Here, we present a protocol for studying orthodontic tooth movement (OTM), serving as a suitable model for investigating the mechanisms of bone adaptation, root resorption, and the response of bone cells to mechanical stimuli. This comprehensive guide provides detailed information on the OTM model, micro-computed tomography acquisition, and subsequent analysis.
Orthodontic tooth movement (OTM) represents a dynamic process in which the alveolar bone undergoes resorption at compression sites and deposition at tension sites, orchestrated by osteoclasts and osteoblasts, respectively. This mechanism serves as a valuable model for studying various aspects of bone adaptation, including root resorption and the cellular response to mechanical force stimuli. The protocol outlined here offers a straightforward approach to investigate OTM, establishing 0.35 N as the optimal force in a mouse model employing a nickel-titanium (NiTi) coil spring. Utilizing micro-computed tomography analysis, we quantified OTM by assessing the discrepancy in the linear distance at the cement-enamel junction. The evaluation also included an analysis of orthodontic-induced inflammatory root resorption, assessing parameters such as root mineral density and the percentage of root volume per total volume. This comprehensive protocol contributes to advancing our understanding of bone remodeling processes and enhancing the ability to develop effective orthodontic treatment strategies.
Bone remodeling is an ongoing process orchestrated by osteoclasts, osteoblasts, bone lining cells, and osteocytes, essential for maintaining the integrity of the adult skeleton1,2. Primarily driven by the differentiation and activity of osteoclasts and osteoblasts, this dynamic process involves the resorption and deposition of bone, triggered by mechanical stress and loading3,4,5.
Animal experiments play a pivotal role in elucidating the intricate biological and cellular mechanisms underpinning orthodontic tooth movement (OTM)6,7. This process involves a diverse array of cell types, such as osteoblasts, osteoclasts, osteocytes, fibroblasts, and immune cells like macrophages and T cells, situated within the jawbone and periodontal ligament7,8. These cells dynamically respond to mechanical stimuli and changes in the local milieu, influencing the composition and architecture of the surrounding bone7,8. Moreover, they also trigger an inflammatory response at a cellular level, even though there are no pathogens present. This inflammatory response plays a role in increasing the turnover of bone tissue9.
Various animal models, including mice, rats, rabbits, dogs, and monkeys, have been utilized in experimental studies of OTM7,8,10. Among these, rodents, particularly mice, are favored for investigating the initial phases of tooth movement and bone remodeling6. Previous research has emphasized the advantages of using mouse models over rat models, primarily due to the widespread availability of genetically modified strains, enabling detailed exploration of genetic influences in OTM7,11. Currently, two main models are employed to induce tooth movement in mice. The first method entails inserting a nickel-titanium (NiTi) coil spring between the first upper molar and upper incisors4,12. The second approach involves placing an elastic band within the interdental space between the first and second upper molars13. The primary outcomes analyzed typically include the magnitude of the tooth movement and bone microarchitecture, preferably evaluated using micro-computed tomography (micro-CT)14. Ideally, assessing the integrity of dental roots is important to ensure that appropriate forces are employed to produce OTM4.
While micro-CT is widely acknowledged as the gold standard for evaluating the microarchitecture of mineralized tissues14, the absence of standardized methodologies and protocols for scanning, analyzing, and reporting data often presents challenges in discerning the precise procedures employed, interpreting results, and facilitating comparisons between different OTM models14,15.
Here, we present a step-by-step guide to the OTM mouse model, including micro-CT acquisition and analysis of OTM, bone microstructure, and dental roots. This method entails applying controlled mechanical force to the first molar to induce movement within the jawbone. The selection of this method stems from several factors, including feasibility, relevance, and precision. Such an approach enables detailed quantitative analysis, providing valuable insights into the biological processes underlying orthodontic tooth movement and facilitating the development of improved orthodontic treatment strategies in the future.
All procedures strictly adhered to the ethical standards established by the Universidade Federal de Minas Gerais Ethics Committee (No. 166/2022). Before each experiment, a sample size calculation is mandatory. Use 8-10-week-old male C57BL6/J wild-type mice weighing approximately 20-30 g. The mice must be housed in a cage within a room maintained at 25 °C, adhering to a 12 h light/12 h dark cycle. Following coil attachment, the animal should be fed with a soft diet. Daily monitoring should include assessments of body weight and overall health.
1. Mechanically-induced alveolar bone remodeling
2. Micro-CT measurements
This protocol enables the investigation of an OTM mouse model using a NiTi coil spring. With a force of 0.35 N applied, the mean CEJ distance on the control side between the first and second molars was 243.69 µm (Figure 1A, line A), whereas on the OTM side was measured at 284.66 µm (Figure 1A, line B). The difference between the OTM and control sides was 40.97 µm (Figure 1B). The linear distance between the CEJ between the first and second molars of the right hemi-maxilla (OTM side) and the left hemi-maxilla (control side) was measured in at least six different sections with the line tool using the proper microCT analyzer software. The mean value of the six measurements of the OTM side was subtracted from the mean value of the control side to generate the OTM result (Figure 1).
Regarding OIIRR, values of dental RV/TV and RMD were demonstrated on both the control side (71.02 ± 10.11% and 1.071 ± 0.1536 g/cm2, respectively) and the OTM side (73.38 ± 6.162% and 1.048 ± 0.2119 g/cm2 µm, respectively; Figure 2).
Figure 1: Orthodontic tooth movement (OTM). (A) Representative image of the control and OTM sides. (B) Results of the CEJ distance of the control and OTM sides and the difference between the distance resulting in the OTM distance. A force of 0.35 N was applied in the mesial direction of the upper right first molar using a NiTi coil spring, and the endpoint of the study was the 12th day. Mean ± standard deviation of the OTM results generated from 10 mice. Please click here to view a larger version of this figure.
Figure 2: Representative image of the selected region of interest (ROI). The ROI of the disto-vestibular root of the first maxillary molar is shown, which was selected by using an irregular, anatomic region of interest drawn using the manual contouring method to analyze root mineral density (RMD; g/cm3) and percentage of root volume per total volume (RV/TV; %). Mean ± standard deviation results were generated from 10 mice. Please click here to view a larger version of this figure.
Here, we describe a standardized protocol designed to elucidate the cellular and molecular mechanisms underlying bone remodeling during OTM. A thorough understanding of these mechanisms in mice requires a meticulously planned protocol to ensure accuracy and reliability7,11. Studies conducted by our research group have shown that this protocol effectively reduces operator variability by incorporating a tension gauge and a specially designed apparatus, establishing 0.35 N as the optimal force for OTM in a mouse model4,16,17,18,19,20. To improve the efficacy of experimental procedures and maximize the use of animal samples, specimens utilized for micro-CT analysis can also undergo processing for routine histology. Subsequently, OTM can be evaluated in 5 µm sections, utilizing the CEJ as a reference point for measurements16,19. Hematoxylin and eosin staining, alongside specialized techniques such as tartrate-resistant acid phosphatase (TRAP) and Masson's trichrome, also serve as effective methods for assessing root integrity and quantifying osteocytes, osteoclast, and osteoblasts within the regions of interest4,5,16.
The dynamic alterations in the microstructure of alveolar bone during OTM in rodents have been investigated using micro-CT systems. These evaluations aim to offer valuable insights for clinical orthodontic treatment14,15. Micro-CT is an analytical technique capable of capturing internal structures with high resolution and micron-level precision. This method allows for the reconstruction of small-scale specimens into detailed 3D images, enabling accurate qualitative and quantitative analysis of samples22. Consistent with previous studies, OTM was assessed by quantifying the linear discrepancy between the CEJ of the first and second molars of the right hemi-maxilla compared to the left hemi-maxilla17,18,19,20. In the context of dental imaging, micro-CT presents several advantages. It can identify small defects on root surfaces, precisely measure linear dental changes, and assess the trabecular and cortical bone morphology22,23. It is noteworthy that the current guideline for assessing bone microstructure in rodents using micro-CT emphasizes the minimal set of variables recommended for describing trabecular and cortical bone morphometry23. Additionally, it is crucial to analyze the occurrence of OIIRR, a serious complication during orthodontic treatment16,21. Researchers should evaluate the intensity of root resorption, as the presence of OIIRR indicates that the applied force may be excessive, necessitating adjustments16,21.
Adhering to critical steps in OTM research in animal models is essential for obtaining reliable results. This includes careful selection of animals, considering factors such as strain, age, bone metabolism, growth rate, and genetic background. The choice of animal models significantly influences study outcomes, as variations in these factors affect bone physiology and response to orthodontic forces6. Timing the euthanasia of animals is also critical because it captures specific stages of bone remodeling in response to orthodontic forces. For instance, Taddei et al.4 conducted molecular analysis at 0, 12, and 72 hours, with histopathological analysis at 6 days, enabling the assessment of temporal changes in bone remodeling markers during OTM. In addition, age-related variations in OTM have been investigated, shedding light on how aging impacts bone remodeling processes23.
The use of a NiTi coil spring, while effective in inducing tooth movement, presents certain drawbacks that may affect animal welfare and experimental outcomes. Inserting a NiTi coil spring is noted to be more time-consuming and technically demanding compared to alternative methods, such as using elastic bands7. This increased complexity may lead to a higher risk of injury during insertion, which in turn can result in adverse effects such as a higher loss of body weight after the procedure and an elevated mortality rate among the animals7. In our experience, daily monitoring of animals and the implementation of supportive measures, such as softened feed, have proven instrumental in minimizing adverse effects associated with the use of NiTi coil springs for OTM in animal models. These measures not only contribute to the welfare of the animals but also enhance the reliability and validity of experimental outcomes by reducing confounding factors and ensuring consistency in research protocols4,16,17,18,19,20.
Employing OTM in mice, combined with micro-CT analysis, offers a suitable model for probing mechanisms of bone adaptation, root resorption, and cellular responses to mechanical force stimuli. This integrated approach provides valuable insights into the intricate processes underlying orthodontic treatment and facilitates the development of novel therapeutic strategies and interventions.
The authors have nothing to disclose.
We wish to express our sincere appreciation to Miss Beatriz M. Szawka for her contribution to the schematic diagram and to Mrs. Ilma Marçal de Souza for her technical support. J.A.A.A. is the recipient of a fellowship granted by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, E-26/200.331/2024), Brazil. This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (406928/2023-1), Fundação de Amparo a Pesquisa do Estado de Minas Gerais and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Finance code 001), Brazil. The authors thank Prof. Dr. Eduardo H. M. Nunes from LabBio/UFMG for the X-ray microtomography analysis.
Acetone | Sigma-Aldrich | 67-64-1 | |
Distal cut pliers | Quinelato | QO.700.00 | |
Dynamometer | SHIMPO | FGE-5XY | |
Fiber Optic Illuminator | Cole-Parmer | N/A | |
ketamine | Syntec | 100477-72-3 | |
NiTi open-coil spring 0.25 x 0.76 | Lancer Orthodontics | ||
Ø 0.20 mm round chrome-nickel (CrNi) | Morelli | 55.01.208 | |
Round CrNi Hard Elastic Orthodontic Wire Ø0.50 mm (.020 inch) | Morelli | 55.01.050 | |
Round CrNi Tie Wire Ø0.20 mm (.008 inch) | Morelli | 55.01.208 | |
Stereomicroscope | Quimis | Q7740SZ | |
Transbond Plus Self Etching Primer | 3M | LE-Q100-1004-7 | |
Weingart Plier | Quinelato | QO.120.00 | |
Xylazine | Syntec | 23076-35-9 | |
MicroCT Analysis | |||
Skyscan 1174v2 | Bruker | 1174v2 | |
Software | |||
NRecon | Skyscan | N/A | |
DataViewer | Skyscan | N/A | |
CTAn | Skyscan | N/A | |
Mimics | Materialise | N/A |
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