Because mechanical stimulation of the periodontal ligament by low-intensity pulsed ultrasound (LIPUS) has been shown to increase the speed of bone remodeling, this study aimed to examine the effects of LIPUS stimulation on the rate of tooth movement and bone remodeling during lateral tooth movement.
Twelve-week-old Wistar rats were divided into 2 groups. The LIPUS group received experimental tooth movement with LIPUS stimulation, and the tooth movement (TM) group were provided experimental tooth movement without LIPUS. For each group, the upper right first molars were moved buccally with fixed appliances. LIPUS exposure was placed in the region corresponding to the right maxillary first molar. Three days after tooth movement, tartrate-resistant acid phosphatase was examined. Fourteen days after tooth movement, the intermolar width, bone mineral content, and bone volume fraction were analyzed by micro-computed tomography, and newly formed bone was measured histomorphometrically.
The number of TRAP-positive cells in the compressed region was higher in the LIPUS group. The intermolar width was significantly higher in the LIPUS group than in the TM group. The alveolar bone around the maxillary first molar showed no differences in bone mineral content and bone volume fraction between the LIPUS and TM groups. The LIPUS group exhibited a more significant amount of newly formed alveolar bone than the TM group.
The present study provides evidence of the beneficial effects of LIPUS on the lateral tooth movement.
LIPUS enhances osteoclastogenesis in the pressure zone of the alveolar bone during lateral tooth movement.
LIPUS increases compensatory bone formation on the buccal surface of the alveolar bone during lateral tooth movement.
LIPUS improves the rate of lateral tooth movement.
Tissue remodeling surrounding tooth roots is essential to the rate of orthodontic tooth movement. Therefore, it is critical to control the molecular mechanisms by which the behaviors of cells in the alveolar bone and periodontal ligament (PDL) are regulated. The duration of orthodontic treatment is the primary concern for most patients and orthodontists. However, long-term orthodontic treatment induces several disadvantages, such as a higher predisposition to dental caries, gingival recession, and root resorption. Consequently, much attention has been paid to find the possible remedies that increase the rate of tooth movement with the fewest potential disadvantages.
To date, several novel modalities have been reported to accelerate orthodontic tooth movement. Surgical modalities including corticotomy, dentoalveolar distraction, and periodontal distraction are based on the principle that when the bone is irritated, which causes increased osteoclastogenesis, the tooth moves faster. Several nonsurgical modalities have also been reported, such as low-level laser therapy, electromagnetic fields, and mechanical vibration. In addition, mechanical stimulation of the PDL by low-intensity pulsed ultrasound (LIPUS) has been reported to increase the speed of bone remodeling. Thus, LIPUS is also considered a nonsurgical modality for accelerating tooth movement. LIPUS has been shown to act by inducing osteoclastogenesis through stimulation of the receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) pathway, and activation of signaling molecules such as mitogen-activated protein kinase. Although the use of LIPUS is safe, there is limited research-based evidence to suggest it accelerates orthodontic tooth movement.
Xue et al reported that LIPUS might promote alveolar bone remodeling by increasing the gene expression of the human growth factor/Runx2/bone morphogenetic protein 2 signaling pathway molecules, resulting in the rapid movement of teeth during orthodontic treatment. A recent rodent study also demonstrated that LIPUS enhanced the amount of tooth movement and bone remodeling during orthodontic tooth movement. However, these results were only found in a mesial orthodontic tooth movement model. Although clinical lateral tooth movement is frequently conducted, the effect of LIPUS on lateral orthodontic tooth movement has not been thoroughly examined. As such, this study aimed to investigate the effects of LIPUS stimulation on the rate of tooth movement and bone remodeling during lateral tooth movement.
Material and methods
All of the procedures described in this study were performed following the guidelines and regulations of the Tsurumi University for Animal Research (29A038). A total of twenty-six 12-week-old male Wistar rats, weighing 320-350 g, were randomly divided into 2 groups. The LIPUS group received experimental tooth movement with LIPUS stimulation, and the tooth movement (TM) group had experimental tooth movement without LIPUS. Each animal was anesthetized with a mixture of 3 types of anesthetic agents at a dose of 2.5 ml/kg body weight. The combination anesthetic was prepared with 0.15 mg/kg medetomidine (Domitor, Nippon Zenyaku Kogyo, Tokyo, Japan), 2 mg/kg midazolam (Dormicum, Astellas Pharma Inc, Tokyo, Japan), and 2.5 mg/kg butorphanol (Vetorphale, Meiji Seika Pharma, Tokyo, Japan). For the LIPUS and TM groups, the upper right first molars were moved buccally with fixed appliances ( Fig 1 , A and B ). The initial force magnitude was approximately 10 g.
An ultrasound exposure machine (Osteotron D2, ITO, Tokyo, Japan) was employed in this study. This system was equipped with transducers with a circular surface area of 9.6 cm 2 . The sound head of this device had an average beam nonuniformity ratio of 3.2-3.6:1.0 and an effective radiating area of 90%. A pulsed ultrasound signal was transmitted at a frequency of 1.5 MHz (pulse repetition frequency, 1000 Hz), with an average spatial intensity of 30 mW/cm 2 and a pulse of 1:4 (2 ms on and 8 ms off). The stimulation protocol, used in this study, consisted of a 20-min LIPUS stimulation repeated every day. The rats were kept in a fixed position under anesthesia, and the ultrasound transducer was placed in contact with 1 side of the face, in the region corresponding to the right maxillary first molar. The fur was shaved in the exposure region, and coupling gel was always in place to optimize penetration of the ultrasound waves into the tissues.
Three days after tooth movement, 4% paraformaldehyde in 0.1 M phosphate-buffered saline (pH 7.4) was perfused for 15 min through the ascending aortae of 10 rats from the experimental and control groups, respectively. After fixation, the maxillae were dissected and trimmed into small blocks containing the first molar, decalcified with disodium ethylenediaminetetraacetate dihydrate (5.0%, pH 7.2, 4 °C) solution containing 7.0% sucrose for 4 weeks, dehydrated with a graded ethanol series, and embedded in paraffin. The serial sections (7 μm) were cut perpendicular to the root axis. Tartrate-resistant acid phosphatase (TRAP) activity was examined in the sections, using a TRAP staining kit (Wako, Tokyo, Japan). An area measuring 700 × 2400 μm 2 was selected from the section for light microscopic examination (BZ-9000, Keyence, Osaka, Japan) according to a previous study ( Fig 2 , A and B ). TRAP-positive multinucleated osteoclasts on the pressure zone of the upper first molar were counted on 3 sections for each specimen, and the mean value was presented.
Sixteen rats served to measure tooth movement and to analyze the bone properties in micro-computed tomography (micro-CT) analysis. After 14 days of tooth movement, the micro-CT (inspeXio SMX-225CT, Shimadzu, Kyoto, Japan) images were taken. The tube voltage was set at 160 kV, and the current was constant at 70 μA. The resolution was set at 20 μm per voxel and 1024 × 1024 pixels. On the 3D models, the distance between the distolingual cusps of the maxillary first molars was measured as the intermolar width ( Fig 3 , A ). The regions of interest (ROI) were alveolar bone proper of the maxillary first molar ( Fig 3 , B – D ).
Each ROI was measured for bone mineral content (BMC) and bone volume fraction (BV/TV). Tissue volume was defined as the volume of tissue in the enlarged ROI. Bone volume was excluded from the teeth ( Fig 3 , D ). The intermolar width and bone parameters were measured by 3D image-analysis software (TRI/3D-BON, Ratoc System Engineering, Tokyo, Japan).
Sixteen rats were intraperitoneally injected with 0.1 mL of calcein (1.6 mg/kg) solution one day before tooth movement and with xylenol orange (50 mg/kg) one day before the end of tooth movement as fluorochrome labels. On day 15, the animals were killed under anesthesia with pentobarbital sodium at a fatal overdose of 50 mg/kg. After the micro-CT images were taken, the maxillae were dissected, cut in half along the sagittal plane, and immersed rapidly in liquid nitrogen. The frozen tissues were embedded with optimal cutting temperature compound (Miles Inc, Torrance, CA). Frozen blocks from the rats were frontally sectioned, and 7-μm thick serial sections were used for histomorphometric analysis. The sections were prepared according to Kawamoto’s film method. Newly formed bone was measured as the distance between the calcein and xylenol orange lines under a fluorescence microscope (BZ-9000, Keyence, Osaka, Japan), which were visible as green and red marks, respectively, at 50 μm, 150 μm, and 300 μm from the alveolar crest, the average of these 3 morphometric values was used for evaluation.
To determine the sample size of the experiments, a power analysis was performed to detect statistically significant differences in each experiment between lateral tooth movement with or without LIPUS exposure during the experimental period was determined (α = 0.05 and β = 0.80). The power analysis was performed by S Plus version 6.0 (NTT Data, Tokyo, Japan). For intermolar width and number of osteoclasts, the minimum sample size to detect the statistically significant differences was 5. For BMC, the minimum sample size was 1613. For newly formed alveolar bone, the minimum sample size by the condition α = 0.05 and β = 0.80 were 13 samples. When calculating the power under the conditions of α = 0.05 and 5 samples, β was 0.65.
The normality of variables was assessed by the Shapiro-Wilk test separately by the control and LIPUS groups. The statistical difference between 2 groups was determined by t tests or Mann-Whitney U tests.
For the assessment of reliability intermolar width measurements, samples were measured by 2 examiners under masking experimental conditions. Measurements were repeated after 2 weeks by the same examiner. A paired-samples t test showed no significant differences between the 2 repeated measures ( P = 0.689). And intraclass correlation coefficients evaluated by measured value was 0.967 (95% CI: 0.912-0.989). The level of significance was set at P <0.05. All analysis (excluding power analysis) was carried out by SPSS version 25.0 (IBM, Tokyo, Japan).