Atorvastatin inhibits osteoclastogenesis and arrests tooth movement

Este é o segundo artigo de minha Tese, no AJODO. Nele falamos um pouco de biologia óssea e também de como medicamentos podem afetar o movimento dentário.

Introduction

In addition to their cholesterol-lowering effects, the statin class of drugs appears to enhance osteogenesis and suppress bone resorption, which could be a clinical concern during orthodontic treatment. In this animal study, we aimed to determine whether atorvastatin (ATV) affects orthodontic tooth movement (OTM) through osteoclast inhibition. Furthermore, we analyzed the potential adverse effects of ATV on long-bone turnover and endochondral ossification.

Methods

Rats were administered ATV (15 mg/kg) or saline solution via gavage (n = 12 animals/group), starting 2 weeks before initial OTM. Tooth displacement was measured after 7, 14, and 21 days. Histologic sections of the maxilla and femur were obtained after 14 and 21 days of OTM and stained (hematoxylin and eosin; TRAP assay) for histomorphometric analysis.

Results

ATV was associated with significant (P <0.05) reductions in OTM and osteoclast counts. Independently of drug administration, OTM increased the number of osteoclasts and reduced the bone-volume ratio compared with the control maxillae without OTM. Long-term statin administration did not appear to affect femoral endochondral ossification.

Conclusions

This experimental study showed that the long-term use of ATV can significantly promote osteoclast inhibition and slow the OTM in the first week in rats. Under physiologic conditions, the drug did not affect bone turnover and endochondral ossification.

Epidemiologic studies have shown a significant increase in the prevalence of diseases such as obesity and hyperlipidemia in adults,1 ;  2 with the latter regarded as the main cause of coronary atherosclerosis.3 According to Mercado et al,2 36.7% of adults, or 78.1 million persons aged 21 years or over, in the United States alone were on or eligible for lipid-lowering treatment; 55.5% of them were taking cholesterol-lowering medications. The drugs most widely used for this purpose are the statins, which lower cholesterol levels by inhibiting hydroxymethylglutaryl-coenzyme A reductase, the rate-controlling enzyme of the mevalonate pathway.4 ;  5 Studies suggest that, in addition to their cholesterol-lowering effects, statins may influence bone turnover, enhancing osteogenesis and suppressing bone resorption.678910111213 ;  14 Attempts to ascertain the mechanism of statin-regulated bone anabolism have suggested 3 aspects: promotion of osteogenesis, inhibition of osteoblast apoptosis, and suppression of osteoclastogenesis.15 Although the effects of statins on bone anabolism have been widely demonstrated in laboratory studies,678910111213 ;  14 their clinical effects are not convincing.16

The biologic reaction of periodontal tissues during orthodontic tooth movement (OTM) is characterized by an aseptic acute inflammatory response in the early stages, followed by a transient, aseptic chronic inflammation. Chemokines, cytokines, and growth factors are the main molecules that orchestrate this inflammatory response, which is followed by osteoclastogenesis and bone resorption, and then by osteoblast and new bone formation.17 Current biologic knowledge raises the possibility that pharmacologic modulation of these periodontal cellular and molecular responses may affect OTM, as shown in various experimental models.1118192021 ;  22 Research has demonstrated that statins reduce OTM and relapse; however, the biologic mechanisms underlying these clinical effects are unknown.11 ;  12 Since statins are among the most commonly prescribed pharmaceutic agents for prevention of cardiovascular diseases, their plausible effect of arresting tooth movement could be a concern in orthodontic practice.4 ;  5

Clinical trials have shown that statins are well tolerated in adult and younger populations,2324 ;  25 but long-term data regarding the impact of statin therapy on growth and development are limited.25 ;  26 In a study by Macpherson et al,23 treatment of children and adolescents with statins over 2 years had no impact on height, weight, body mass index, and sexual maturation. On the other hand, preclinical studies have suggested that these drugs increase chondrocyte proliferation and longitudinal bone growth, which could have implications for their clinical use in the pediatric population.27 ;  28 These discrepancies between clinical trials, in-vitro experiments, and in-vivo studies are puzzling, and further research is necessary to investigate the potential adverse effects of statins on bone turnover and endochondral ossification.2324252627 ;  28

In an attempt to mimic the clinical perspective of orthodontic treatment in patients taking statins, we developed an experimental animal model in which high-dose statin administration was started before OTM, simulating a protocol used in humans. We hypothesized that atorvastatin (ATV) treatment in rats might reduce OTM by inhibiting osteoclastogenesis. Furthermore, we analyzed its potential adverse effects on long-bone turnover and endochondral ossification.

Material and methods

Twenty-four male Wistar rats (age, 6 weeks; weight, approximately 330-340 g) was the sample size estimated by the resource equation method. They were housed four to a cage, under a 12-hour light and dark cycle, at a controlled ambient temperature of 23°C, and given food and water ad libitum. All animal care and use procedures were conducted in keeping with the internationally accepted guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals29 and were approved by the relevant institutional ethics committee of the School of Dentistry of Federal University of Rio Grande do Sul (number 28401) in Brazil.

The animals were randomly divided into 2 groups: experimental (ATV, n = 12) and control (saline solution [SAL], n = 12). Those in the experimental group received atorvastatin (ATV; Medley Farmacêutica, Suzano, Brazil), 15 mg per kilogram daily via gavage, and those in the control group received 0.1 mL of phosphate-buffered saline solution via the same route. Saline solution or drug administration continued until the animals were killed (Fig 1).

Figura 1. Experimental study design. Saline solution or ATV (15 mg/kg) was administered daily via gavage from day 0 through day 35. At day 14, mesial displacement of the right first molars was begun. Total displacements were evaluated at 7, 14, and 21 days of OTM (T7, T14, and T21, respectively); histologic analyses (henatoxylin and eosin and TRAP) were performed at days 14 and 21. The left maxilla served as the internal control (no OTM).

After 14 days of saline solution or drug administration, the animals were anesthetized with ketamine and xylazine (80 mg/kg and 5 mg/kg, respectively) for orthodontic appliance placement. This procedure consisted of placing a superelastic nickel-titanium closed-coil spring between the maxillary right first molar and incisors, as described elsewhere.12303132 ;  33 Our protocol was based on previous studies demonstrating that 50 cN of force is sufficient to provide substantial OTM.112232 ;  33 The device was kept in place for 21 days (Fig 1) to generate mesial displacement of the first molar. In a split-mouth design, the maxillary right side of each rat served as the experiment (OTM), and the maxillary left side, without OTM, served as internal control. Throughout the study, the animals were evaluated weekly for weight gain or loss, appliance breakage, and gingival or other soft tissue inflammation.

Precise plaster models of the maxilla were obtained from impressions made with silicone material (Perfil; Vigodent, Rio de Janeiro, Brazil) and dental stone (Durone; Dentsply Sirona, York, Pa). Impressions were made every 7 days under anesthesia (Fig 1). The occlusal surfaces were photographed (DSC-H10; Sony, Tokyo, Japan) at 300 dpi and magnified (4 times) in ImageJ software (version 1.44; National Institutes of Health, Bethesda, Md). For each photograph, a 100-mm ruler was placed next to the cast to calibrate measurements. The mean distance between the distal surface of the first molar and the mesial surface of the second molar, measured at 3 points on each cast, was calculated for each animal and averaged for the 2 groups. The validity of these data was confirmed by comparing them with the data from a well-described methodology, carried out by measuring the distance between the first and second molars with a digital caliper. The intraclass correlation between both methods was considered satisfactory (>0.8).

Tooth displacements were evaluated after 7, 14, and 21 days of OTM; histologic analysis was done 14 and 21 days after orthodontic appliance insertion, as shown in Figure 1.

At each time point of analysis (days 14 and 21), 12 animals, 6 per group, were killed by overdose of ketamine and xylazine. Maxillae and distal left femurs were immediately resected and fixed by immersion in 10% buffered formalin for 24 hours. The specimens were demineralized in 10% ethylenediaminetetraacetic acid, pH 7, for 30 to 60 days. Then the samples were dehydrated through an ethanol series, embedded in paraffin, cut into 5-μm longitudinal sections, stained with hematoxylin and eosin, and prepared for a TRAP assay. TRAP is an enzyme expressed in mature osteoclasts and in their precursors, which develop an osteoclastic phenotype at early stages and promote bone resorption.34 Briefly, for TRAP staining, histologic sections were selected and incubated in acetate buffer (pH, 5.0) containing naphthol AS-MX phosphate (Sigma, St Louis, Mo), Fast Red Violet LB Salt (Sigma), and 50 mmol per liter of sodium tartrate. The sections were counterstained with hematoxylin.

Histomorphometric analyses of the maxilla specimens were performed with 4 subgroups: ATV + OTM, experimental hemimaxillae (with OTM) from animals in the ATV group; control ATV, control hemimaxillae (without OTM) from animals in the ATV group; SAL + OTM, experimental hemimaxillae from animals in the SAL group; and control SAL, control hemimaxillae from animals in the SAL group. Comparisons across subgroups were performed at each time point of analysis (14 and 21 days) or by analysis of the overall mean. For evaluation of femur specimens, the sample was divided into 2 groups: ATV (n = 18) and SAL (n = 18). In this case, between-group comparisons were done considering overall mean values.

Hematoxylin and eosin and TRAP slides were visualized in an Eclipse 90i microscope (Nikon, Tokyo, Japan) coupled to a Coolsnap EZ camera (Photometrics, Tucson, Ariz). Microphotographs were captured using NIS Elements Imaging 3.10 Sp2 software (Nikon). All histomorphometric measurements and terminology were in accordance with the American Society for Bone and Mineral Research recommendations.35

For maxillary bone turnover, the supporting structures of the molars were evaluated using the NIS Elements Imaging 3.10 Sp2 software (Nikon). Under high magnification (100 times), the number of osteoclasts was counted on the most mesial root of the first molars. The region of interest was the periodontal tissue at the distal surface of the mesial root of the first molar.18 ;  19 The mesial root of the first molar was chosen because it is the largest of the 5 roots of rat teeth, thus allowing histologic evaluation of the entire root structure. Furthermore, it is commonly used for analysis in tooth-movement studies.11121819 ;  31 According to Gonzalez et al,36 during OTM in rats, a major stress occurs in the periodontal tissues adjacent on the middle part of the mesial root. Then we tend to argue that the region of interest could represent an important remodeling zone during OTM. Moreover, in a previous study in our laboratory, a high level of data reproducibility of this histologic field (osteoclast number and bone volume) was verified.37 Cells were considered to be osteoclasts if they were TRAP-positive, multinucleated, and located on the bone surface or in Howship’s lacunae.33

The maxillary bone-volume ratio, expressed as the ratio of cancellous bone volume to total tissue volume, was defined in the region of interest of the stained sections using Photoshop (CS6; Adobe Systems, San Jose, Calif) and ImageJ software, following the method suggested by Egan et al.38 The total osteoclast count and bone volume were recorded for each animal and then averaged for each group.

For femoral bone turnover, using NIS Elements Imaging 3.10 Sp2 software, the number of osteoclasts was counted in 10 fields chosen randomly in the metaphyseal region of the femur.39 The bone-volume ratio of 5 rectangular areas of subchondral bone tissue was calculated according to the protocol outlined by Ho et al.14 The total number of osteoclasts and the bone-volume ratio were recorded for each animal and then averaged for each group, For endochondral ossification, growth-plate cartilage and hypertrophic zone thickness were calculated as the means of 10 measurements obtained at randomly chosen locations in the hematoxylin and eosin-stained sections, again using NIS Elements Imaging 3.10 Sp2 software.40 ;  41

All histologic sections and plaster models were identified with a random numeric sequence to codify experimental periods and groups. The acquired digital images were analyzed by an examiner (G.S.D.) blinded to the experimental groups and periods. To obtain data reproducibility, a calibration process was performed in 10% of the specimens (plaster models and histologic sections), randomly chosen and evaluated twice, with a 15-day interval between the first and second evaluations. Then the random error was assessed according to Dahlberg’s equation,42 S2 = Ʃ2d/2n, where S2 is the error variance, and d is the difference between 2 determinations of the same variable. When acceptable values were obtained (<10%), the examiner was considered to be accurate, and the measurements were performed in the overall samples.

Statistical analysis

Data are presented as means and standard deviations or standard errors as appropriate. A linear mixed model with repeated measures for the factor of time was used to evaluate the main effects and interactions of group (ATV or SAL) and time (days 7, 14, and 21) on OTM. Between-group comparisons of maxillary histomorphometric parameters were performed using 1-way analysis of variance followed by the LSD multiple comparison test (for homogeneous variances) or the Games-Howell test (for heterogeneous variances). When comparisons were made between 2 groups, the independent Student ttest was used. Results were processed in Statistics for Windows (version 18.0; SPSS, Chicago, Ill), and the significance level was set at 5% (P <0.05) for all tests.

Results

There was no difference in weight changes between groups during the experimental period. The mean weights at the end of the study were 327.18 g (SD, ±86.54 g) in the SAL animals and 344.56 g (SD, ±55.44 g) in ATV animals.

After 7, 14, and 21 days of OTM, the first molar displacements were 310.13 ± 14.79, 381.38 ± 33.33, and 485.85 ± 68.94 μm, respectively, in the SAL group vs 263.53 ± 14.79, 334.78 ± 33.33, and 439.25 ± 68.94 μm, respectively, in the ATV group. Statistical analysis indicated that administration of ATV decreased tooth movement significantly (P <0.05), as shown in Figure 2A and B. From day 0 to day 7, the rate of OTM decreased in the ATV group, compared with the SAL group (263.53 vs 310.13 μm, respectively). After that, this rate increased progressively in the SAL and ATV groups (176 vs 175 μm from days 7 to 21, respectively). From day 0 to day 7, the rate of tooth movement decreased in the ATV group, compared with the SAL group (263.53 vs 310.13 μm, respectively). After that, this rate increased progressively in the SAL and ATV groups (176 vs 175 μm from days 7 to 21, respectively).

Figura 2. A, Linear mixed model used to compare overall tooth displacement in the SAL and ATV groups, showing significantly reduced tooth displacement in the ATV group. Data are expressed as means and standard errors. B, Occlusal photographic views of representative animals in the SAL and ATV groups. Note the difference in interdental distance (overall mean) between the first and second molars in the SAL vs ATV-treated animals. C, Overall mean osteoclast counts were reduced in the ATV group compared with the SAL (independent Student t test). Data are expressed as means and standard deviations. D, The overall bone-volume ratio was not affected by ATV administration (P >0.05; independent t test). Data are expressed as means and standard deviations. *P <0.05.

During OTM, ATV administration reduced the overall osteoclast count but did not affect the overall bone-volume ratio compared with SAL (Fig 2C and D). However, when analyzing the drug effect at each time point separately, only after 14 days of OTM did ATV promote osteoclast inhibition ( Fig 3A and B). At this time point, the number of TRAP-positive cells in the ATV group was significantly smaller than that in experimental maxillae of the SAL animals (with OTM) and equal to that observed in control maxillae of the SAL animals (without OTM). During OTM, there was a trend toward increased osteoclast counts only in the SAL group when compared with its respective control maxillae (without OTM), as shown in Figure 3A and B. After 21 days, there were no statistical differences in osteoclast count across study groups.

Figura 3. A, TRAP staining of control maxillae (without OTM) and experimental maxillae (after 14 days of OTM) in representative animals from the ATV and SAL groups. During OTM, osteoclasts were clearly inhibited by statin administration. In addition, these cells were frequently observed in the periphery of blood vessels, independently of drug administration or time point of analysis. B, Osteoclast counts in control maxillae (without OTM) and experimental maxillae (after 14 and 21 days of OTM) of the ATV and SAL groups. Under physiologic conditions (control maxillae, without OTM), ATV did not affect osteoclast counts at either time point (14 and 21 days; 1-way analysis of variance). After 14 days of OTM, animals in the ATV group had reduced numbers of TRAP-positive cells. C, Hematoxylin and eosin stained sections of control and experimental maxillae in ATV and SAL animals. Descriptive analysis indicated that ATV did not affect the bone-volume ratio under physiologic conditions. During OTM, rats treated with ATV had a slight increase in bone-volume ratio. D, Statistical comparisons of bone-volume ratios between the ATV and SAL groups (1-way analysis of variance). OTM reduced bone volume in both groups (ATV and SAL) compared with their respective controls without OTM. Atorvastatin prevented bone loss after 14 days of OTM, as shown by the statistically similar bone-volume ratios of ATV animals and control maxillae of SAL animals. Small arrows indicate osteoclasts. The scale bar represents 100 μm at an original magnification of 200 times. Yellow arrows represents the direction of OTM. Bo, Bone; Ro, root. Data are expressed as means and standard deviations.

Regarding osteoclast distribution, our data indicated that, independently of drug or saline solution administration, these cells were observed in the periphery around bone blood vessels (Haversian canals) or scattered through the periodontal ligament (PDL) (Fig 3A). After 14 days, chronic inflammatory cells were observed in the region of interest; after 21 days, there appeared to be resolution of the cellular infiltrate, with increasing amounts and maturation of bone. However, in some specimens, the width of the PDL remained enlarged, especially in the SAL group ( Fig 3C).

After 14 and 21 days of OTM, the bone-volume ratios were significantly reduced in the SAL and ATV groups compared with their respective controls (without OTM). However, after 14 days of OTM, the bone-volume ratio in the ATV group was statistically similar to that in the control maxillae of the SAL group, as shown in Figure 3C and D. Without orthodontic force (maxillae without OTM), osteoclast count and bone volume were statistically similar between the ATV and SAL groups ( Fig 3, B and D).

ATV did not affect long-bone turnover or endochondral ossification. As shown in the Table, ATV did not affect femur osteoclastogenesis or bone volume. Growth plate cartilage and hypertrophic zone thickness were increased in the ATV group when compared with the SAL group; however, the differences between the groups were not significant (P = 0.259 and P = 0.09, respectively).

Table 1. Histomorphometric analysis of femur specimens.

An independent Student t test was performed to compare osteoclast count, bone volume, and thickness of the hypertrophic zone and growth plate cartilage between the ATV and SAL groups. No parameter was significantly affected by ATV administration (all P >0.05). NS, Not significant. Data are expressed as means and standard deviations.

Discussion

In a previous study conducted in our laboratory, we demonstrated that osteoclast counts in the alveolar bones of rat molars were inhibited by statin administration, which was also correlated with decreased orthodontic relapse.37 However, we also found that ATV can affect long-bone endochondral ossification, because it led to enlargement of the growth plate cartilage and chondrocytic hypertrophic zone of femurs after short-term therapy (7-21 days). These results highlight the clinical relevance of pharmacologic bone modulation during orthodontic treatment and the adverse effects of statins on the femoral growth plate; this seems to limit their potential for use as a pharmacologic strategy to enhance orthodontic treatment in children. In this context, we designed this study to confirm the plausible effects of statins on OTM through osteoclast inhibition, while simultaneously analyzing their potentially adverse effects on endochondral ossification in longer-term therapy (28-35 days).

Our study was conducted with Wistar rats, which are recognized as an adequate model with some translational potential and are widely used in preclinical research to evaluate the cellular aspects of OTM.91112193032 ;  33 To reproduce oral statin therapy as usually prescribed in humans, ATV was administered daily, via gavage, at a dose of 15 mg per kilogram. Unlike in previous studies,91112 ;  43 we administered a high dose because 73% of the orally administered dose is expected to be excreted in bile.44 Furthermore, according to Elewa et al,45 the peak plasma concentration of ATV after administering 15 mg per kilogram by the oral route in rats was similar to that reported after daily administration of 80 mg of ATV in humans.46 Our study protocol was designed to allow statin therapy to reach its full potency, since we started drug administration 2 weeks before OTM. Because each day of life in a rat corresponds to 30 days of human life,47 our investigation period of 2 weeks would correspond to 1 year of ATV therapy in a human. Reamy48 noted that statins achieve full clinical potency after 6 weeks of administration in humans but can require up to 12 weeks in some patients.

In our study, the majority of total molar movement in both groups occurred in the first 7 days. In the SAL group, we observed escalating tooth displacement from days 0 through 7; in the ATV group, OTM was reduced at this time point. After the first week, the rates of OTM were almost identical in the ATV and SAL groups (176 vs 175 μm from days 7 to 21, respectively), but the tooth displacements observed in the ATV group never reached the values of the SAL group. Although the statistical model pointed out a significant difference of tooth displacement between the SAL and ATV groups after 7, 14, and 21 days, these results seemed to have a straight relationship with the initial effects of ATV on the rate of OTM observed at day 7, in accordance with the previous remarks.

Our findings tend to corroborate previous studies that reported significant prevention of OTM and relapse after statin administration.11 ;  12 MirHashemi et al12 noted that ATV, administered at a dose of 5 mg per kilogram per day via gavage, decreased the rate of OTM in Wistar rats after 21 days. Han et al11 also reported a significant reduction of tooth relapse in rats treated with intraperitoneal simvastatin (2.5 mg/kg/day) 7 and 28 days after appliance removal. If we extrapolate this potential action of statins to human bone turnover, hindered OTM should be considered a possibility in patients taking these drugs. Therefore, our results may suggest an anabolic effect of statins on bone, increasing osteogenesis and suppressing osteoclastogenesis, as previously described.67891011;1213 ;  14

Considering the 3 phases of OTM (displacement, lag, and postlag), the hindered initial tooth displacement observed in the ATV group could arguably be related to a major effect on bone turnover. In the first phase, the OTM would be mainly caused by PDL compression. However, other studies also demonstrated a high shift of first molars during the first 7 days of OTM. According to Lee et al,49 the mesial displacement of first molars tends to increase during the first 6 days of OTM, in a time-dependent manner, with a relatively rapid increment at day 1. Moreover, according to Gonzalez et al,36 just a minimal compression of PDL was verified (0.03 mm) after 3 days of rat tooth movement. Therefore, we suggest that additional studies to evaluate the effects of statins on OTM should be encouraged, because they could demonstrate whether these clinical effects are sustained.

Also, the controversial pharmacologic inhibition of OTM may represent a clinical advance, since it could be used to improve tooth anchorage during treatment. Several strategies have been investigated to prevent undesirable OTM.111218192122 ;  33However, our findings do not allow us to state a plausible statin application with this therapeutic aim. We suggest that future studies focusing on this purpose should be performed; this seems to be necessary to surpress any speculations regarding statin use in these therapeutic proposals.

Another important finding of our study was that ATV inhibits osteoclasts. The overall number of TRAP-positive cells was significantly decreased in the ATV group compared with the SAL group. Running counter to our findings, other authors have suggested that these drugs cannot affect osteoclastogenesis during OTM or relapse.11 ;  12 On the other hand, we used a histochemical technique appropriate for osteoclast counting (TRAP), whereas the aforementioned investigations used hematoxylin and eosin sections, which could explain this significant difference between results. According to Kirstein et al,50TRAP expression in osteoclasts correlates directly with bone resorptive activity; moreover, agents that stimulate or inhibit bone resorption also stimulate and inhibit osteoclast expression of TRAP. It has been suggested that, during the bone resorption process, TRAP generates free radicals that help to dissolve collagen fragments in vacuoles of the osteoclastic cell.51

In addition, the inhibition of osteoclasts by statins is widely supported in the literature.67;911 ;  13 However, the exact mechanisms involved in this process are unknown. It was suggested that the ability of statins to inhibit hydroxymethylglutaryl-coenzyme A reductase, thus suppressing the mevalonate pathway, correlates with their ability to inhibit bone resorption, since this prevents the activation of guanosine triphosphate-binding proteins to molecules that play crucial roles in osteoclast development and function (actin cytoskeleton regulation, apoptosis, membrane ruffling, and vesicular trafficking).6 ;  13 Pan et al52 conducted a study to further elucidate the role of the small GTPase-Rho signaling pathway in the regulation of periodontal tissues while responding to mechanical stimuli. They observed, in vitro, that human periodontal cells subjected to a cyclic strain protocol exhibit upregulation of Rho. They suggested that this molecular mechanism promotes actin polymerization, which may be responsible for cytoskeletal rearrangement, thus stimulating OTM and alveolar bone remodeling. Therefore, it is reasonable that statin-induced downregulation of small GTPases would affect osteoclastogenesis during OTM.

Another plausible hypothesis is blockade of activation of the nuclear factor-kappa B pathway,79 ;  11 which involves modulation of the cell receptor activator of nuclear kappa B (RANK), the extracellular and cell receptor activator of nuclear kappa B ligand (RANKL), and the extracellular decoy receptor osteoprotegerin, ultimately promoting suppression of osteoclastogenesis.911 ;  12 We sought to investigate the cellular effects of long-term ATV administration on bone turnover, disregarding molecular aspects, which we believed was a limitation. Further studies associating the cellular and molecular effects of ATV on the skeletal system during OTM are warranted.

In the SAL group, peak osteoclast counts were observed after 14 days of OTM, with a decline after 21 days; this corroborates previous reports.53 However, this trend was not obvious in the ATV group, which had only fewer osteoclasts at day 14 compared with the SAL group. This transient inhibition of osteoclasts may be due to a reduction of the effects of statins on alveolar bone over time. In this regard, we suggest that an in-vivo compensatory mechanism might stimulate osteoclasts to overcome statin-induced blockade at day 21.6 Moreover, the anti-inflammatory effects of statin would be expected to reduce the acute inflammation observed at the early stages of OTM, thus inhibiting the recruitment, maturation, and activation of osteoclasts during OTM. However, despite the transient effect of ATV on bone that we observed, our results highlight osteoclastogenesis as a cellular candidate target modulated by ATV, which prevented OTM in rats, thereby providing mechanistic clues for future studies in humans.

Remarkably, although ATV affected osteoclast counts, our observational data showed a quite similar distribution of these cells across the studied groups. Most osteoclasts were localized around blood vessels in the prospective Haversian canals. These findings seem to confirm that, during OTM, early mononuclear precursors in the bone marrow differentiate into committed mononuclear precursors, fuse, and migrate into the PDL, where they differentiate into active multinuclear osteoclasts.54 The importance of angiogenesis during bone remodeling must also be stressed. It has been shown that microvascular pericytes—undifferentiated cells located around the periphery of blood vessels55—have pluripotent potential for differentiation into osteoblastic bone cells and chondroblasts.545556 ;  57 Further preclinical investigations are needed to confirm the plausibility of osteoclast differentiation from pericytes; this could explain some observations in our study (Fig 3A).

To assess the effects of ATV administration on periodontal tissues under physiologic conditions, a split-mouth design was used, as in other studies.123032 ;  33 Our data indicate that long-term ATV administration did not affect the bone remodeling cycle in the control maxillae (without OTM), since the overall osteoclast count and bone-volume ratio were statistically similar between the ATV and SAL groups. During OTM, although the overall bone-volume ratio was higher in the ATV group, it did not reach statistical significance when compared with the SAL group. Although the inefficacy of oral administration of statins to improve bone formation has been previously described,58 we considered this an unexpected finding; because ATV administration had inhibited initial tooth displacement and osteoclastogenesis. Intending to elucidate this question, we analyzed the bone-volume ratio at each time point separately (Fig 3D). After 14 days of OTM in the SAL group, there was a trend toward a marked decrease in alveolar bone-volume ratio compared with the control maxillae (without OTM), as already described by others. 18 ;  22 On the other hand, in the ATV group, after 14 days of OTM, the bone-volume ratio was similar to that in the control maxillae of the SAL animals, apparently confirming that at this time point statin-mediated osteoclast inhibition prevented bone resorption, promoted bone formation, or both. After 21 days of OTM, both groups had reduced bone-volume ratios, when compared with their control maxillae. In short, our results seem to indicate that statins promote a transient effect on bone volume. In addition, although the osteoclastic activity has been significantly reduced by the drug, this reduction did not reflect the overall alveolar bone volume. On the other hand, the OTM in the ATV group was decreased in the first 7 days, highlighting that other factors in addition to bone turnover should to be considered to explain this outcome.

In this study, long-term statin administration demonstrated no adverse effects on long-bone turnover or endochondral ossification. This is consistent with several previous studies.2324 ;  25 Although reduced hypertrophic zone and growth plate cartilage thickness were observed in animals given ATV, there were no statistical differences between the ATV and SAL groups at the 5% significance level. In contrast, a previous study in our laboratory found that ATV elongates the growth plate cartilage and chondrocytic hypertrophic zone after short-term administration (7-21 days)37; this may constitute a major clinical limitation to its use in children and adolescents.27 ;  28 This divergence between results appears to be related to the duration of ATV administration (mean, 31.5 days in this study vs 14 days in our previous investigation). Consequently, in this study, the animals were older when the were killed than in our previous investigation. Thus, the physiologic reduction in growth plate cartilage and hypertrophic zone thickness that is normal for this age could account for our findings. Corroborating this hypothesis, Walker and Kember59 found that, over time, cellular proliferation slows in the growth plate, causing the rate of longitudinal bone growth to decrease and approach zero as the organism reaches adult size. Further preclinical studies are warranted to elucidate the effects of statins on endochondral ossification and confirm their safety for use in children and adolescents.

Taken together, our data suggest that statins can affect osteoclastogenesis and inhibit OTM in the first week. Whether these findings will translate to clinical practice is unknown; however, considering the widespread use of statins, clinical trials should be performed to evaluate this bone-anabolic role of these agents during OTM.4 ;  5 Finally, among the expected effects of statins as applied to orthodontic practice and science, we underline 2 conflicting aspects: (1) hindered tooth displacement in patients taking these drugs, which is a clinical concern; and (2) additional tooth anchorage obtained by pharmacologic bone modulation, which could be a clinical adjunct.

Conclusions

This experimental study showed that long-term use of ATV can significantly inhibit osteoclastogenesis and slow OTM in rats in the first week. Arguably, this clinical effect would be due to its inhibition of bone responses; in this first phase of OTM, the squeezing of periodontal fibers also might be considered. The alveolar bone volume was only transiently affected by drug usage, suggesting that other factors, in addition to osteoclastogenesis, should be considered to better explain the effects of statins on OTM. Under physiologic conditions, however, the drug did not affect bone turnover and endochondral ossification.

Acknowledgment

We thank Julia Walter and Andreia Lara for their contributions to the laboratory procedures.

References

1
H.J. Kim, Y. Kim, Y. Cho, B. Jun, K.W. Oh
Trends in the prevalence of major cardiovascular disease risk factors among Korean adults: results from the Korea National Health and Nutrition Examination Survey, 1998-2012
Int J Cardiol, 174 (2014), pp. 64–72

2
C. Mercado, A.K. DeSimone, E. Odom, C. Gillespie, C. Ayala, F. Loustalot
Prevalence of cholesterol treatment eligibility and medication use among adults—United States, 2005-2012
MMWR Morb Mortal Wkly Rep, 64 (2015), pp. 1305–1311

3
J.C. LaRosa, D. Hunninghake, D. Bush, M.H. Criqui, G.S. Getz, A.M. Gotto, et al.
The cholesterol facts. A summary of the evidence relating dietary fats, serum cholesterol, and coronary heart disease. A joint statement by the American Heart Association and the National Heart, Lung, and Blood Institute. The Task Force on Cholesterol Issues, American Heart Association
Circulation, 81 (1990), pp. 1721–1733

4
T.R. Pedersen, J. Kjekshus, K. Berg, T. Haghfelt, O. Faergeman, G. Faergeman, et al.
Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S)
Atheroscler Suppl, 2004 (5) (1994), pp. 81–87

5
C. Baigent, A. Keech, P.M. Kearney, L. Blackwell, G. Buck, C. Pollicino, et al.
Efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins
Lancet, 366 (2005), pp. 1267–1278

6
A. Staal, J.C. Frith, M.H. French, J. Swartz, T. Gungor, T.W. Harrity, et al.
The ability of statins to inhibit bone resorption is directly related to their inhibitory effect on HMG-CoA reductase activity
J Bone Miner Res, 18 (2003), pp. 88–96

7
J.Y. Kim, E.Y. Lee, E.B. Lee, Y.J. Lee, H.J. Yoo, J. Choi, et al.
Atorvastatin inhibits osteoclastogenesis by decreasing the expression of RANKL in the synoviocytes of rheumatoid arthritis
Arthritis Res Ther, 14 (2012), p. R187

8
T. Maeda, A. Matsunuma, T. Kawane, N. Horiuchi
Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells
Biochem Biophys Res Commun, 280 (2001), pp. 874–877

9
R.F. de Araujo Junior, T.O. Souza, L.M. de Moura, K.P. Torres, L.B. de Souza, S. Alves Mdo, et al.
Atorvastatin decreases bone loss, inflammation and oxidative stress in experimental periodontitis
PLoS One, 8 (2013), p. e75322

10
L.B. Ferreira, V. Bradaschia-Correa, M.M. Moreira, N.D. Marques, V.E. Arana-Chavez
Evaluation of bone repair of critical size defects treated with simvastatin-loaded poly(lactic-co-glycolic acid) microspheres in rat calvaria
J Biomater Appl, 29 (2015), pp. 965–976

11
G. Han, Y. Chen, J. Hou, C. Liu, C. Chen, J. Zhuang, et al.
Effects of simvastatin on relapse and remodeling of periodontal tissues after tooth movement in rats
Am J Orthod Dentofacial Orthop, 138 (2010), pp. 550.e1–550.e7 discussion, 550-551

12
A.H. MirHashemi, M. Afshari, M. Alaeddini, S. Etemad-Moghadam, A. Dehpour, S. Sheikhzade, et al.
Effect of atorvastatin on orthodontic tooth movement in male wistar rats
J Dent (Tehran), 10 (2013), pp. 532–539

13
W.A. Grasser, A.P. Baumann, S.F. Petras, H.J. Harwood Jr., R. Devalaraja, R. Renkiewicz, et al.
Regulation of osteoclast differentiation by statins
J Musculoskelet Neuronal Interact, 3 (2003), pp. 53–62

14
M.L. Ho, Y.H. Chen, H.J. Liao, C.H. Chen, S.H. Hung, M.J. Lee, et al.
Simvastatin increases osteoblasts and osteogenic proteins in ovariectomized rats
Eur J Clin Invest, 39 (2009), pp. 296–303

15
F. Ruan, Q. Zheng, J. Wang
Mechanisms of bone anabolism regulated by statins
Biosci Rep, 32 (2012), pp. 511–519

16
J. Yue, X. Zhang, B. Dong, M. Yang
Statins and bone health in postmenopausal women: a systematic review of randomized controlled trials
Menopause, 17 (2010), pp. 1071–1079

17
I. Andrade Jr., S.R. Taddei, P.E. Souza
Inflammation and tooth movement: the role of cytokines, chemokines, and growth factors
Semin Orthod, 18 (2012), pp. 257–269

18
J.B. Hudson, N. Hatch, T. Hayami, J.M. Shin, M. Stolina, P.J. Kostenuik, et al.
Local delivery of recombinant osteoprotegerin enhances postorthodontic tooth stability
Calcif Tissue Int, 90 (2012), pp. 330–342

19
N. Zhao, J. Lin, H. Kanzaki, J. Ni, Z. Chen, W. Liang, et al.
Local osteoprotegerin gene transfer inhibits relapse of orthodontic tooth movement
Am J Orthod Dentofacial Orthop, 141 (2012), pp. 30–40

20
Y. Hirate, M. Yamaguchi, K. Kasai
Effects of relaxin on relapse and periodontal tissue remodeling after experimental tooth movement in rats
Connect Tissue Res, 53 (2012), pp. 207–219

21
A.H. Hassan, A. Al-Hubail, A.A. Al-Fraidi
Bone inductive proteins to enhance postorthodontic stability
Angle Orthod, 80 (2010), pp. 1051–1060

22
D.A. Schneider, S.M. Smith, C. Campbell, T. Hayami, S. Kapila, N.E. Hatch
Locally limited inhibition of bone resorption and orthodontic relapse by recombinant osteoprotegerin protein
Orthod Craniofac Res, 18 (Suppl 1) (2015), pp. 187–195

23
M. Macpherson, B. Hamrén, M.J. Braamskamp, J.J. Kastelein, T. Lundström, P.D. Martin
Population pharmacokinetics of rosuvastatin in pediatric patients with heterozygous familial hypercholesterolemia
Eur J Clin Pharmacol, 72 (2016), pp. 19–27

24
H.J. Avis, B.A. Hutten, C. Gagné, G. Langslet, B.W. McCrindle, A. Wiegman, et al.
Efficacy and safety of rosuvastatin therapy for children with familial hypercholesterolemia
J Am Coll Cardiol, 55 (2010), pp. 1121–1126

25
M.L. Miller, C.C. Wright, B. Browne
Lipid-lowering medications for children and adolescents
J Clin Lipidol, 9 (5 Suppl) (2015), pp. S67–S76

26
L.S. Eiland, P.K. Luttrell
Use of statins for dyslipidemia in the pediatric population
J Pediatr Pharmacol Ther, 15 (2010), pp. 160–172

27
K. Leem, S.Y. Park, D.H. Lee, H. Kim
Lovastatin increases longitudinal bone growth and bone morphogenetic protein-2 levels in the growth plate of Sprague-Dawley rats
Eur J Pediatr, 161 (2002), pp. 406–407

28
A. Yamashita, M. Morioka, H. Kishi, T. Kimura, Y. Yahara, M. Okada, et al.
Statin treatment rescues FGFR3 skeletal dysplasia phenotypes
Nature, 513 (2014), pp. 507–511

29
Committee to Revise the Guide for the Care and Use of Laboratory Animals
The 1996 Guide for the care and use of laboratory animals
National Academies Press, Washington DC (1996)

30
C. Verna, M. Dalstra, B. Melsen
The rate and the type of orthodontic tooth movement is influenced by bone turnover in a rat model
Eur J Orthod, 22 (2000), pp. 343–352

31
M. Shirazi, H. Alimoradi, Y. Kheirandish, S. Etemad-Moghadam, M. Alaeddini, A. Meysamie, et al.
Pantoprazole, a proton pump inhibitor, increases orthodontic tooth movement in rats
Iran J Basic Med Sci, 17 (2014), pp. 448–453

32
T.J. Franzen, P. Brudvik, V. Vandevska-Radunovic
Periodontal tissue reaction during orthodontic relapse in rat molars
Eur J Orthod, 35 (2013), pp. 152–159

33
T.J. Franzen, S.E. Zahra, A. El-Kadi, V. Vandevska-Radunovic
The influence of low-level laser on orthodontic relapse in rats
Eur J Orthod, 57 (2015), pp. 111–117

34
N. Takahashi, N. Udagawa, S. Tanaka, H. Murakami, I. Owan, T. Tamura, et al.
Postmitotic osteoclast precursors are mononuclear cells which express macrophage-associated phenotypes
Dev Biol, 163 (1994), pp. 212–221

35
A.M. Parfitt, M.K. Drezner, F.H. Glorieux, J.A. Kanis, H. Malluche, P.J. Meunier, et al.
Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee
J Bone Miner Res, 2 (1987), pp. 595–610

36
C. Gonzales, H. Hotokezaka, Y. Arai, T. Ninomiya, J. Tominaga, I. Jang, et al.
An in vivo 3D micro-CT evaluation of tooth movement after the application of different force magnitudes in rat molar
Angle Orthod, 79 (2009), pp. 703–714

37
G.S. Dolci, L.V. Portela, D. Onofre de Souza, A.C. Medeiros Fossati
Atorvastatin-induced osteoclast inhibition reduces orthodontic relapse
Am J Orthod Dentofacial Orthop, 151 (2017), pp. 528–538

38
K.P. Egan, T.A. Brennan, R.J. Pignolo
Bone histomorphometry using free and commonly available software
Histopathology, 61 (2012), pp. 1168–1173

39
T. El Khassawna, W. Böcker, P. Govindarajan, N. Schliefke, B. Hürter, M. Kampschulte, et al.
Effects of multi-deficiencies-diet on bone parameters of peripheral bone in ovariectomized mature rat
PLoS One, 8 (2013), p. e71665

40
A.C. Aybar Odstrcil, S.N. Carino, J.C. Ricci, P.M. Mandalunis
Effect of arsenic in endochondral ossification of experimental animals
Exp Toxicol Pathol, 62 (2010), pp. 243–249

41
C.M. Fan, B.K. Foster, S.K. Hui, C.J. Xian
Prevention of bone growth defects, increased bone resorption and marrow adiposity with folinic acid in rats receiving long-term methotrexate
PLoS One, 7 (2012), p. e46915

42
G. Dahlberg
Statistical methods for medical and biological students
Interscience Publications, New York (1940)

43
G. Mundy, R. Garrett, S. Harris, J. Chan, D. Chen, G. Rossini, et al.
Stimulation of bone formation in vitro and in rodents by statins
Science, 286 (1999), pp. 1946–1949

44
A.E. Black, R.N. Hayes, B.D. Roth, P. Woo, T.F. Woolf
Metabolism and excretion of atorvastatin in rats and dogs
Drug Metab Dispos, 27 (1999), pp. 916–923

45
H.F. Elewa, A. Kozak, A.B. El-Remessy, R.F. Frye, M.H. Johnson, A. Ergul, et al.
Early atorvastatin reduces hemorrhage after acute cerebral ischemia in diabetic rats
J Pharmacol Exp Ther, 330 (2009), pp. 532–540

46
R.L. Lins, K.E. Matthys, G.A. Verpooten, P.C. Peeters, M. Dratwa, J.C. Stolear, et al.
Pharmacokinetics of atorvastatin and its metabolites after single and multiple dosing in hypercholesterolaemic haemodialysis patients
Nephrol Dial Transplant, 18 (2003), pp. 967–976

47
N.A. Andreollo, E.F. Santos, M.R. Araújo, L.R. Lopes
Rat’s age versus human’s age: what is the relationship?
Arq Bras Cir Dig, 25 (2012), pp. 49–51

48
B.V. Reamy
Hyperlipidemia management for primary care: an evidence-based approach
Springer, Berlin, Germany (2009)

49
S. Lee, H. Yoo, S. Kim
CCR5-CCL axis in PDL during orthodontic biophysical force application
J Dent Res, 94 (2015), pp. 1715–1723

50
B. Kirstein, T.J. Chambers, K. Fuller
Secretion of tartrate-resistant acid phosphatase by osteoclasts correlates with resorptive behavior
J Cell Biochem, 98 (2006), pp. 1085–1094

51
J.M. Halleen, S. Räisänen, J.J. Salo, S.V. Reddy, G.D. Roodman, T.A. Hentunen, et al.
Intracellular fragmentation of bone resorption products by reactive oxygen species generated by osteoclastic tartrate-resistant acid phosphatase
J Biol Chem, 274 (1999), pp. 22907–22910

52
J. Pan, T. Wang, L. Wang, W. Chen, M. Song
Cyclic strain-induced cytoskeletal rearrangement of human periodontal ligament cells via the Rho signaling pathway
PLoS One, 9 (2014), p. e91580

53
Y. Ren, A.M. Kuijpers-Jagtman, J.C. Maltha
Immunohistochemical evaluation of osteoclast recruitment during experimental tooth movement in young and adult rats
Arch Oral Biol, 50 (2005), pp. 1032–1039

54
R. Xie, A.M. Kuijpers-Jagtman, J.C. Maltha
Osteoclast differentiation during experimental tooth movement by a short-term force application: an immunohistochemical study in rats
Acta Odontol Scand, 66 (2008), pp. 314–320

55
S.M. Chim, J. Tickner, S.T. Chow, V. Kuek, B. Guo, G. Zhang, et al.
Angiogenic factors in bone local environment
Cytokine Growth Factor Rev, 24 (2013), pp. 297–310

56
M.J. Doherty, B.A. Ashton, S. Walsh, J.N. Beresford, M.E. Grant, A.E. Canfield
Vascular pericytes express osteogenic potential in vitro and in vivo
J Bone Miner Res, 13 (1998), pp. 828–838

57
L. Diaz-Flores, R. Gutierrez, A. Lopez-Alonso, R. Gonzalez, H. Varela
Pericytes as a supplementary source of osteoblasts in periosteal osteogenesis
Clin Orthop Relat Res, 275 (1992), pp. 280–286

58
B. Ma, S.A. Clarke, R.A. Brooks, N. Rushton
The effect of simvastatin on bone formation and ceramic resorption in a peri-implant defect model
Acta Biomater, 4 (2008), pp. 149–155

59
K.V. Walker, N.F. Kember
Cell kinetics of growth cartilage in the rat tibia. II. Measurements during ageing
Cell Tissue Kinet, 5 (1972), pp. 409–419

Link original da publicação: https://authors.elsevier.com/a/1X7gA3AGXGaJ3v