The Effect of Statins on Orthodontic Tooth Movements: A Meta-analysis of Animal Studies

Iran. 2Assistant Professor, Dental Research Center, Department of Orthodontics, Dental Research Institute, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran. 3Assistant Professor, Dental Research Center, Department of Orthodontics, Dental Research Institute, School of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran. Background Nowadays, the prevalence of obesity-related diseases such as hyperlipidemia has increased, especially in adults and causes atherosclerosis and other coronary diseases. Lipidlowering medications such as the statin family of drugs are mostly used by these patients. Clinical trials have shown that statins are well-tolerated in adult and younger populations (1-3). The statin family of drugs is a safe therapeutic agent for the treatment of arteriosclerotic cardiovascular disease. They act as potential inhibitors of 3-hydroxy-3methylglutaryl reductase A (HMG-CoA), which is a ratelimiting enzyme in the mevalonate pathway of cholesterol biosynthesis, thus preventing the synthesis of cholesterol in the liver and reducing the levels of blood cholesterol and triglycerides (4). In addition to their cholesterollowering effects, it is reported that statins have several promising effects on human health, including pleiotropic effects, improvement of endothelial function, and antiThe Effect of Statins on Orthodontic Tooth Movements: A Meta-analysis of Animal Studies

inflammatory, antioxidant, and immunomodulatory effects (5)(6)(7)(8)(9)(10)(11)(12)(13). It has been clarified that statins affect bone metabolism in different ways. They stimulate the osteoblastic differentiation of bone marrow stem cells through the increased gene expression of bone morphogenic protein-2 and angiogenesis. Statins may also stimulate bone formation by preventing osteoblastic apoptosis (14)(15)(16)(17)(18). Furthermore, statins inhibit bone resorption through suppressing osteoclastogenesis (19,20). Thus, they could influence orthodontic tooth movements (OTM) and orthodontic relapse in adult patients by their stimulatory effects on bone formation and pleiotropic effects such as antiinflammatory and immunomodulatory ones (21). Factors affecting the remodeling process will influence OTM (22). Pharmacological factors could potentially affect tooth movements either for reducing (when anchorage strengthening is desirable) or increasing the movement (23)(24)(25)(26). Various experimental trials evaluated the effects of pharmacologic factors on the periodontal responses of the OTM. Various animal studies have proved the stimulatory effects of statins on bone formation during its use by different carriers (9,15,(27)(28)(29)(30)(31)(32)(33)(34)(35). Statins are one of the most commonly prescribed therapeutic agents for the prevention of cardiovascular diseases, thus their plausible effect of arresting tooth movements in adult patients could justify their relevance in orthodontic practice. Considering these facts, the present study aimed to systematically review the efficacy of statin delivery in orthodontic movements.

Protocol
This systematic review and meta-analysis was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement guidelines (36). The protocol of this study was registered on PROSPERO (https://www.crd.york.ac.uk/PROSPERO/) with the ID # CRD42020164155.

Focus Question
The present systematic review and meta-analysis sought to assess the null hypothesis that "there would be no difference in the amount of the OTM by the administration of statins". The focused question according to the PICO format (i.e., Population, Intervention, Control group, and Outcome) was "whether there is a significant decrease in the OTM of statin receivers compared to the control group in animal models". Population (P) indicated animals who underwent orthodontic treatments; Interventions (I) represented orthodontic treatments with statin administration. Finally, control intervention (C) included orthodontic treatments without adjunct statin administration and outcome measured (O) denoted the amount of the OTM.

Search Strategy
An electronic search was performed until 1 st January 2020 to acquire potentially eligible studies with no time or language restrictions in several electronic bibliographic databases such as PubMed, EMBASE, Web of Science, Scopus, and Cochrane. The search strategies of each database are presented in Table 1. The reference part of the retrieved full-text articles (cross-referencing) was also searched for further papers. Non-English translatable papers were included in this study.

Eligibility Criteria
The following selection criteria were applied for this systematic review: 1. Inclusion Criteria: Randomized experimental trials and parallel and split-mouth groups were considered in this study. Articles providing data regarding the effects of statins on the OTM were considered eligible in the first analysis. 2. Exclusion Criteria: (a) in vitro histological studies, (b) review articles, case reports, and letters to editors.

Study Selection
The titles and abstracts of the searched studies were independently screened by two authors. Studies were excluded if they were either irrelevant to the current study and duplicates and/or failed to address the focused question. The controversies were resolved through discussion. Publications were included for full-text  blinded induction of ischemia, blinded assessment of outcomes, and use of anesthetic without significant intrinsic neuroprotective activity. Other domains were animal model (i.e., aged, diabetic, or hypertensive), sample size calculation, compliance with animal welfare regulations, and statement of the potential conflict of interests. Each domain included one specific entry in a study quality table and assigned a "Yes" or "No" judgment for that entry. Then, each study was given a quality score out of a possible total of 10 points.

Summary Measures and Synthesis of Results
The amount of the OTM was used as the outcome measure of this meta-analysis. The mean difference (MD) with 95% confidence intervals was calculated for the continuous data (the OTM amount in millimeter). Statistical analyses were conducted using Comprehensive Meta-analysis, version 2.2. A P value less than 0.05 was considered statistically significant. The random-effect method for analysis was used to compare the outcome measure due to high heterogeneity. The subgroup analysis for different amounts of the OTM in different methods of statin administration, different types, and dosage of statins was not performed due to the lack of sufficient similar studies.

Study Selection
In total, 244 studies were found after a comprehensive search of five online databases. After removing duplicates, the titles and abstracts of the remaining 240 studies were independently screened by two authors. Two hundred and thirty-four studies were excluded based on the eligibility criteria and the PICO model in this step. Moreover, one study was a review article and thus was excluded from the investigation. The full texts of the remaining 5 studies were assessed by the same two authors.
Study characteristics varied in most of the included studies. Variations in animal models, animal age, type and dosage of the administered statins, the method, frequency and duration of statin administration, duration and force of orthodontic treatments, and methods of outcome assessments resulted in heterogeneity among the studies.
Different subjects such as rats, dogs, and rabbits were evaluated in studies. Among the included animal studies, three trials were performed on rat models (40,41,43), one evaluations if they met the inclusion criteria in the first analysis or if insufficient information was provided in the title and abstract so that to make a decision on. A third review author was consulted where the resolution was impossible. The review authors were not blinded to the author(s), institution, or the site of the publication of all studies.
All eligible studies then underwent validity assessments and data extraction. Data were independently extracted by at least two review authors. Any disagreement was discussed and a third review author consulted where necessary. In papers that included inadequate or limited information about the OTM in the statin receivers, the corresponding authors were contacted via an e-mail for making clarification and a request for the missing data, and a reminder e-mail was sent twice after. Finally, several data were extracted from the eligible studies using extraction forms by one of the reviewers, including study design, sample size, animal spices, type and dosage of statin, method of statin administration, active tooth movement period, time of final analysis, the outcome measurement method, and the measured outcome (the amount of the OTM).

Risk of Bias in Individual Studies
The assessment of the risk of bias and the study quality of the included trials were undertaken as part of the data extraction process by at least two review authors independently and in duplicate. The risk of bias assessment was conducted using the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) Tool (37). It is a two-part tool addressing ten specific domains (i.e., random sequence generation, baseline characteristics, allocation concealment, random housing, blinding of caregivers and/or investigators, random outcome assessment, blinding of assessors, incomplete outcome data, selective outcome reporting, and 'other biases'). Each domain includes one specific entry in a 'Risk of bias' table. Within each entry, the first part of the tool involves reporting the events occurring in the study. The second part of the tool includes assigning a judgment relating to the risk of bias for that entry. Consequently, it was judged in terms of having a low, unclear, or high risk of bias. "Yes" and "No" judgments indicate a low and high risk of bias, respectively. The judgment will be "unclear" if insufficient details have been reported to assess the risk of bias properly. Any differences regarding the reviewers' judgments were resolved by a third review author.

Quality of Evidence
The q uality of evidence was evaluated using the Collaborative Approach to Meta-Analysis and Review of Animal Data from Experimental Studies (CAMARADES) tool (38). It addresses ten specific domains including peer-reviewed publication, control of temperature, random allocation to treatment or control, study was conducted on rabbits 39, and one study used dogs as animal models (42). The number of applied rats in trials ranged from 24 to 36. Rats aged 6 and 8-10 weeks were used in 2 studies (40,41). The mean age of the rats was not reported in one study (43). The age of rabbits was 16 weeks in one study and that of the applied dogs in another study was 10-12 months (39,42) ( Table 2).
The dosage of the applied SMV in animal model studies was 0.5 mg/0.48 mL, 0.6 mg/mL, and 0.5 mg/kg/mL (39,41,42). Two studies performed their experiments by oral administration of ATV (15 mg/kg) and 5 mg/kg ATV in carboxymethyl cellulose (CMC) (40,43). Two studies administered the statin drug locally near the teeth under investigation (39,42). Overall, the exposure duration of the subjects to statin drugs in animal studies ranged between 14 and 37 days.
The control groups in all studies underwent orthodontic treatments and were administered to non-statins such as phosphate-buffered saline, CMC, and normal saline (40-42) ( Table 3).

Method of Orthodontic Force Application
The method of orthodontic force application was similar in three trials and they used Nickel-Titanium closed coil springs stretched between maxillary first molar and  and 21 days. The time and method of outcome assessments also varied between the trials. The amount of the OTM was reported in micrometer in one study (40), thus this amount was reported in millimeter in our analysis ( Table 5).

Risk of Bias Within Studies
A summary of the risk of bias in the included studies is presented in Figure 2. Although this study included five animal trials and 108 animals randomized to the Stains group, each of these trials had at least one domain at a high risk of bias. Four of the studies did not report the method of sequence generation (selection bias) (40)(41)(42)(43). All studies reported maxillary incisor (40,41,43). In one trial, animals received an orthodontic appliance consisting of nickel-titanium closed coil spring between the mandibular first premolar and mandibular incisor (39). One study used the maxillary and mandibular canines (anchorage unit) and second premolars (movement unit) to place the ligation wires (42). The applied forces ranged between 50 cN and 200 cN ( Table 4).

Duration of Orthodontic Tooth Movements, Time, and Method of Outcome Assessments
These parameters also varied between the included trials in this review. The duration of the OTM ranged between 7   (39)(40)(41)(42)(43). In terms of assessing allocation concealment, only one trial clearly indicated an adequate means of allocation concealment (39). Conversely, no study described random housing (performance bias) (39)(40)(41)(42)(43). Only three studies announced about the blinding of caregivers, outcome assessors, or investigators (performance and detection bias) (39,40,43). Two studies reported random outcome assessment (detection bias) (39,40). Only one study adequately addressed incomplete outcome (attrition bias) (39). Finally, none of the trials had selective reporting bias.
The quality assessment of the included studies is presented in Table 6.

Results of Individual Studies and Result Synthesis
The five included studies are inconsistent in terms of their methods and the frequency of statin administration, and the type and dosage of statins. In addition, different animal models were used and different final assessment times existed between the studies. However, four studies were consistent in their methodologies regarding the measurement and comparison of the amount of the OTM between the intervention (statin receivers) and control groups (39)(40)(41)43). The heterogeneity decreased from 90.65 to 66.37 when one of these four studies was excluded from meta-analysis (39) (Figure 3), and a meta-analysis of three studies was performed for differences in the amount of OTM (40,42,43).
Data were available comparing the outcomes at the end of the OTM. A random-effect model was performed due to differences in terms of methodologies (intervention) and the obvious heterogeneity of the data. The intervention group had a statistically insignificant lower amount of tooth movements compared to the control group (P = 0.087, MD = 0.134 (-0.020 -0.288), degree of freedom (df) = 2, I 2 = 66.37, Figure 4). This results from analyzing 92 participants in three studies.

Discussion
The aim of this study was to systematically review and

Experimental (Statin) Group
AlSwafeeri et al (39) 21 The linear distance between the first plane drawn on the distal contact area of the distal surface of the mandibular first premolar and a second plane drawn on the mesial contact area of the mesial surface of the mandibular second premolar     Note. CAMARADES: Collaborative approach to meta-analysis and review of animal data from experimental studies.

Dolci et al Alswafeeri et al Mirhashemi et al Esnaashari Esfahani et al Feizbakhsh et al
evaluate whether statins decrease the OTM in animals undergoing orthodontic treatments. The literature search was comprehensive and included several databases and a hand search in the reference lists of relevant articles. Initial evidence from four of the included studies with a high risk of bias suggested a reduction in the OTM by statin administration (39)(40)(41)43). This effect could be explained by the role of Statins in stimulating alveolar bone formation (35,(44)(45)(46). The delivery of statins during OTM inhibits bone resorption through the inhibition of osteoclast cells and increases in osteoblast differentiation (19,47,48). Moreover, statins exhibit anti-inflammatory properties by inhibiting the production of certain proinflammatory cytokines such as IL-6 and IL-8 which are responsible for the biological reaction of periodontal tissues during OTM (49,50). Only one study reported that OTM did not decrease following statins administration (42). This finding may be due to the frequency of statin administration, which was only twice. The number of evaluated samples in this study was also less than that of other studies. This lower sample size may result in insignificant findings.
The overall effect of statin (i.e., the type of drugs as HMGA-CoA inhibitors) on OTM magnitude was also quantitatively analyzed in this study. It should be noted that only three trials were evaluated in this meta-analysis due to heterogeneity among the included studies and their limited number, and sub-group analysis could not be undertaken in this study. The main finding of this meta-analysis is that, on average, statins do not reduce the magnitude of the OTM. This finding may be due to the limited number of included studies which results in insignificant findings.
Although the results of this study showed no difference between statin and control groups, statins reduced bone resorptive lacunas, number of osteoclasts, and generation of inflammatory interleukins such as IL-6 and IL-8 in most of our included studies and the ones related to periodontology (51,52).
Nowadays, adult patients receiving comprehensive orthodontic treatment compromise a significant number of orthodontic patients. The previously discussed properties of statins could affect OTM in adult patients who are under the treatment with statins due to their cardiovascular diseases.
In recent years, several studies have evaluated the effect of the local and systemic administration of pharmacological agents on the control of OTM (anchorage or relapse) (23)(24)(25)(26). Thus, the local administration of the statin type of drugs could have promising effects on anchorage in orthodontic treatments if they could be evaluated in further animal and human studies.
However, reliable conclusions about statins effect on the magnitude of the OTM could not be drawn with the insufficient evidence based on the current literature, and it is important to take caution when extrapolating animal studies to humans.

Limitation of the Study
Although this study was performed carefully following PRISMA guidelines, several limitations remain which deserve further discussion. First, the shortage of highquality trials is evident. Although a comprehensive literature search was performed, only three studies could be included in this analysis. Future well-designed animal and human studies are needed to obtain a more reliable conclusion. When future animal studies are planned, greater consideration should be given to study design (i.e., sequence generation, allocation concealment, random housing, blinding, and the like) in order to reduce bias. Second, methodological heterogeneity is another limitation. Different types and dosages of statins were administered in each study. ATV and SMV, which were administered in most trials, are both lipophilic ones but have different chemical structures (53) and dosages (ranging between 2.5 mg/kg and 5 mg/kg of SMV, as well as 5 mg/kg and 15 mg/kg of ATV). The comparison of the effect of these two types of statins on OTM could not be performed due to the limited number of studies. Therefore, well-designed trials are needed to compare different types of statins and determine the most appropriate types and dosages of these drugs.
Additionally, different animal models were used in the included animal studies. Bone turnover and structure vary in different species of animals and from humans. Particularly, the bone and the root structure of rabbits are different from those of humans (54)(55)(56). In our meta-analysis, the three included trials were performed on rats. To the best of our knowledge, no human study has so far evaluated the effect of statins on OTM. Only one human study was found regarding the effect of statins on orthodontic relapse (57). Accordingly, further evaluations of Stains should consider their potential side-effects on the turnover of other bones. Only one of the trials in this review reported both the benefits (regarding the amount of tooth movement) and the possible adverse effects (on long bone turn-over) associated with the administration of statins (40).

Conclusions
In general, the results of this meta-analysis showed that the reduction in the amount of OTM after the administration of statins was not statistically significant. Based on the information provided from the animal studies eligible for inclusion in this study, a cautionary perspective should be provided due to the low quality of evidence in this meta-analysis.
Conclusive confirmation regarding the effect of these drugs on OTM in humans is lacking since no human studies were conducted and investigations on animals cannot currently provide plausible explanations for the effects of these drugs.