Biomarkers of Orthodontic Tooth Movement With Fixed Appliances and Vibration Appliance Therapy

Specific Aims

1. To determine if combined vibration-fixed appliance treatment alters the expression of specific bone remodeling factors in saliva compared to fixed orthodontic treatment alone.
2. To further elucidate the role of vibration treatment on the degree of tooth mobility during fixed appliance treatment compared to control.
3. To determine if combined vibration-fixed appliance treatment increases the speed of orthodontic tooth movement during the alignment phase of treatment.
4. To evaluate the role of vibration treatment in the control of pain in patients undergoing orthodontic treatment.

Screening \& Recruitment Procedures Approximately 600 patients begin treatment every year in the University of Connecticut Orthodontic Clinic. The pilot project aim is to recruit a total of 40 patients, with 20 male and 20 female subjects, equally divided between control and vibration groups. Based on the projected starts, we anticipate meeting the recruitment goals during the first 12 months of the study.

Prospective subjects will be screened for this study after completing all the usual initial screening procedures for patients in the University of Connecticut Orthodontic Clinic with their appointed primary orthodontic provider. The primary orthodontic provider will determine if the patient is likely to qualify (pre-screen) for the study based on the inclusion/exclusion criteria. The initial clinical indicators of eligibility evaluated during the pre-screen are a healthy patient undergoing non-extraction treatment with good oral hygiene. Male and female subjects of all ethnicities will be included if between the ages of 15-35 years of age.

If the prospective patient meets the initial criteria, the orthodontic provider will notify the study coordinator and PI to screen the patient's existing dental records, including screening forms, models and/or radiographs for confirmation of likely eligibility for participation in the study.

Study Procedures I. Standardized Orthodontic Treatment Protocol The orthodontic clinic treatment protocol will be standardized to minimize variability in the sequence of treatment. All subjects will be bonded with 0.022"X0.028" brackets passive self-ligating brackets with maximal band tension (MBT) prescription from 2nd premolar to 2nd premolar in both arches and bonded tubes on the 1st molars at the baseline (T0) visit. Each subject will have a 0.014" copper nickel titanium (NiTi) archwire placed at the initial visit. At T2, the next archwire in the sequence (0.014"X0.025" copper NiTi) will be placed.

Subjects will be seen at normally appointed visits every 4-6 weeks. The primary orthodontic provider will direct the alignment and leveling phase of treatment and proceed through the wire sequence as indicated. No specific procedure/timeframe will be assigned to placement of each archwire. Providers are expected to proceed to the next archwire when it can be placed comfortably based on the degree of alignment achieved.

In the event of bracket breakages, the subject is to be seen within 7 days to have the bracket rebonded, in standard care as well as the study. The primary orthodontic provider will reposition the bracket in the ideal position and continue with alignment using the study archwires. If the subject does not present with 7 days of the breakage, he/she will be disqualified from the study.

II. Randomization Procedures Once recruited to the study and after baseline measures are taken at T0 by the study coordinator, the randomization will be performed. Since study groups will be subdivided by gender, separate randomization will be performed for males and females. Block randomization will be used. Based on gender, 20 opaque envelopes will be generated containing the group allocation (10 for the vibration study group, 10 for the non-vibration control group). At the time of randomization, the subject will choose an envelope and disclose the group assignment. For subjects allocated to the vibration group, the study coordinator will provide detailed instructions as to how to operate the appliance and will instruct those subjects to use it for 20 minutes per day.

III. Data Collection Procedures On the day of bonding (T0), prior to the placement of any appliances, baseline unstimulated whole saliva will be collected and Periotest will performed on selected teeth in the mandibular arch. In addition, alginate model impressions will be taken. These procedures will be described in more detail in the next sections. Once baseline measures have been taken, the PI will perform the randomization and notify the study coordinator as to which group the subject is assigned to ("vibration treatment" or "no vibration"). The patients allocated to the vibration treatment group will be provided with the vibration appliance (Acceledent) appliance and instructed to use the 20 minutes per day according to the manufacturer's instructions.

After fixed appliances are placed and tooth movement begins, subjects will proceed with standard orthodontic treatment with or without vibration treatment based on allocation. Collection of saliva samples, Periotest and alginate impressions will be taken at T1 (5-6 weeks post-T0), T2 (10-12 weeks post-T0) and T3 (15-17 weeks post-T0). Subjects will be seen between noon and 5pm to help minimize possible confounding of salivary markers by circadian rhythms.

Salivary Biomarkers Collection of unstimulated whole saliva will be performed using the same methods described by Navazesh and Kumar. Unstimulated whole saliva will be collected by passive drooling into sterile centrifuge tubes on ice for 15 minutes or once 2-10 milliliters (mL) of saliva is collected at baseline and each visit, whichever occurs first. Once saliva is collected, samples will be kept on ice and supplemented with proteinase inhibitor. Samples will then be centrifuged for 10 minutes at 3000 rpm to remove cellular debris and supernatants will be collected and stored at -80°C until they are ready to be analyzed for biomarkers.

Biomarkers will be evaluated by ELISA assay by a direct sandwich method using a standard protocol.

Periotest Measurement Procedures This measurement is obtained at baseline and each visit. The degree of tooth mobility will be used using a Periotest device (Siemens, Bensheim, Germany) on the central incisors, canines and 2nd premolars in both mandibular quadrants as previously described by Liou et al. The archwire will be removed and the Periotest measurements will be taken in triplicate, with means recorded.

Dental Model Analysis

Two blinded clinical evaluators will assess the rate of alignment of the lower incisors for both groups. Analyses of dental casts will be done on the mandibular cast using a method based on Little's irregularity index. Little's irregularity index was developed to assess amount of anterior tooth displacement from canine to canine. Contact displacement will be measured in millimeters around the arch from canine to canine. Irregularity will be measured on models from each of the four time points and be evaluated based on percentage change over the 6-month evaluation period.

Orthodontic Pain Assessment

Subjects will be given a pain diary on the baseline (T0), visits T1 \& T2 to record the level of orthodontic pain each evening for the first 7 days after each study visit. The degree of pain will be assessed using a Visual Analog Scale (VAS) ranging from 1 (least pain) to 10 (highest pain). In addition, patients will report if the pain is constant or only present on biting/chewing. The subjects will return the completed diary to the next visit to be collected by the study coordinator and kept in the study record.

Oral Health Quality of Life (OHQoL)

To assess the impact of the vibration appliance on the overall oral health quality of life of the subject, subjects will be given an Oral Health Impact Profile (OHIP-14) questionnaire. The OHIP-14 consists of 14 questions divided into specific dimensions including functional limitation, physical pain, psychological discomfort, physical disability, psychological and social disability and overall life handicap from orthodontic treatment with or without the vibration appliance. The OHIP-14 will be administered at each study visit and the profile changes will be compared across time points in data analyses.

Individual scores are multiplied by the weights to give a subscale score for each category.

IV. Sample size and justification Few studies exist evaluating salivary biomarkers during orthodontic tooth movement. Our primary outcome is changes in the expression of specific biomarkers of cellular activation and bone remodeling. No randomized clinical trials (RCTs) are currently available to predict treatment effects on biomarker expression. This study will serve as a pilot project with 40 patients total in 4 groups divided by gender and vibration/no-vibration treatment arms.

We estimate 600 patients in the target age range beginning orthodontic treatment at the University of Connecticut Orthodontic Clinic each year. We anticipate meeting our recruitment goal for this study.

1. Subject Participation The timing of visit activity is planned to coincide with normally scheduled clinic provider/patient visits. Typically, treatment for non-extraction cases last from 12-30 months in the Orthodontic clinic using fixed appliances. However, the subjects will only be evaluated during the first 6 months of their clinical appliance treatment for the study purpose and data. Providers will follow patients every 5-6 weeks and the study coordinator will ensure/confirm the patients for study appointments. After completion of the observation and data collection phase ( up to 17 weeks), the patients will continue to proceed with treatment as directed by the their primary orthodontic provider. Data collection will occur at normally scheduled visits with no additional appointments specific to the study.

In the event that the PI determines that a subject is not suited to continue in the study (eg. the subject shows consistent failure to keep appointed visits, consistent breakage of appliances, patient safety considerations, etc.) he/she will be withdrawn by the PI from the study and no further data will be collected. Specifically, poor compliance with attending appointments during the allotted time frame for each study visit will result in subjects being disqualified from the study. For example, the follow-up time frame for T1 is 5-6 weeks after T0 (baseline). Therefore if the patient misses an appointment and cannot or does not reschedule to attend his/her appoint during the allotted time from (by 6 weeks after T0), then he/she will be disqualified from the study. Also, in the event of bracket breakages (as stated earlier), the subject is to be seen within 7 days to have the bracket rebounded. The primary orthodontic provider will reposition the bracket in the ideal position and continue with alignment using the study archwires. If the subject does not present with 7 days of the breakage, he/she will be disqualified from the study.

The data collected to that point will be utilized in analyses. The patient's treatment will continue with the primary orthodontic provider consistent with the standard of care.

VI. Statistical/Data Analysis Intra and inter-rater reliability of the irregularity measurements will be assessed using the T0 and T3 models evaluated by the two blinded evaluators one week apart for all patients. A Cronbach alpha analysis will be used to assess reliability of the measurements. A single calibrated evaluator (the study coordinator) will collect all tooth mobility measurements with the Periotest and means will be evaluated using non-parametric statistical analyses.

A Wilcoxon Rank Sum test for independent samples will be used to assess differences between groups for all the continuous variables with an α=0.05 for irregularity index changes-Periotest measurements, VAS \&OHIP-14 measures.

Salivary biomarker expression will be analyzed by ANOVA to assess differences between the four study groups at each time point. The study coordinator and principal investigator will perform statistical analyses.