Introduction
This study was performed to evaluate the effect of isotretinoin on tooth displacement and tissues related to induced tooth movement (ITM) in rats.
Methods
Wistar rats were randomly divided into 4 groups: vegetable oil (O; n = 40), 7.5 mg/kg isotretinoin (I; n = 40), vegetable oil + ITM (OM; n = 44), and 7.5 mg/kg isotretinoin and ITM (IM; n = 39). After the daily application of the solutions for 30 days, an orthodontic appliance was installed to mesially displace the maxillary first right molar (30 cN) of rats in the OM and IM groups. The animals were killed 2, 7, 14, or 21 days after placement of the devices. The animals in the O and I groups did not undergo ITM but were killed simultaneously. The animals were examined for tooth displacement, the neoformation of mature collagen, bone and root resorption, the presence of hyalinized areas, and trabecular bone modeling by microcomputed tomography.
Results
There was no difference in tooth displacement, the number of osteoclasts, the presence of hyalinized areas, or trabecular bone among the O, I, OM, and IM groups across the periods tested ( P >0.05). A lower percentage of mature collagen was found in the IM group than in the OM group on day 7 ( P <0.05). A lower frequency of root resorption was found in the IM group than in the OM group on days 2 and 21 ( P <0.05).
Conclusions
Isotretinoin at 7.5 mg/kg decreased root resorption in rats subjected to ITM.
Highlights
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We evaluated the effect of isotretinoin (7.5 mg/kg) on tooth movement in rats.
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Isotretinoin did not affect tooth displacement and collagen neoformation.
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Isotretinoin did not affect bone resorption and the presence of hyaline areas.
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Isotretinoin did not affect bone microarchitecture.
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Isotretinoin (7.5 mg/kg) decreased root resorption in rats subjected to tooth movement.
Acne is the most common skin disease among adolescents, with a prevalence of 80%-90%. Isotretinoin, or 13-cis-retinoic acid, is the drug of choice for the treatment of this disease, and its use among adolescents and young adults is considerable. Isotretinoin is derived from vitamin A, and studies have shown that vitamin A antagonizes the actions of vitamin D , because both require the same binding proteins, the retinoid X receptors, to carry out their transcription activity. Consequently, high doses of vitamin A may cause side effects on bone. ,
Induced tooth movement (ITM) is caused by biomechanical forces on the teeth. This action generates a complex biological process characterized by sequential reactions in 2 regions of the periodontal tissue, the areas of compression and tension. Tissue remodeling is induced by activation of alveolar bone resorption in the compression area and the resulting bone apposition in the tension area. , On entering the systemic circulation, several drugs can alter the biological reactions related to ITM.
Several side effects of isotretinoin on bone tissue have been reported, including reduced bone mineral density, , , the calcification of tendons and ligaments, increased bone resorption, increased number and size of osteoclasts, and changes in the differentiation of osteoblasts. , Two studies found no differences in bone mineral density in patients treated with isotretinoin at 1 mg/kg/d , ; thus, the treatment time and dose of isotretinoin seem to influence its effects on bone tissue. , , Other authors found the acceleration of the alveolar bone repair process after tooth extraction in rats. ,
Although the use of isotretinoin for ITM has been previously studied, its effects on root resorption and hyalinized areas have not been evaluated. Evidence shows that the presence of isotretinoin may alter bone metabolism; however, definitive studies of its effects in ITM are lacking. Thus, the present study evaluated tooth displacement, collagen neoformation, bone and root resorption, the presence of hyalinized areas, and bone microarchitecture in rats exposed to ITM and isotretinoin. No studies that evaluated the aspects considered in the present study were found in the literature.
Because of the changes in bone tissue caused by isotretinoin and considering the widespread use of this drug among adolescents and young adults, who represent most of the patients who undergo orthodontic treatment, it is necessary to investigate the possibility of isotretinoin-mediated effects on ITM.
Material and methods
A total of 163 male Wistar rats ( Rattus norvegicus albinus ) aged 6-8 weeks and weighing approximately 200 g were used. The rats were weighed with a precision electronic scale (BG 4001; Gehaka, São Paulo, Brazil) on the first day of the experiment and weekly until they were killed for drug dose adjustment. The animals were randomly divided into 4 groups: vegetable oil (O; n = 40), isotretinoin (I; n = 40), vegetable oil + ITM (OM; n = 44), and isotretinoin and ITM (IM; n = 39). Animals in groups O and OM received 0.5 mL of vegetable oil daily by gavage; vegetable oil was used as a control because of the liposoluble properties of isotretinoin. Rats in the I and IM groups received 7.5 mg/kg isotretinoin daily by gavage. At the time of isotretinoin administration, the powdered drug was diluted in vegetable oil according to the weight of the animal. The dose and route of isotretinoin administration chosen were equivalent to those used for its clinical use in humans. , ,
After 30 days of daily vegetable oil or isotretinoin administration, each rat in the OM and IM groups received an orthodontic appliance to induce ITM, as used by Ribeiro et al. The appliance consisted of a closed nickel-titanium coil spring (G&H Wire, Franklin, Ind) fixed to the maxillary first right molar with a 0.010-in stainless steel ligature wire (Morelli, São Paulo, Brazil) and was connected to the maxillary central incisors, positioned in the most cervical region of the teeth, causing mesial movement of the first molar. The reciprocal force produced by the closed spring was 30 cN and standardized using a tensiometer (Haag-Streit, Köniz, Switzerland) ( Fig 1 ). After the installation, the spring was not reactivated; however, its position was checked daily.
The administration of vegetable oil or isotretinoin continued until the animals were killed by intraperitoneal injection of sodium pentobarbital (100 mg/kg, Thiopentax; Cotia, Brazil) on days 32, 37, 44, or 51, corresponding to 2, 7, 14, and 21 days after placement of the orthodontic appliance, respectively. The number of animals was chosen according to the release standards of the Ethics Committee on Animal Use and based on previously published studies. , Even in the absence of the sample calculation, the power of the test revealed that the sample size was sufficient. Five animals were lost during the experiment because of gavage, as the animals aspirated the medicine during the procedure. The animals were restored, with 40 animals remaining in the O and I groups (10 animals per subgroup according to the evaluation time), 44 animals in the OM group, and 39 animals in the IM group. Therefore, 10 animals were killed in each subgroup per test period, except on day 21 in the OM (n = 14) and IM (n = 9) subgroups. The animals in the O and I groups, which did not undergo ITM, were killed simultaneously.
Tooth displacement in animals in the OM and IM groups was measured in millimeters with a digital caliper (Absolute; Mitutoyo, Kawasaki, Japan), according to Frigotto el al. The displacement of the tooth corresponded to the initial measurement (measured before placement of the orthodontic device) with the final measurement (measured after the animals were killed) subtracted.
The right hemimaxilla of each animal was dissected and decalcified. The specimens were processed and embedded in paraffin, and adjacent 3-μm-thick cross-sections (for picrosirius red staining, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining) of the mesiobuccal root were cut starting from the cervical third in the apical direction with intervals of 60 μm between each section. The procedure was repeated 5 times in each specimen; therefore, 5 sections were obtained from each animal at each time point.
The neoformation of collagen was determined by polarization microscopy using picrosirius red-stained sections. The analysis was performed by a single examiner (A.X.G.P.). The bone adjacent to the periodontal ligament (PDL) in the distal portion of the mesiobuccal root was selected for analysis during ITM because bone tissue is deposited in the alveolar cortex in the traction area and because this strategy allowed observation of the stretching of the fibers of the PDL. The percentage of mature and immature collagen was quantified on the basis of color differences. Mature collagen is known as “type I collagen,” and immature collagen is known as “type III collagen.” The average percentages of each type of collagen were calculated on the basis of the 5 sections. The methodology was based on a study by Araujo et al.
Histochemical tartrate-resistant acid phosphatase staining was performed with a TRAP 387 kit (Sigma-Aldrich, St Louis, Mo) to identify osteoclasts and cementoclasts and thus determine resorption of bone and cementum, respectively. From each of the 5 sections, images of the entire PDL of the mesiobuccal root of the first molar were captured at magnifications of 40× and 400×. For images captured at 400×, the number of osteoclasts was determined by a single examiner (A.X.G.P.) in 2 periods, with a minimum interval of 3 weeks between examinations. The reliability of the examiner was assessed using an intraexaminer test. For images captured at 40×, the PDL area was measured. To obtain the number of osteoclasts per μm 2 of the PDL, the number of osteoclasts was divided by the area of the PDL. Areas of root resorption in the images magnified at 400× were noted. The average percentage of the number of osteoclasts and cementoclasts per μm 2 of the PDL was calculated on the basis of 5 sections.
The presence of hyalinized areas throughout the PDL was evaluated with the hematoxylin and eosin staining method at a magnification of 40×. Hyalinized areas were measured using Image-Pro Plus software (version 4.5; Media Cybernetics, Rockville, Md) and then quantified as the percentage of hyalinized area per μm 2 of PDL.
The distal epiphysis of the femur was analyzed using the method described by Frigotto et al to determine whether oral administration of isotretinoin had a systemic effect with equal impact on all bones, a computerized microtomograph (SkyScan 1172; Bruker, Kontich, Belgium, 2010) was used. Twelve slices from the central area of the distal epiphysis of each femur were imaged at a resolution of 12.8 μm/pixel. The raw data were then reconstructed using the software NRecon (version 1.6.9; Bruker). A volume of interest was selected for analysis and digitized. The 3-dimensional analysis was performed using CT-Analyzer software (version 1.14.4; Bruker), and the following morphometric indexes were calculated: trabecular volume, trabecular thickness, trabecular separation, and trabecular number. , Because of the impossibility of carrying out microtomography with all the study samples, samples collected on days 7 and 21—corresponding to the most significant tissue and bone changes during ITM and the final ITM phase, respectively—were selected. ,
Statistical analysis
Statistical analysis was performed using SPSS (version 23.0; IBM, Armonk, NY) and Statistica (version 7; Stat Soft, Tulsa, Okla). Data normality and homogeneity were tested using the Kolmogorov-Smirnov and Shapiro-Wilk tests and the Levene test, respectively.
Comparisons between groups and time points were performed using a 2-way analysis of variance with a full factorial design. When the results of the analysis of variance indicated a difference, the treatments were compared 2 × 2 with the Games-Howell parametric test for heterogeneous variances. When the tests did not show a normal distribution, the Mann-Whitney and Kruskal-Wallis tests were used, followed by Dunn multiple nonparametric comparison test. For variables with dichotomous categorical scales, analyses of independence were performed using the chi-square test. When there was a dependence between the variables, the Z-test for the difference between 2 proportions was applied. The significance level adopted for all tests was 0.05.
A power test was used to calculate the power on the basis of the sample size to confirm differences between the mean values of dependent variables according to the factors of group and time.
Results
The intraexaminer test evaluated the reproducibility of osteoclast and cementoclast counts. Dahlberg maximum error was 0.6947%, less than 1%, and the reliability coefficient was greater than 99%. Student t test showed no significant difference in the scores ( P >0.05).
No significant difference was found in tooth displacement between the OM and IM groups ( P >0.05) ( Table I ). There was a significantly lower percentage of mature collagen in the I group than in the O group on day 21 ( P <0.05). There was a significantly lower percentage of mature collagen in the IM group than in the OM group on day 7 ( P <0.05). No significant difference was found in the number of osteoclasts between the groups ( P >0.05) ( Fig 2 ; Table II ).
| Variable, d | Groups | P value ∗ | Power test | |
|---|---|---|---|---|
| OM | IM | |||
| Tooth displacement (mm) | ||||
| 2 | 0.84 ± 0.56 | 1.11 ± 0.67 | 0.0586 | 0.7572 |
| 7 | 1.58 ± 0.58 | 0.90 ± 0.44 | ||
| 14 | 1.48 ± 0.63 | 1.28 ± 0.76 | ||
| 21 | 0.93 ± 0.67 | 1.05 ± 0.56 | ||
There was a significantly lower number of cementoclasts in the IM group than in the OM group on days 2 and 21 ( P <0.05). On day 7, the number of cementoclasts was higher in the IM group than in the OM group ( P <0.05) ( Table III ; Fig 3 ).
| Variable, d | Groups | Games-Howell test | Power test | ||||
|---|---|---|---|---|---|---|---|
| O × I | OM × IM | ||||||
| O | I | OM | IM | P value ∗ | P value ∗ | ||
| Mature collagen (%) | |||||||
| 2 | 95.93 ± 3.29 | 91.33 ± 6.63 | 94.84 ± 4.59 | 89.29 ± 10.49 | 0.8420 | 0.9556 | 0.9934 |
| 7 | 97.02 ± 2.61 | 93.38 ± 5.48 | 96.74 ± 1.13 | 90.35 ± 6.72 | 0.8382 | 0.0047 ∗ | |
| 14 | 94.77 ± 2.35 | 91.83 ± 6.75 | 96.25 ± 2.22 | 96.82 ± 2.52 | 0.9866 | 0.9999 | |
| 21 | 97.74 ± 1.20 | 92.01 ± 4.84 | 96.39 ± 3.57 | 94.17 ± 5.93 | 0.0111 ∗ | 0.9984 | |
| Osteoclasts per μm 2 of PDL (n) | |||||||
| 2 | 7.81 ± 1.96 | 8.18 ± 5.99 | 19.99 ± 3.77 | 19.60 ± 5.96 | 0.9999 | 0.9999 | 0.9988 |
| 7 | 6.52 ± 2.69 | 5.22 ± 1.83 | 11.49 ± 5.63 | 13.60 ± 3.98 | 0.9919 | 0.9995 | |
| 14 | 7.77 ± 2.35 | 8.36 ± 3.64 | 9.26 ± 4.94 | 9.89 ± 5.40 | 0.9999 | 0.9999 | |
| 21 | 6.29 ± 2.43 | 5.73 ± 1.61 | 8.34 ± 4.44 | 7.47 ± 2.61 | 0.9999 | 0.9999 | |
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