The European Journal of Orthodontics Advance Access published September 23, 2013

European Journal of Orthodontics doi:10.1093/ejo/cjt066

The Author 2013. Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please email: journals.permissions@oup.com

A preliminary study of the effects of low-intensity pulsed ultrasound exposure on the stability of orthodontic miniscrews in growingrats
KyokoMiura, MitsuruMotoyoshi, MizukiInaba, HiroakiIwai, YokoKarasawa, NoriyoshiShimizu
Department of Orthodontics, Nihon University School of Dentistry, Tokyo, Japan Correspondence to: Mitsuru Motoyoshi, Department of Orthodontics, Division of Clinical Research, Dental Research Center, Nihon University School of Dentistry, 1-8-13, Kanda-surugadai, Chiyoda-ku, Tokyo 1018310, Japan. E-mail: motoyoshi.mitsuru@nihon-u.ac.jp
Downloaded from http://ejo.oxfordjournals.org/ at Semmelweis Ote on October 27, 2013 Summary BackGround: Orthodontic

miniscrews placed in growing subjects often loosen during orthodontic treatment. The ability to place miniscrews, regardless of age, would be clinically benecial. ObjectiVes: To assess the effects of low-intensity pulsed ultrasound (LIPUS) on the stability of orthodontic miniscrews in growing rats. Materials/Methods: The mobility of miniscrews after placement was recorded and the miniscrewbone interface was examined histomorphometrically using tibiae of seven male Sprague-Dawley rats (aged 6 weeks). Field-emission scanning electron microscopic images were used to evaluate the boneminiscrew interface, and a mobility test device was used to assess the stiffness of miniscrew placement. Fourteen custom-made miniscrews with 1.4mm diameters and 4.0mm lengths were placed in the right and left tibiae. LIPUS was used to stimulate right tibiae at the sites of miniscrew placement, and left tibiae were left untreated as controls. Results: Signicantly lower mobility was observed in the LIPUS-treated group compared with the control group (P < 0.05). Histomorphometric evaluation indicated that boneminiscrew adhesion was signicantly better in the LIPUS-treated group than in the control group (P<0.05). Limitations: This in vivo study used tibiae rather than jaw bones because the jaw bones of 6-week-old rats were too small to allow miniscrew placement. Conclusions: LIPUS was able to increase the boneminiscrew contact and reduce the mobility of miniscrews in growing subjects. Implications: LIPUS may accelerate the bone healing process after miniscrew placement in growing subjects and can reduce the latent period.

Introduction Miniscrews are used to provide denite anchorage in orthodontic tooth procedures (Kanomi, 1997; Umemori et al., 1999; Freudenthaler et al., 2001; Lee et al., 2001; Bae etal., 2002; Park etal., 2002; Sugawara etal., 2004; Tseng et al., 2006). Because orthodontic miniscrews are small and are associated with a lower surgical burden, they can be placed at various locations in the alveolar bone and can be subjected to traction immediately after placement. Although one notable problem is failure of miniscrews, the success rate has risen recently in association with the resolution of various risk factors, better placement technique, and improvements in implant design (Cheng etal., 2004). In a meta-analysis, Schtzle etal. (2009) estimated that the success rate of miniscrews was 83.6 per cent (95 per cent condence interval, 79.986.6 percent). Young patients are frequently treated in the orthodontic eld. Miniscrew placement in alveolar bones of adolescent

patients is effective, even in cases with skeletal problems. De Clerck etal. (2012) used miniplates in patients with classIII malocclusion and reported signicant skeletal changes in patients treated with bone-anchored maxillary protraction. However, miniscrews placed in adolescents often loosen during orthodontic treatment. Motoyoshi etal. (2007) noted that the success rate was 63.8 per cent in an early-loading group of adolescents. They proposed a longer latent period to increase the ultimate success rate for adolescent patients, although immediate or early loading of miniscrews is preferred to reduce the treatment period and to respond to patient demands. The ability to place miniscrews, regardless of age and timing of loading, would be clinically benecial. Low-intensity pulsed ultrasound (LIPUS) has been used to induce healing after bone fracture and surgical osteotomy in the orthopaedic eld (Malizos etal., 2006; Dudda etal., 2011). In dentistry, some researchers have investigated the effects of LIPUS on periodontal tissue (Shiraishi et al.,

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2011) and bone regeneration after oral surgery (Kim and Hong, 2010), and reported consistently positive effects of LIPUS on the organs in the dentofacial eld. Studies have also demonstrated the potential of LIPUS to induce bone formation around dental implants. Hsu et al. (2011) and Liu et al. (2012) investigated the effects of LIPUS on the osseointegration of endosseous dental implants. Hsu etal. (2011) reported that blood ow and mature type I collagen bres were more prevalent around titanium implants, and bone formation was accelerated in pulsed ultrasound groups. Using a mechanical pull-out test, Liu etal. (2012) found that the strength of implants on the LIPUS-treated side was greater than that on the control side. However, the effects of LIPUS on bone formation surrounding small titanium implants in growing subjects are not well understood. The aims of this study were to evaluate the stability of orthodontic miniscrews placed in young rats, and to ensure that LIPUS exerts positive effects on this stability in an attempt to reduce the latent period after placement in growing subjects. To evaluate the stability of miniscrews, we used a mobility test device and examined the miniscrew bone interface histomorphometrically using a eld-emission scanning electron microscope (FE-SEM). Materials and methods Animals and miniscrews Seven male Sprague-Dawley rats (aged 6 weeks; body weight at the beginning of the experiment=17010g) were used. In our primary investigation using cone-beam computed tomography, the amounts of bone growth were greatest before and after the age of 6 weeks, and bone growth became more gradual after 20 weeks of age. We estimated that a 6-week-old rat corresponded to an approximate 10- to 12-year-old human and used 6-week-old rats to represent actively growing subjects. The experiments were approved by the Animal Experimentation Committee of XXXXX. The rats were used for measurements of miniscrew stability and FE-SEM observations. Small titanium miniscrews were produced for insertion into the rat tibiae (diameter=1.4mm, spearhead=1.2mm, thread length = 4.0mm; Figure 1). Custom-made miniscrews (n = 14) were placed in both tibiae. A computed tomographic survey was conducted prior to the experiment to evaluate cortical bone thickness in the observation areas on the right and left tibiae; this thickness did not differ signicantly between sides. Miniscrew placement sites in the right tibiae (test group) were stimulated with LIPUS and the left tibiae was used as controls, with no LIPUS stimulation. Surgical procedure The rats were anaesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight,

K. MIURA ET AL.

Figure1 Custom-made miniscrews used in this study.

Somnopentyl; Kyoritsu Seiyaku Co. Ltd, Tokyo, Japan). Surgical incisions were made in the skin of both legs along the tibial crests, and the surfaces of the tibiae were exposed. Ahole for miniscrew insertion was made in each tibia, 10.0mm inferior to the knee joint, using a bone drill (diameter=1.0mm, length=4.0mm; Dentsply-Sankin Co. Ltd, Tokyo, Japan) perpendicular to the bone surface under physiological saline ow. Then, miniscrews were inserted into the right and left holes using a hand driver. To avoid inuences of infection after placement, we applied tetracycline hydrochloride paste and embedded the miniscrews under the membrane. This procedure of enveloping the miniscrews also prevented the rats from biting and scratching the screw heads. LIPUS stimulation After surgery, LIPUS (BR-Sonic Pro; Ito Chou-tampa Co. Ltd, Tokyo, Japan) was used to stimulate the right tibiae at the sites of miniscrew placement. LIPUS was applied with an average spatial intensity of 30 mW/cm2, frequency 3.0 MHz, and duty cycle of 20 per cent with medical ultrasound gel on the shaved skin. The side of each right tibia was stimulated with LIPUS, perpendicular to the long axis of the miniscrew, for 15 minutes/day beginning immediately after placement until the rats were sacriced (Figure2) with sodium pentobarbital (50mg/kg body weight, Somnopentyl)

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Figure2 Low-intensity pulsed ultrasound (LIPUS) was applied perpendicular to the long axis of the miniscrew in the right tibia using medical ultrasound gel.

Figure 3 The tibia was xed tightly in plaster to ensure the precision of mobility measurement perpendicular to the long axis of the miniscrew using a mobility test device (Periotest; Hakuhou, Tokyo, Japan).

under anaesthesia. The left tibiae were left untreated as controls. Mobility test and histological procedure All rats were sacriced with sodium pentobarbital after 2 weeks, and tibiae with miniscrews were resected at the knee and ankle joints. Before histological analysis, miniscrew mobility testing was performed. Resected tibiae were anchored tightly with plaster (Figure3). Then, the mobility of the miniscrews was measured perpendicular to their long axes using a mobility test device (Periotest; Medizintechnik Gulden, Bensheim, Germany). After the mobility test, the resected tibiae were washed in water with ethanol dehydration and acetone degreasing after xation in 10 per cent formalin (Wako Pure Chemical Industries Ltd, Osaka, Japan) for 48 hours. The tibiae were embedded in polyester resin (Rigolac; Showa Highpolymer Co. Ltd, Tokyo, Japan) at a constant temperature of 60 degree C for 8 hours, and blocks (10.0 10.0 6.0 mm) were prepared. After the surface of the specimen was ground with water proof grinding papers (800, 1200, and 2000 grit), the blocks were cut in the perpendicular direction to the bone surface using a crystal cutter (Maruto Instrument Co. Ltd) at the centre of the miniscrew along the axis of the miniscrew. Then the blocks were ground with a hard grinding cloth and liquid containing 1 m diamond particles. The condition of contact between the miniscrew and surrounding cortical bone was observed using FE-SEM (Miniscope TM-1000; Hitachi High-Technologies Co. Ltd, Tokyo, Japan). Evaluation of boneminiscrew interface and statistical analysis The boneminiscrew contact ratio was calculated using enlarged 30 FE-SEM micrographs, applying the method established by Yano et al. (2006). After tracing the

micrographs, the boneminiscrew contact ratio was calculated using the following equation:
Bone - miniscrew pitch length displaying bone contact contact ratio (% ) = 100 total pitch length

The measuring area was the span from one thread to the next thread in the cortical bone, on both sides of the miniscrew (Figure4). One examiner (KM) traced and took measurements on all photographs under blind conditions to eliminate bias, at least three times at 2day intervals to eliminate intra-examiner error; mean values were used. In cases of greater than 5 per cent difference among the three bone miniscrew contact ratio values, measurement was repeated and the mean value was used. The paired t-test was used to compare the boneminiscrew contact ratio between the test (right tibiae) and control (left tibiae) groups using the PSS software (version 16.0 for Windows; SPSS Inc., Chicago, Illinois, USA). P-values greater than 0.05 were considered to indicate statistical signicance. Results No rat died and no screw was lost or became infected during the experimental period. Mobility test evaluation Periotest values (PTVs) were 16 [standard deviation (SD), 2.5] in the control group and 10 (SD, 2.5) in the test group, suggesting signicantly lower mobility in the test group (P<0.05; Figure5). FE-SEM evaluation Mono-cortical insertion was conrmed in all miniscrews. Typical SEM images are shown in Figure6. Bone contacted

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Figure4 The boneminiscrew contact ratio was calculated using the following equation: Boneminiscrew contact ratio (%)=pitch length displaying bone contact/total pitch length 100 The area of measurement was dened as the span of one thread length in the cortical bone.

Figure 7 Boneminiscrew contact ratios in the LIPUS and control groups. Asterisk, P-value < 0.05.

Discussion Maeda etal. (2013) reported the practical use of a 3 MHz frequency for oral soft-tissue treatment and suggested optimization of the LIPUS frequency according to the target. Miniscrews are generally placed into shallow tissue, and the penetration of a 3 MHz sine wave is shallower than that of a 1.5 MHz wave. This, a 3 MHz sine wave was used in thisstudy. A method involving use of the tibial bone has been established, and its reliability and stability have been determined (Yano etal., 2006, Uemura etal., 2012). Inaba (2009) performed an animal study to investigate the initial stability of miniscrews placed at an angle or perpendicular to the bone surface. She concluded that the boneminiscrew interface was inuenced by miniscrew inclination, as slanted implants showed signicantly longer boneminiscrew contact lengths. To standardize the condition of the boneminiscrew interface, we placed miniscrews perpendicular to bone surfaces. Furthermore, to avoid inuence of the degree of root proximity and to examine the effect of LIPUS under a simple condition, we used tibiae, which have sufficient bone quantity and lack complex anatomy, instead of jaw bones. Motoyoshi et al. (2007) reported that low miniscrew stability in adolescent patients is likely related to active bone metabolism and a low degree of bone maturation, including in the maxilla and mandible, in these growing subjects. The quality of tibial bone would also be weakened, due to its immaturity. Although the characteristics of jaw bones and tibiae differ, this study unavoidably used tibiae instead of jaw bones because the jaw bones of 6-week-old rats were too small for miniscrew placement. Because the anatomical structure of mandibular bone was thought to be similar to that of long bones, tibiae were expected to serve

Figure 5 Periotest values in the LIPUS-stimulated groups and control groups. Astersik, P-value < 0.05.

Figure6 Field-emission scanning electron microscope images from the LIPUS and control groups. Some spaces (arrows) were observed at the boneminiscrew interface in controls.

the implant surfaces well in LIPUS-treated subjects, but some gaps were observed in the boneminiscrew interfaces in controls (Figure6, arrows). Boneminiscrew contact ratios were 72.9 per cent (SD, 10.2 per cent; range, 58.988.5 per cent) in the test group and 52.3 per cent (SD, 9.0 per cent; range, 40.063.4 per cent) in the control group (P<0.05; Figure7).

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the experimental and control groups was 20 per cent. This difference is expected to reect an increased success rate in adolescent patients, although this proposal should be veried in a future clinicalstudy. Yano etal. (2006) documented boneminiscrew contact ratios greater than or equal to 80 per cent in adult (aged 20 weeks) rats. In growing rats, we observed boneminiscrew contact ratios greater than or equal to 72 per cent. This ratio was less than that in adult rats but indicated signicant improvement by LIPUS stimulation. This positive effect of LIPUS simultaneously implies the possibility of excessive adhesion between the bone and screw, which can cause problems with miniscrew removal. However, LIPUS stimulation is properly applied for a short duration immediately after placement. Motoyoshi et al. (2010) reported average placement and removal torques of approximately 8 and 4 N cm, respectively, and found that the removal torque decreased signicantly in the high-torque groups during orthodontic treatment. Thus, in our opinion, LIPUS stimulation should cause no problem with miniscrew removal. In a clinical study, Miyawaki etal. (2003) inserted miniscrews in 51 orthodontic patients and reported that the success rate in patients aged less than 20 years (80 per cent) was slightly lower than that in those aged more than 20years (8588 per cent), but the difference was not signicant. Park etal. (2005) placed miniscrews in 11 patients; implant failure occurred in three patients aged less than 14 years, but no failure occurred in patients aged 1428years. Motoyoshi etal. (2007) indicated that the success rate following early loading was signicantly lower in adolescents than in adults, whereas a latent period of 3 months before loading signicantly improved the success rate of miniscrews placed in the alveolar bone of adolescent patients. However, a 3 month latent period is not short from the point of view of patientsand their parentsand a reduction and consequent shortening of the duration of orthodontic treatment would be preferable. Our ndings suggest that LIPUS could accelerate the bone healing process after miniscrew placement in growing subjects, thereby reducing the duration of the latent period. In future studies, histochemical and genetic examinations would elucidate the biological and metabolic responses of bony tissue and the mechanism of bone remodeling in growing subjects associated with LIPUS stimulation. Miniscrew stability should be examined after orthodontic force application in a future study because the orthodontic force characteristics that preserve the intact boneminiscrew interface achieved with LIPUS remain unidentied. Nevertheless, this preliminary study indicated the efficacy of LIPUS for the stimulation of bone formation around miniscrews; this potential value of LIPUS warrants further study. Conclusions LIPUS increased boneminiscrew contact and reduces miniscrew mobility in growing subjects. Thus, LIPUS may

as substitutes for jaw bones. However, in addition to differences in the characteristics of tibiae and jaw bones, the presence of food in the mouth and mastication might affect the evaluation of miniscrew stability. Thus, the results of this preliminary study indicate the need for a future clinical study to verify the efficacy of LIPUS in human jawbones. The mobility test is an objective and readily applied method of judging successful implantation (Oliv and Aparicio, 1990; Inaba, 2009; Uemura et al., 2012). The Periotest device is useful for predicting the boneminiscrew contact condition, as Inaba (2009) reported a strong correlation between the boneminiscrew contact ratio and the PTV. Uemura etal. (2012) used a Periotest device to assess miniscrews before and after traction, and found that mobility measurements were useful for evaluating the prognosis of the miniscrew. Thus, the PTV is an appropriate index of primary implant stability. No report has described the effects of LIPUS on orthodontic miniscrews in growing subjects, although some previous studies of the effects of endosseous dental implants on stability have been published. Ustun etal. (2008) indicated that LIPUS may have positive effects on the stability of dental implants, although they used skeletally mature rabbits. Hsu et al. (2011) reported that pulsed ultrasound groups exhibited enhanced cell migration and new bone regeneration more effectively than did controls, and bone formation was accelerated in pulsed ultrasound groups in vivo and in vitro. Although the mechanism underlying the acceleration of new bone formation by LIPUS is unknown (Xie etal., 2011), theoretically, uid shear ow, the micro-streaming ow derived from LIPUS, may provide moderate stimulation to cells (Khan and Laurencin, 2008), such as periosteal cells (Leung etal., 2004), chondrocytes (Yang etal., 1996), and osteoblasts (Chen etal., 2003). Our mobility evaluation data suggest that LIPUS has positive effects on the stability of miniscrews; mobility was signicantly lower in the test group than in the control group. The effects of LIPUS on the stability of miniscrews were veried, and positive effects were identied in growing animals. A mechanical histomorphometric evaluation by Ustun etal. (2008) showed that bonescrew contact values were signicantly higher in LIPUS-treated subjects than in control subjects. Zhou etal. (2011) investigated the effects of LIPUS on implant osseointegration using rats and concluded that LIPUS therapy accelerates bone healing and osseointegration at the interface between titanium implants and bone and promotes trabecular bone remodeling during the early stage. FE-SEM images showed that bone surrounding the miniscrews contacted well in LIPUS-treated subjects, compared with control subjects, and the boneminiscrew contact ratio was signicantly greater in the LIPUS group than in controls (P<0.05). Histomorphometric evaluation also showed positive effects of LIPUS on boneminiscrew adhesion in growing rats. The difference in bone contact between

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accelerate the bone healing process after miniscrew placement and reduce the latent period in growing rats. Funding Grants-in-Aid for Scientic Research (21592613; 2011). References
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