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Effect of low-frequency mechanical vibration on orthodontic tooth movement
ORIGINAL ARTICLE
Effect of low-frequency mechanical vibration on orthodontic tooth movement Sumit Yadav,a Thomas Dobie,b Amir Assefnia,c Himank Gupta,c Zana Kalajzic,d and Ravindra Nandae Farmington, Conn
Introduction: Our objective was to investigate the effect of low-frequency mechanical vibration (LFMV) on the rate of tooth movement, bone volume fraction, tissue density, and the integrity of the periodontal ligament. Our null hypothesis was that there would be no difference in the amount of tooth movement between different values of LFMV. Methods: Sixty-four male CD1 mice, 12 weeks old, were used for orthodontic tooth movement. The mice were randomly divided into 2 groups: control groups (baseline; no spring 1 5 Hz; no spring 1 10 Hz; and no spring 1 20 Hz) and experimental groups (spring 1 no vibration; spring 1 5 Hz; spring 1 10 Hz; and spring 1 20 Hz). In the experimental groups, the first molars were moved mesially for 2 weeks using nickeltitanium coil springs delivering 10 g of force. In the control and experimental groups, LFMV was applied at 5, 10, or 20 Hz. Microfocus x-ray computed tomography analysis was used for tooth movement measurements, bone volume fraction, and tissue density. Additionally, immunostaining for sclerostin, tartrate-resistant acid phosphatase (TRAP) staining, and picrosirius red staining were used on the histologic sections. Simple descriptive statistics were used to summarize the data. Kruskal-Wallis tests were used to compare the outcomes across treatment groups. Results: LFMV did not increase the rate of orthodontic tooth movement. Microfocus x-ray computed tomography analysis showed increases in bone volume fractions and tissue densities with applications of LFMV. Sclerostin expression was decreased with 10 and 20 Hz vibrations in both the control and experimental groups. Additionally, the picrosirius staining showed that LFMV helped in maintaining the thickness and integrity of collagen fibers in the periodontal ligament. Conclusions: There was no significant increase in tooth movement by applying LFMV when compared with the control groups (spring 1 no vibration). (Am J Orthod Dentofacial Orthop 2015;148:440-9)
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ooth movement involves both remodeling and modeling of bone.1,2 Remodeling and modeling involve a coordinated action of osteoclasts and osteoblasts in response to mechanical loading.3 Moreover, inflammatory mediators (interleukin [IL]-1, IL-2, IL-6, IL-8, and tumor necrosis factor-alpha) are released after mechanical stimulus or injury, triggering the biologic process associated with orthodontic tooth movement (OTM).4–7
From the Health Center, University of Connecticut, Division of Orthdontics, Farmington, Conn. a Assistant professor. b Visiting assistant professor. c Resident. d Research associate. e Professor and head. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Supported by the American Association of Orthodontists Foundation and the Division of Orthodontics of the University of Connecticut. Address correspondence to: Sumit Yadav, Division of Orthodontics, University of Connecticut Health Center, 263 Farmington Ave, Room L7063 MC1725, Farmington, CT 06030; e-mail, [email protected]. Submitted, November 2014; revised and accepted, March 2015. 0889-5406/$36.00 Copyright Ó 2015 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2015.03.031
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Currently, orthodontic treatment requires approximately 24 to 30 months of intervention to complete the treatment.8–10 The longer duration of treatment is a great concern and poses high risks for caries, root resorption, and decreased patient compliance and satisfaction.8,10,11 Thus, accelerating OTM and shortening the total treatment duration is a primary goal of the orthodontist, and it can prevent detrimental effects of longer treatment time and increase patient satisfaction. Nishimura et al have shown that the application of cyclical forces (60 Hz) on the maxillary first molar increases the rate of OTM.12 However, the main drawback of the Nishimura study was the method of force application (transpalatal expansion spring). The force was applied to accelerate tooth movement in the first order (buccolingually) rather than in the second order (mesiodistally), which comprises the majority of OTM. Moreover, it may confound the actual tooth movement because of its skeletal effects.12 Studies have shown that whole body vibration (30, 45, and 90 Hz) may have an anabolic response on bone mass and architecture.13–15 Miles et al,16 in their randomized controlled trial, showed that
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application of 111 Hz of vibrational frequency for 20 minutes per day did not speed tooth movement when compared with the controls. Kalajzic et al17 showed the inhibitory effect of cyclical forces (30 Hz and 40 g of force applied with an electromechanical actuator) on OTM in rats; moreover, they showed the deleterious effects of the cyclical forces on the periodontal ligament (PDL). The major drawback of their model was higher cyclical force (40 g). The effect and mechanism of lowfrequency mechanical vibrations (LFMV) (#20 Hz) on OTM and paradental tissues still remain unclear. To our understanding, this is the first in-vivo study regarding the effect of LFMV (frequency, #20 Hz) on OTM. Recently, LFMV has gained interest in accelerating OTM by increasing alveolar bone turnover. The osteocytes in the bone tissue are thought to orchestrate “mechanotransduction” by reacting to different forms of mechanical loading through biologic signals.18,19 The role of osteocytes in bone remodeling and modeling has been well documented.18,19 It has been shown that osteocytes are the major source of sclerostin (product of SOST gene), and they antagonize the canonical Wnt signaling pathway, thus exhibiting an inhibitory effect on bone formation.20,21 Matsumoto et al22 demonstrated the role of osteocytes in resorption modeling during OTM (mechanotransduction) using osteocyteablated mice. Our null hypothesis was that there would be no difference in the amount of tooth movement between different amounts of LFMV. We had 3 specific aims: (1) to determine the effect of LFMV on the rate of tooth movement; (2) to quantify bone modeling and remodeling in both the control and experimental groups using microfocus computed tomography (micro-CT) and immunostaining; and (3) to determine the effect of LFMV on the PDL. MATERIAL AND METHODS
The Institutional Animal Care Committee of the University of Connecticut Health Center approved this study, which conformed to the ARRIVE guidelines. Data were obtained from 64 male CD1 mice (Charles River Laboratories, Wilmington, Mass; body weight, 24-30 g). The 12-week-old mice were randomly divided into 2 groups: control and experimental. The control group was further subdivided into 4 groups; each control group had 5 mice: (1) group 1 (baseline), no spring and no mechanical vibration; (2) group 2, no orthodontic spring, but 5 Hz vibration was applied to the maxillary first molars; (3) group 3, no orthodontic spring, but 10 Hz vibration was applied to the maxillary first molars; and (4) group 4, no orthodontic spring, but 20 Hz
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vibration was applied to the maxillary first molars. The experimental group was also subdivided into 4 groups, each with 11 mice: (1) group 5, orthodontic spring only but no vibration; (2) group 6, orthodontic spring and 5 Hz vibration applied to the maxillary first molars; (3) group 7, orthodontic spring and 10 Hz vibration applied to the maxillary first molars; (4) group 8, orthodontic spring and 20 Hz vibration applied to the maxillary first molars. The animals were allowed at least a week of acclimatization at our Health Center to compensate for their different origins. The animals were placed under general anesthesia with xylazine (13 mg/kg) and ketamine (87 mg/kg). A custom mouth-prop was fabricated from 0.036-in stainless steel wire and placed between the maxillary and mandibular incisors to hold the mouth open. A 0.004-in stainless steel ligature wire was passed beneath the contact between the maxillary first and second molars and threaded to the maxillary first molar (Fig 1, A). A low force/deflection rate nickel-titanium coil spring (Ultimate Wireforms, Bristol, Conn) delivering 10 g of force was attached to the 0.004-in stainless steel ligature around the first molar, and the other end of the spring was attached to the incisors with 0.004-in stainless steel wire. The force/deflection rate for the spring was determined to calibrate the amount of force produced by activation of the nickel-titanium coil spring. Additionally, grooves 0.5 mm from the gingival margin were prepared on the facial, mesial, and distal surfaces of the maxillary central incisors to prevent the ligatures from dislodging from the incisor because of their lingual curvature and eruption pattern. Selfetching primer (Transbond Plus; 3M Unitek, Monrovia, Calif) and light-cured adhesive resin cement (Transbond; 3M Unitek) were applied to the lingual surfaces of the maxillary first molars and incisors to secure the ligature wire. Moreover, to minimize the distal movement of the right incisor and to reinforce the anterior anchorage, the right and left incisors were joined together to act as a unit (Fig 1, A). After appliance insertion, the mice were allowed to recover with an incandescent light for warmth; they were returned to their cages once full ambulation and self-cleansing returned. The appliances were checked every other day, and additional light-cured bonding material was added if necessary. After anesthesia with ketamine (87 mg/kg) and xylazine (13 mg/kg), a custom mouth-prop fabricated from 0.017 3 0.025-in titanium-molybdenum alloy wire was placed between the maxillary and mandibular incisors to hold the mouth open. A feedback loop–controlled electromechanical actuator (model 3230; Bose Enduratec,
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Fig 1. A, Inserted nickel-titanium coil spring; B, application of LFMV on the maxillary right molar (the electromechanical actuator applies mechanical vibration at 5, 10, and 20 Hz at a compression force of 1 g); C, schematic of the tooth movement model with the spring attached to the maxillary first molar and maxillary incisors; D, intermolar distance (M1-M2) at day 14 in the experimental groups; E-H, sagittal micro-CT sections used for measuring intermolar distance in the control groups (E, baseline; F, no spring 1 5 Hz vibration; G, no spring 1 10 Hz vibration; H, no spring 1 20 Hz vibration); I-L, sagittal micro-CT sections used for measuring intermolar distance in the experimental groups (I, spring 1 no vibration; J, spring 1 5 Hz vibration; K, spring 1 10 Hz vibration; L, spring 1 20 Hz vibration).
Minnetonka, Minn) was used to apply unilateral LFMV to the occlusal surface of the maxillary right first molar along its long axis (Fig 1, B). Loading protocols for each animal consisted of 15 minutes of LFMV at 1 cN of force with the electromechanical actuator at a frequency of 5, 10, or 20 Hz (cycles/second) depending on the mouse's group. LFMV was applied to the maxillary right first molar at 3-day intervals (days 1, 4, 7, 10, and 13).
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After the 14 days of the experimental period, the animals were killed by inhalation of carbon dioxide followed by cervical dislocation. The maxilla was hemisected, and the attached soft tissues and muscles were removed. Subsequently, the hemisected maxilla was placed in 10% formalin for 5 days at 4 C. The samples were then decalcified in 14% ethylenediaminetetraacetic acid for 3 weeks and then processed for standard paraffin embedding. Serial
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Fig 2. Micro-CT data showing BVF and tissue density. Each value in each graph represents the mean 6 the standard deviation: A, region of interest where the bone parameters (BVF and tissue density) were measured; B, BVF values in the control groups (significantly [*] higher BVF in the no spring 1 5 Hz vibration group compared with the no spring 1 20 Hz vibration group); C, BVF values in the experimental groups; BVF decreases with tooth movement; baseline had a significantly higher BVF compared with the spring 1 no vibration group, the spring 1 5 Hz vibration group, the spring 1 10 Hz vibration group, and the spring 1 20 Hz vibration group; moreover, the spring 1 no vibration group had significantly less BVF than did the spring 1 5 Hz vibration (*) and the spring 1 10 Hz vibration (#) groups. D, Tissue density in the control groups; note the trend for an increase in tissue density with vibration; the spring 1 5 Hz group had significantly higher bone density compared with baseline (*); a,b,c,d signifies P \ 0.5. E, tissue density in the experimental groups.
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Fig 3. Sclerostin expression in the control and experimental groups on the compression and tension sides in alveolar bone. Upper panel, control groups; lower panel, experimental groups. A-D, Tension side in the control groups (A, baseline; B, no spring 6 5 Hz; C, no spring 6 10 Hz; D, no spring 6 20 Hz); E-H, tension side in experimental groups (E, spring 1 no vibration; F, spring 15 Hz; G, spring 110 Hz; H, spring 120 Hz); I-L,compression side in the control groups (I, baseline; J, no spring 6 5 Hz; K, no spring 6 10 Hz; L: no spring 6 20 HzA); M-P, compression side in the experimental groups (M, spring 1 no vibration; N, spring 15 Hz; O, spring 1 10 Hz; P, spring 120 Hz). Note the decrease in sclerostin expression in the 10 Hz (C, G, K, and O) and 20 Hz (D, H, L, and P) vibrations in both the control and experimental groups. Q, with no expression of sclerostin, is a negative control. The double white arrows signify the cells positive for sclerostin.
sagittal sections (5 mm) were obtained from the mesial and distal roots of the maxillary right first molar. All the animals in the different control (5 in each group) and experimental (11 in each group) groups were used to measure tooth movements. Threedimensional image arrays of the hemisected right maxillae were collected using micro-CT. OTM was defined and measured as the distance between the maxillary first and second molars at the most mesial point of the second molar crown and the most distal point of the first molar crown.17,23 The distance at day 0 was 0 mm in all groups (ie, the convex crown surfaces were touching). The OTM measurements were
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done in the sagittal plane, locating the image plane that showed the most root structure. The original 2dimensional image was then magnified 10 times for more precise line drawings, which were made at the closest proximity of the 2 convex molar crown surfaces. The OTM was measured in the 3 sagittal sections of each animal in all groups. Micro-CT images were used for quantitative analyses of bone changes occurring in the region of the maxillary first molar. Changes in the alveolar bone were studied by analyzing the furcation area of the maxillary first molar. The region of interest for the alveolar bone analysis was defined vertically as the
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Fig 4. Histologic examination of PDL collagen fibers on the tension side by polarized light microscopy. The sirius red stained PDL shows a more organized PDL in the control groups (A, baseline; B, no spring 1 5 Hz; C, no spring 1 10 Hz; D, no spring 1 20 Hz) compared with the experimental groups (E, spring 1 no vibration; F, spring 1 5 Hz vibration; G, spring 1 10 Hz vibration; H, spring 1 20 Hz vibration). Note that vibration only does not lead to disorganization of PDL collagen fibers. The spring 1 no vibration group had disorganized and thin PDL fibers. Note that the spring 1 vibration (F, G, and H) had more thickened and organized fibers (shown by the red staining) compared with the spring 1 no vibration group. A.B., Alveolar bone; P, periodontal ligament; De, dentin.
most occlusal point of the furcation to the apex of the maxillary first molar roots; transversely, it was defined as the space between the buccal and lingual cortical bone; sagittally, it included 50 sections (10 mm) beginning at the mesial root and continuing distally (Fig 2, A). The parameters studied and measured using the established algorithms were bone volume fraction (BVF) and tissue density. Histology, immunohistochemistry, tartrate-resistant acid phosphatase (TRAP), and picrosirius red staining histology were performed on each section. After blocking for 10 minutes at room temperature with 1x universal blocking reagent (HK085-5k; BioGenex, Fremont, Calif), deparaffinized histologic sections were incubated with goat polyclonal anti-SOST antibody (AF1589; R&D Systems, Minneapolis, Minn) overnight at 140 C at a concentration of 5 mg per milliliter. Subsequently, the sections were washed with phosphate-buffered saline and incubated with Alexa Fluor 594 donkey anti-goat IgG (A-11058; Life Technologies, Grand Island, NY) at
a concentration of 1:300 for 1 hour at room temperature and mounted with a suspension of 50% glycerol in phosphate-buffered saline containing the nuclear stain (Hoechst H3570; Life Technologies) at a concentration of 1:5000. TRAP staining was performed using a leukocyte acid phosphatase (TRAP) kit (386-1 KT; Sigma-Aldrich, St Louis, Mo) according to the manufacturer's instructions. TRAP positive, multinucleated cells were counted on the alveolar bone surfaces on the mesial sides of the distobuccal roots. The area for quantification included a square with 1 side extending from the apex to the bifurcation, and the other side extending 200 mm from the PDL border inside the alveolar bone. The osteoclast numbers were counted in 6 sections from 4 mice in each group, and the values were then averaged for each animal to run a statistical test. Deparaffinized sections were also stained with 1% picrosirius red for 1 hour, washed with acidified water (0.5% acetic acid water), and dehydrated with serial
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ethanol washes before mounting. The sections were examined with a fluorescent microscope (Carl Zeiss, Thornwood, NY). Statistical analysis
Simple descriptive statistics were used to summarize the data. The outcomes examined included intermolar distance, BVF, and tissue density. Because of the sample size, nonparametric tests were used to examine the association between the outcome variables and the treatment groups (spring 1 no vibration, spring 1 5 Hz, spring 1 10 Hz, and spring 1 20 Hz). The Kolmogorov-Smirnov test was used to examine the distribution of the outcome variables. Kruskal-Wallis tests were used to compare the outcomes across treatment groups. Pairwise comparisons between different groups were conducted with the Mann-Whitney U test. All statistical tests were 2 sided, and a P value of \0.05 was deemed to be statistically significant for the KruskalWallis test. Because of the multiple pairwise comparisons used, to minimize type 1 errors, Bonferroni corrections were used. RESULTS
All mice in the study remained healthy and had a slight increase in body weight. The delta displacement of the molar was the same throughout the vibration to maintain 1 g of force with the vibrator tip. The distance between the first and second molars in the control groups was 0 mm (Fig 1, C), whereas there was no statistically significant difference in the experimental groups after 14 days of orthodontic force application (Fig 1, D). However, the maximum tooth movement was observed in the spring 1 10 Hz group (0.248 6 0.074 mm), and the least was in the spring 1 20 Hz group (0.200 6 0.075 mm). For the bone parameters, the micro-CT analysis showed a statistically significant (P \0.05) decrease in BVF when the control groups were compared with the experimental groups (baseline vs spring 1 no vibration
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[P \0.05]; no spring 1 5 Hz vs spring 1 5 Hz [P \0.05]; no spring 1 10 Hz vs spring 1 10 Hz [P \0.05]; and no spring 1 20 Hz vs spring 1 20 Hz [P \0.05]) (Fig 2, C and D). Moreover, in the control groups, the no spring 1 5 Hz group had a significantly greater BVF than did the no spring 1 20 Hz group. In the experimental groups, the BVF was significantly (P \0.05) lower for the spring 1 no vibration group, the spring 1 5 Hz group, the spring 1 10 Hz group, and the spring 1 20 Hz group, when compared with the baseline (Fig 2, B and C). In the control groups, there was a trend for an increase in tissue density with mechanical vibration; however, the no spring 1 5 Hz group had significantly (P \0.05) more tissue density than did the baseline control. Similarly, in the experimental groups, there was a trend for an increase in bone density with mechanical vibration, but it was not statistically significant among the experimental groups (Fig 2, D and E). Our data show reductions in sclerostin expression on the tension and compression sides with 10 and 20 Hz in both the control (no spring 1 10 Hz, and no spring 1 20 Hz) and the experimental (spring 1 10 Hz, and spring 1 20 Hz) groups (Fig 3). However, there was no decrease in sclerostin expression in either the control or the experimental group with 5 Hz vibration (Fig 3). Our data show that LFMV in the control group at 5, 10, and 20 Hz did not affect the quality of the collagen fibers. However, orthodontic loading does make a huge impact on the integrity and the quality of the fibers (Fig 4, E). Our experimental groups showed that LFMV at 5, 10, and 20 Hz brings back the integrity and the thickness of collagen fibers that were lost due to orthodontic loading. The spring-only group (orthodontic loading) had the least thickened and wavy fibers, whereas the other experimental groups (spring 1 5 Hz, spring 1 10 Hz, and spring 1 20 Hz) had thick collagen fibers, as shown by the dark red stain. Our experimental groups showed significantly increased numbers of osteoclasts when compared with
Fig 5. Quantification and histologic examination of osteoclast numbers at day 14: quantification of osteoclast numbers in A, different control groups and B, different experimental groups; *significantly higher osteoclast numbers in the spring 1 no vibration group compared with the baseline control. Histologic examinations in: C, the different control groups at baseline; D, no spring 1 5 Hz; E, no spring 1 10 Hz; F, no spring 1 20 Hz; and experimental groups: G, spring 1 no vibration; H, spring 1 5 Hz vibration; I, spring 1 10 Hz vibration; J, spring 1 20 Hz vibration. TRAP positive cells were higher in the PDL and alveolar bone in the spring 1 no vibration group (G). A.B., Alveolar bone; P, periodontal ligament; De, dentin.
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the control groups (spring 1 no vibration [P \0.05] vs baseline; spring 1 5 Hz [P \0.05] vs no spring 1 5 Hz; spring 1 10 Hz [P \0.05] vs no spring 1 10 Hz; and spring 1 20 Hz [P \0.05] vs no spring 1 20 Hz [P \0.05]) (Fig 5). However, in the control groups, there were no significant differences in osteoclast numbers, implying that LFMV does not affect the osteoclast numbers in molars not subjected to tooth movement. In the experimental groups, there was a trend toward a decrease in osteoclast numbers with LFMV, although it was not statistically significant. DISCUSSION
Our null hypothesis that there would be no difference in the amount of tooth movement between the experimental groups (spring 1 no vibration, spring 1 5 Hz vibration, spring 1 10 Hz vibration, and spring 1 20 Hz vibration) was accepted. We selected LFMV (5, 10, and 20 Hz) for our study because Kalajzic et al17 showed that higher cyclical forces inhibit OTM and are deleterious to the PDL. Our results of OTM with LFMV were contrary to those of Liu et al24 and Nishimura et al12; a plausible reason was a different tooth movement model. Nishimura et al and Liu et al used transpalatal expansion springs (orthodontic load in the first order), whereas we used nickel-titanium coil springs (orthodontic load in the second order), and the orthodontic force was in the mesial direction. In the transpalatal model, the increase in OTM can be due to both skeletal and dental effects, whereas in our model, the OTM was primarily due to dental effects because we used adult mice, in which growth of the alveolar bone was complete. OTM primarily depends on bone volume and bone density (quantity and quality of bone). In this study, there was a trend toward an increase in tissue density with LFMV in both the control and experimental groups. Moreover, a similar trend toward an increase in BVF in the experimental group was noted after the application of LFMV. Orthodontic loading decreased the BVF (baseline, 79.24%; spring 1 no vibration, 53.45%), which was recovered by LFMV (spring 1 5 Hz, 64.45%; spring 1 10 Hz, 66.28%; and spring 1 20 Hz, 59.54%). Moreover, the spring 1 no vibration group had significantly less (P \0.05) BVF than did the spring 1 5 Hz and the spring 1 10 Hz groups. Similarly, the results regarding BVF were shown by Kalajzic et al,17 and probably the reason could be inhibition of osteoclastogenesis with LFMV. Furthermore, Vij and Mao25 showed that cyclical loading (4 Hz and 300 mN) can cause sutural growth, and Alikhani et al26 showed that application of high-frequency
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acceleration significantly increases the rate of alveolar bone formation. Moreover, our study showed that there were decreased osteoclast numbers with LFMV (spring 1 no vibration [4.658] was greater than spring 1 5 Hz [1.233], spring 1 10 Hz [3.875], and spring 1 20 Hz [2.738]). To our knowledge, this is the first study to show a decrease in osteoclastic activity (although it was not statistically significant) in the alveolar bone with LFMV on the maxillary first molar. However, similar results were obtained by Rubin et al27 and Xie et al28 with whole body vibration in tibiae, and they attributed this to kinase-dependent inhibition of RANKL expression in the bone stromal cells. The sclerostin expression was decreased on both the tension and compression sides with 10 and 20 Hz vibrations in the control and experimental groups (Fig 3). Tu et al29 showed that sclerostin is secreted by osteocytes and inhibits bone formation through Wnt signaling. Moreover, they showed that the loss of sclerostin expression shows a high bone mass phenotype. Our experimental group showed a trend toward an increase in BVF with mechanical vibration, but for an unknown reason the sclerostin expression was not decreased in the 5 Hz group in both the control and experimental animals (Fig 3). The microscopic observation of the collagen fibers showed no detrimental effect in the control group with different LFMV (Fig 4). However, the collagen fibers look thin and wavy after orthodontic loading in the spring 1 no vibration group. Moreover, we found that LFMV (spring 1 5 Hz, spring 1 10 Hz, and spring 1 20 Hz) after orthodontic loading was beneficial for the fibers, and they look organized and thick (Fig 4). Because this was an animal study, extrapolation of our findings to the clinical situation must be done with caution as there is no osteonal remodeling (secondary remodeling) in mice, unlike in humans. Moreover, a frictionless space closure mechanism was used in this study. Nevertheless, this in-vivo study helped us to understand the effect of LFMV on the surrounding alveolar bone. Our future studies will focus on understanding the signaling pathways associated with LFMV and in-vitro gene expression of the vibrated osteoblasts, osteoclasts, and cementoblasts. CONCLUSIONS
1. There was no difference in the rate of tooth movement between the different experimental groups. However, the maximum tooth movement was observed in the spring 1 10 Hz group, and the least was in the spring 1 20 Hz group.
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2. Tooth movement significantly decreased the BVF. The baseline control group had significantly more BVF compared with the spring 1 no vibration group. 3. LFMV at 5, 10, and 20 Hz had no deleterious effect on the integrity of the PDL. LFMV helped in maintaining the thickness and integrity of the PDL after application of the orthodontic load.
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dependent on the applied frequency but not on the strain magnitude. J Biomech 2007;40:1333-9. Oxlund BS, Ortoft G, Andreassen TT, Oxlund H. Low-intensity, high-frequency vibration appears to prevent the decrease in strength of the femur and tibia associated with ovariectomy of adult rats. Bone 2003;32:69-77. Miles P, Smith H, Weyant R, Rinchuse DJ. The effects of a vibrational appliance on tooth movement and patient discomfort: a prospective randomised clinical trial. Aust Orthod J 2012;28: 213-8. Kalajzic Z, Peluso EB, Utreja A, Dyment N, Nihara J, Xu M, et al. Effect of cyclical forces on the periodontal ligament and alveolar bone remodeling during orthodontic tooth movement. Angle Orthod 2014;84:297-303. Nishiyama Y, Matsumoto T, Lee JW, Saitou T, Imamura T, Moriyama K, et al. Changes in the spatial distribution of sclerostin in the osteocytic lacuno-canalicular system in alveolar bone due to orthodontic forces, as detected on multimodal confocal fluorescence imaging analyses. Arch Oral Biol 2015; 60:45-54. Bonewald LF. The amazing osteocyte. J Bone Miner Res 2011;26: 229-38. van Bezooijen RL, ten Dijke P, Papapoulos SE, Lowik CW. SOST/ sclerostin, an osteocyte-derived negative regulator of bone formation. Cytokine Growth Factor Rev 2005;16:319-27. van Bezooijen RL, Roelen BA, Visser A, van der Wee-Pals L, de Wilt E, Karperien M, et al. Sclerostin is an osteocyte-expressed negative regulator of bone formation, but not a classical BMP antagonist. J Exp Med 2004;199:805-14. Matsumoto T, Iimura T, Ogura K, Moriyama K, Yamaguchi A. The role of osteocytes in bone resorption during orthodontic tooth movement. J Dent Res 2013;92:340-5. Murphy CA, Chandhoke T, Kalajzic Z, Flynn R, Utreja A, Wadhwa S, et al. Effect of corticision and different force magnitudes on orthodontic tooth movement in a rat model. Am J Orthod Dentofacial Orthop 2014;146:55-66. Liu D, Genetos DC, Shao Y, Geist DJ, Li J, Ke HZ, et al. Activation of extracellular-signal regulated kinase (ERK1/2) by fluid shear is Ca(21)- and ATP-dependent in MC3T3-E1 osteoblasts. Bone 2008;42:644-52. Vij K, Mao JJ. Geometry and cell density of rat craniofacial sutures during early postnatal development and upon in vivo cyclic loading. Bone 2006;38:722-30. Alikhani M, Khoo E, Alyami B, Raptis M, Salgueiro JM, Oliveira SM, et al. Osteogenic effect of high-frequency acceleration on alveolar bone. J Dent Res 2012;91:413-9. Rubin J, Murphy TC, Fan X, Goldschmidt M, Taylor WR. Activation of extracellular signal-regulated kinase is involved in mechanical strain inhibition of RANKL expression in bone stromal cells. J Bone Miner Res 2002;17:1452-60. Xie L, Jacobson JM, Choi ES, Busa B, Donahue LR, Miller LM, et al. Low-level mechanical vibrations can influence bone resorption and bone formation in the growing skeleton. Bone 2006;39: 1059-66. Tu X, Rhee Y, Condon KW, Bivi N, Allen MR, Dwyer D, et al. Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading. Bone 2012; 50:209-17.
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Effect of low-frequency mechanical vibration on orthodontic tooth movement Sumit Yadav,a Thomas Dobie,b Amir Assefnia,c Himank Gupta,c Zana Kalajzic,d and Ravindra Nandae Farmington, Conn
Introduction: Our objective was to investigate the effect of low-frequency mechanical vibration (LFMV) on the rate of tooth movement, bone volume fraction, tissue density, and the integrity of the periodontal ligament. Our null hypothesis was that there would be no difference in the amount of tooth movement between different values of LFMV. Methods: Sixty-four male CD1 mice, 12 weeks old, were used for orthodontic tooth movement. The mice were randomly divided into 2 groups: control groups (baseline; no spring 1 5 Hz; no spring 1 10 Hz; and no spring 1 20 Hz) and experimental groups (spring 1 no vibration; spring 1 5 Hz; spring 1 10 Hz; and spring 1 20 Hz). In the experimental groups, the first molars were moved mesially for 2 weeks using nickeltitanium coil springs delivering 10 g of force. In the control and experimental groups, LFMV was applied at 5, 10, or 20 Hz. Microfocus x-ray computed tomography analysis was used for tooth movement measurements, bone volume fraction, and tissue density. Additionally, immunostaining for sclerostin, tartrate-resistant acid phosphatase (TRAP) staining, and picrosirius red staining were used on the histologic sections. Simple descriptive statistics were used to summarize the data. Kruskal-Wallis tests were used to compare the outcomes across treatment groups. Results: LFMV did not increase the rate of orthodontic tooth movement. Microfocus x-ray computed tomography analysis showed increases in bone volume fractions and tissue densities with applications of LFMV. Sclerostin expression was decreased with 10 and 20 Hz vibrations in both the control and experimental groups. Additionally, the picrosirius staining showed that LFMV helped in maintaining the thickness and integrity of collagen fibers in the periodontal ligament. Conclusions: There was no significant increase in tooth movement by applying LFMV when compared with the control groups (spring 1 no vibration). (Am J Orthod Dentofacial Orthop 2015;148:440-9)
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ooth movement involves both remodeling and modeling of bone.1,2 Remodeling and modeling involve a coordinated action of osteoclasts and osteoblasts in response to mechanical loading.3 Moreover, inflammatory mediators (interleukin [IL]-1, IL-2, IL-6, IL-8, and tumor necrosis factor-alpha) are released after mechanical stimulus or injury, triggering the biologic process associated with orthodontic tooth movement (OTM).4–7
From the Health Center, University of Connecticut, Division of Orthdontics, Farmington, Conn. a Assistant professor. b Visiting assistant professor. c Resident. d Research associate. e Professor and head. All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest, and none were reported. Supported by the American Association of Orthodontists Foundation and the Division of Orthodontics of the University of Connecticut. Address correspondence to: Sumit Yadav, Division of Orthodontics, University of Connecticut Health Center, 263 Farmington Ave, Room L7063 MC1725, Farmington, CT 06030; e-mail, [email protected]. Submitted, November 2014; revised and accepted, March 2015. 0889-5406/$36.00 Copyright Ó 2015 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2015.03.031
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Currently, orthodontic treatment requires approximately 24 to 30 months of intervention to complete the treatment.8–10 The longer duration of treatment is a great concern and poses high risks for caries, root resorption, and decreased patient compliance and satisfaction.8,10,11 Thus, accelerating OTM and shortening the total treatment duration is a primary goal of the orthodontist, and it can prevent detrimental effects of longer treatment time and increase patient satisfaction. Nishimura et al have shown that the application of cyclical forces (60 Hz) on the maxillary first molar increases the rate of OTM.12 However, the main drawback of the Nishimura study was the method of force application (transpalatal expansion spring). The force was applied to accelerate tooth movement in the first order (buccolingually) rather than in the second order (mesiodistally), which comprises the majority of OTM. Moreover, it may confound the actual tooth movement because of its skeletal effects.12 Studies have shown that whole body vibration (30, 45, and 90 Hz) may have an anabolic response on bone mass and architecture.13–15 Miles et al,16 in their randomized controlled trial, showed that
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application of 111 Hz of vibrational frequency for 20 minutes per day did not speed tooth movement when compared with the controls. Kalajzic et al17 showed the inhibitory effect of cyclical forces (30 Hz and 40 g of force applied with an electromechanical actuator) on OTM in rats; moreover, they showed the deleterious effects of the cyclical forces on the periodontal ligament (PDL). The major drawback of their model was higher cyclical force (40 g). The effect and mechanism of lowfrequency mechanical vibrations (LFMV) (#20 Hz) on OTM and paradental tissues still remain unclear. To our understanding, this is the first in-vivo study regarding the effect of LFMV (frequency, #20 Hz) on OTM. Recently, LFMV has gained interest in accelerating OTM by increasing alveolar bone turnover. The osteocytes in the bone tissue are thought to orchestrate “mechanotransduction” by reacting to different forms of mechanical loading through biologic signals.18,19 The role of osteocytes in bone remodeling and modeling has been well documented.18,19 It has been shown that osteocytes are the major source of sclerostin (product of SOST gene), and they antagonize the canonical Wnt signaling pathway, thus exhibiting an inhibitory effect on bone formation.20,21 Matsumoto et al22 demonstrated the role of osteocytes in resorption modeling during OTM (mechanotransduction) using osteocyteablated mice. Our null hypothesis was that there would be no difference in the amount of tooth movement between different amounts of LFMV. We had 3 specific aims: (1) to determine the effect of LFMV on the rate of tooth movement; (2) to quantify bone modeling and remodeling in both the control and experimental groups using microfocus computed tomography (micro-CT) and immunostaining; and (3) to determine the effect of LFMV on the PDL. MATERIAL AND METHODS
The Institutional Animal Care Committee of the University of Connecticut Health Center approved this study, which conformed to the ARRIVE guidelines. Data were obtained from 64 male CD1 mice (Charles River Laboratories, Wilmington, Mass; body weight, 24-30 g). The 12-week-old mice were randomly divided into 2 groups: control and experimental. The control group was further subdivided into 4 groups; each control group had 5 mice: (1) group 1 (baseline), no spring and no mechanical vibration; (2) group 2, no orthodontic spring, but 5 Hz vibration was applied to the maxillary first molars; (3) group 3, no orthodontic spring, but 10 Hz vibration was applied to the maxillary first molars; and (4) group 4, no orthodontic spring, but 20 Hz
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vibration was applied to the maxillary first molars. The experimental group was also subdivided into 4 groups, each with 11 mice: (1) group 5, orthodontic spring only but no vibration; (2) group 6, orthodontic spring and 5 Hz vibration applied to the maxillary first molars; (3) group 7, orthodontic spring and 10 Hz vibration applied to the maxillary first molars; (4) group 8, orthodontic spring and 20 Hz vibration applied to the maxillary first molars. The animals were allowed at least a week of acclimatization at our Health Center to compensate for their different origins. The animals were placed under general anesthesia with xylazine (13 mg/kg) and ketamine (87 mg/kg). A custom mouth-prop was fabricated from 0.036-in stainless steel wire and placed between the maxillary and mandibular incisors to hold the mouth open. A 0.004-in stainless steel ligature wire was passed beneath the contact between the maxillary first and second molars and threaded to the maxillary first molar (Fig 1, A). A low force/deflection rate nickel-titanium coil spring (Ultimate Wireforms, Bristol, Conn) delivering 10 g of force was attached to the 0.004-in stainless steel ligature around the first molar, and the other end of the spring was attached to the incisors with 0.004-in stainless steel wire. The force/deflection rate for the spring was determined to calibrate the amount of force produced by activation of the nickel-titanium coil spring. Additionally, grooves 0.5 mm from the gingival margin were prepared on the facial, mesial, and distal surfaces of the maxillary central incisors to prevent the ligatures from dislodging from the incisor because of their lingual curvature and eruption pattern. Selfetching primer (Transbond Plus; 3M Unitek, Monrovia, Calif) and light-cured adhesive resin cement (Transbond; 3M Unitek) were applied to the lingual surfaces of the maxillary first molars and incisors to secure the ligature wire. Moreover, to minimize the distal movement of the right incisor and to reinforce the anterior anchorage, the right and left incisors were joined together to act as a unit (Fig 1, A). After appliance insertion, the mice were allowed to recover with an incandescent light for warmth; they were returned to their cages once full ambulation and self-cleansing returned. The appliances were checked every other day, and additional light-cured bonding material was added if necessary. After anesthesia with ketamine (87 mg/kg) and xylazine (13 mg/kg), a custom mouth-prop fabricated from 0.017 3 0.025-in titanium-molybdenum alloy wire was placed between the maxillary and mandibular incisors to hold the mouth open. A feedback loop–controlled electromechanical actuator (model 3230; Bose Enduratec,
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Fig 1. A, Inserted nickel-titanium coil spring; B, application of LFMV on the maxillary right molar (the electromechanical actuator applies mechanical vibration at 5, 10, and 20 Hz at a compression force of 1 g); C, schematic of the tooth movement model with the spring attached to the maxillary first molar and maxillary incisors; D, intermolar distance (M1-M2) at day 14 in the experimental groups; E-H, sagittal micro-CT sections used for measuring intermolar distance in the control groups (E, baseline; F, no spring 1 5 Hz vibration; G, no spring 1 10 Hz vibration; H, no spring 1 20 Hz vibration); I-L, sagittal micro-CT sections used for measuring intermolar distance in the experimental groups (I, spring 1 no vibration; J, spring 1 5 Hz vibration; K, spring 1 10 Hz vibration; L, spring 1 20 Hz vibration).
Minnetonka, Minn) was used to apply unilateral LFMV to the occlusal surface of the maxillary right first molar along its long axis (Fig 1, B). Loading protocols for each animal consisted of 15 minutes of LFMV at 1 cN of force with the electromechanical actuator at a frequency of 5, 10, or 20 Hz (cycles/second) depending on the mouse's group. LFMV was applied to the maxillary right first molar at 3-day intervals (days 1, 4, 7, 10, and 13).
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After the 14 days of the experimental period, the animals were killed by inhalation of carbon dioxide followed by cervical dislocation. The maxilla was hemisected, and the attached soft tissues and muscles were removed. Subsequently, the hemisected maxilla was placed in 10% formalin for 5 days at 4 C. The samples were then decalcified in 14% ethylenediaminetetraacetic acid for 3 weeks and then processed for standard paraffin embedding. Serial
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Fig 2. Micro-CT data showing BVF and tissue density. Each value in each graph represents the mean 6 the standard deviation: A, region of interest where the bone parameters (BVF and tissue density) were measured; B, BVF values in the control groups (significantly [*] higher BVF in the no spring 1 5 Hz vibration group compared with the no spring 1 20 Hz vibration group); C, BVF values in the experimental groups; BVF decreases with tooth movement; baseline had a significantly higher BVF compared with the spring 1 no vibration group, the spring 1 5 Hz vibration group, the spring 1 10 Hz vibration group, and the spring 1 20 Hz vibration group; moreover, the spring 1 no vibration group had significantly less BVF than did the spring 1 5 Hz vibration (*) and the spring 1 10 Hz vibration (#) groups. D, Tissue density in the control groups; note the trend for an increase in tissue density with vibration; the spring 1 5 Hz group had significantly higher bone density compared with baseline (*); a,b,c,d signifies P \ 0.5. E, tissue density in the experimental groups.
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Fig 3. Sclerostin expression in the control and experimental groups on the compression and tension sides in alveolar bone. Upper panel, control groups; lower panel, experimental groups. A-D, Tension side in the control groups (A, baseline; B, no spring 6 5 Hz; C, no spring 6 10 Hz; D, no spring 6 20 Hz); E-H, tension side in experimental groups (E, spring 1 no vibration; F, spring 15 Hz; G, spring 110 Hz; H, spring 120 Hz); I-L,compression side in the control groups (I, baseline; J, no spring 6 5 Hz; K, no spring 6 10 Hz; L: no spring 6 20 HzA); M-P, compression side in the experimental groups (M, spring 1 no vibration; N, spring 15 Hz; O, spring 1 10 Hz; P, spring 120 Hz). Note the decrease in sclerostin expression in the 10 Hz (C, G, K, and O) and 20 Hz (D, H, L, and P) vibrations in both the control and experimental groups. Q, with no expression of sclerostin, is a negative control. The double white arrows signify the cells positive for sclerostin.
sagittal sections (5 mm) were obtained from the mesial and distal roots of the maxillary right first molar. All the animals in the different control (5 in each group) and experimental (11 in each group) groups were used to measure tooth movements. Threedimensional image arrays of the hemisected right maxillae were collected using micro-CT. OTM was defined and measured as the distance between the maxillary first and second molars at the most mesial point of the second molar crown and the most distal point of the first molar crown.17,23 The distance at day 0 was 0 mm in all groups (ie, the convex crown surfaces were touching). The OTM measurements were
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done in the sagittal plane, locating the image plane that showed the most root structure. The original 2dimensional image was then magnified 10 times for more precise line drawings, which were made at the closest proximity of the 2 convex molar crown surfaces. The OTM was measured in the 3 sagittal sections of each animal in all groups. Micro-CT images were used for quantitative analyses of bone changes occurring in the region of the maxillary first molar. Changes in the alveolar bone were studied by analyzing the furcation area of the maxillary first molar. The region of interest for the alveolar bone analysis was defined vertically as the
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Fig 4. Histologic examination of PDL collagen fibers on the tension side by polarized light microscopy. The sirius red stained PDL shows a more organized PDL in the control groups (A, baseline; B, no spring 1 5 Hz; C, no spring 1 10 Hz; D, no spring 1 20 Hz) compared with the experimental groups (E, spring 1 no vibration; F, spring 1 5 Hz vibration; G, spring 1 10 Hz vibration; H, spring 1 20 Hz vibration). Note that vibration only does not lead to disorganization of PDL collagen fibers. The spring 1 no vibration group had disorganized and thin PDL fibers. Note that the spring 1 vibration (F, G, and H) had more thickened and organized fibers (shown by the red staining) compared with the spring 1 no vibration group. A.B., Alveolar bone; P, periodontal ligament; De, dentin.
most occlusal point of the furcation to the apex of the maxillary first molar roots; transversely, it was defined as the space between the buccal and lingual cortical bone; sagittally, it included 50 sections (10 mm) beginning at the mesial root and continuing distally (Fig 2, A). The parameters studied and measured using the established algorithms were bone volume fraction (BVF) and tissue density. Histology, immunohistochemistry, tartrate-resistant acid phosphatase (TRAP), and picrosirius red staining histology were performed on each section. After blocking for 10 minutes at room temperature with 1x universal blocking reagent (HK085-5k; BioGenex, Fremont, Calif), deparaffinized histologic sections were incubated with goat polyclonal anti-SOST antibody (AF1589; R&D Systems, Minneapolis, Minn) overnight at 140 C at a concentration of 5 mg per milliliter. Subsequently, the sections were washed with phosphate-buffered saline and incubated with Alexa Fluor 594 donkey anti-goat IgG (A-11058; Life Technologies, Grand Island, NY) at
a concentration of 1:300 for 1 hour at room temperature and mounted with a suspension of 50% glycerol in phosphate-buffered saline containing the nuclear stain (Hoechst H3570; Life Technologies) at a concentration of 1:5000. TRAP staining was performed using a leukocyte acid phosphatase (TRAP) kit (386-1 KT; Sigma-Aldrich, St Louis, Mo) according to the manufacturer's instructions. TRAP positive, multinucleated cells were counted on the alveolar bone surfaces on the mesial sides of the distobuccal roots. The area for quantification included a square with 1 side extending from the apex to the bifurcation, and the other side extending 200 mm from the PDL border inside the alveolar bone. The osteoclast numbers were counted in 6 sections from 4 mice in each group, and the values were then averaged for each animal to run a statistical test. Deparaffinized sections were also stained with 1% picrosirius red for 1 hour, washed with acidified water (0.5% acetic acid water), and dehydrated with serial
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ethanol washes before mounting. The sections were examined with a fluorescent microscope (Carl Zeiss, Thornwood, NY). Statistical analysis
Simple descriptive statistics were used to summarize the data. The outcomes examined included intermolar distance, BVF, and tissue density. Because of the sample size, nonparametric tests were used to examine the association between the outcome variables and the treatment groups (spring 1 no vibration, spring 1 5 Hz, spring 1 10 Hz, and spring 1 20 Hz). The Kolmogorov-Smirnov test was used to examine the distribution of the outcome variables. Kruskal-Wallis tests were used to compare the outcomes across treatment groups. Pairwise comparisons between different groups were conducted with the Mann-Whitney U test. All statistical tests were 2 sided, and a P value of \0.05 was deemed to be statistically significant for the KruskalWallis test. Because of the multiple pairwise comparisons used, to minimize type 1 errors, Bonferroni corrections were used. RESULTS
All mice in the study remained healthy and had a slight increase in body weight. The delta displacement of the molar was the same throughout the vibration to maintain 1 g of force with the vibrator tip. The distance between the first and second molars in the control groups was 0 mm (Fig 1, C), whereas there was no statistically significant difference in the experimental groups after 14 days of orthodontic force application (Fig 1, D). However, the maximum tooth movement was observed in the spring 1 10 Hz group (0.248 6 0.074 mm), and the least was in the spring 1 20 Hz group (0.200 6 0.075 mm). For the bone parameters, the micro-CT analysis showed a statistically significant (P \0.05) decrease in BVF when the control groups were compared with the experimental groups (baseline vs spring 1 no vibration
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[P \0.05]; no spring 1 5 Hz vs spring 1 5 Hz [P \0.05]; no spring 1 10 Hz vs spring 1 10 Hz [P \0.05]; and no spring 1 20 Hz vs spring 1 20 Hz [P \0.05]) (Fig 2, C and D). Moreover, in the control groups, the no spring 1 5 Hz group had a significantly greater BVF than did the no spring 1 20 Hz group. In the experimental groups, the BVF was significantly (P \0.05) lower for the spring 1 no vibration group, the spring 1 5 Hz group, the spring 1 10 Hz group, and the spring 1 20 Hz group, when compared with the baseline (Fig 2, B and C). In the control groups, there was a trend for an increase in tissue density with mechanical vibration; however, the no spring 1 5 Hz group had significantly (P \0.05) more tissue density than did the baseline control. Similarly, in the experimental groups, there was a trend for an increase in bone density with mechanical vibration, but it was not statistically significant among the experimental groups (Fig 2, D and E). Our data show reductions in sclerostin expression on the tension and compression sides with 10 and 20 Hz in both the control (no spring 1 10 Hz, and no spring 1 20 Hz) and the experimental (spring 1 10 Hz, and spring 1 20 Hz) groups (Fig 3). However, there was no decrease in sclerostin expression in either the control or the experimental group with 5 Hz vibration (Fig 3). Our data show that LFMV in the control group at 5, 10, and 20 Hz did not affect the quality of the collagen fibers. However, orthodontic loading does make a huge impact on the integrity and the quality of the fibers (Fig 4, E). Our experimental groups showed that LFMV at 5, 10, and 20 Hz brings back the integrity and the thickness of collagen fibers that were lost due to orthodontic loading. The spring-only group (orthodontic loading) had the least thickened and wavy fibers, whereas the other experimental groups (spring 1 5 Hz, spring 1 10 Hz, and spring 1 20 Hz) had thick collagen fibers, as shown by the dark red stain. Our experimental groups showed significantly increased numbers of osteoclasts when compared with
Fig 5. Quantification and histologic examination of osteoclast numbers at day 14: quantification of osteoclast numbers in A, different control groups and B, different experimental groups; *significantly higher osteoclast numbers in the spring 1 no vibration group compared with the baseline control. Histologic examinations in: C, the different control groups at baseline; D, no spring 1 5 Hz; E, no spring 1 10 Hz; F, no spring 1 20 Hz; and experimental groups: G, spring 1 no vibration; H, spring 1 5 Hz vibration; I, spring 1 10 Hz vibration; J, spring 1 20 Hz vibration. TRAP positive cells were higher in the PDL and alveolar bone in the spring 1 no vibration group (G). A.B., Alveolar bone; P, periodontal ligament; De, dentin.
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the control groups (spring 1 no vibration [P \0.05] vs baseline; spring 1 5 Hz [P \0.05] vs no spring 1 5 Hz; spring 1 10 Hz [P \0.05] vs no spring 1 10 Hz; and spring 1 20 Hz [P \0.05] vs no spring 1 20 Hz [P \0.05]) (Fig 5). However, in the control groups, there were no significant differences in osteoclast numbers, implying that LFMV does not affect the osteoclast numbers in molars not subjected to tooth movement. In the experimental groups, there was a trend toward a decrease in osteoclast numbers with LFMV, although it was not statistically significant. DISCUSSION
Our null hypothesis that there would be no difference in the amount of tooth movement between the experimental groups (spring 1 no vibration, spring 1 5 Hz vibration, spring 1 10 Hz vibration, and spring 1 20 Hz vibration) was accepted. We selected LFMV (5, 10, and 20 Hz) for our study because Kalajzic et al17 showed that higher cyclical forces inhibit OTM and are deleterious to the PDL. Our results of OTM with LFMV were contrary to those of Liu et al24 and Nishimura et al12; a plausible reason was a different tooth movement model. Nishimura et al and Liu et al used transpalatal expansion springs (orthodontic load in the first order), whereas we used nickel-titanium coil springs (orthodontic load in the second order), and the orthodontic force was in the mesial direction. In the transpalatal model, the increase in OTM can be due to both skeletal and dental effects, whereas in our model, the OTM was primarily due to dental effects because we used adult mice, in which growth of the alveolar bone was complete. OTM primarily depends on bone volume and bone density (quantity and quality of bone). In this study, there was a trend toward an increase in tissue density with LFMV in both the control and experimental groups. Moreover, a similar trend toward an increase in BVF in the experimental group was noted after the application of LFMV. Orthodontic loading decreased the BVF (baseline, 79.24%; spring 1 no vibration, 53.45%), which was recovered by LFMV (spring 1 5 Hz, 64.45%; spring 1 10 Hz, 66.28%; and spring 1 20 Hz, 59.54%). Moreover, the spring 1 no vibration group had significantly less (P \0.05) BVF than did the spring 1 5 Hz and the spring 1 10 Hz groups. Similarly, the results regarding BVF were shown by Kalajzic et al,17 and probably the reason could be inhibition of osteoclastogenesis with LFMV. Furthermore, Vij and Mao25 showed that cyclical loading (4 Hz and 300 mN) can cause sutural growth, and Alikhani et al26 showed that application of high-frequency
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acceleration significantly increases the rate of alveolar bone formation. Moreover, our study showed that there were decreased osteoclast numbers with LFMV (spring 1 no vibration [4.658] was greater than spring 1 5 Hz [1.233], spring 1 10 Hz [3.875], and spring 1 20 Hz [2.738]). To our knowledge, this is the first study to show a decrease in osteoclastic activity (although it was not statistically significant) in the alveolar bone with LFMV on the maxillary first molar. However, similar results were obtained by Rubin et al27 and Xie et al28 with whole body vibration in tibiae, and they attributed this to kinase-dependent inhibition of RANKL expression in the bone stromal cells. The sclerostin expression was decreased on both the tension and compression sides with 10 and 20 Hz vibrations in the control and experimental groups (Fig 3). Tu et al29 showed that sclerostin is secreted by osteocytes and inhibits bone formation through Wnt signaling. Moreover, they showed that the loss of sclerostin expression shows a high bone mass phenotype. Our experimental group showed a trend toward an increase in BVF with mechanical vibration, but for an unknown reason the sclerostin expression was not decreased in the 5 Hz group in both the control and experimental animals (Fig 3). The microscopic observation of the collagen fibers showed no detrimental effect in the control group with different LFMV (Fig 4). However, the collagen fibers look thin and wavy after orthodontic loading in the spring 1 no vibration group. Moreover, we found that LFMV (spring 1 5 Hz, spring 1 10 Hz, and spring 1 20 Hz) after orthodontic loading was beneficial for the fibers, and they look organized and thick (Fig 4). Because this was an animal study, extrapolation of our findings to the clinical situation must be done with caution as there is no osteonal remodeling (secondary remodeling) in mice, unlike in humans. Moreover, a frictionless space closure mechanism was used in this study. Nevertheless, this in-vivo study helped us to understand the effect of LFMV on the surrounding alveolar bone. Our future studies will focus on understanding the signaling pathways associated with LFMV and in-vitro gene expression of the vibrated osteoblasts, osteoclasts, and cementoblasts. CONCLUSIONS
1. There was no difference in the rate of tooth movement between the different experimental groups. However, the maximum tooth movement was observed in the spring 1 10 Hz group, and the least was in the spring 1 20 Hz group.
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2. Tooth movement significantly decreased the BVF. The baseline control group had significantly more BVF compared with the spring 1 no vibration group. 3. LFMV at 5, 10, and 20 Hz had no deleterious effect on the integrity of the PDL. LFMV helped in maintaining the thickness and integrity of the PDL after application of the orthodontic load.
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September 2015 Vol 148 Issue 3