Trial design and any changes after trial commencement
This single-center, open-label, pilot randomized clinical trial was conducted with a 1:1 allocation ratio. The study received ethical approval from the Institutional Ethics Committee, AIIMS Jodhpur (AIIMS/IEC/2019-20/977), Rajasthan, India, and was registered prospectively at the Clinical Trials Registry India under CTRI/2020/12/029660. The methodology remained the same after the trial had commenced.
Participants, eligibility criteria, and settings
The recruitment of patients was done in the graduate orthodontic clinic, Department of Dentistry, AIIMS Jodhpur, Rajasthan, India, from January 2020 to April 2021. Inclusion criteria were as follows: age range of 12–30 years, requiring maxillary first premolar extractions primarily for retraction of protruded anterior maxillary teeth, minimal arch length tooth size discrepancy (≤ 3 mm), full permanent dentition with sound first and second molars and good periodontal health. Exclusion criteria were: moderate to severe crowding in maxillary arch, history of previous extractions or those requiring asymmetric extractions, any systematic condition that could affect periodontal status, previous history of orthodontic treatment, patients with disorders of bone metabolism (e.g., osteoporosis) or under any medications for same, patients with systemic diseases, cleft lip and palate, and other craniofacial abnormalities or smoking. All patients or parents (in the case of minors) signed the informed consent before initiation of the treatment.
Interventions
After recruitment, patients were randomly assigned into two groups. Each patient received a 0.022-inch pre-adjusted edgewise appliance (3M Unitek™ Gemini metal brackets, MBT prescription, California, USA). Alignment and leveling was performed with nickel-titanium (G4™ Nickel-titanium, G&H, Franklin, Indiana, USA) archwires ligated in the sequence of 0.014-inch, 0.016-inch, 0.018-inch, and 0.019 x 0.025-inch, respectively. After completion of alignment and leveling, the arch form was stabilized with 0.019 x 0.025-inch stainless-steel (SS) archwire.
In the healed extraction group (HE group), maxillary first premolars were extracted at the beginning of treatment (before initiation of alignment). The extraction site was allowed to heal during the alignment and leveling phase. In the recent extraction group (RE group) maxillary first premolar extraction was performed after leveling phase when the passivity of the stabilized archwire was ascertained.
En-masse retraction of anterior maxillary teeth was performed on a 0.019 x 0.025-inch SS archwire, aided by an elastomeric chain (Power Chain, Ormco, Glendora, California, USA) with forces of 150 grams, extended between the second molar and the archwire hook crimped between maxillary lateral incisor and canine. In the RE group, retraction was initiated within one week of extraction.
Data collection procedure
The salivary sample and study models were collected at five time intervals; at the start of orthodontic treatment (T0; baseline, before any extraction was performed), after completion of alignment and leveling (T1), and at 2-week (T2), 8-week (T3), and 12-week (T4), respectively, after the application of retractive forces. In the RE group, samples at T1 were collected after alignment and leveling but before extraction of premolars. These time intervals usually correspond to different phases of orthodontic tooth movement (14). This study chose a 12-week endpoint for space closure because it has been previously studied that at 8 weeks post-extraction, the extraction socket gets replaced by a provisional matrix and immature bone [15]. The clinical interventions of the study were performed by NV. Outcomes from saliva samples and study models were measured by MB and RS.
Saliva collection procedure
Saliva samples were collected in the morning before breakfast by the same procedure as Florez et al. [10] in their study. The saliva sample was obtained using a sterile tube through passive drooling for 5 minutes or until 5 ml was reached. The accumulated samples were centrifuged at 4000 rpm for eight minutes, after which the supernatant was extracted, divided into 500 µL portions, and stored in a freezer at -80ºC until further processing. Salivary samples were later analyzed for RANKL and OPG concentration using human Enzyme-linked immunoassay (Bioassay Technology Laboratory, Jiaxing, Zhejiang).
Sampling and Processing Preparation
At the time of biomarker analysis, the salivary samples and kits were equilibrated at room temperature. 100 µL of standard working buffer was added to each sample and gradually diluted, which was incubated at 37ºC for 80 minutes. The liquid from the plate was removed and replaced with 200 µL of wash buffer in each vial. The plate was washed thrice using an automated ELISA plate washer before spin-drying. Next, 100 µL of biotinylated antibody working solution was added to each vial and incubated at 37ºC for 50 minutes. The plate was washed three more times and allowed to dry before adding 100 µL of Streptavidin-horseradish peroxide (HRP) working solution to each vial, followed by incubation at 37°C for 50 minutes. The plate was then washed five times and spin-dried before adding 90 µL of tetramethyl benzidine (TMB) to each vial and incubating at 37°C for 20 minutes. A stop solution of approximately 50 µl was added to each vial, and plates were read immediately at 450 nm using an absorption reader, followed by which calculation of the results was done.
Outcomes (primary and secondary)
The primary outcome was to evaluate the concentration of salivary biomarkers RANKL, OPG, and RANKL/OPG, during en-masse retraction of anterior maxillary teeth into RE and HE sites. The concentration of RANKL and OPG in saliva samples was determined by comparing the optical density of the samples to the standard curve given by RANKL human Enzyme-linked immunoassay (RANKL-ELISA) and OPG human Enzyme-linked immunoassay (OPG-ELISA). The concentrations were calculated using Chromate Manager software through a nonlinear regression model. A 4-parameter logistic calibration curve was used to construct the standard curves, which were further used for determining the actual concentration of each protein in the samples, standards, and internal controls. The R-squared values for RANKL and OPG were 0.9995 and 0.9998, respectively, which is typical for standard curves.
Secondary outcomes were the amount and rate of en-masse retraction measured from study models. The distance between the maxillary canine cusp tip and the mesiobuccal cusp tip of the molar at different time points (T1, T2, T3, and T4) was measured using a sharpened fine-edge standard caliper with an accuracy of up to 0.02 mm. The amount of retraction was calculated by subtracting the distances at each time point. The rate of en-masse retraction was calculated by dividing the amount of retraction by the duration of time taken (12 weeks) in mm/week.
Sample size calculation
We conducted a pilot study as a precursor to a more considerable randomized clinical trial. Before the start of the trial, there were no published estimates on the concentration of salivary biomarkers in recent and healed extraction sites during en-masse retraction. For this study, we selected a convenient sample of 20 participants from the target population.
Method error
The Dahlberg error (16) for study model measurements was found to be in the range of 0.12 to 0.28 mm, suggesting no significant measurement error. Additionally, excellent intra-examiner reproducibility was observed for all study model measurements, with an intraclass correlation coefficient of 1.000 (95% CI, 0.999-1.000) when repeated after two weeks.
Interim analyses and stopping guidelines
Not applicable
Randomization (random number generation, allocation concealment, and implementation)
Eligible patients were randomly assigned in a 1:1 ratio to RE and HE sites using a variable block randomization scheme. The randomization sequence was computer-generated. To ensure unbiased allocation, opaque, sealed, and sequentially numbered envelopes were used for concealment. The data analyst who was not involved in any trial stage was responsible for randomization concealment.
Blinding
The study participants and clinician rendering treatment could not be blinded. The independent expert (MB) who performed the ELISA test for RANKL and OPG concentration, the research personnel (RS) who measured the study models, and the data analyst (SS) were blinded to treatment allocation.
Statistical Analysis
The data analysis used the Statistical Package for Social Sciences for Windows, version 23.0 (Armonk, NY: IBM Corp.). The intention-to-treat (ITT) principle was used for analyses. Descriptive statistics were calculated, including means and standard deviations (SDs). Baseline categorical data were compared using the chi-square test. Before running the t-test, data were tested for normality using the Kolmogorov-Smirnov test, yielding insignificant results. A one-way repeated-measures ANOVA was used to assess the changes in salivary concentrations of RANKL, OPG, and RANKL/OPG from T1 to T4. An independent t-test was used to compare the differences in changes between the groups. The significance level was P < 0.05 with 95% confidence intervals (CIs).