The effect of high concentration of zoledronic acid on tooth induced movement and its repercussion on root, periodontal ligament and alveolar bone tissues in rats

Zoledronic acid (ZA) is often prescribed for osteoporosis or resorptive metabolic bone disease. This study aims to evaluate the effect of ZA on orthodontic tooth movement (OTM) and root and bone resorption and its repercussion on root, periodontal ligament and alveolar bone tissues. The experimental group consisted of 72 Wistar rats divided in four subgroups: Naive, Saline and Zoledronic Acid groups at the concentration of 0.2 mg/kg [ZA (0.2)] or 1.0 mg/kg [ZA (1.0)]. The animals were subjected to i.v (dorsal penile vein) administrations of ZA or saline solution, on days 0, 7, 14 and 42. Under anesthesia, NiTi springs were installed in the first left maxillary molar with 50gf allowing the OTM, except for the negative control group (N) for mesial movement of the left first maxillary teeth. The animals were sacrificed and maxillae were removed for macroscopic and histopathological analyzes, scanning electron microscopy, computerized microtomography and confocal microscopy. Treatment with ZA decreased the OTM and the number of osteoclasts and loss of alveolar bone when compared to the naive and saline groups. Reduction of radicular resorption, increased necrotic areas and reduced vascularization in the periodontal ligament were observed in the ZA groups. ZA interferes with OTM and presents anti-resorptive effects on bone and dental tissues associated with a decreased vascularization, without osteonecrosis.


Materials and methods
Animals. All experimental protocols were approved by the Federal University of Ceará Committee on the Ethical Treatment of Research Animals (CEUA. No. 95/15) and performed in accordance with the ARRIVE ethical guidelines.
In order to determine the sample size, it was performed a power calculation. The sample size was determined to provide 80% power to recognize a significant difference of 20% among groups and the standard deviation of 15% with a 95% confidence interval (p = 0.05). Based on this study, a sample size of 6 rats per group was required. The animals were kept in cages with temperature-controlled rooms, with food and water ad libitum throughout the experiment.
After two weeks of acclimation to the laboratory enviroment, seventy-two male Wistar rats, weighing 200-250 g (mean age of 10 weeks), were divided, in a blind manner, into 2 groups treated with Zoledronic Acid, diluted with 0.9%w/v sodium chloride solution, following the manufacturer's instructions, (CRISTÁLIA, Itapira, SP, Brazil) at 0.2 mg/kg ZA(0.2) or 1.0 mg /kg ZA(1.0) concentrations 16 . The vehicle control group was submitted to experimental orthodontic tooth movement and received weekly intravenous administrations of 0.9%w/v sodium chloride solution (0.1 ml/kg; Saline). The control naive group was not subjected to any procedure (Naive).
The animals were subjected to i.v (dorsal penile vein) administrations of ZA or saline solution, under anestesia with ketamine (60 mg/kg; i.p) and xylazine (5 mg/kg; i.p), on days 0, 7, 14 and 42. The last administration was performed immediately after the installation of an orthodontic tooth movement device. On day 46, 24 animals were euthanized (n = 6/group) and had their maxillae collected and processed for histopathological analyzes. On day 63, 48 animals were euthanized (n = 12/group) and their maxillae were collected and processed for scanning electron microscopy (SEM) (n = 6/group) and computerized microtomography (n = 6/group) examination ( Fig. 1).

Experimental orthodontic tooth movement (OTM). For the induction of experimental orthodontic
tooth movement (OTM) it was used nickel-titanium (NiTi) closed-coil springs and a 0.25-mm (MORELLI, Brazil) thick stainless steel tying wire to move the maxillary left molars mesially 7,9,11 . The animals were previously anesthetized with ketamine (70 mg/kg administered i.p., 10% Quetamina, VETNIL, São Paulo, SP, Brazil) and xylazine (10 mg/kg administered i.p., 2% CALMIUM, São Paulo, SP, Brazil). All groups, except the naïve group, were submitted to NiTi springs installation in the first left maxillary molar (force of 50gf; Fig. 2A) 9 allowing OTM for a period of 4 or 21 days. The other end was attached to the maxillary left incisor using a tying wire and composite resin (COLTENE, Brazil). The force produced by the spring was measured and standardized using a tensiometer (MORELLI, Brazil).
Measurement of the amount of tooth movement. The amount of tooth movement (ΔTM) was measured using a digital calliper (DIGIMESS, Brazil), positioned in the most cervical point of the molar and incisor 21 , prior to installation of coin spring and immediately following euthanasia, on day 63. The distance between the first upper left molar and the upper central incisor on the same side, measured prior to placement of the orthodontic appliance (initial measurent [im]) and after euthanasia (final measurement [fm]) were obtained. The amount of tooth movement was calculated using the following formula: ΔTM (%) = (im/fm − 1) × 100.
Histological analysis. Four days after the installation of the springs, 24 animals (n = 6/group) were euthanized by an overdose of ketamine (300 mg/kg) and zylazine (30 mg/kg). The maxillae were removed, fixed in 10% neutral buffered formalin for 24 h, and then demineralized in 10% EDTA for 60 days, as previously described 7 . When decalcification was completed, the tissues were well rinsed in buffer and dehydrated in a series of increasing concentrations of alcohol solutions: 70% (2 changes, 30 min each), 80% (1 h), 90% (1 h) and abso-   Picrosirius red staining) were consecutively cut, starting at the cervical third in the apical direction, showing the mesiobuccal and distobuccal roots 11 . The histological stainings were performed following the manufacture's instructions: hematoxylin and eosin (Dinâmica Química, Indaiatuba, SP, Brazil), Mallory (Bio-Optica Milano S.p.A, Milano, Italy) and Picrosirius red (Scytek, Logan, UT, USA). The distobuccal root was used to identify the periodontal ligament (PDL) areas undergoing tension and compression forces (Fig. 2B,C). The slides were examined under light microscope to evaluate the periodontal ligament and root surface in the tension and compression sides 7 . For this, 10 fields/ slide (n = 6/group) were randomly photographed, under a final magnification of 200 ×, as previously described 7 .
At the tension side, blood vessels and collagen fibers were analyzed. The number of blood vessels was determined from the acquisition of images (400×) over a select area of interest (areas of intense vasculature, measuring 6 × 10 4 μm 2 ), from Mallory's trichrome stained slides (n = 6/group) 15 . The percentage of the area occupied by these vessels in relation to the total area (6 × 10 4 μm 2 ) were also evaluated using the IMAGE J 1.51 j8 software (NIH, Bethesda, MD, USA). Data were expressed as number of vessels and (%) vessel area, respectively 15 .
Anaysis of collagen fibers in PDL (at the tension side) of the first maxillary left molars were performed in slices stained with Picrosirius Red (n = 6/group) to identify the presence and type of fibrilar collagen under polarized light 22 . The data was expressed as the mean percentage ± E.P.M. of collagen content per group.
The presence or absence of hyaline areas in PDL and root resorption were identified in the compression side in slices stained with hematoxylin and eosin. The hyaline areas were delimited and measured using IMAGE J 1.51 j8 software (NIH, Bethesda, MD, USA). The data is presented as percentage of hyaline areas in relation to the total PDL area, as previouls described 21 .
In order to evaluate the resorption of root dental tissue, a line was drawn through the center of the DV root pulp using IMAGEJ 1.51 j8 software (NIH, Bethesda, MD, USA), separating the tension and compression sides. The compression side was then divided into 6 equal parts, each part corresponding to an angle of 30° (whose vertex lies at the center in the pulp). The root resorption was evaluated according to scores, based on the number of sextants that present foci of root resorption.
Histomorphometric analysis of alveolar bone. Osteocytes and osteoclasts (cells containing more than 3 nuclei) along the margins of the DV roots were counted in 10 fields/slide (200 × magnification) using IMAGE J 1.51 j8software (NIH, Bethesda, MD, USA) 23 .
The alveolar bone resorption on the compression side was evaluated through the preservation of alveolar bone collagen from images captured from the slices stained with the HE under a confocal microscope (at 100 × magnification) (LSM 710, ZEISS, Jena, Thuringia, Germany) using the manufacturer's software (ZEN 2.1 LITE BLACK, 64-bit version, 758 MB, ZEISS, Jena, Thuringia, Germany). The excitation wavelength of collagen was taken at 488 nm (FITC-green fluorescence emission channel) 24 . The images were processed in the IMAGE J 1.51 j8 software (NIH, Bethesda, MD, USA) to calculate the percentage of resorbed area. To evaluate the alveolar bone, a rectangle (3 mm thick × 0.5 mm height) was drawn in order to determine the limits of the evaluation. The rectangle was positioned parallel to the periodontal ligament and adjacent to the frontal wall of the alveolar bone, in order to measure the structural changes in the alveolar bone 9 . The following data were obtained on the tension and compression sides: fraction of bone volume and total volume (BV/TV), trabecular thickness (Tb.Th), number of trabeculae (Tb.N) and separation between trabeculae (Tb.Sp). These data were quantified using the CT Analyzer software (Bruker Micro CT, Version 1.15.4.0, Kontich, Belgium).

Scanning electron microscopy (SEM) of evaluation of the roots.
After euthanasia, 21 days after the installation of orthodontic appliances, the first upper left molars were removed and fixed in Karnovisky for at least 6 h, then kept in a Cacodylate buffer. All molars were washed in 1% sodium hypochlorite 9 . The tooth was placed in an eppendorf tube and left in the desiccator drying for 24 h. The fragments were assembled into stubs for metallization with gold powder (Quorum Metallist QT150ES, Quorum Technologies, Laughton, England) for scanning electron microscopy (MEV Inspect-50, FEI, Hillsboro, Oregon, USA) 25 . The root resorption gaps on mesiovestibular roots were qualitatively evaluated from images obtained at 600 × magnification 2 .

Statical analysis.
Values are given as means, and error bars are standard deviations. Normality was evaluated by Shapiro-Wilk test. Outliers were removed. The Student's t-test or one-way ANOVA with Tukey / Games Howell post-hoc test were used to compare all measurements. The homogeneity of variances was tested by Levene's test. Statistical analyses were performed using the GraphPad Prism 6.0 (Graphpad Prism SOFTWARE, La Jolla, CA, USA) and SPSS 20.0 (SPSS Inc., Chicago, IL, USA) computer software programs. Probability level (p-value) < 0.05 was assumed.
Ethical approval. All applicable international, national, and institutional guidelines for the care and use of animals were followed. No statistically significant difference in the number of vessels on the tension side was observed between naive and saline groups. The ZA(1.0), but not ZA(0.2), showed reduced vascularization of periodontal tissues when compared to the saline group (p = 0.010; 95%CI = 6.72-10.28) (Fig. 4B,F). Figure 4G shows that ZA treatment reduced the total cross-sectional area of blood vessels when compared to the saline group (ZA(0,2) versus Saline, p < 0.001, 95%CI = 3.91-6.09; ZA(1,0) versus Saline, p < 0.001, 95%CI = 2.89-5.56).
There were no statistically significant differences (p > 0.05) in the percentage of collagen fibers of the periodontal ligament among the groups, but a reduction was observed (p < 0.05) in the type I/type III collagen ratio in the groups submitted to orthodontic movement when compared to the naive group (Fig. 4C,D,H,I).
Effect of ZA on alveolar bone. A higher number of osteoclasts was found in the saline group (p = 0.008; 95%CI = 21.05-35.35) when compared to the naive. Treatment with ZA, at 1.0 mg/kg, but not at 0.2 mg/kg, sig- www.nature.com/scientificreports/  www.nature.com/scientificreports/ nificantly reduced the number of osteoclastic cells when compared to the saline group (p = 0.003; 95%CI = 1.18-6.02) (Fig. 5A,D). There was no significant difference in the number of osteocytes among the experimental groups (Fig. 5B,E). A greater loss of bone collagen was observed in the saline group when compared to the naive group (p = 0.018; 95%CI = 1.46-14.38). Zoledronic acid significantly prevented the alveolar bone matrix resorption (Fig. 5C,F).
Effect of ZA on root resorption. It was observed 21 days after the installation of orthodontic appliances significant differences in the microscopic analysis of tooth roots between the groups submitted to orthodontic movement and the naive group, as shown in Table 1 and Fig. 6A,B. In the naive group, the cementum and dentin exhibited intact connective tissue, with no resorption areas on the cementum surface (Fig. 6A,B). On the other hand, resorption is evident on the compression side in the saline group (Fig. 6A). SEM revelead the orthodontically induced root craters (Fig. 6B), much larger in the saline group than in the AZ treated groups. The SEM evaluation shows a normal smooth topography of the root surface of the naive group. . Treatment with ZA prevented the reduction in bone volume during OTM, characterizing a decrease in bone resorption when compared to the saline group (Fig. 7A). An increased trabeculae separation (Tb.Sp) was observed in the saline group when compared to the naive group (p = 0.001; 95%CI = 0.01-0.036). Treatment with ZA decreased trabeculae separation when compared to the saline group (Fig. 7D). There were no significant differences in the trabeculae number (Tb.N) or in the trabeculae thickness (Tb.Th) among the groups (Fig. 7B,C).
There were no significant differences in the bone composition on the tension side among the experimental groups ( Fig. 7E-H).

Discussion
The concentrations of zoledronic acid (ZA) used in the present study were selected from previous research described in the literature that investigated whether the continuous use of bisphosphonates, including ZA, predisposes to osteonecrosis of the jaws after tooth extraction 16 . The authors used an interspecies allometric scaling for dose conversion between animals and human, based on the differences in body surface area and body weight. According to this study, the monthly administration of 4 mg of ZA, used to treat multiple myeloma in humans, corresponds to 0.60 mg/kg in Wistar rats. Based on that study, we investigated the influence of this concentration of ZA, divided into three weekly intravenously administrations of 0.20 mg/kg each, on accelerated bone metabolism induced by tooth movement and its repercussion on periodontal tissues. In addition, we also investigated the effects of a five times greater concentration, 1.00 mg/kg.
Osteonecrosis of the jaws, due to unclear mechanisms that involve changes in vascularization, is a described clinical condition that has been associated with the use of bisphosphonates, including ZA, a potent third generation bisphosphonate 26 . It has been reported that patients treated with intravenous bisphosphonate are at a higher risk compared to patients receiving oral bisphosphonate 26 . In the present study, however, despite the decrease in blood vessel area observed in the periodontal ligament of the groups treated with ZA, no macroscopic www.nature.com/scientificreports/ or microscopic clinical signs of necrosis were found, according to the parameters described by Ruggiero and collaborators 27 , which include fistulas and/or abscesses. On the contrary, bone integrity was verified, without necrotic areas and empty osteocytic gaps, verified by histopathological analysis. The present study clearly showed that ZA interfered with tooth displacement, since a lower rate of orthodontic tooth movement (OTM) was found in the ZA groups compared to the untreated group. This can be explained by the decrease in osteoclasts observed in the ZA groups. It is well established the crucial role of osteoclast, as the first step for the orthodontic movement. Any interference in the function of these cells results in decreased efficiency of orthodontic treatment. In fact, ZA acts as a regulator of bone metabolism, through the inibition of osteoclastic activity, mainly by inducing apoptosis of osteoclasts and inhibiting the maturation of these cells [15][16][17][18][19][20][21][22] . Studies described in the literature reinforce our findings 21,[23][24][25][26][27][28] . Different results, however, have been described by Seifi et al. 17 , who evaluated the effect of local administration (injected into the vestibule of the mesial root of the first maxillary molars) of a ZA made in Iran (called zolena) on orthodontic tooth movement and bone and root resorption in rats. Zolena did not decrease OTM but it significantly inhibited bone and root resorption and angiogenesis. The authors speculate that zolena might be less potent than its foreing counterparts.
The anti-resorptive effect of ZA treatment on alveolar bone can be seen on the compression side, where the periodontal ligament is compressed by the tooth against the alveolar bone. It is the place where the highest rate of bone resorption was observed in the untreated group, submitted to OTM (saline group). Accordinly, the literature reports that during induced-tooth movement, periodontal ligament responds to mechanical force stimulation and provides a microenvironment for cellular reactions and tissue remodeling 29 . The accumulation of M1 and M2 macrophages on the compression side of periodontal tissues has been described 30,31 . Activated M1 macrophages may secrete proinflammatory cytokines that are associated with bone loss and root resorption, while M2 macrophages release antiinflammatory cytokines. These studies suggest that the balance between M1 and M2 macrophages is the key point in root resorption and bone loss induced by orthodontic mechanical force 30,31 .
The periodontal ligament space thickness at the compression side were wider in the saline group than in AZ groups, as a consequence of the greater bone resorption observed in the untreated control group, which results in more space to be occupied by the periodontal ligament. In accordance, the number of osteoclasts was significanly higher at the compression side of the saline group compared to the group treated with the highest concentration of ZA. This finding explains the significant reduction in tooth displacement observed in the ZA groups when compared to the untreated group. The non-resorbed alveolar bone acts as a physical barrier, preventing tooth movement.
In addition to the anti-osteoclastic actions of ZA, we especulate that the smaller number of osteoclasts found in the ZA groups may be related to the lower vascularization observed in the periodontal ligament of these animals, considering that osteoclasts are formed from the fusion of monocytes that reach the bone through the bloodstream 32 . The greater compression of the periodontal ligament in the ZA group may contributes to the decrease in the number and area of blood vessels in the periodontal ligament observed in the ZA groups when compared to saline. The bone anti-resorptive effect of ZA was confirmed by confocal microscopy, as greater collagen integrity was found in the alveolar bone of ZA groups when compared to the saline group.
The computerized microtomographs (Micro-CT) analysis also confirms the anti-resorptive action of ZA in bone tissue during OTM. This analysis is extremely relevant, since the micro-CT allows a three-dimensional assessment of the alveolar bone microarchitecture 33 . In the evaluation of the trabecular bone of the saline group, it was observed that the induced tooth movement increases the separation between the trabeculae (Tb.Sp), without changing the number of these trabeculae (Tb.N) and neither the thickness of trabeculae (Tb. Th) on the compression side. Treatment with ZA maintains the number and thickness of the trabeculae, but reduces, however, the separation of these trabeculae, when compared to the saline group. This finding reflects the greater compression in the alveolar bone observed in the ZA groups compared to the saline group. The maintenance of bone matrix integrity, observed in all groups, without alteration in the microarchitecture of the alveolar bone, confirms histopathological analyzes, which did not show osteonecrosis of the alveolar bone, reinforcing that ZA did not induce bone necrosis in the present study.
In the tension side, where there is distension of the periodontal ligament, there was no significant difference between the ZA and saline groups regarding periodontal ligament space thickness. This result reinforces the fact that the decrease in tooth movement observed in the ZA group is due to the lack of bone resorption at the compression side, and not to morphological changes at the tension side, such as the lack of stretching of periodontal fibers. The confocal microscopy analyses did not show differences in the morphology and organization of the periodontal fibers at the tension side between ZA and saline groups. The micro-CT based image analyses, on the tension side, did not show differences in the microarchitecture of the alveolar bone between the saline and ZA groups, confirming that the effects of ZA on OTM are evident on the compression side.
To investigate the effects of ZA on the periodontal ligament during OTM, we evaluated the presence of hyalinic areas and also the composition and distribution of collagen fibers, the main component of the periodontal ligament, in slices stained with picrosirius red, a dye that allows visualization and differentiation of type I collagen fibers, the most abundant in the periodontal ligament, and type III 34 . This analysis was motivated by studies suggesting toxic/ inflammatory effects of ZA on periodontal ligament cells 35,36 .
The hyaline areas, described in the literature as a cell-free area, observed in the periodontal ligament of ZA groups were significantly higher when compared to the saline group, which corroborates with Kubek and collaborators 32 . This finding might be associated with greater compression of the periodontal fibers (on the compression side) observed in the ZA groups. We also found a significant decrease in vascularization in the periodontal ligament of ZA groups. Accordingly, vascular injuries have been demonstrated in the compressed areas of the periodontal ligament which undergo hyalinization of the fibrous tissue 37 .
The analysis of the periodontal ligament performed in the present study in slices stained with picrosirius red reinforce data from the literature demonstrating that during orthodontic movement, the alveolar bone and www.nature.com/scientificreports/ periodontal fibers are continuously remodeled and periodontal structures are dynamically reconstructed through biological reactions in the periodontal ligament 38 . In fact, no statistical differences on the collagen content between the groups submitted to OTM (saline and ZA groups) and the naive group (animals are submitted to OTM) were found, suggesting the active and continuous repair of collagen fibers during OTM. However, under polarized light, it was clearly observed, on the traction side, a significant reduction in the type I / type III collagen ratio in the periodontal ligament of the groups submitted to orthodontic movement when compared with the naive group.
According to the literature, mature periodontal fiber collagen, type I collagen, induces metalloproteinase (MMP-1) expression in fibroblasts and osteoblasts in response to orthodontic force applied to the tooth 38 , which results in collagen degradation. During the repair of the periodontal ligament, the amount of collagen III increases considerably and is gradually replaced by mature collagen type I 39 . Our results reinforce that active and constant reconstruction of the fibers of the periodontal ligament occurs in orthodontically moved teeth. Treatment with ZA did not interfere with the repair of periodontal ligament fibers in the present work, contradicting studies that show toxic effects of ZA on periodontal ligament 35,36 .
We also evaluated, in the present work, the effects of ZA on the tooth root. This analysis was motivated by previous studies that demonstrate root resorption in experimental models of orthodontic movement. Dentin and cementum are two mineralized tissues, both with a composition similar to bone tissue, with a predominance of type I collagen. Like bone tissue, dentin and cement are degraded by clastic cells, called odontoclasts. However, the odontoclast is, morphologically and functionally, very similar to the osteoclast 28 . OTM resulted in significant root resorption and ZA prevented this effect. The inhibitory effects of ZA on osteoclasts formation and activation may explain the lower root resorption, observed in the group treated with the highest concentration of this drug. In the presente study, optical microscopy and scanning electron microscopy analyses cleary show a reduction in the number and depth of root resorption gaps in the ZA group, when compared to saline. Root resorption is an undesirable effect of orthodontic treatment, usually associated with forces of high magnitude, which promote a greater number of hyalinic (necrotic) areas and, consequently, greater formation and activation of clastic cells 17,40 . In the present study, a force of 50gf was used, based on previous studies 9,11 , which corresponds to a force that exceeds the intensity of force necessary to move a tooth with minimal tissue damage (optimal strength). In experimental tooth movement, the use of a force that exceeds the optimum force allows the occurrence of all tissue events involved in orthodontic movement in a viable time frame for experimental studies.

Conclusion
In summary, our results contribute to a better understanding of the cellular effects of ZA and its repercussion on root, periodontal ligament and alveolar bone tissues, which is crucial for the indication of orthodontic treatment to patients who use high doses of this drug. Although in the present study the antiangiogenic effect was associated with the treatment of ZA, the bone structure was preserved with no signs of osteonecrosis.