Randomized clinical trial on the effect of intermittent vibrational force application during orthodontic treatment with aligners on RANKL and OPG concentrations in crevicular fluid

Abstract Application of intermittent forces by vibration is proposed as an easy‐to‐use accelerator of dental movement. The purpose of this study was to determine the effect of intermittent vibrational force application during orthodontic aligner treatment on receptor activator of nuclear factor‐kappa B ligand (RANKL) and osteoprotegerin (OPG) concentrations in crevicular fluid as markers of bone remodeling. This three‐arm parallel randomized clinical trial included 45 candidates for malocclusion treatment with aligners, randomly assigned to: Group A (vibrational forces from onset of treatment); Group B (vibrational forces at 6 weeks after treatment onset); or Group C (no vibration). The frequency of aligner adjustment also differed among groups. At different time points, a paper tip was used to draw crevicular fluid samples from a moving lower incisor for RANKL and OPG analysis using ELISA kits. Mixed‐model ANOVA found no significant differences in RANKL (A: p = 0.31, B: p = 0.8, C: p = 0.49) or OPG (A: p = 0.24, B: p = 0.58, C: p = 0.59) over time in any group or as a function of the application/non‐application of vibration or the frequency of aligner adjustments. Application of this accelerator device did not significantly affect bone remodeling in patients undergoing orthodontic treatment with aligners. However, a nonsignificant improvement in biomarker concentrations was observed when aligners were changed every 7 days and vibration was also applied. Further research is warranted to establish protocols for the application of vibration and the timing of aligner adjustments.


| BACKGROUND
The growing number of adults seeking orthodontic therapy has focused interest on the acceleration of tooth movement to reduce treatment times. Longer treatments carry a higher risk of gingivitis, decalcifications, caries, and possible root resorption and have a greater negative impact on the quality of life and facial esthetics of patients. 1,2 Surgical approaches to acceleration have been associated with elevated morbidity, and the application of intermittent forces by vibration offers an alternative easy-to-use option that is well accepted by patients and may have similar cellular effects.
Interactions of numerous inflammation mediators and growth factors have been related to cellular activation and responses after tooth movement onset. These biomediators and inflammatory markers are involved not only in activating osteoclasts and osteoblasts during bone remodeling but also in regulating the speed of tooth movement. 3 The main mediators reported to regulate bone remodeling during orthodontic treatment are interleukin 1β, tumor necrosis factor, receptor activator of nuclear factor-kappa B ligand (RANKL), receptor activator of nuclear factor-kappa B (RANK), and osteoprotegerin (OPG). [4][5][6] Bone remodeling is a balance between RANK-RANKL and OPG activation. In addition, the RANK signaling pathway is crucial for differentiating and activating osteoclasts. Both OPG and RANKL are expressed by osteoblastic cells. The binding of RANKL to RANK promotes osteoclast activation, whereas the binding of OPG to RANKL inhibits this activation, promoting bone formation. This mechanism is triggered by the application of forces. 3 In a rat study, Nishimura et al. 7 described a relationship between movement acceleration and the application of vibrational forces, finding that utilization of a vibration device increased RANKL expression and osteoclastic activation. Likewise, Nakao et al. 8 reported that RANKL expression of human periodontal ligament cells was stimulated by the application of intermittent vibratory forces. In contrast, Woodhouse et al. 9 found that intermittent vibratory forces had no effect on the speed of initial orthodontic movement, measured as shifts in tooth position on study models; however, they did not investigate associated biochemical markers.
The aim of this study was to explore whether the application of intermittent vibratory force modifies RANKL and OPG concentrations in patients undergoing orthodontic treatment with clear aligners. The specific objective was to compare gingival crevicular fluid concentrations of RANKL and OPG among groups according to the application or not of Acceledent ® treatment at different time points and the frequency of aligner changes.

| Trial design and changes after trial commencement
This three-arm parallel randomized clinical trial (allocation ratio of 1

| Participants, eligibility criteria, and setting
Participants were recruited from among patients attending the Clinic of a university department of orthodontics and two private orthodontic clinics. All patients were treated by the same orthodontist. Inclusion criteria were age between 21 and 50 years (inclusive) and treatment of malocclusion with Invisalign ® (Align Technology, San Jose, CA), using at least 14 sets of aligners. Exclusion criteria were smoking habit, poor oral hygiene, the presence of periodontal disease (plaque indices >3 and community periodontal index of treatment needs >2; periodontal pockets >4 mm), or any other chronic or systemic diseases that could affect bone metabolism or inflammation, and previous or current receipt of medication that could influence bone metabolism (e.g., bisphosphonates).

| Interventions
Patients who met the eligibility criteria were invited to participate in the study. All participants signed their informed consent.
Treatments were all carried out using Invisalign ® aligners. Intermittent vibratory force was applied with an Acceledent ® device (Acceledent

| Outcomes
The primary outcomes of this study were the gingival crevicular fluid concentrations of RANKL and OPG at different measurement time points. Secondary outcomes were Silness and Loe 10 plaque index, Lobenne et al. 11 modified gingival index, and bleeding on probing scores (in teeth under study and two adjacent teeth) at the same time points. Cotton rolls were used to isolate the tooth and clean the plaque remains, keeping the area dry and free of bleeding while the paper tip was inserted 1 mm into the gingival sulcus for 30 s. All samples were immediately frozen at À20 C and transferred to the UCM Research Laboratory for storage at À80 C with associated patient number and the time and date of sample collection. Consequently, an effect size (mean difference divided by its SD) of 1.108 was established, and a minimum sample of 14 patients was calculated for a statistical power of 80% and significance level of 5%. 6

| Randomization and blinding
A computer-generated randomization list (GraphPad Software, Inc., USA) was used to randomly assign patients to study groups. Blinding was ensured by using preprepared sequentially numbered, opaque, sealed envelopes containing treatment allocation cards. A single researcher (CM) was responsible for opening the envelopes in sequence and implementing the randomization process. Neither patients nor orthodontist could be blinded to group membership because of the differences in treatment protocols. Nevertheless, assessments of clinical and laboratory outcomes were blinded because it was not possible to distinguish among the groups.

| Statistical methods
The demographic characteristics of patients in each group were expressed as means with standard deviations and as frequencies.
ANOVA and chi-square tests were used to compare age and sex Between-group comparisons OPG. Table 2 Table 2

| Secondary outcomes: plaque and gingival indices
No statistically significant between-group or within-group differences were found in gingival index, plaque index, or bleeding on probing scores (data not shown).

| DISCUSSION
Various techniques have been developed to accelerate tooth movement and thereby reduce the duration of orthodontic treatments. 12 A key research question is whether the association of higher tooth movement with increased RANKL concentrations and decreased OPG concentration found in rats can also be observed in humans. [4][5][6] In this study on the effects of vibration with a specific device and  Regarding OPG concentrations, a nonsignificant progressive reduction was observed when vibration was used from treatment onset, and an increase was recorded when it was not. In contrast, Alikhani reported that vibration has an anabolic effect on nonmoving teeth, [12][13][14] although Yadav 15 showed that the effect of vibration at 30 Hz may be more anabolic than catabolic, given the increase in OPG during phases when it should be reduced. These effects may vary according to the timing of vibration application and the length of interval between aligner changes at the start of treatment. 15 Data were gathered on the concentrations of RANKL and OPG (pg) rather than the volume in each sample, applying the same dilution to homogenize the results. According to Leethanakul et al. knowledge of the volume of the sample or the concentration of protein is equally valid to determine changes in the concentration of these biomarkers. 16 Comparisons with previous studies on the effects of vibration are limited because most examined clinical but not molecular changes.
The purpose of the present investigation was to associate possible effects of vibration with increases and decreases in biomarker con-

| Adverse effects
No adverse effects were observed in any patient.

| Limitations and generalizability
Study limitations include the relatively small sample size which may be responsible for the lack of statistically significant differences. Interpretation of these results should also take into account that the value of ranges of RANKL and OPG concentrations for periodontal diagnostics has not yet been validated. It would also have been useful to gather samples more frequently during initial phases of treatment and leave longer intervals after the third month to determine the cytokine response not only at the beginning but also at the end of treatment and in the retention phase.
Protocols should ideally be standardized in future research in terms of the application of vibration, length of treatment, and vibration frequency, which can determine the cells that are activated. Finally, the periodontal effect of vibration should be studied in vivo in periodontally healthy individuals, as well as in patients with periodontal disease.

| CONCLUSIONS
• The application of intermittent vibratory force using Acceledent ® does not appear to significantly alter crevicular fluid RANKL and OPG concentrations in patients undergoing orthodontic treatment with Invisalign ® .
• Although statistically significant results were not obtained, the application of vibration was associated with an increase in RANKL and a decrease in OPG concentrations, observing a greater increase in RANKL and decrease in OPG when applied at the initiation of treatment, before the onset of tooth movement.

ACKNOWLEDGMENTS
The authors are grateful with Ana O'Connor de la Oliva and Marta García Chac on for their invaluable support in laboratory analysis.

FUNDING INFORMATION
This study was funded by the Spanish Society of Orthodontics (BIJC-2017-0001).