Studying Orthodontic Tooth Movement in Mice

Summary

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.

Abstract

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.

Introduction

Bone remodeling is an ongoing process orchestrated by osteoclasts, osteoblasts, bone lining cells, and osteocytes, essential for maintaining the integrity of the adult skeleton^1,2. 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 loading^3,4,5.

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 ligament^7,8. These cells dynamically respond to mechanical stimuli and changes in the local milieu, influencing the composition and architecture of the surrounding bone^7,8. 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 OTM^7,8,10. 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 OTM^7,11. 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 incisors^4,12. 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 OT^M4.

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 models^14,15.

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.

Protocol

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…

Representative Results

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 betwe…

Discussion

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 reliability^7,11. 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…

Materials

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