , T . ( 1984 ) Clinical application of prostaglandin E1 (PGE1) upon orthodontic tooth movement . American Journal of Orthodontics , 85 , 508 – 518 . Google Scholar CrossRef Search ADS PubMed 14. Marie , P.J . ( 2008 ) Transcription factors controlling osteoblastogenesis . Archives of Biochemistry and Biophysics , 473 , 98 – 105 . Google Scholar CrossRef Search ADS PubMed 15. Zhang , C. , Li , J. , Zhang , L. , Zhou , Y. , Hou , W. , Quan , H. , Li , X. , Chen , Y. and Yu , H . ( 2012 ) Effects of mechanical vibration on proliferation and osteogenic differentiation of human periodontal ligament stem cells . Archives of Oral Biology , 57 , 1395 – 1407 . Google Scholar CrossRef Search ADS PubMed 16. Römer , P. , Köstler , J. , Koretsi , V. and Proff , P . ( 2013 ) Endotoxins potentiate COX-2 and RANKL expression in compressed PDL cells . Clinical Oral Investigations , 17 , 2041 – 2048 . Google Scholar CrossRef Search ADS PubMed 17. Lallier , T.E. , Spencer , A. and Fowler , M.M . ( 2005 ) Transcript profiling of periodontal fibroblasts and osteoblasts . Journal of Periodontology , 76 , 1044 – 1055 . Google Scholar CrossRef Search ADS PubMed 18. Han , X. and Amar , S . ( 2002 ) Identification of genes differentially expressed in cultured human periodontal ligament fibroblasts vs. human gingival fibroblasts by DNA microarray analysis . Journal of Dental Research , 81 , 399 – 405 . Google Scholar CrossRef Search ADS PubMed 19. Lau , E. , Al-Dujaili , S. , Guenther , A. , Liu , D. , Wang , L. and You , L . ( 2010 ) Effect of low-magnitude, high-frequency vibration on osteocytes in the regulation of osteoclasts . Bone , 46 , 1508 – 1515 . Google Scholar CrossRef Search ADS PubMed 20. Rubin , C. , Judex , S. and Qin , Y.X . ( 2006 ) Low-level mechanical signals and their potential as a non-pharmacological intervention for osteoporosis . Age and Ageing , 35 Suppl 2 , ii32 – ii36 . Google Scholar CrossRef Search ADS PubMed 21. Manokawinchoke , J. , Limjeerajarus , N. , Limjeerajarus , C. , Sastravaha , P. , Everts , V. and Pavasant , P . ( 2015 ) Mechanical Force-induced TGFB1 Increases Expression of SOST/POSTN by hPDL Cells . Journal of Dental Research , 94 , 983 – 989 . Google Scholar CrossRef Search ADS PubMed 22. Hayata , K. , Weissbach , L. , Kawashima , M. , Rubah , H. and Shanbhag , A . ( 2005 ) Bisphosphonates modulate RANKL and OPG expression in human osteoblasts . The Orthopaedic Journal at Harvard Medical School , 7 , 81 – 83 . 23. Jiang , S.Y. , Shu , R. , Song , Z.C. and Xie , Y.F . ( 2011 ) Effects of enamel matrix proteins on proliferation, differentiation and attachment of human alveolar osteoblasts . Cell Proliferation , 44 , 372 – 379 . Google Scholar CrossRef Search ADS PubMed 24. Judex , S. , Lei , X. , Han , D. and Rubin , C . ( 2007 ) Low-magnitude mechanical signals that stimulate bone formation in the ovariectomized rat are dependent on the applied frequency but not on the strain magnitude . Journal of Biomechanics , 40 , 1333 – 1339 . Google Scholar CrossRef Search ADS PubMed 25. Nakashima , T. , Kobayashi , Y. , Yamasaki , S. , Kawakami , A. , Eguchi , K. , Sasaki , H. and Sakai , H . ( 2000 ) Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-kappaB ligand: modulation of the expression by osteotropic factors and cytokines . Biochemical and Biophysical Research Communications , 275 , 768 – 775 . Google Scholar CrossRef Search ADS PubMed 26. Fukushima , H. , Jimi , E. , Okamoto , F. , Motokawa , W. and Okabe , K . ( 2005 ) IL-1-induced receptor activator of NF-kappa B ligand in human periodontal ligament cells involves ERK-dependent PGE2 production . Bone , 36 , 267 – 275 . Google Scholar CrossRef Search ADS PubMed 27. Nettelhoff , L. , Grimm , S. , Jacobs , C. , Walter , C. , Pabst , A.M. , Goldschmitt , J. and Wehrbein , H . ( 2016 ) Influence of mechanical compression on human periodontal ligament fibroblasts and osteoblasts . Clinical Oral Investigations , 20 , 621 – 629 . Google Scholar CrossRef Search ADS PubMed 28. Kang , Y.G. , Nam , J.H. , Kim , K.H. and Lee , K.S . ( 2010 ) FAK pathway regulates PGE₂ production in compressed periodontal ligament cells . Journal of Dental Research , 89 , 1444 – 1449 . Google Scholar CrossRef Search ADS PubMed 29. Kim , J.W. , Lee , K.S. , Nahm , J.H. and Kang , Y.G . ( 2009 ) Effects of compressive stress on the expression of M-CSF, IL-1B, RANKL and OPG mRNA in periodontal ligament cells . The Korean Journal of Orthodontics , 39 , 248 – 256 . Google Scholar CrossRef Search ADS 30. Nakajima , R. , Yamaguchi , M. , Kojima , T. , Takano , M. and Kasai , K . ( 2008 ) Effects of compression force on fibroblast growth factor-2 and receptor activator of nuclear factor kappa B ligand production by periodontal ligament cells in vitro . Journal of Periodontal Research , 43 , 168 – 173 . Google Scholar CrossRef Search ADS PubMed 31. Tripuwabhrut , P. , Mustafa , M. , Gjerde , C.G. , Brudvik , P. and Mustafa , K . ( 2013 ) Effect of compressive force on human osteoblast-like cells and bone remodelling: an in vitro study . Archives of Oral Biology , 58 , 826 – 836 . Google Scholar CrossRef Search ADS PubMed 32. Zhou , S. , Zhang , J. , Zheng , H. , Zhou , Y. , Chen , F. and Lin , J . ( 2013 ) Inhibition of mechanical stress-induced NF-κB promotes bone formation . Oral Diseases , 19 , 59 – 64 . Google Scholar CrossRef Search ADS PubMed 33. Ota , T. , Chiba , M. and Hayashi , H . ( 2016 ) Vibrational stimulation induces osteoblast differentiation and the upregulation of osteogenic gene expression in vitro . Cytotechnology , 68 , 2287 – 2299 . Google Scholar CrossRef Search ADS PubMed 34. Zhou , Y. , Guan , X. , Zhu , Z. , Gao , S. , Zhang , C. , Li , C. , Zhou , K. , Hou , W. and Yu , H . ( 2011 ) Osteogenic differentiation of bone marrow-derived mesenchymal stromal cells on bone-derived scaffolds: effect of microvibration and role of ERK1/2 activation . European Cells and Materials , 22 , 12 – 25 . Google Scholar CrossRef Search ADS PubMed 35. Sen , B. , Xie , Z. , Case , N. , Styner , M. , Rubin , C.T. and Rubin , J . ( 2011 ) Mechanical signal influence on mesenchymal stem cell fate is enhanced by incorporation of refractory periods into the loading regimen . Journal of Biomechanics , 44 , 593 – 599 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

The European Journal of OrthodonticsOxford University Press

Published: Aug 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off