The Establishment of a Murine Maxillary Orthodontic Model

Summary

Here we demonstrate step-by-step a manageable, orthodontic tooth movement protocol operated on a murine maxillary model. With the explicit explanation of each step and visual demonstration, researchers can master this model and apply it to their experimental needs with a few modifications.

Abstract

Due to the lack of reproducible protocols for establishing a murine maxillary orthodontic model, we present a reliable and reproducible protocol to provide researchers with a feasible tool to analyze mechanical loading-associated bone remodeling. This study presents a detailed flowchart in addition to different types of schematic diagrams, operation photos, and videos. We performed this protocol on 11 adult wide-type C57/B6J mice and harvested samples on postoperative days 3, 8, and 14. The micro-CT and histopathological data have proven the success of tooth movements coupled with bone remodeling using this protocol. Furthermore, according to the micro-CT results on days 3, 8, and 14, we have divided bone modeling into three stages: preparation stage, bone resorption stage, and bone formation stage. These stages are expected to help researchers concerned with different stages to set sample collection time reasonably. This protocol can equip researchers with a tool to carry out regenerative analysis of bone remodeling.

Introduction

Bone is a highly active reconstructed tissue that adapts its size, shape, and properties through the lifetime of the individual^1,2. In addition to hormones, aging, nutrition, and other biological or biochemical factors³, the idea that mechanical load is the most determining factor has garnered general acceptance^4,5. Under some circumstances with abnormal mechanical load, the imbalance between bone resorption and bone formation may lead to abnormal bone remodeling and bone disorders. Bone diseases such as disuse osteoporosis and bone loss during long-term bed rest or in the presence of microgravity at spaceflight have a close relationship with abnormal mechanical load^6,7,8.

Mechanical load has also been used to treat bone-related diseases such as distraction treatment and orthodontic treatment. Distraction treatment has been used in developmental diseases such as craniosynostosis and mandibular hypoplasia^9,10, while orthodontic treatment has been widely used to rectify abnormal teeth position and any malocclusion¹¹. The core of orthodontic treatment is also the management of mechanical load. When the bone tissue is subjected to mechanical load, a highly coordinated bone remodeling process is induced by coupling of bone resorption followed by bone formation, which can move teeth to achieve the orthodontic purpose^12,13.

Although orthodontic treatment has been widely applied for clinical practice, as our knowledge of the biological effects of mechanical load is limited, the results of orthodontic treatment are uncontrollable. To overcome these limitations, several animal models such as mouse, rat, rabbit, cat, dog, monkey, and pig have been established to investigate the underlying mechanism of mechanical load-induced bone remodeling (Table 1)^{14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32}. Large animals such as dogs, monkeys, and pigs have some advantages over small animals in orthodontic operation-they have more human-like teeth and dentition so that the surgical procedure is easy to replicate in humans. Additionally, a wide view can reduce the operation difficulty and make it possible to apply a variety of orthodontic schemes^33,34. However, large animals are difficult to obtain, leading to challenges related to sample size, and they are subject to ethical restrictions³⁵. Furthermore, routine extraction procedures and complex instruments make the experiments difficult to perform due to which large animals are rarely used.

Under such circumstances, rodents are mainly used to establish orthodontic models. Among these models, rats and rabbits have lower operating difficulty and more tooth movement schemes compared with mice. However, the murine model has the unique advantage that there are a large number of genetically modified mice available, which is especially crucial for investigating the underlying mechanisms³⁶. However, the murine model is the most difficult model to manipulate because of its small size. Reviewing the current methods, moving the first molar in the mesial direction is the only practical method for an orthodontic model. Two devices are mainly used to move the tooth-coil spring and elastic band. Using an elastic band is easier, but the orthodontic force varies greatly, which makes it difficult to obtain stable results.

Xu et al.¹⁵ have established a murine model with a coil spring on the mandible. However, due to the mobility of the mandible and the obstructive nature of the tongue, operation on the maxilla is always the first choice for both intraoperative and postoperative considerations. Taddei et al.¹⁶ described a more detailed protocol on the murine maxilla 10 years ago and more visual and pellucid details should be added. In summary, this protocol has systematically described a detailed orthodontic tooth movement protocol in a murine maxillary model to help researchers master the modeling method in a standardized way and enable the comparative evaluation between different studies.

Protocol

The animal procedures in this study were reviewed and approved by the Ethical Committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2017-041). Adult C57BL/6 mice were used in this study (see the Table of Materials). This protocol adds mechanical load to the right maxillary first molar (M1) for mesial movement where a highly coordinated bone remodeling process is induced by coupling of bone resorption and bone formation (Figure 1).

1. Preoperative preparation

1. Surgical items
   1. Prepare the following surgical items for the operation: surgical platform (Figure 2A), fastener (Figure 2B), surgical instruments (Figure 2C and Supplementary Figure S1), orthodontic supplies (Figure 2C), and dental restoration supplies (Figure 2D).
      NOTE: The customized coil spring is custom-made and provides a force of 10cN when stretched to 10 mm.
2. Sterilization
   1. Sterilize the surgical instruments by autoclaving and all the surgical items with ultraviolet irradiation for at least 30 min.
3. Anesthesia
   1. Anesthetize the mouse by administering ketamine (100 mg/kg) and diazepam (5 mg/kg) by intraperitoneal injection.
   2. Apply vet ointment to the eyes of the murine with a cotton stick to avoid eye dryness.
   3. Proceed with the surgery only when the mouse does not respond when its toes are pinched with forceps.

2. Surgical process

1. Spread and tape the limbs of the anesthetized mouse in a supine position to the surgical platform using adhesive tape.
2. Pin a 27 G needle on either side above the head and another 27 G needle on either side below the axilla.
3. Wind a rubber band around the above two needles and the upper incisors and another one around another two needles and the lower incisors. Change the needle positions to control the degree of opening and the orientation of the mouth (Figure 3A).
   NOTE: For the orthodontic tooth movement operation, keep the mouth open to the maximum extent before the buccinator becomes completely tight. The tongue should be pulled towards the non-operative side to expose the surgical field and prevent ischemia.
4. Bend the 1.5 mm end of a 3 cm 304 stainless steel wire and push the bent end through the interproximate space between the M1 and the maxillary second molar (M2) from the buccal side with curved ophthalmic tweezers (Figure 3B). When the palatal end of the ligature wire is seen from the palatal side, pull it out up to about half of its length and pass it through one end of the customized coil spring.
5. Tie a square knot with the two ends of the ligature wire in the mesial direction of the maxillary M1 until the spring is firmly fixed to the tooth (Figure 3C). Subtract the excess wire.
6. Similarly, pierce a second 3 cm 304 stainless steel wire through the other end of the coil spring.
7. Clean and dry the incisors' surfaces with cotton balls. Apply adhesives on all those surfaces with cotton sticks and light-cure them.
8. Push the second stainless steel wire through the interproximate space between the maxillary incisors and tie a slip knot in the labial direction (Figure 3D). Subtract the excess wire and make the rest of the wire lie close to the tooth surface.
9. Inject light-cured resin to cover the knot and incisors; light-cure the resin (Figure 3E).

3. Postoperative management

1. After surgery, inject the mice with 0.05 mg/kg buprenorphine intraperitoneally for postoperative analgesia.
2. Place the anesthetized mouse on a 37 °C thermostatic electric blanket. When the murine regains consciousness with ambulation, return it to a separate housing cage.
3. Due to the limited functioning of the incisors after the surgery, replace regular hard fodder with only a soft diet.
4. Check the orthodontic appliances every day. If any condition are observed during the inspection that affects the conduction of orthodontic force, such as spring deformation, spring loosening, and device falling off, the mouse should be excluded from the experiment.
5. In order to maintain the comparability of experiments, assess the weight of the mice daily post-surgery. Any mice exhibiting a weight loss exceeding 30% of their preoperative weight must be excluded from the experiment.

Results

We have performed the OTM surgery on 11 adult male mice (C57/BL6, 3 months old). They were euthanized for results on days 3, 8, and 14 post surgery. In these experiments, the right maxillary side is the operation side, while the left maxillary side is the control side. The micro-CT showed that there was a temporal consecutive increase in the distance between M1 and M2: 30 µm, 70 µm, and 110 µm at days 3, 8, and 14 post surgery, respectively (Figure 4). The low-density periodon...

Discussion

In this paper, we tried to describe the simplest orthodontic tooth movement protocol on murine maxillary model step by step to study the latent mechanisms of mechanical load-induced bone remodeling. Apart from research on bone remodeling, there are some other mainstream applications of this method: 1) methodological research on the acceleration of orthodontic tooth movement; 2) research on orthodontic root resorption; 3) biological mechanisms of orthodontic tooth movement and pain; 4) research on the transgenic model.

Materials

Name	Company	Catalog Number	Comments
Experimental Models: Mouse Lines
C57/B6J	Gempharmatech Experimental Animals Company	C57/B6J
Critical Commercial Assays
Hematoxylin and Eosin Stain Kit	Biosharp	BL700B
Masson’s Trichrome Stain Kit	Solarbio	G1340
Instruments
27 G needle	Chengdu Xinjin Shifeng Medical Apparatus & Instruments Co. LTD.	SB1-074(IV)
Adhesives	Minnesota Mining and Manufacturing Co., Ltd.	41282
Corkboard	DELI Group Co., Ltd.	8705
Cotton balls	Haishi Hainuo Group Co.,  Ltd.	20120047
Cotton sticks	Lakong Medical Devices Co., Ltd.	M6500R
Customized coil spring	Chengdu Mingxing Spring Co., Ltd.	1109-02
Forceps	Chengdu Shifeng Co., Ltd.	none
Light-cured fluid resin	Shofu Dental Trading (SHANGHAI) Co., Ltd.	518785
Light curer	Liang Ya Dental Equipment Co., Ltd.	LY-A180
Medical adhesive tapes	Haishi Hainuo Group Co.,  Ltd.	0008-2014
Medical non-woven fabric	Henan Yadu Industrial Co., Ltd.	01011500018
Needle holders	Chengdu Shifeng Co., Ltd.	none
Rubber bands	Haishi Hainuo Group Co.,  Ltd.	32X1
Surgical scissors	Chengdu Shifeng Co., Ltd.	none
Tweezers	Chengdu Shifeng Co., Ltd.	none