Influence of isotretinoin on orthodontic tooth movement in rats: microtomographic and histomorphometric analysis

doi:10.21203/rs.3.rs-3111108/v1

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Influence of isotretinoin on orthodontic tooth movement in rats: microtomographic and histomorphometric analysis

https://doi.org/10.21203/rs.3.rs-3111108/v1

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published 16 May, 2024

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Objective

Isotretinoin, also known as 13-cis-retinoic acid, is an isomer of tretinoin, the oxidized form of Vitamin A. Orthodontic tooth movement (OTM) is the result of a cascade of inflammatory responses stimulated by a physical element that is the force generated by orthodontic appliances. Isotretinoin is mainly used among adolescents and young adults, and coincidentally it is this age group that also undergoes orthodontic treatment.

Materials and Methods

Thirty-five animals were used, and they were randomly divided into 7 groups, containing 5 animals in each group. Group 1: Control; Group 2: OTM for 7 days; Group 3: OTM for 14 days; Group 4: Treated with isotretinoin for 14 days; Group 5: Treated with isotretinoin for 21 days; Group 6: Treated with isotretinoin for 14 days and undergoing OTM for 7 days; Group 7: Treated with isotretinoin for 21 days and undergoing OTM for 14 days. In Groups 6 and 7, the animals were treated with isotretinoin at a dosage of 1.0mg/kg/day for 7 days before OTM and maintained during the movement period in the respective groups.

Results

There was no significant difference on microtomographic parameters [Trabecular Volume (BV/TV), Trabecular Thickness (Tb.Th), Number of Trabeculae (Tb.N) and Trabecular Separation (Tb.Sp)] and tooth displacement between groups.

Conclusion

Isotretinoin did not cause a reduction in tooth displacement during OTM when administered at a dose of 1.0mg/kg/day and isotretinoin did not change the microtomographic parameters of animals treated with isotretinoin + OTM.

isotretinoin

drug response

bone remodeling

orthodontic tooth movement

acne

retinoids

Isotretinoin, also known as 13-cis-retinoic acid, is an isomer of Tretinoin, the oxidized form of Vitamin A. It was synthesized for therapeutic use in the treatment of severe inflammatory acne and for cases of acne that are resistance to antibiotics or topical drugs [1].

Acne is a common condition that affects a significant percentage of teenagers and young adults in the Western world, with estimates ranging from 79–95% [1–3]. Isotretinoin is believed to act on the sebaceous gland by binding to specific retinoid receptors and modifying gene transcription [1]. It reduces the activity and size of the sebaceous gland, thereby decreasing sebum production [1]. Some studies have shown that after 8 weeks of isotretinoin treatment in humans, there is a decrease in the genes involved in steroids, cholesterol, and fatty acids metabolism, as well as an increase in genes that encode structural proteins like collagen and fibronectin [4, 5]. Additionally, retinoids, including isotretinoin, normalize sebaceous follicle keratinization and the population of Propionibacterium acnes [6].

Animal studies have demonstrated that an overdose of the active form of Vitamin D, Calcitriol, increases the orthodontic tooth movement (OTM) displacement [7, 8]. Isotretinoin affects Vitamin D metabolism and antagonizes its role in calcium homeostasis [9]. In rats, an overdose of Vitamin A can lead to accelerated bone resorption due to increased osteoclast activity. Hypercalcemia induced by Vitamin A may cause the removal of calcium ions from bones, increasing their concentration in the bloodstream [10, 11]. A study on the association of isotretinoin and OTM in animals observed that tooth extraction facilitated OTM without interfering with the quality of bone remodeling [12]. In a literature review with clinical studies, it was found that, apart from its teratogenic effects, the adverse effects of isotretinoin are generally mucocutaneous, such as dryness of the lips, eyes and skin. These effects are dose-dependent and reversible upon discontinuation of the drug [13, 14]. However, there is a lack of literature regarding reports of intraoral alterations in patients during isotretinoin treatment.

OTM occurs as a result of a cascade of inflammatory responses stimulated by the force generated by orthodontic appliances. It involves remodeling of the alveolar bone, changes in the periodontal ligament, and sometimes in the root cementum [12]. Specific factors, such as drug use and changes in metabolism, can influence the cellular events involved in OTM, leading to changes in the quality and quantity of tooth displacement [12]. The influence of drug use on the amount of tooth displacement and biological responses has been explored by several authors [15, 16].

The interrelation between isotretinoin and tooth movement has been the subject of previous studies, but the results remain inconclusive, and there are still unresolved issues [12, 17]. Isotretinoin is primarily used among teenagers and young adults, coincidentally the age group that also often undergoes orthodontic treatment [18–21]. Considering that there are individuals undergoing orthodontic treatment while also using isotretinoin, and that there is limited information on the effects of this combination in the literature, we are proposing this study to evaluate the influence of isotretinoin on OTM.

Animals and experimental conditions

The project was approved by the Ethics Committee on the Use of Animals (CEUA) under protocol number: 00462–2020. All experiments were conducted in compliance with the Ethical Principles for Animal Experimentation [22]. The experiment involved the use of 35 male rats (Rattus norvergicus albinus, Wistar) at the age of 75 days, weighing between 300g − 350g at the beginning of the experiments. The rats were housed in a controlled environment with a temperature of 25 ± 2 ºC, a 12-hour light/12-hour dark cycle, and provided with food (Purina®, Paulínia-SP, Brazil) and water ad libitum throughout the experiment.

Experimental delineation

The sample calculation for the experiment was performed based on a previous study, using SigmaPlot 14.0 software [38]. The following test details were used: a significance level (α) = 0.05, test power (1- β) = 0.80, and dropout (β) = 0.2. Based on these parameters, a minimum sample size of 5 rats per group was determined.

A total of thirty-five animals were used and randomly assigned to 7 groups, with each group consisting of 5 animals. The composition of the groups and the procedures performed will be described in detail below:

Group 1 - CON: Not subjected to any kind of treatment
Group 2 - OTM-7: Undergoing orthodontic tooth movement for 7 days
Group 3 - OTM-14: Undergoing orthodontic tooth movement for 14 days
Group 4 - ISO-14: Treated with isotretinoin for 14 days
Group 5 - ISO21: Treated with isotretinoin for 21 days
Group 6 - OTM7-ISO14: Treated with isotretinoin for 14 days and undergoing orthodontic tooth movement for 7 days
Group 7 - OTM14-ISO21: Treated with isotretinoin for 21 days and undergoing orthodontic tooth movement for 14 days

In Groups 4, 5, 6 and 7, the animals were treated with isotretinoin at a dosage of 1.0mg/kg/day for 7 days prior to OTM, and the treatment was continued throughout the movement period in their respective groups. The weight of all animals was measured daily using a precision electronic scale (Bel 0.01g, 2200g S2202H, Bel Engineering, Piracicaba, São Paulo, Brazil) until they were euthanized.

Application of isotretinoin

Isotretinoin was obtained in the form of a 20mg Roaccutane® capsule (Roaccutane, Roche, Basel, Switzerland) and stored at room temperature, between 59 to 86°F, in its original packaging, protected from light and humidity. Each solution was prepared in the morning, in a dark room, immediately before administration, due to the high sensitivity of Vitamin A to light [23]. The capsule contents were dissolved in soybean oil to facilitate gavage administration. Soybean oil was chosen as it is a component of Roaccutane®. The solution was prepared in an amber bottle, taking into account the animal's weight and the desired concentration. To prepare the solution, a 20mg Roaccutane® capsule was pierced using a hypodermic needle (30x0.70mm, Descarpack 22G, Santa Catarina, Brazil), and its contents were added to the vial. The recommended daily dose for humans, based on the literature and patient weight, ranges from 0.5 to 2mg/kg/day [24]. When adjusting the dosage to the equivalent between animals and humans, a dosage of 1.0mg/kg/day was chosen, which closely resembles the recommended human dosage [25, 26]. The vial containing the solution was shaken to ensure proper dilution, and the solution was administered to the animals via oral gavage using a suitable short needle for rats (Bonther, Reference: Steel INOX BD-12, Ribeirão Preto, São Paulo). The dosage was adjusted according to each animal's body weight, which was measured daily until the time of euthanasia.

Orthodontic Tooth Movement (OTM) protocol

For the installation of the mechanical appliance, the muscle relaxant Xylazine Hydrochloride (DOPASER, Caleir S.A., Barcelona, Spain) was administered at a proportion of 0.03 ml/100g of body weight, and the anesthetic Ketamine Hydrochloride (VETASET, Fort Dodge Animal Health, Iowa, USA) was administered at a proportion of 0.07 ml/100g of body weight. Both drugs were administered intramuscularly. In this research, a mechanical system was used based on the method adapted by Cuoghi and colleagues [27]. The appliance consisted of a nickel-titanium spring (Sentalloy, GAC, USA) with an effective length of 4mm and a constant force of 50cN. The force magnitude and the amount of spring stretch were predetermined by the manufacturer. To adapt the springs to the molars and incisors, 0.2 mm diameter wires (Morelli, Sorocaba, SP, Brazil) were utilized. After installing the spring, resin (Z100™ − 3M, Minnesota, USA) was applied to the incisors to enhance retention. Orthodontic tooth movement (OTM) was performed on the upper right first molar, with the upper right incisor serving as anchorage (Fig. 1).

Euthanasia and specimen preparation

After the experimental periods of each group, the animals were euthanized via transcardiac perfusion. This involved the injection of 100 mL of physiological saline solution containing 0.1% heparin, followed by 800 mL of 4% formaldehyde in saline phosphate buffer (PBS) at a temperature of 4ºC and pH 7.4. The purpose of this perfusion was to preserve the tissues for subsequent analysis. The right hemimaxillas (half of the upper jaw) were then dissected and subjected to fixation for 12 hours. After fixation, they were washed for 12 hours in running water to remove excess chemicals and prepare them for three-dimensional (3D) imaging. The 3D images were obtained using a Skyscan 1272 scanner (Bruker, Billerica, Massachusetts, USA). High-resolution images were captured at 70 KvP (142 µA) with a field of view (FOV) of 20.5 mm. Once the images were acquired, the specimens were returned to the fixative solution and kept there for an additional 72 hours. Next, the samples were demineralized in a 10% ethylenediaminetetraacetic acid (EDTA) solution (Sigma Chemical®) in PBS for a period of 60 days. This process helps to remove mineral content from the tissues. After demineralization, the samples underwent conventional histological processing, including embedding in paraffin and subsequent microtomy to obtain 5 µm thick sections. Finally, the histological sections were stained with hematoxylin-eosin (HE), a common staining method used in histology to visualize cellular structures and tissue architecture.

Tooth displacement analysis

The 3D image reconstructions were generated using the NRecon Reconstruction Software from Micro Photonics Inc. (Allentown, Pennsylvania). The specific region of interest analyzed was the space between the distal surface of the first molar and the mesial surface of the second molar. The 3D image was obtained with the aim of capturing a sagittal view that allows for the observation of the entire mesiobuccal and distobuccal roots of the first molar (including the cervical, middle, and apical thirds), the pulp, the curvature of the furcation region, and the integrity of the mesial alveolar bone crest. This was achieved to assess the relevant structures and changes in the area (Fig. 2C). To facilitate image orientation and analysis, specific lines were adjusted in the Skyscan Dataviewer Software (Bruker, Billerica, Massachusetts, USA). These lines included the axial line (blue), sagittal line (green), and coronal line (red). In the coronal view, the blue line passed through the long axis of the first molar in the mesiodistal direction (Fig. 2A). In the axial view, the image was appropriately angled (Fig. 2B) to allow for a clear sagittal view (Fig. 2C) that encompassed the mesiobuccal and distobuccal roots of the first molar in their entirety. In the sagittal view, a dashed yellow line was positioned at the tip of the molar cusps, serving as a reference for locating the most convex point on the enamel of the first molar, indicated by the green line. The space between the first molar and the second molar at its greatest convexity on the distal and mesial surface, as determined by the green line, was used for the linear measurement of tooth displacement. This measurement was performed using the Image J Software (National Institute of Health, NIH, U.S).

Analysis of bone morphology

To assess the bone morphology in the region between the roots of the first molar, coronal view images were reconstructed for all groups (Fig. 3). In the coronal plane, the axial line was aligned with the long axis of the maxillary first molar in the mesiodistal direction (Fig. 3A). For bone density analysis, the 3D images were imported into the CT-Analyser Software (CTAn version 1.13, Bruker MicroCT, Kontich, Belgium). To define the area analyzed for bone architecture, the image of the first molar was evaluated in the occlusal-apical direction. The most cervical level analyzed corresponded to the floor of the pulp chamber (Fig. 3B-C), determined by the green line. The most apical level analyzed was close to the middle third of the mesiobuccal root, ensuring that it did not exceed the middle third of the root. A total of 80 image slices were analyzed for each sample. Using the CTAn Software, several parameters related to bone structure were calculated and compared between groups. These parameters included bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). These measurements provided quantitative information on the bone density and architecture in the analyzed region.

Periodontal ligament and alveolar bone analysis

The analysis of the periodontal ligament architecture involved the examination of various areas and histological characteristics. These areas included the interradicular septum (the space between the roots), as well as the mesiobuccal and distobuccal roots. For the mesiobuccal root, the analysis covered the mesial and distal surfaces, as well as the cervical, middle, and apical thirds. Similarly, for the distobuccal root, the mesial and distal surfaces, as well as the cervical, middle, and apical thirds, were examined. In each of these areas of the periodontal ligament, several histological features were assessed. These included the presence of hemorrhage (bleeding), inflammatory infiltrate (immune cells present in the tissue), and the arrangement of collagen fiber bundles. These parameters provided insights into the condition and health of the periodontal ligament in the different areas analyzed. Regarding the architecture of the alveolar bone, the analysis focused on the characteristics of the bone located between the mesiobuccal and distobuccal roots. The criteria used for analysis included assessing areas of bone resorption (loss of bone tissue), areas of new bone formation, and overall bone quantity. These assessments helped to understand the changes in alveolar bone structure and remodeling that occurred during the experimental period.

Statistical analysis

In order to assess the error of the method used to measure tooth displacement, a comparison was made between ten images in the sagittal microtomographic norm and their respective measurements performed by different evaluators. These evaluations were conducted with a 20-day interval between them. The average measurements from the two evaluations were compared using the paired t-test, with a significance level of 5%. The results of this analysis showed no significant differences between the different measurement moments, indicating a low error in the measurement method.

For the statistical analysis of the data, the GraphPad Prism Software (Version 9.5, Boston, MA) was used. The normality and homogeneity of the data were assessed using the Shapiro-Wilk test. Comparisons between groups and different time points were performed using the Kruskal-Wallis test, followed by Dunn's non-parametric multiple comparison test. The significance level for all statistical tests was set at 5%. These analyses helped to determine any significant differences between the groups and time points being compared, providing valuable insights into the experimental findings.

Effects of isotretinoin on bone morphology

Based on the results of the microtomographic parameters (Trabecular Volume - BV/TV, Trabecular Thickness - Tb.Th, Trabecular Number - Tb.N, and Trabecular Separation - Tb.Sp) obtained from groups 1, 4, and 5, the statistical analysis showed no significant differences between these groups (P > 0.05). This indicates that the administration of isotretinoin did not have a significant impact on bone morphology under the specific experimental conditions of this study. The results suggest that the dosage and duration of isotretinoin used in the experiment did not lead to noticeable changes in the microtomographic parameters measured in the study groups (Table 1; Chart 1).

Effects of tooth movement on bone morphology

Based on the results of the microtomographic parameters (Trabecular Volume - BV/TV, Trabecular Thickness - Tb.Th, Trabecular Number - Tb.N, and Trabecular Separation - Tb.Sp) obtained from groups 1, 2, and 3, the statistical analysis revealed a significant difference in Tb.Th between group 1 and group 3 (P = 0.0024). Specifically, group 1 exhibited a decrease in Tb.Th compared to group 3. This suggests that the OTM performed for 14 days had an effect on bone morphology under the specific experimental conditions of this study. (Table 1; Chart 1).

Effects of tooth movement and isotretinoin on bone morphology

Based on the results of the microtomographic parameters (Trabecular Volume - BV/TV, Trabecular Thickness - Tb.Th, Trabecular Number - Tb.N, and Trabecular Separation - Tb.Sp) obtained from groups 1, 6, and 7, the statistical analysis revealed the following comparisons: Group 6 had a significant increase in BV/TV compared to group 1 (P = 0.0426). Group 1 had a significant increase in Tb.Sp compared to group 6 (P = 0.0250). Based on these findings, it can be concluded that the combination of OTM and isotretinoin treatment did not cause significant changes in bone morphology under the specific experimental conditions of this study (Table 1; Chart 1).

Table 1

Bone Volume Fraction (BV/TV), Trabecular Thickness (Tb.Th), Trabecular Number (Tb.N) e Trabecular Separation (Tb.Sp) in all groups.
	GROUPS
	CON	OTM-7	OTM-14	ISO14	ISO21	OTM7-ISO14	OTM14-ISO21
BV/TV (%)	15.83 ± 0.6843	22.35 ± 2.095	22.66 ± 2.415	16.21 ± 0.8499	18.15 ± 3.458	22.39 ± 0.7087	22.12 ± 1.990
Tb.Th (mm)	2.291 ± 0.05735	2.549 ± 0.1280	2.721 ± 0.1446	2.434 ± 0.1774	2.930 ± 1.098	2.453 ± 0.04103	2.523 ± 0.08242
Tb.N (1/mm)	0.06834 ± 0.0050	0.08754 ± 0.004	0.08316 ± 0.006	0.06990 ± 0.0063	0.06116 ± 0.0083	0.08912 ± 0.003	0.008762 ± 0.006
Tb.Sp (mm)	13.77 ± 0.5575	10.23 ± 0.7573	10.31 ± 1.043	13.02 ± 0.9189	14.58 ± 0.9793	9.458 ± 0.2686	9.844 ± 0.8079

Effects of isotreinoin on the amount of tooth displacement

Based on the results obtained from groups 2, 3, 6, and 7, the statistical analysis revealed no significant difference between these groups (P > 0.05) regarding the effects of isotretinoin on OTM. This indicates that isotretinoin, administered at a dose of 1.0mg/kg/day, did not influence tooth displacement, regardless of the duration of movement (7 and 14 days) under the conditions of this experiment (Table 2; Chart 2).

Table 2

Amount of Tooth Displacement in Groups 2, 3, 6 and 7, respectively.
	GROUPS
	OTMI7	OTM14	OTM7-ISO14	OTM14-ISO21
µm	53.86 ± 8.294	106.0 ± 26.74	76.94 ± 15.13	62.02 ± 20.10

Quantitative and qualitative analysis of the effects of isotretinoin on the periodontal ligament

In groups 1, 4, and 5, where OTM was not performed, the histological characteristics of the periodontal tissues were similar. Cementum was present in all specimens, with little or no focus of resorption. The cementum was covered by cementoblasts and fibroblasts, with the incorporation of Sharpey's fibers into the cement matrix. In cellular cementum, gaps containing cementocytes were observed. The periodontal ligament consisted of a fibrous connective tissue with thick bundles of collagen fibers, forming the main fibers of the periodontal ligament. Among these fibers, a network of finer collagen fibers, known as secondary fibers, was present. Fibroblasts and blood vessels were abundant in the periodontal ligament, while inflammatory cells were rare. The bone tissue showed the presence of osteoblasts or bone lining cells on its surface, Sharpey's fibers incorporated into the bone matrix, rare active osteoclasts, and numerous osteocytes within the matrix (Figs. 4 and 5).

In groups 2 and 6, an area of stretching of the periodontal ligament fibers was observed on the distal surface of the distobuccal root. On the mesial face of the distobuccal root, areas of hyalinization of the periodontal ligament and the presence of a few macrophage cells were observed, with more extensive hyalinization observed in the groups treated with isotretinoin. A slight stretching of the periodontal ligament fibers was observed on the distal surface of the mesiobuccal root, while on the mesial face of the mesiobuccal root, a slight compression of the periodontal ligament fibers and the presence of active clastic cells were observed. In groups 3 and 7, the distal face of the distobuccal root showed a slight stretching of the periodontal ligament fibers. On the mesial face of the distobuccal root in group 3, a small area of hyalinization was observed, along with reabsorption of cementum, macrophage activity, and the presence of clastic cells in the alveolar bone tissue. In group 7, an extensive area of hyalinization was observed, similar to that seen in group 6. In groups 2, 3, 6, and 7, intact cementum, periodontal ligament restructuring, and the presence of clastic cells in the alveolar bone tissue, especially on the mesial surface, were observed on the distal and mesial faces of the mesiobuccal root. These histological findings provide insights into the effects of orthodontic tooth movement and isotretinoin treatment on the periodontal tissues, including changes in the periodontal ligament and bone (Figs. 4 and 5).

In this study, isotretinoin was administered at a dosage of 1.0mg/kg/day for either 14 or 21 days. The dosage used in the study was determined based on equivalent dosage criteria between animals and humans [24–26]. The recommended daily dose of isotretinoin for acne treatment in humans, as stated in the literature and guidelines, is calculated based on the patient's weight and ranges from 0.5 to 2mg/kg/day for up to 24 weeks [24–26, 28, 29]. Considered that the side effects of isotretinoin are dose-duration dependent, meaning that higher doses used for longer periods can lead to more significant side effects [26, 28, 29], in this study, we aimed to evaluate the effects of low doses of isotretinoin, which correspond to the dosage commonly used in humans, on bone morphology in animals. The results of the study indicated that low doses of isotretinoin, did not significantly alter bone morphology in the treated animals. This finding is consistent with the findings of Graciano Parra et al., who evaluated the systemic effects of isotretinoin on the femurs of animals and found no relevant effects on bone morphology compared to animals treated only with soy oil [17]. Similarly, Nishio et al. did not observe significant changes in bone morphology in the tibia of their animal model with isotretinoin treatment [12]. However, it is worth noting that Lind et al. showed a significant reduction in bone density in the calvaria of rats with an overdose of Vitamin A, which is chemically related to isotretinoin [30]. Additionally, Johansson & Melhus suggested that isotretinoin may indirectly cause a decrease in bone density by interfering with the actions of vitamin D and directly causing bone resorption [31]. Given these conflicting results, further studies are needed to thoroughly investigate the effects of isotretinoin on bone morphology under the same experimental conditions. It is important to consider the potential risks and side effects associated with isotretinoin treatment and to monitor bone health in patients receiving long-term and high-dose therapy.

In this study, we utilized NiTi helical springs with a constant force of 50cN to perform OTM. The results of the study demonstrated a significant difference in microtomographic parameters when comparing the animals undergoing OTM for 7 and 14 days with the control group. Specifically, there was a significant increase in bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), accompanied by a significant decrease in trabecular separation (Tb.sp). Previous research has indicated that different magnitudes of force, ranging from 3 to 100cN, can produce similar tooth movements [32], knowing that, in a study by Gudhimella et al., using a constant force of 3cN, a decrease in BV/TV was observed during the first 7 days and after 28 days of OTM when compared to a group without OTM [33]. Another study by Lu et al., employing a constant force of 50cN, revealed a significant decrease in BV/TV in the furcation area of the first molar when comparing a group treated with sclerostin to a control group [34]. Although there is a lack of studies analyzing microtomographic parameters under the exact experimental conditions described in this study (50cN force), it is crucial to conduct further research where a force of 50cN is exerted and the microtomographic parameters are evaluated. This will allow for a comparison of the results with animals that have not undergone OTM, providing a deeper understanding of the effects of this specific force magnitude on bone morphology during orthodontic tooth movement.

The investigation of the effects of both OTM and isotretinoin on bone morphology showed no significant impact on the microtomographic parameters in groups 8 and 9. The only existing study found in the literature that examined OTM with isotretinoin and analyzed the same microtomographic parameters (BV/TV, Tb.Th, Tb.N, and Tb.Sp) also reported no significant difference in bone morphology [17]. However, it is important to note that this particular study analyzed the parameters in the distal epiphysis of the femur, rather than between the roots of the maxillary first molar, which was the focus of our study. To gain a better understanding of the effects of OTM and isotretinoin on bone morphology in the specific area between the roots of the moved tooth, further research is necessary. Conducting studies under the same experimental conditions, with a focus on analyzing the microtomographic parameters in the desired area, will provide valuable insights into the potential effects of these interventions on bone morphology. This will contribute to a more comprehensive understanding of the subject matter.

In your study, we found that isotretinoin did not have a significant influence on tooth displacement when compared to animals that underwent tooth movement alone for the same duration. These findings are consistent with the results reported by Graciano Parra et al., the only study found in the literature that performed tooth movement with a force of 30cN in conjunction with isotretinoin treatment [17]. In their study, they also did not observe significant differences in tooth displacement between animals treated with isotretinoin and those undergoing OTM alone. It is worth noting that the dose of isotretinoin used in their study was 7.5mg/kg/day, which is higher than the dose utilized in our study. The fact that both our study and the study by Graciano Parra et al. did not find significant differences in tooth displacement suggests that isotretinoin, even at different doses, may not exert a substantial effect on this aspect of orthodontic treatment. However, it is important to consider the limitations of available research and the need for further studies to confirm these findings and explore potential dose-dependent effects of isotretinoin on tooth displacement during OTM.

The inflammatory process plays a crucial role in tooth displacement during orthodontic treatment, as it involves new bone formation and remodeling of collagen fibers in the periodontal tissue. Previous studies have indicated that hyalinized areas, which are indicative of tissue remodeling, appear around the third day of induced tooth movement and gradually decrease thereafter [35–37]. In our study, when examining the periodontal ligament of animals treated with isotretinoin at a dosage of 1.0mg/kg/day for 14 days in combination with 7 days of OTM, it was observed a greater expression of hyalinized areas in the mesial and middle thirds of the mesial phase of the distobuccal root of the first molar, compared to the group that underwent tooth movement alone for 7 days. This finding contrasts with the results reported by Graciano Parra et al., who did not find significant differences in the presence of hyalinized areas between their groups treated with isotretinoin and OTM on days 2, 7, 14, and 21. These discrepancies findings regarding the presence of hyalinized areas could be attributed to several factors, including variations in experimental protocols, treatment duration, and possibly the dosage of isotretinoin. It is important to further investigate and understand the specific effects of isotretinoin on the inflammatory response and tissue remodeling during orthodontic tooth movement to gain a comprehensive understanding of its impact on treatment outcomes.

Based on the methodology and results of our study, the following conclusions can be drawn:

Isotretinoin at a dosage of 1.0mg/kg/day did not reduce tooth displacement during orthodontic tooth movement (OTM). This suggests that the administration of isotretinoin did not have a significant inhibitory effect on the process of tooth movement.
Isotretinoin did not result in significant changes in the microtomographic parameters of animals treated with isotretinoin in combination with OTM. This indicates that the use of isotretinoin at the given dosage did not have a notable impact on the evaluated bone morphology parameters.

Acknowledgments
Nothing to declare.

Author Contribution
Mayra Fernanda Ferreira, DDS, MSc.
Contribution of the author: Project Administration, Carrying out the experiments, Conceptualization, Writing

Cristina Antoniali Silva, DDS, MSc, Ph.D
Contribution of the author: Methodology, Writing – Review & Editing

Edilson Ervolino, DDS, MSc, Ph.D
Contribution of the author: Methodology, Writing – Review & Editing

Luy de Abreu Costa, DDS, MSc
Contribution of the author: Writing – Review & Editing

José Ricardo Prando dos Santos, DDS, MSc, Ph.D
Contribution of the author: Writing – Review & Editing

Alberto Carlos Botazzo Delbem, DDS, MSc, Ph.D
Contribution of the author: Project Administration, Resources, Data Curation

Marcos Rogério de Mendonça, DDS, MSc, Ph.D
Contribution of the author: Conceptualization, Methodology, Supervision, Project Administration

Ethics Approval and Consent to Participate
The protocol of this research received the approval of the Ethics Committee on the Use of Animals at the Faculty of Dentistry – campus Aracatuba, Sao Paulo State University (Process FOA nº 2020-0462). Ethical approval is found in the supplementary information (Online Resource 1).

Funding
This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP [Grant number: 2020/06608-4].

Conflict of Interests
The authors have no competing interests to declare that are relevant to the content of this article.

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Chart 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

Chart1.png
Bone Volume Fraction (BV/TV), Trabecular Thickness (Tb.Th), Trabecular Number (Tb.N) e Trabecular Separation (Tb.Sp) in all groups. Note that group 6 had an increase in BV/TV compared to group 1 (P = 0.0426); Group 1 had a decrease in Tb.Th compared to group 3 (P=0.0024); Group 5 had a decrease in Tb.N compared to group 2, 6 and 7 (P = 0.0250, P = 0.0050 and P = 0.0383, respectively); Group 1 had an increase in Tb.Sp compared to group 6 (P = 0.0250) and group 6 and 7 had an decrease compared to Group 5 (P = 0.0040 and P = 0.0144, respectively).
Chart2.png
Amount of Tooth Displacement in Groups 2, 3, 6 and 7, respectively.
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Influence of isotretinoin on orthodontic tooth movement in rats: microtomographic and histomorphometric analysis

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Abstract

Objective

Materials and Methods

Results

Conclusion

Figures

Introduction

Methodology

Animals and experimental conditions

Experimental delineation

Application of isotretinoin

Orthodontic Tooth Movement (OTM) protocol

Euthanasia and specimen preparation

Tooth displacement analysis

Analysis of bone morphology

Periodontal ligament and alveolar bone analysis

Statistical analysis

Results

Effects of isotretinoin on bone morphology

Effects of tooth movement on bone morphology

Effects of tooth movement and isotretinoin on bone morphology

Effects of isotreinoin on the amount of tooth displacement

Quantitative and qualitative analysis of the effects of isotretinoin on the periodontal ligament

Discussion

Conclusion

Declarations

References

Chart

Additional Declarations

Supplementary Files

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