Evaluation of salivary biomarkers during en-masse retraction of protruded teeth into recent and healed extraction sites– a Randomized Clinical Trial

Evaluation of salivary biomarkers during en-masse retraction of protruded teeth into recent and healed extraction sites– a Randomized Clinical Trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of salivary biomarkers during en-masse retraction of protruded teeth into recent and healed extraction sites– a Randomized Clinical Trial Vaghela Niraj, Rinkle Sardana, Mithu Banerjee, Navleen Kaur Bhatia, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4562549/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives The study aimed to evaluate levels of salivary biomarkers during en-masse retraction of anterior maxillary teeth into recent and healed extraction sites. Materials and methods Twenty patients with protruded anterior maxillary teeth requiring en-masse retraction were randomly allocated into two groups. In the healed extraction (HE) group, first premolar extraction was performed at the beginning of treatment while in the recent extraction (RE) group, it was done just before the initiation of en-masse retraction. Saliva and study model collection was done at start of treatment, after leveling, and after 2, 8 and 12-weeks of retraction, respectively. The primary outcome was to evaluate concentrations of salivary biomarkers; receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). Rate of en-masse retraction was secondary outcome. One-way repeated-measures ANOVA was applied for within-group changes, and an independent t-test for the intergroup comparison (P < 0.05). Results An increase in concentration of RANKL and decrease in OPG concentration was observed in both the RE and HE groups at 12 weeks; however, the intergroup comparison showed no statistically significant differences (P = 0.517). RANKL/OPG ratio was significantly higher in the RE group (P = 0.015) at 12 weeks. A significantly higher rate of en-masse retraction was found in the RE group (P < 0.001). Conclusion Variations in RANKL/OPG ratio can be considered a better predictor of orthodontic tooth movement. A significant higher rate of retraction in the RE group suggests that immediate application of retractive forces towards the RE site may hasten space closure. Clinical Relevance Findings of the present study suggest that immediate application of activation forces at the recent extraction site may hasten initial orthodontic tooth movement by preventing excessive strain on the alveolar bone, which has had time to heal during the tooth movement at healed extraction site. Additionally, the study contributes to existing literature that highlights saliva as an easily accessible alternative to crevicular fluid for sampling purposes. Salivary biomarkers Receptor activator of nuclear kappa B ligand Osteoprotegerin RANKL/OPG Recent extraction site Healed extraction site En-masse retraction Figures Figure 1 Figure 2 INTRODUCTION Extraction space closure is the foremost time taking phase in orthodontic treatment, especially in patients with protruded maxillary anterior teeth. The movement of teeth into an extraction space depends not just on the biomechanical procedure but also on biological factors. Application of orthodontic forces post-extraction can cause variation in the structure of the alveolar process into which the anterior teeth move. The teeth can either be moved into the extraction space with sufficient time to heal or into the recently extracted socket. Studies [ 1 – 3 ] have been done on animals where surgically accelerated tooth movement and histological characteristics of extraction sockets were investigated. Hasler et al. [ 1 ] and Liou [ 4 ] found that tooth movement was faster at sites of recent extraction. In contrast, another animal study by Samruajbenjakun et al. [ 5 ] found no difference in the rate of tooth movement in rats between the recent and healed extraction socket group. While it has been reported that tooth movement is accelerated towards recent extraction sockets, the underlying biological mechanism has yet to be extensively examined. The different cells of the bone multicellular unit (BMU) act in a specific sequence composed of four phases [ 6 ]. During the activation phase, the receptor activator of nuclear factor K ligand (RANKL), a homotrimeric protein present on the cell surface of the preosteoclasts from the bone marrow, interacts with the receptor activator of nuclear factor K (RANK), a homotrimeric transmembrane protein member of the tumor necrosis factor (TNF) family [ 6 , 7 ]. Their linkage leads to bone resorption, lasting about two weeks. In the reversal phase, preosteoblasts that have migrated into the resorption lacunae mature and differentiate into mature osteoblasts. These osteoblasts secrete osteoprotegerin (OPG), a free-floating soluble decoy receptor belonging to the tumor necrosis factor family, that binds with the RANKL and, thus, prevents further activation of the preosteoclast which in turn inhibits resorption activity by the osteoclast [ 6 , 7 ]. One way to measure the activity of the bone remodeling process is by analyzing biochemical markers. Previous studies have shown that saliva provided a sensitive and inexpensive detection technique for determining analytes in the periodontal microenvironment [ 8 , 9 ]. According to Florez et al. [ 10 ], the ratio of sRANKL to OPG varies over time during orthodontic alignment. This suggests that sRANKL/OPG may be associated with the various stages of orthodontic tooth movement. Reiss et al. [ 11 ] recently observed salivary biomarkers for three months and found no difference in salivary biomarkers' expression and the mandibular anterior alignment rate when supplemented with the vibratory force. Although many experimental human and animal studies have studied salivary biomarkers, these studies have examined the biochemical markers in the whole saliva of patients undergoing fixed appliance therapy during the initial stages of treatment [ 10 , 11 ]. However, to our knowledge, no study has evaluated these biomarkers during extraction space closure based on the type of extraction socket. In addition, many studies that concentrate on the rate of en-masse retraction of anterior teeth have yet to distinguish between healed and recent extraction sites [ 12 , 13 ]. This study aimed to evaluate concentrations of salivary biomarkers (RANKL and OPG) during en-masse retraction of anterior maxillary teeth into RE and HE sites. Additionally, the amount and rate of en-masse retraction were also evaluated. The null hypothesis was that there was no significant difference between the groups. MATERIALS AND METHODS 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). RESULTS Participant flow During recruitment, 24 patients were assessed for eligibility. Three patients were deemed ineligible as inclusion criteria were not met, while one declined to participate. Twenty patients were randomly assigned to the RE, or HE groups in a 1:1 ratio. All participants completed the study. Ten patients in each group were analyzed. The CONSORT flowchart depicting the participants in the trial is presented in Fig. 1 . Recruitment Patient recruitment began in January 2020 and concluded in April 2021. Baseline data Table 1 presents a comparison of the baseline characteristics between the groups. The groups showed no statistically significant differences in any parameter. No significant difference was found between the groups in the concentration of RANKL and OPG at T0. The final sample size for the RE and HE groups was the same. Each group had ten subjects (7 females, 3 males). Table 1 Baseline characteristics of participants in each study group Variable RE group, n = 10 HE group, n = 10 P value Significance Initial age, years 23.2 ± 5.28 19.8 ± 3.56 0.101 NS Gender Male 3 (30) 3 (30) 1.000 NS Female 7 (70) 7 (70) Duration of alignment and leveling (months) 5.6 ± 0.91 5.7 ± 0.68 0.703 NS Salivary biomarkers RANKL (pg/ml) at T0 71.7 ± 4.69 71.4 ± 5.88 0.891 NS OPG (pg/ml) at T0 2.6 ± 0.72 2.4 ± 0.48 0.514 NS RANKL/OPG ratio at T0 29.8 ± 11.39 36.4 ± 6.32 0.129 NS Distance between cusp tip of canine and mesiobuccal cusp tip of first molar at T1 (mm) 21.6 ± 0.81 20.7 ± 1.09 0.158 NS Note. Values are mean ± SD or n (%). Abbreviations. RE; recent extraction, HE; healed extraction, SD; standard deviation, n; number, NS; non-significant, T0; start of orthodontic treatment, before extraction (baseline). T1; after completion of alignment and leveling. Primary Outcome. Table 2 gives the result for changes in RANKL, OPG, and RANKL/OPG concentrations at the different sampling times in the RE and HE group during en-masse retraction of maxillary anterior teeth. The salivary biomarker RANKL showed a linear trend in their expression throughout the 12 weeks in both groups. The change from T1 to T2, T3, and T4 was statistically significant in the RE group (P < 0.001). However, in the HE group, the change was statistically significant only from T1 to T4 (P = 0.003). On comparison between the groups, salivary RANKL concentration showed no statistically significant difference at any time point (P = 0.205, P = 0.472, P = 0.517, respectively; Table 2 ). Table 2 Comparison of salivary concentration of RANKL (pg/ml), OPG (pg/ml), RANKL/OPG ratio at different time points during en-masse retraction of maxillary anterior teeth into recent and healed extraction sites. Outcome Timepoint RE group, n = 10 HE group, n = 10 Within-group P value Between-group P value RE group, n = 10 HE group, n = 10 RE group, n = 10 HE group, n = 10 RANKL T1 71.8 ± 6.37 76.5 ± 20.67 --- --- 0.496 T2 99.1 ± 21.63 87 ± 26.28 0.003* 0.361 0.205 T3 99.5 ± 12.04 96 ± 25.89 0.000* 0.081 0.472 T4 115.5 ± 20.08 112.9 ± 18.24 0.000* 0.003* 0.517 OPG T1 2.7 ± 0.53 2.6 ± 1.32 --- --- 0.755 T2 2.1 ± 0.41 2.4 ± 0.98 0.012* 0.693 0.445 T3 2.43 ± 0.88 2.7 ± 0.56 0.430 0.813 0.477 T4 1.8 ± 0.64 2.3 ± 0.67 0.007* 0.486 0.142 RANKL/OPG T1 27.4 ± 6.37 34.6 ± 10.87 --- --- 0.090 T2 45.3 ± 11.45 44.5 ± 23.98 0.001* 0.261 0.393 T3 44.9 ± 13.69 34.2 ± 13.13 0.009* 0.949 0.031* T4 72.9 ± 26.77 52.1 ± 14.83 0.001* 0.004* 0.015* Note. Values are mean ± SD. Within-group and between group comparison P- values for change from T1 to T2, T3, or T4 against no change measured using repeated-measures ANOVA with results of Greenhouse-Geisser. P value for comparison between group means done by Student’s t-test. *Statistically significant at P < 0.05. Abbreviations. RE; recent extraction, HE; healed extraction, SD; standard deviation, n; number, NS; non-significant, T1; after completion of alignment and leveling, T2; 2 weeks after retraction, T3; 8 weeks after retraction, T4; 12 weeks after retraction The change in salivary concentration of OPG from T1 to T2 and T4 was statistically significant in RE group (P = 0.012, P = 0.007, respectively). In the HE group the changes were statistically insignificant at all time points (P = 0.693, P = 0.813, P = 0.486, respectively; Table 2 ). On comparison between the groups, salivary OPG concentration showed no statistically significant difference at any time point (P = 0.445, P = 0.477, P = 0.142, respectively; Table 2 ). Due to variation for each biomarker, RANKL/OPG ratio showed a nonlinear trend. Two weeks after the initial force application, the ratio increased from T1 to T2 in both groups. It was observed that the ratio decreased at 8 weeks followed by an increase again at 12 weeks in both groups. The change from T1 to T2, T3, and T4 was statistically significant in the RE group (P = 0.001, P = 0.009, P = 0.001, respectively). However, in the HE group, the change was statistically significant only from T1 to T4 (P = 0.004). On comparison between the groups, RANKL/OPG ratio showed a statistically significant difference from T1 to T3 and T4 (P = 0.031, P = 0.015, respectively; Table 2 ). Figure 2 provides a visual representation of the changes in concentration for each biomarker over time, which helps to clarify the results. Each protein had its peak at 12 weeks. Secondary Outcome. The total amount of space closure was approximately 3.3 mm in the RE group and 2.46 mm in the HE group. The amount of space closure was found to be significantly higher (P < 0.001) in the RE group as compared to the HE group at the end of 12 weeks (Table 3 ). The rate of en-masse retraction was 0.27 mm/week in the RE group and 0.20 mm/week in the HE group. A significantly higher rate of retraction was found in the RE group (P < 0.001) when compared to the HE group during en-masse retraction of maxillary anterior teeth (Table 3 ). Table 3 Comparison of amount and rate of en-masse (mm/week) during during en-masse retraction of maxillary anterior teeth into recent and healed extraction sites. Outcome Timepoint RE group, n = 10 HE group, n = 10 Within-group P value Between-group P value RE group, n = 10 HE group, n = 10 RE group, n = 10 HE group, n = 10 Amount of space closure (mm) Diff T1 – T2 0.72 ± 0.15 0.35 ± 0.30 0.001* 0.005* 0.003* Diff T1 – T3 2.00 ± 0.24 1.38 ± 0.38 0.001* 0.001* 0.001* Diff T1 – T4 3.3 ± 0.26 2.46 ± 0.29 0.001* 0.001* < 0.001* Rate of en-masse retraction (mm/weeks) T1 – T4 0.27 ± 0.02 0.20 ± 0.02 --- --- < 0.001* Note. Values are mean ± SD. P value of difference between groups is calculated using student’s t test. Rate of tooth movement is calculated by T1-T4/12 Weeks. *Statistically significant at P < 0.05. Abbreviations. RE; recent extraction, HE; healed extraction, SD; standard deviation, n; number, NS; non-significant, T1; after completion of alignment and leveling, T2; 2 weeks after retraction, T3; 8 weeks after retraction, T4; 12 weeks after retraction. Harms No harm was observed during the study. DISCUSSION Saliva has great potential as a non-invasive method for detecting markers of orthodontic tooth movement [ 17 ]. It has been used in studies to monitor changes in OPG and RANKL levels during orthodontic tooth movement [ 10 , 11 , 18 ]. These salivary RANKL and OPG levels correspond well with those investigated in gingival crevicular fluid samples and, hence unstimulated whole saliva was considered an easy alternative compared to crevicular fluid for sampling [ 9 ]. This can help the clinician understand the biology behind orthodontic space closure towards RE and HE sites. In the present study, it was observed that during en-masse retraction of anterior maxillary teeth, the mean salivary concentration of RANKL increased and that of OPG decreased in both the RE and HE group at 2 weeks. However, the change was significant only in the RE group. The result corresponds well with the initial phase of tooth movement that takes place after the application of orthodontic forces. In-vitro studies have shown that the periodontal ligament (PDL) can either upregulate RANKL or OPG, depending on the type of force applied [ 19 ]. In cases where the PDL cells were subjected to compression forces, RANKL expression was upregulated while OPG was downregulated [ 20 ]. The significant change in the RE group could be due to significant inflammation and active bone remodeling at the tooth socket site. The results are in agreement with the study of Florez et al. [ 10 ] where they observed that the salivary concentration of RANKL increased and that of OPG decreased after two weeks of force application although they evaluated salivary biomarkers only during the initial phase of alignment. At 2 weeks, the RE group experienced a significant increase in RANKL concentration and a decrease in OPG concentration, resulting in a significant increase in the RANKL/OPG ratio. This shift favored osteoclastogenesis and led to the movement of teeth in the periodontal ligament space. The results at 8 weeks showed an increase in the mean concentration of RANKL in both the RE and HE groups. However, the intergroup differences were insignificant. The salivary concentration of OPG at 8 weeks showed a non-linear trend. There was decrease in the OPG concentration in the RE group while a slight increase in the HE group. However, both the intragroup and intergroup differences were found to be insignificant. The insignificant change in concentration of RANKL and OPG in saliva at 8 weeks could be due to the reactivation of retractive forces that causes another period of acute inflammation which may overlap with ongoing chronic inflammation. This correlates well with the lag phase and post-lag phase of orthodontic tooth movement. During the lag phase, bone resorption slows down as osteoclasts have to be recruited from nearby marrow spaces to reach the periodontal ligament and alveolar bone through the vascular system, to create a transit inflammation in order to remove the hyalinized tissue [ 21 ]. The post-lag phase follows, where tooth movement accelerates due to periodontal ligament remodeling and localized apposition and resorption of alveolar bone. The RANKL/OPG ratio was significantly increased at 8 weeks in the RE group when compared to the HE group. This is mainly because of a significant increase in the concentration of RANKL at 8 weeks, favoring bone resorption. After 12 weeks of en-masse retraction of maxillary anterior teeth, it was observed that there was a significant increase in the salivary concentration of RANKL in both the RE and HE group. The salivary concentration of OPG decreased in both the RE and HE group at 12 weeks, however significant difference was observed only in the RE group. The result was in agreement with the trial of Reiss et al. [ 11 ]. where the salivary concentration of RANKL was increased and that of OPG was decreased at 10–12 weeks after reactivation of orthodontic forces although their study focused only the initial phase of alignment. The study by Naoumova et al. [ 18 ] has also shown a decrease in the concentration of OPG during space closure. The results correspond well with the linear phase of orthodontic tooth movement. An interesting finding in the present study was that at 12 weeks post retraction, the RANKL/OPG ratio showed a significant difference between RE and HE group. The concentration of RANKL was increased, and the level of OPG was decreased, thus leading to a significant increase in RANKL/OPG ratio in both the recent (P = 0.001) and HE group (P = 0.004). Our finding was similar to Reiss et al. [ 11 ]. The increase in the RANKL/OPG ratio is in concurrence with the increased rate of tooth movement observed during the linear phase. The significant changes in both RANKL and OPG salivary concentrations, as well as the RANKL/OPG ratio observed at 12 weeks, may be linked to the enlistment of new osteoclasts, and the activation and further differentiation of pre-existing osteoclasts, causing bone resorption and a negative bone balance at the basic multicellular unit level [ 22 ]. So, it can be inferred that the RANKL /OPG ratio is a more important indicator in defining the rate of orthodontic tooth movement than the individual RANKL and OPG concentrations. In the present study, the amount of space closure was significantly greater in the RE group (3.3 mm) than in the HE group (2.4 mm) at the end of 12 weeks. Our findings were similar to Bauer [ 23 ], who reported from animal experiments that tooth movement was more into a recent extraction site than into a healed extraction site. Hasler [ 1 ] also reported that individual canines retracted more into the RE site than into the HE site by 1.14 mm. However, either the studies were animal studies or reported the amount of canine retraction, which cannot be compared directly to the present study. In the current study, the rate of retraction of maxillary anterior teeth was significantly higher (0.27 mm/week) for the RE group when compared to HE group (0.20 mm/week). Recently, Alhaija et al. [ 24 ] also reported a significantly higher amount of space closure and retraction rate (3.02 mm and 1.01 mm per month, respectively) towards the recent socket when compared to the healed socket (1.98 mm and 0.66 mm per month, respectively) although they studied the amount and rate of space closure in the mandibular arch. The difference in the rate of tooth movement between the RE and HE groups could be explained by the histological and histochemical study of Amler and Johnson on undisturbed alveolar socket healing [ 25 ]. They found that at the tooth extraction site, the osteoid starts to appear at the base of the socket by the seventh day, and at least two-thirds of the socket gets filled with trabecular bone by thirty-eight days after exodontia. Murphey [ 26 ] also concluded that there were more osteoclastic activities at recent sites and new bone formation at healed sites that can cause greater restrained tooth movement into healed sockets than into recent sockets. Earlier studies have stated that the duration of the regional acceleratory phenomenon lasts for maximum up to 4 months [ 27 , 28 ]. The present study is in concurrence with previous studies that the immediate application of retractive forces towards the recent extraction site can facilitate faster space closure due to immediate tissue inflammatory response and the regional acceleratory phenomenon [ 29 ]. This in turn helps to prevent overtaxing of the alveolar bone, which has already been allowed to heal. Limitations: A small sample size was used to determine whether the extraction timing could profoundly affect orthodontic tooth movement. Future studies with a larger sample size should be undertaken. The salivary biomarkers were evaluated during the initial retraction phase of 12 weeks. Further evaluation of these salivary biomarkers beyond twelve weeks and after completion of retraction phase could provide a better understanding of their role in orthodontic tooth movement. CONCLUSIONS The following conclusions were drawn from the study: A significant increase in the salivary concentration of RANKL was observed at 12 weeks in RE and HE groups during en-masse retraction of maxillary anterior teeth; however, no significant difference was found between groups at all time points. A statistically significant decrease in the salivary concentration of OPG was observed at 12 weeks only in the RE group during en-masse retraction of anterior maxillary teeth. Both groups showed no statistically significant differences at any time point. There was a significant increase in the RANKL/OPG ratio at 12 weeks in both RE and HE groups. The ratio was significantly higher in the RE group when compared to the HE group at 8 and 12 weeks. Significantly higher RANKL/OPG ratio in RE group suggests faster tooth movement in the RE group during the evaluation period. The amount and rate of en-masse retraction of anterior maxillary teeth were significantly higher in the RE group. Abbreviations RE: Recent extraction; HE: Healed extraction; RANKL: Receptor activator of nuclear factor kappa-B ligand; OPG: osteoprotegerin; ELISA: Enzyme-linked immunoassay; CI: Confidence interval; SD: Standard deviation; n: number; NS: Non-significant; T0: start of orthodontic treatment, before extraction (baseline); T1: after completion of alignment and leveling; T2: 2 weeks after retraction; T3: 8 weeks after retraction; T4: 12 weeks after retraction. Declarations Ethical Approval 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. All patients or parents (in the case of minors) signed the informed consent before initiation of the treatment. Funding This work was supported as a part of thesis research grant by All India Institute of Medical Sciences, Jodhpur, Rajasthan, India. Availability of data and materials The data supporting this study's findings are available from the corresponding author upon reasonable request. Conflict of Interests The authors have no conflict of interest to declare. References Hasler R, Schmid G, Ingervall B, Gebauer U (1997) A clinical comparison of the rate of maxillary canine retraction into healed and recent extraction sites-a pilot study. Eur J Orthod 19:711-719. Baumrind S (1969) A reconsideration of the propriety of the "pressure-tension" hypothesis. Am J Orthod 55(1):12-22. Van Leeuwen EJ, Maltha JC, Kuijpers‐Jagtman AM (1999) Tooth movement with light continuous and discontinuous forces in beagle dogs. Eur J Oral Sci 107(6):468-474. Liou EJ, Huang CS (1998) Rapid canine retraction through distraction of the periodontal. Am J Orthod Dentofacial Orthop 114(4):372-382. Samruajbenjakun B, Kanokpongsak K, Leethanakul C (2018) Comparison of clinical and histological characteristics of orthodontic tooth movement into recent and healed extraction sites combined with corticotomy in rats. Korean J Orthod 48(6):405-411. Verna C (2016) Regional Acceleratory Phenomenon. Front Oral Biol 18:28-35. Boyce BF, Xing L (2008) Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch Biochem Biophys 473(2):139-146. Maltha JC, Krishnan V, Kuijpers‐Jagtman AM (2021) Cellular and Molecular Biology of Orthodontic Tooth Movement. Biol Mech Tooth Mov 33-48. Nunes LA, Mussavira S, Bindhu OS (2015) Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid: a systematic review. Biochem Med (Zagreb) 25(2):177-192. Florez-Moreno GA, Isaza-Guzman DM, Tobon-Arroyave SI (2013) Time-related changes in salivary levels of the osteotropic factors sRANKL and OPG through orthodontic tooth movement. Am J Orthod Dentofacial Orthop 143(1):92-100. Reiss S, Chouinard MC, Landa DF, Nanda R, Chandhoke T, Sobue T, et al (2020) Biomarkers of orthodontic tooth movement with fixed appliances and vibration appliance therapy: A pilot study. Eur J Orthod 42(4):378-386. Upadhyay M, Yadav S, Patil S (2008) Mini-implant anchorage for en-masse retraction of maxillary anterior teeth: A clinical cephalometric study. Am J Orthod Dentofacial Orthop 134(6):803-810. Basha AG, Shantaraj R, Mogegowda SB (2010) Comparative study between conventional En-masse retraction (Sliding Mechanics) and En-masse retraction using orthodontic micro implant. Implant Dent 19(2):128-136. Krishnan V, Davidovitch Z (2006) Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop 129(4):469.e1-32. Cohen N, Cohen-Levy J (2014) Healing processes following tooth extraction in orthodontic cases. J Dentofacial Anom Orthod 17:304. Galvao MCS, Sato JR, Coelho EC (2012) Dahlberg formula- a novel approach for its evaluation. Dental Press J Orthod 17:115-124. Allen RK, Edelmann AR, Abdulmajeed A, Bencharit S (2019) Salivary protein biomarkers associated with orthodontic tooth movement: A systematic review. Orthod Craniofac Res 22 Suppl 1:14-20. Naoumova J, Olofsson R, Almstahl A, Cevik-Aras H (2023) Salivary levels of Osteoprotegerin and receptor activator of nuclear factor-kappa ligand during orthodontic tooth movement-A prospective pilot study. Orthod Craniofac Res doi: 10.1111/ocr.12687. Tyrovola JB, Spyropoulos MN, Makou M, Perrea D (2008) Root resorption and the OPG/RANKL/RANK system: a mini review. J Oral Sci 50(4):367-376. Nishijima Y, Yamaguchi M, Kojima T, Aihara N, Nakajima R, Kasai K (2006) Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro Structured. Orthod Craniofacial Res 9(2):63-70. Iwasaki LR, Crouch LD, Tutor A, et al (2005) Tooth movement and cytokines in gingival crevicular fluid and whole blood in growing and adult subjects. Am J Orthod Dentofacial Orthop 128(4):483-491. Pellegrini GG, Gonzales CM, Somoza JC, Friedman SM, Zeni SN (2008) Correlation between salivary and serum markers of bone turnover in osteopenic rats. J Periodontol 79(1):158-165. Bauer Jr (1969) The rate of orthodontic retraction into extraction wounds undergoing various stages of healing: A radiographic study. [Master Thesis]: St Louis University: MS. Abu Alhaija ES, Al Shayeb RA, Al-Khateeb S, Daher HO, Daher SO (2022) A comparative assessment of the amount and rate of orthodontic space closure toward a healed vs recent lower premolar extraction site. Angle Orthod 92(4):463-470. Amler MH, Johnson PL, Salman I (1960) Histological and histochemical investigation of human alveolar socket healing in undisturbed extraction wounds. J Am Dent Assoc 61(1):32-44. Murphey WH (1970) Oxytetracycline microfluorescent comparison of orthodontic retraction into recent and healed extraction sites. Am J Orthod 58(3):215-239. Sanjideh PA, Rossouw PE, Campbell PM, Opperman LA, Buschang PH (2010) Tooth movements in foxhounds after one or two alveolar corticotomies. Eur J Orthod 32:106-113. Aboul-Ela SM, El-Beialy AR, El-Sayed KM, Selim EM, El-Mangoury NH, Mostafa YA (2011) Miniscrew implant-supported maxillary canine retraction with and without corticotomy-facilitated orthodontics. Am J Orthod Dentofacial Orthop 139:252-259. Frost HM (1989) The biology of fracture healing, an overview for clinicians, Part II. Clin Orthop Relat Res 248:294-309. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4562549","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":323842773,"identity":"c1e35386-48a5-4999-8490-d82e8aa744c8","order_by":0,"name":"Vaghela Niraj","email":"","orcid":"","institution":"All India Institute of Medical Sciences Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Vaghela","middleName":"","lastName":"Niraj","suffix":""},{"id":323842776,"identity":"57ed6b11-4e3d-4b79-b30b-2341a20c248c","order_by":1,"name":"Rinkle Sardana","email":"","orcid":"","institution":"All India Institute of Medical Sciences Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Rinkle","middleName":"","lastName":"Sardana","suffix":""},{"id":323842779,"identity":"74582844-fd1e-4114-9acb-d6f57a4c207f","order_by":2,"name":"Mithu Banerjee","email":"","orcid":"","institution":"All India Institute of Medical Sciences Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Mithu","middleName":"","lastName":"Banerjee","suffix":""},{"id":323842781,"identity":"91b85d73-6e82-4b56-b439-63e9305b009f","order_by":3,"name":"Navleen Kaur Bhatia","email":"","orcid":"","institution":"All India Institute of Medical Sciences Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Navleen","middleName":"Kaur","lastName":"Bhatia","suffix":""},{"id":323842782,"identity":"a951a746-76d9-4735-a9fb-cf247fb10dd9","order_by":4,"name":"Priyawati Moungkhom","email":"","orcid":"","institution":"All India Institute of Medical Sciences Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Priyawati","middleName":"","lastName":"Moungkhom","suffix":""},{"id":323842783,"identity":"2ed5b3e9-12de-4d5e-a8cb-8790c30aeb5f","order_by":5,"name":"Pravin Kumar","email":"","orcid":"","institution":"All India Institute of Medical Sciences Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Pravin","middleName":"","lastName":"Kumar","suffix":""},{"id":323842787,"identity":"3a45ce6c-fcb6-4e67-8b92-bf1dfd7ddd99","order_by":6,"name":"Surjit Singh","email":"","orcid":"","institution":"All India Institute of Medical Sciences Jodhpur","correspondingAuthor":false,"prefix":"","firstName":"Surjit","middleName":"","lastName":"Singh","suffix":""},{"id":323842788,"identity":"87b81215-8c4c-451a-be58-165196d54a92","order_by":7,"name":"Vinay Kumar Chugh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYDCCA2DEwGDAkHyAgbGBNC1pCcRrYYBoyTEgTgvf7TOGhwtqDsubs+d8k/i5w0aOgf3w0Q34tEieyzE4POPYYcOdPW+3SfaeSTNm4ElLu4FPi8EZtoTDPGyHEwxu5G6T4G07nNggwWNGhJZ/IC05zyT/EqeF+cBhoOEgLWzSRNkiCdbSl2644cwzY2vZtjRjNkJ+4TvD2PyZ55u1vMHx5Ic337bZyPGzHz6GVwsUNIMIFgkQyUaEchCoAxHMH4hUPQpGwSgYBSMMAADOMVHiacOXiAAAAABJRU5ErkJggg==","orcid":"","institution":"All India Institute of Medical Sciences Jodhpur","correspondingAuthor":true,"prefix":"","firstName":"Vinay","middleName":"Kumar","lastName":"Chugh","suffix":""}],"badges":[],"createdAt":"2024-06-11 08:38:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4562549/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4562549/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60602221,"identity":"4b5f8b2b-d491-4aae-96e7-41dc5696b0a0","added_by":"auto","created_at":"2024-07-18 16:11:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61226,"visible":true,"origin":"","legend":"\u003cp\u003eCONSORT flowchart\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4562549/v1/84535cc9e4201bfc9b3882f0.png"},{"id":60600860,"identity":"5beaaf20-9074-4f3e-b1ab-b59f33ddc31b","added_by":"auto","created_at":"2024-07-18 16:03:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61756,"visible":true,"origin":"","legend":"\u003cp\u003eLine graphs displaying the changes in salivary concentrations of RANKL, OPG, and RANKL/OPG over time during the en-masse retraction process after the activation visit. The x-axis on each graph represents the sampling time period, while the y-axis shows the quantitative immunoenzymatic data.\u003c/p\u003e","description":"","filename":"Fig2..png","url":"https://assets-eu.researchsquare.com/files/rs-4562549/v1/e9601695b381c2459c4c9d76.png"},{"id":61968385,"identity":"1b1c6934-8cf3-456f-b63c-04e97da24724","added_by":"auto","created_at":"2024-08-07 16:13:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":718024,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4562549/v1/5bac8bc5-d015-4749-944c-592201607ea1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of salivary biomarkers during en-masse retraction of protruded teeth into recent and healed extraction sites– a Randomized Clinical Trial","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eExtraction space closure is the foremost time taking phase in orthodontic treatment, especially in patients with protruded maxillary anterior teeth. The movement of teeth into an extraction space depends not just on the biomechanical procedure but also on biological factors. Application of orthodontic forces post-extraction can cause variation in the structure of the alveolar process into which the anterior teeth move. The teeth can either be moved into the extraction space with sufficient time to heal or into the recently extracted socket. Studies [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] have been done on animals where surgically accelerated tooth movement and histological characteristics of extraction sockets were investigated. Hasler et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and Liou [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] found that tooth movement was faster at sites of recent extraction. In contrast, another animal study by Samruajbenjakun et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] found no difference in the rate of tooth movement in rats between the recent and healed extraction socket group.\u003c/p\u003e \u003cp\u003eWhile it has been reported that tooth movement is accelerated towards recent extraction sockets, the underlying biological mechanism has yet to be extensively examined. The different cells of the bone multicellular unit (BMU) act in a specific sequence composed of four phases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. During the activation phase, the receptor activator of nuclear factor K ligand (RANKL), a homotrimeric protein present on the cell surface of the preosteoclasts from the bone marrow, interacts with the receptor activator of nuclear factor K (RANK), a homotrimeric transmembrane protein member of the tumor necrosis factor (TNF) family [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Their linkage leads to bone resorption, lasting about two weeks. In the reversal phase, preosteoblasts that have migrated into the resorption lacunae mature and differentiate into mature osteoblasts. These osteoblasts secrete osteoprotegerin (OPG), a free-floating soluble decoy receptor belonging to the tumor necrosis factor family, that binds with the RANKL and, thus, prevents further activation of the preosteoclast which in turn inhibits resorption activity by the osteoclast [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne way to measure the activity of the bone remodeling process is by analyzing biochemical markers. Previous studies have shown that saliva provided a sensitive and inexpensive detection technique for determining analytes in the periodontal microenvironment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. According to Florez et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], the ratio of sRANKL to OPG varies over time during orthodontic alignment. This suggests that sRANKL/OPG may be associated with the various stages of orthodontic tooth movement. Reiss et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] recently observed salivary biomarkers for three months and found no difference in salivary biomarkers' expression and the mandibular anterior alignment rate when supplemented with the vibratory force.\u003c/p\u003e \u003cp\u003eAlthough many experimental human and animal studies have studied salivary biomarkers, these studies have examined the biochemical markers in the whole saliva of patients undergoing fixed appliance therapy during the initial stages of treatment [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, to our knowledge, no study has evaluated these biomarkers during extraction space closure based on the type of extraction socket. In addition, many studies that concentrate on the rate of en-masse retraction of anterior teeth have yet to distinguish between healed and recent extraction sites [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to evaluate concentrations of salivary biomarkers (RANKL and OPG) during en-masse retraction of anterior maxillary teeth into RE and HE sites. Additionally, the amount and rate of en-masse retraction were also evaluated. The null hypothesis was that there was no significant difference between the groups.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTrial design and any changes after trial commencement\u003c/h2\u003e \u003cp\u003eThis 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eParticipants, eligibility criteria, and settings\u003c/h2\u003e \u003cp\u003eThe 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\u0026ndash;30 years, requiring maxillary first premolar extractions primarily for retraction of protruded anterior maxillary teeth, minimal arch length tooth size discrepancy (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eInterventions\u003c/h2\u003e \u003cp\u003eAfter recruitment, patients were randomly assigned into two groups. Each patient received a 0.022-inch pre-adjusted edgewise appliance (3M Unitek\u0026trade; Gemini metal brackets, MBT prescription, California, USA). Alignment and leveling was performed with nickel-titanium (G4\u0026trade; Nickel-titanium, G\u0026amp;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.\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e \u003cp\u003eEn-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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData collection procedure\u003c/h2\u003e \u003cp\u003eThe 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 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The clinical interventions of the study were performed by NV. Outcomes from saliva samples and study models were measured by MB and RS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSaliva collection procedure\u003c/h2\u003e \u003cp\u003eSaliva samples were collected in the morning before breakfast by the same procedure as Florez et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] 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 \u0026micro;L portions, and stored in a freezer at -80\u0026ordm;C until further processing. Salivary samples were later analyzed for RANKL and OPG concentration using human Enzyme-linked immunoassay (Bioassay Technology Laboratory, Jiaxing, Zhejiang).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSampling and Processing Preparation\u003c/h2\u003e \u003cp\u003eAt the time of biomarker analysis, the salivary samples and kits were equilibrated at room temperature. 100 \u0026micro;L of standard working buffer was added to each sample and gradually diluted, which was incubated at 37\u0026ordm;C for 80 minutes. The liquid from the plate was removed and replaced with 200 \u0026micro;L of wash buffer in each vial. The plate was washed thrice using an automated ELISA plate washer before spin-drying. Next, 100 \u0026micro;L of biotinylated antibody working solution was added to each vial and incubated at 37\u0026ordm;C for 50 minutes. The plate was washed three more times and allowed to dry before adding 100 \u0026micro;L of Streptavidin-horseradish peroxide (HRP) working solution to each vial, followed by incubation at 37\u0026deg;C for 50 minutes. The plate was then washed five times and spin-dried before adding 90 \u0026micro;L of tetramethyl benzidine (TMB) to each vial and incubating at 37\u0026deg;C for 20 minutes. A stop solution of approximately 50 \u0026micro;l 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eOutcomes (primary and secondary)\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003cp\u003eSecondary 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.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003eSample size calculation\u003c/h2\u003e \u003cp\u003eWe 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.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMethod error\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInterim analyses and stopping guidelines\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRandomization (random number generation, allocation concealment, and implementation)\u003c/h2\u003e \u003cp\u003eEligible 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBlinding\u003c/h2\u003e \u003cp\u003eThe 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05 with 95% confidence intervals (CIs).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eParticipant flow\u003c/h2\u003e \u003cp\u003eDuring recruitment, 24 patients were assessed for eligibility. Three patients were deemed ineligible as inclusion criteria were not met, while one declined to participate. Twenty patients were randomly assigned to the RE, or HE groups in a 1:1 ratio. All participants completed the study. Ten patients in each group were analyzed. The CONSORT flowchart depicting the participants in the trial is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRecruitment\u003c/h2\u003e \u003cp\u003ePatient recruitment began in January 2020 and concluded in April 2021.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBaseline data\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents a comparison of the baseline characteristics between the groups. The groups showed no statistically significant differences in any parameter. No significant difference was found between the groups in the concentration of RANKL and OPG at T0. The final sample size for the RE and HE groups was the same. Each group had ten subjects (7 females, 3 males).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline characteristics of participants in each study group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRE group,\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSignificance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial age, years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23.2\u0026thinsp;\u0026plusmn;\u0026thinsp;5.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 (30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7 (70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7 (70)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of alignment and leveling (months)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.7\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eSalivary biomarkers\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRANKL (pg/ml) at T0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e71.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.4\u0026thinsp;\u0026plusmn;\u0026thinsp;5.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOPG (pg/ml) at T0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.514\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRANKL/OPG ratio at T0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.8\u0026thinsp;\u0026plusmn;\u0026thinsp;11.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistance between cusp tip of canine and mesiobuccal cusp tip of first molar at T1 (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.6\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.7\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote. Values are mean\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;SD or n (%).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eAbbreviations. RE; recent extraction, HE; healed extraction, SD; standard deviation, n; number, NS; non-significant, T0; start of orthodontic treatment, before extraction (baseline). T1; after completion of alignment and leveling.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePrimary Outcome.\u003c/b\u003e Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e gives the result for changes in RANKL, OPG, and RANKL/OPG concentrations at the different sampling times in the RE and HE group during en-masse retraction of maxillary anterior teeth. The salivary biomarker RANKL showed a linear trend in their expression throughout the 12 weeks in both groups. The change from T1 to T2, T3, and T4 was statistically significant in the RE group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, in the HE group, the change was statistically significant only from T1 to T4 (P\u0026thinsp;=\u0026thinsp;0.003). On comparison between the groups, salivary RANKL concentration showed no statistically significant difference at any time point (P\u0026thinsp;=\u0026thinsp;0.205, P\u0026thinsp;=\u0026thinsp;0.472, P\u0026thinsp;=\u0026thinsp;0.517, respectively; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of salivary concentration of RANKL (pg/ml), OPG (pg/ml), RANKL/OPG ratio at different time points during en-masse retraction of maxillary anterior teeth into recent and healed extraction sites.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTimepoint\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRE group,\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHE group,\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eWithin-group P value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eBetween-group P value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eRANKL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71.8\u0026thinsp;\u0026plusmn;\u0026thinsp;6.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e76.5\u0026thinsp;\u0026plusmn;\u0026thinsp;20.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.496\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.1\u0026thinsp;\u0026plusmn;\u0026thinsp;21.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87\u0026thinsp;\u0026plusmn;\u0026thinsp;26.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.003*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.361\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.205\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96\u0026thinsp;\u0026plusmn;\u0026thinsp;25.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.472\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e115.5\u0026thinsp;\u0026plusmn;\u0026thinsp;20.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e112.9\u0026thinsp;\u0026plusmn;\u0026thinsp;18.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.000*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.003*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.517\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eOPG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.755\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.012*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.693\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.445\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.813\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.477\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.007*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.486\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.142\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eRANKL/OPG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;6.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.090\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.3\u0026thinsp;\u0026plusmn;\u0026thinsp;11.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.5\u0026thinsp;\u0026plusmn;\u0026thinsp;23.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.393\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e44.9\u0026thinsp;\u0026plusmn;\u0026thinsp;13.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.2\u0026thinsp;\u0026plusmn;\u0026thinsp;13.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.009*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.949\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.031*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72.9\u0026thinsp;\u0026plusmn;\u0026thinsp;26.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.1\u0026thinsp;\u0026plusmn;\u0026thinsp;14.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.004*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.015*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote. Values are mean\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;SD. Within-group and between group comparison P- values for change from T1 to T2, T3, or T4 against no change measured using repeated-measures ANOVA with results of Greenhouse-Geisser. P value for comparison between group means done by Student\u0026rsquo;s t-test. *Statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eAbbreviations. RE; recent extraction, HE; healed extraction, SD; standard deviation, n; number, NS; non-significant, T1; after completion of alignment and leveling, T2; 2 weeks after retraction, T3; 8 weeks after retraction, T4; 12 weeks after retraction\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe change in salivary concentration of OPG from T1 to T2 and T4 was statistically significant in RE group (P\u0026thinsp;=\u0026thinsp;0.012, P\u0026thinsp;=\u0026thinsp;0.007, respectively). In the HE group the changes were statistically insignificant at all time points (P\u0026thinsp;=\u0026thinsp;0.693, P\u0026thinsp;=\u0026thinsp;0.813, P\u0026thinsp;=\u0026thinsp;0.486, respectively; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). On comparison between the groups, salivary OPG concentration showed no statistically significant difference at any time point (P\u0026thinsp;=\u0026thinsp;0.445, P\u0026thinsp;=\u0026thinsp;0.477, P\u0026thinsp;=\u0026thinsp;0.142, respectively; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to variation for each biomarker, RANKL/OPG ratio showed a nonlinear trend. Two weeks after the initial force application, the ratio increased from T1 to T2 in both groups. It was observed that the ratio decreased at 8 weeks followed by an increase again at 12 weeks in both groups. The change from T1 to T2, T3, and T4 was statistically significant in the RE group (P\u0026thinsp;=\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.009, P\u0026thinsp;=\u0026thinsp;0.001, respectively). However, in the HE group, the change was statistically significant only from T1 to T4 (P\u0026thinsp;=\u0026thinsp;0.004). On comparison between the groups, RANKL/OPG ratio showed a statistically significant difference from T1 to T3 and T4 (P\u0026thinsp;=\u0026thinsp;0.031, P\u0026thinsp;=\u0026thinsp;0.015, respectively; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e provides a visual representation of the changes in concentration for each biomarker over time, which helps to clarify the results. Each protein had its peak at 12 weeks.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSecondary Outcome.\u003c/b\u003e The total amount of space closure was approximately 3.3 mm in the RE group and 2.46 mm in the HE group. The amount of space closure was found to be significantly higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the RE group as compared to the HE group at the end of 12 weeks (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The rate of en-masse retraction was 0.27 mm/week in the RE group and 0.20 mm/week in the HE group. A significantly higher rate of retraction was found in the RE group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) when compared to the HE group during en-masse retraction of maxillary anterior teeth (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of amount and rate of en-masse (mm/week) during during en-masse retraction of maxillary anterior teeth into recent and healed extraction sites.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOutcome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTimepoint\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eHE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eWithin-group P value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eBetween-group P value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHE group, n\u0026thinsp;=\u0026thinsp;10\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eAmount of space closure (mm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiff T1 \u0026ndash; T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.72\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.35\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.005*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.003*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiff T1 \u0026ndash; T3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.00\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.38\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiff T1 \u0026ndash; T4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.3\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.46\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRate of en-masse retraction (mm/weeks)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT1 \u0026ndash; T4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.27\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.20\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote. Values are mean\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;SD. P value of difference between groups is calculated using student\u0026rsquo;s t test. Rate of tooth movement is calculated by T1-T4/12 Weeks. *Statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eAbbreviations. RE; recent extraction, HE; healed extraction, SD; standard deviation, n; number, NS; non-significant, T1; after completion of alignment and leveling, T2; 2 weeks after retraction, T3; 8 weeks after retraction, T4; 12 weeks after retraction.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eHarms\u003c/h2\u003e \u003cp\u003eNo harm was observed during the study.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSaliva has great potential as a non-invasive method for detecting markers of orthodontic tooth movement [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It has been used in studies to monitor changes in OPG and RANKL levels during orthodontic tooth movement [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These salivary RANKL and OPG levels correspond well with those investigated in gingival crevicular fluid samples and, hence unstimulated whole saliva was considered an easy alternative compared to crevicular fluid for sampling [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This can help the clinician understand the biology behind orthodontic space closure towards RE and HE sites.\u003c/p\u003e \u003cp\u003eIn the present study, it was observed that during en-masse retraction of anterior maxillary teeth, the mean salivary concentration of RANKL increased and that of OPG decreased in both the RE and HE group at 2 weeks. However, the change was significant only in the RE group. The result corresponds well with the initial phase of tooth movement that takes place after the application of orthodontic forces. In-vitro studies have shown that the periodontal ligament (PDL) can either upregulate RANKL or OPG, depending on the type of force applied [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In cases where the PDL cells were subjected to compression forces, RANKL expression was upregulated while OPG was downregulated [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The significant change in the RE group could be due to significant inflammation and active bone remodeling at the tooth socket site. The results are in agreement with the study of Florez et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] where they observed that the salivary concentration of RANKL increased and that of OPG decreased after two weeks of force application although they evaluated salivary biomarkers only during the initial phase of alignment. At 2 weeks, the RE group experienced a significant increase in RANKL concentration and a decrease in OPG concentration, resulting in a significant increase in the RANKL/OPG ratio. This shift favored osteoclastogenesis and led to the movement of teeth in the periodontal ligament space.\u003c/p\u003e \u003cp\u003eThe results at 8 weeks showed an increase in the mean concentration of RANKL in both the RE and HE groups. However, the intergroup differences were insignificant. The salivary concentration of OPG at 8 weeks showed a non-linear trend. There was decrease in the OPG concentration in the RE group while a slight increase in the HE group. However, both the intragroup and intergroup differences were found to be insignificant. The insignificant change in concentration of RANKL and OPG in saliva at 8 weeks could be due to the reactivation of retractive forces that causes another period of acute inflammation which may overlap with ongoing chronic inflammation. This correlates well with the lag phase and post-lag phase of orthodontic tooth movement. During the lag phase, bone resorption slows down as osteoclasts have to be recruited from nearby marrow spaces to reach the periodontal ligament and alveolar bone through the vascular system, to create a transit inflammation in order to remove the hyalinized tissue [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The post-lag phase follows, where tooth movement accelerates due to periodontal ligament remodeling and localized apposition and resorption of alveolar bone. The RANKL/OPG ratio was significantly increased at 8 weeks in the RE group when compared to the HE group. This is mainly because of a significant increase in the concentration of RANKL at 8 weeks, favoring bone resorption.\u003c/p\u003e \u003cp\u003eAfter 12 weeks of en-masse retraction of maxillary anterior teeth, it was observed that there was a significant increase in the salivary concentration of RANKL in both the RE and HE group. The salivary concentration of OPG decreased in both the RE and HE group at 12 weeks, however significant difference was observed only in the RE group. The result was in agreement with the trial of Reiss et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. where the salivary concentration of RANKL was increased and that of OPG was decreased at 10\u0026ndash;12 weeks after reactivation of orthodontic forces although their study focused only the initial phase of alignment. The study by Naoumova et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] has also shown a decrease in the concentration of OPG during space closure. The results correspond well with the linear phase of orthodontic tooth movement. An interesting finding in the present study was that at 12 weeks post retraction, the RANKL/OPG ratio showed a significant difference between RE and HE group. The concentration of RANKL was increased, and the level of OPG was decreased, thus leading to a significant increase in RANKL/OPG ratio in both the recent (P\u0026thinsp;=\u0026thinsp;0.001) and HE group (P\u0026thinsp;=\u0026thinsp;0.004). Our finding was similar to Reiss et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The increase in the RANKL/OPG ratio is in concurrence with the increased rate of tooth movement observed during the linear phase. The significant changes in both RANKL and OPG salivary concentrations, as well as the RANKL/OPG ratio observed at 12 weeks, may be linked to the enlistment of new osteoclasts, and the activation and further differentiation of pre-existing osteoclasts, causing bone resorption and a negative bone balance at the basic multicellular unit level [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. So, it can be inferred that the RANKL /OPG ratio is a more important indicator in defining the rate of orthodontic tooth movement than the individual RANKL and OPG concentrations.\u003c/p\u003e \u003cp\u003eIn the present study, the amount of space closure was significantly greater in the RE group (3.3 mm) than in the HE group (2.4 mm) at the end of 12 weeks. Our findings were similar to Bauer [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], who reported from animal experiments that tooth movement was more into a recent extraction site than into a healed extraction site. Hasler [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] also reported that individual canines retracted more into the RE site than into the HE site by 1.14 mm. However, either the studies were animal studies or reported the amount of canine retraction, which cannot be compared directly to the present study. In the current study, the rate of retraction of maxillary anterior teeth was significantly higher (0.27 mm/week) for the RE group when compared to HE group (0.20 mm/week). Recently, Alhaija et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] also reported a significantly higher amount of space closure and retraction rate (3.02 mm and 1.01 mm per month, respectively) towards the recent socket when compared to the healed socket (1.98 mm and 0.66 mm per month, respectively) although they studied the amount and rate of space closure in the mandibular arch. The difference in the rate of tooth movement between the RE and HE groups could be explained by the histological and histochemical study of Amler and Johnson on undisturbed alveolar socket healing [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. They found that at the tooth extraction site, the osteoid starts to appear at the base of the socket by the seventh day, and at least two-thirds of the socket gets filled with trabecular bone by thirty-eight days after exodontia. Murphey [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] also concluded that there were more osteoclastic activities at recent sites and new bone formation at healed sites that can cause greater restrained tooth movement into healed sockets than into recent sockets.\u003c/p\u003e \u003cp\u003eEarlier studies have stated that the duration of the regional acceleratory phenomenon lasts for maximum up to 4 months [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The present study is in concurrence with previous studies that the immediate application of retractive forces towards the recent extraction site can facilitate faster space closure due to immediate tissue inflammatory response and the regional acceleratory phenomenon [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This in turn helps to prevent overtaxing of the alveolar bone, which has already been allowed to heal.\u003c/p\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLimitations:\u003c/h2\u003e \u003cp\u003eA small sample size was used to determine whether the extraction timing could profoundly affect orthodontic tooth movement. Future studies with a larger sample size should be undertaken. The salivary biomarkers were evaluated during the initial retraction phase of 12 weeks. Further evaluation of these salivary biomarkers beyond twelve weeks and after completion of retraction phase could provide a better understanding of their role in orthodontic tooth movement.\u003c/p\u003e \u003c/div\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe following conclusions were drawn from the study:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eA significant increase in the salivary concentration of RANKL was observed at 12 weeks in RE and HE groups during en-masse retraction of maxillary anterior teeth; however, no significant difference was found between groups at all time points.\u003c/li\u003e\n \u003cli\u003eA statistically significant decrease in the salivary concentration of OPG was observed at 12 weeks only in the RE group during en-masse retraction of anterior maxillary teeth. Both groups showed no statistically significant differences at any time point.\u003c/li\u003e\n \u003cli\u003eThere was a significant increase in the RANKL/OPG ratio at 12 weeks in both RE and HE groups. The ratio was significantly higher in the RE group when compared to the HE group at 8 and 12 weeks. Significantly higher RANKL/OPG ratio in RE group suggests faster tooth movement in the RE group during the evaluation period.\u003c/li\u003e\n \u003cli\u003eThe amount and rate of en-masse retraction of anterior maxillary teeth were significantly higher in the RE group.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRE: Recent extraction; HE: Healed extraction; RANKL: Receptor activator of nuclear factor kappa-B ligand; OPG: osteoprotegerin; ELISA: Enzyme-linked immunoassay; CI: Confidence interval; SD: Standard deviation; n: number; NS: Non-significant; T0: start of orthodontic treatment, before extraction (baseline); T1: after completion of alignment and leveling; T2: 2 weeks after retraction; T3: 8 weeks after retraction; T4: 12 weeks after retraction.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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.\u0026nbsp;All patients or parents (in the case of minors) signed the informed consent before initiation of the treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported as a part of thesis research grant by All India Institute of Medical Sciences, Jodhpur, Rajasthan, India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this study's findings are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflict of interest to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHasler R, Schmid G, Ingervall B, Gebauer U (1997) A clinical comparison of the rate of maxillary canine retraction into healed and recent extraction sites-a pilot study. Eur J Orthod 19:711-719.\u003c/li\u003e\n\u003cli\u003eBaumrind S (1969) A reconsideration of the propriety of the \u0026quot;pressure-tension\u0026quot; hypothesis. Am J Orthod 55(1):12-22.\u003c/li\u003e\n\u003cli\u003eVan Leeuwen EJ, Maltha JC, Kuijpers‐Jagtman AM (1999) Tooth movement with light continuous and discontinuous forces in beagle dogs. Eur J Oral Sci 107(6):468-474.\u003c/li\u003e\n\u003cli\u003eLiou EJ, Huang CS (1998) Rapid canine retraction through distraction of the periodontal. Am J Orthod Dentofacial Orthop 114(4):372-382.\u003c/li\u003e\n\u003cli\u003eSamruajbenjakun B, Kanokpongsak K, Leethanakul C (2018) Comparison of clinical and histological characteristics of orthodontic tooth movement into recent and healed extraction sites combined with corticotomy in rats. Korean J Orthod 48(6):405-411.\u003c/li\u003e\n\u003cli\u003eVerna C (2016) Regional Acceleratory Phenomenon. Front Oral Biol 18:28-35.\u003c/li\u003e\n\u003cli\u003eBoyce BF, Xing L (2008) Functions of RANKL/RANK/OPG in bone modeling and remodeling. Arch Biochem Biophys 473(2):139-146.\u003c/li\u003e\n\u003cli\u003eMaltha JC, Krishnan V, Kuijpers‐Jagtman AM (2021) Cellular and Molecular Biology of Orthodontic Tooth Movement. Biol Mech Tooth Mov 33-48.\u003c/li\u003e\n\u003cli\u003eNunes LA, Mussavira S, Bindhu OS (2015) Clinical and diagnostic utility of saliva as a non-invasive diagnostic fluid: a systematic review. Biochem Med (Zagreb) 25(2):177-192. \u003c/li\u003e\n\u003cli\u003eFlorez-Moreno GA, Isaza-Guzman DM, Tobon-Arroyave SI (2013) Time-related changes in salivary levels of the osteotropic factors sRANKL and OPG through orthodontic tooth movement. Am J Orthod Dentofacial Orthop 143(1):92-100.\u003c/li\u003e\n\u003cli\u003eReiss S, Chouinard MC, Landa DF, Nanda R, Chandhoke T, Sobue T, et al (2020) Biomarkers of orthodontic tooth movement with fixed appliances and vibration appliance therapy: A pilot study. Eur J Orthod 42(4):378-386.\u003c/li\u003e\n\u003cli\u003eUpadhyay M, Yadav S, Patil S (2008) Mini-implant anchorage for en-masse retraction of maxillary anterior teeth: A clinical cephalometric study. Am J Orthod Dentofacial Orthop 134(6):803-810.\u003c/li\u003e\n\u003cli\u003eBasha AG, Shantaraj R, Mogegowda SB (2010) Comparative study between conventional En-masse retraction (Sliding Mechanics) and En-masse retraction using orthodontic micro implant. Implant Dent 19(2):128-136.\u003c/li\u003e\n\u003cli\u003eKrishnan V, Davidovitch Z (2006) Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofacial Orthop 129(4):469.e1-32.\u003c/li\u003e\n\u003cli\u003eCohen N, Cohen-Levy J (2014) Healing processes following tooth extraction in orthodontic cases. J Dentofacial Anom Orthod 17:304.\u003c/li\u003e\n\u003cli\u003eGalvao MCS, Sato JR, Coelho EC (2012) Dahlberg formula- a novel approach for its evaluation. Dental Press J Orthod 17:115-124.\u003c/li\u003e\n\u003cli\u003eAllen RK, Edelmann AR, Abdulmajeed A, Bencharit S (2019) Salivary protein biomarkers associated with orthodontic tooth movement: A systematic review. Orthod Craniofac Res 22 Suppl 1:14-20.\u003c/li\u003e\n\u003cli\u003eNaoumova J, Olofsson R, Almstahl A, Cevik-Aras H (2023) Salivary levels of Osteoprotegerin and receptor activator of nuclear factor-kappa ligand during orthodontic tooth movement-A prospective pilot study. Orthod Craniofac Res doi: 10.1111/ocr.12687. \u003c/li\u003e\n\u003cli\u003eTyrovola JB, Spyropoulos MN, Makou M, Perrea D (2008) Root resorption and the OPG/RANKL/RANK system: a mini review. J Oral Sci 50(4):367-376. \u003c/li\u003e\n\u003cli\u003eNishijima Y, Yamaguchi M, Kojima T, Aihara N, Nakajima R, Kasai K (2006) Levels of RANKL and OPG in gingival crevicular fluid during orthodontic tooth movement and effect of compression force on releases from periodontal ligament cells in vitro Structured. Orthod Craniofacial Res 9(2):63-70.\u003c/li\u003e\n\u003cli\u003eIwasaki LR, Crouch LD, Tutor A, et al (2005) Tooth movement and cytokines in gingival crevicular fluid and whole blood in growing and adult subjects. Am J Orthod Dentofacial Orthop 128(4):483-491. \u003c/li\u003e\n\u003cli\u003ePellegrini GG, Gonzales CM, Somoza JC, Friedman SM, Zeni SN (2008) Correlation between salivary and serum markers of bone turnover in osteopenic rats. J Periodontol 79(1):158-165.\u003c/li\u003e\n\u003cli\u003eBauer Jr (1969) The rate of orthodontic retraction into extraction wounds undergoing various stages of healing: A radiographic study. [Master Thesis]: St Louis University: MS.\u003c/li\u003e\n\u003cli\u003eAbu Alhaija ES, Al Shayeb RA, Al-Khateeb S, Daher HO, Daher SO (2022) A comparative assessment of the amount and rate of orthodontic space closure toward a healed vs recent lower premolar extraction site. Angle Orthod 92(4):463-470.\u003c/li\u003e\n\u003cli\u003eAmler MH, Johnson PL, Salman I (1960) Histological and histochemical investigation of human alveolar socket healing in undisturbed extraction wounds. J Am Dent Assoc 61(1):32-44.\u003c/li\u003e\n\u003cli\u003eMurphey WH (1970) Oxytetracycline microfluorescent comparison of orthodontic retraction into recent and healed extraction sites. Am J Orthod 58(3):215-239.\u003c/li\u003e\n\u003cli\u003eSanjideh PA, Rossouw PE, Campbell PM, Opperman LA, Buschang PH (2010) Tooth movements in foxhounds after one or two alveolar corticotomies. Eur J Orthod 32:106-113.\u003c/li\u003e\n\u003cli\u003eAboul-Ela SM, El-Beialy AR, El-Sayed KM, Selim EM, El-Mangoury NH, Mostafa YA (2011) Miniscrew implant-supported maxillary canine retraction with and without corticotomy-facilitated orthodontics. Am J Orthod Dentofacial Orthop 139:252-259.\u003c/li\u003e\n\u003cli\u003eFrost HM (1989) The biology of fracture healing, an overview for clinicians, Part II. Clin Orthop Relat Res 248:294-309.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Salivary biomarkers, Receptor activator of nuclear kappa B ligand, Osteoprotegerin, RANKL/OPG, Recent extraction site, Healed extraction site, En-masse retraction","lastPublishedDoi":"10.21203/rs.3.rs-4562549/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4562549/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study aimed to evaluate levels of salivary biomarkers during en-masse retraction of anterior maxillary teeth into recent and healed extraction sites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty patients with protruded anterior maxillary teeth requiring en-masse retraction were randomly allocated into two groups. In the healed extraction (HE) group, first premolar extraction was performed at the beginning of treatment while in the recent extraction (RE) group, it was done just before the initiation of en-masse retraction. Saliva and study model collection was done at start of treatment, after leveling, and after 2, 8 and 12-weeks of retraction, respectively. The primary outcome was to evaluate concentrations of salivary biomarkers; receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG). Rate of en-masse retraction was secondary outcome. One-way repeated-measures ANOVA was applied for within-group changes, and an independent t-test for the intergroup comparison (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn increase in concentration of RANKL and decrease in OPG concentration was observed in both the RE and HE groups at 12 weeks; however, the intergroup comparison showed no statistically significant differences (P = 0.517). RANKL/OPG ratio was significantly higher in the RE group (P = 0.015) at 12 weeks. A significantly higher rate of en-masse retraction was found in the RE group (P \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVariations in\u003cstrong\u003e \u003c/strong\u003eRANKL/OPG ratio can be considered a better predictor of orthodontic tooth movement. A significant higher rate of retraction in the RE group suggests that immediate application of retractive forces towards the RE site may hasten space closure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Relevance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFindings of the present study suggest that immediate application of activation forces at the recent extraction site may hasten initial orthodontic tooth movement by preventing excessive strain on the alveolar bone, which has had time to heal during the tooth movement at healed extraction site. Additionally, the study contributes to existing literature that highlights saliva as an easily accessible alternative to crevicular fluid for sampling purposes.\u003c/p\u003e","manuscriptTitle":"Evaluation of salivary biomarkers during en-masse retraction of protruded teeth into recent and healed extraction sites– a Randomized Clinical Trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 16:03:05","doi":"10.21203/rs.3.rs-4562549/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ddae42b-6cf3-413a-b627-a8d19e1ca47e","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-07T16:05:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 16:03:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4562549","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4562549","identity":"rs-4562549","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}