Introduction
This study aimed to build an experimental immature tooth movement model and verify less resorption of incompletely developed roots than those fully developed during the same orthodontic treatment, followed by investigating the cellular and molecular mechanism.
Methods
The development of Wistar rat tooth was investigated using in vivo microcomputed tomography and hematoxylin and eosin staining to decide the optimal ages of rats for immature tooth and mature tooth groups. The rats in the immature tooth and mature tooth groups were divided into experimental, sham control, and blank control groups. After orthodontic treatment for 3 weeks, the mesial root volume, crown movement distance, neck movement distance, root inclination, and apical distance were measured by microcomputed tomography. The expressions of TRAP, Jagged1, Notch2, IL-6, and RANKL were analyzed by immunohistochemical staining and real-time polymerase chain reaction. The repair of root resorption was also investigated after removing orthodontic force for 3 and 6 weeks.
Results
The root achieved the development stage around 10 weeks, so 4-week-old rats and 10-week-old rats were used in the immature tooth group and mature tooth group, respectively. The volume of root resorption in the experimental immature tooth group was 0.0869 ± 0.0244 mm 3 , which was less than that in the mature tooth group (0.1218 ± 0.0123 mm 3 ) ( P <0.001). Immature tooth movement decreased TRAP-positive odontoclasts on the compression side while having no statistically significant effect on osteoclasts. The protein expression of Jagged1, Notch2, IL-6, and RANKL in the mature tooth group increased significantly compared with the immature tooth group, not only on the compression side but also on the tension sides. The mRNA expression of Jagged1 , Notch2 , and RANKL was significantly lower in the immature tooth group, whereas the expression of IL-6 had no significance but a strong tendency. The root volume after repairing for 3 weeks was still less than that of blank control, whereas after repairing for 6 weeks, the difference was not statistically significant.
Conclusions
The experimental immature tooth movement model for the Wistar rat was achieved for the first time. The immature tooth will suffer less root resorption than the mature tooth, which may be due to odontoclastogenesis inhibition by decreased expression of Jagged1/Notch2/IL-6/RANKL signaling.
Highlights
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An experimental immature tooth movement model was achieved for the first time.
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Root resorption of the immature tooth was verified to be less than that of the mature tooth.
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The less root resorption may be caused by decreased Notch signaling expression.
External apical root resorption (EARR) is a common side effect during orthodontic treatment. EARR was a multifactorial problem according to our previous study, including genetic characteristics, biological factors, and orthodontic treatment techniques, among which previous studies had different views on the influence of age, and some indicated that increased age coincided with more root resorption. However, physiological age used by these studies was unable to reflect root development, which made it difficult to evaluate resorption accurately. Thus, considering root development as criteria can be simple for observers to increase repeatability and reproducibility. Clinical experience and a few studies had recommended that the orthodontic treatment be initiated when the roots were partially formed with open apex, but the great limitation of these studies lay on the use of panoramic radiographs and the unknown mechanism.
As root resorption is essentially a kind of volume loss, 3-dimensional quantitative volumetric measurement should be much more precise in the diagnosis of root resorption than qualitative or semiquantitative methods using 2-dimensional images such as x-ray tomography and scanning electron microscopy. Cone-beam computed tomography is used in clinical practice, but the accuracy is not sufficient compared with microcomputed tomography (microCT) to detect the details of the roots. Thus, microCT with extraordinarily high resolution has been widely applied in the medical field and could be considered the reference standard in 3-dimensional dental studies. MicroCT had been used to view the progression and repair of root resorption. Therefore, using microCT to evaluate immature tooth movement, alveolar bone remodeling, and root resorption can be more accurate in this study.
Notch signaling is a highly conserved signaling pathway that plays an important role in cell proliferation, differentiation, and apoptosis. The involvement of the Notch signaling pathway in root resorption has attracted increasing attention. Results from recent studies stated that root resorption was mediated by Jagged1/Notch2 signaling in response to orthodontic forces of high magnitude, stimulating the process of root surface resorption via IL-6 as well as the RANKL production by periodontal ligament cells. Moreover, Liu et al previously demonstrated the enhanced expression of IL-6 in the alveolar bone-modeling process with the application of orthodontic forces, and IL-6 family cytokines have been proposed to stimulate osteoclast formation and contribute to bone resorption, , so it is possible that IL-6 are important factors in the process of EARR because the morphologic and functional characteristics of odontoclasts are extremely similar to those of the osteoclast.
Therefore, this study focused on verifying less resorption of incompletely developed roots than fully developed roots and exploring if the Jagged1/Notch2/IL-6/RANKL signaling pathway played a role in reducing root resorption of the immature tooth by regulating odontoclastic activities.
Material and methods
This study was approved by the ethical board of West China Hospital of Stomatology. A total of 150 male Inbred Wistar rats were purchased from Sichuan University laboratory animal center (Chengdu, Sichuan, China) and supplied with powdered food and tap water. During the experiment, the mice were kept in separate cages in a room maintained at 25°C with a 12-to-24-hour light-and-dark cycle. All animal care and experimental procedures were performed according to the Guidelines for Animal Experimentation of Sichuan University. All animal experiments were approved by the Animal Welfare Committee of Sichuan University.
First, 30 four-week-old rats were chosen randomly and anesthetized using a 10% solution of chloral hydrate (0.03 mL/kg) with intraperitoneal injection and scanned dynamically on each week until 13-week-old by in vivo microCT (Scanco Medical, Zurich, Switzerland) with the voltage of 70 kVp and an image voxel size of 6.0 μm. The data obtained from microCT scanning were reconstructed, and the mesial root volume of the maxillary left first molars was measured to decide the optimal ages of rats for immature tooth group and mature tooth group.
Then 40 rats in the immature tooth group and 40 rats in the mature tooth group were divided into 3 subgroups by simple randomization. Orthodontic force was applied in group a (experimental group; n = 20), the tooth movement device was placed without activation in group b (sham control group; n = 10), and no device was placed in group c (blank control group; n = 10). There were remaining 20 rats in the immature tooth group, which were divided into group d (experimental and repair 3 weeks group; n = 10) and group e (experimental and repair 6 weeks group; n = 10).
The animals were anesthetized as previously described, and then they were fixed in a supine position and given a mouth gag. In groups a, d, and e, the nickel-titanium coil springs (West China Hospital of Stomatology), whose force level of the spring was set to approximately 10 g of force with an electric force gauge (West China Hospital of Stomatology), were inserted between the maxillary left incisors, and the first molar was fixed with 0.1 mm stainless steel wires, moving the maxillary left first molar in the mesial direction. To prevent the detachment of the appliance from the interproximal contact between the maxillary incisors, the anterior end of the spring was reinforced with dental adhesive resin (3M, St Paul, Minn) ( Fig 1 , D ). Group b underwent the same procedures, with the exception that the nickel-titanium coil springs exerted no force. The appliances were checked every day and reinstalled if there was any detachment or damage.
The rats in groups a, b, and c were scanned before the interventions and 3 weeks later by in vivo microCT. In group d and e, the nickel-titanium coil springs were removed after 3 weeks, and the repair effect of root resorption was investigated after another 3 weeks and 6 weeks by microCT, respectively.
The data obtained from microCT scanning were reconstructed. The mesial root volume, crown movement distance, neck movement distance, root inclination, and apical distance were measured ( Supplementary Fig 5 ). Trabecular bone cubes (200 × 200 × 200 μm), which were 100 μm away from the mesial and distal side of the mesial root of the maxillary first molar, were selected for analysis. The trabecular thickness (Tb.Th), trabecular number (Tb.N), the bone volume fraction (BV/TV), and trabecular separation (Tb.Sp) were measured by the affiliated program of the microCT. The measurement procedure was repeated 3 times by the same researcher (doctoral student L.) to obtain the mean value, and the average errors were determined by intraobserver comparison using intraclass correlation coefficients.
Then the rats were killed with an overdose of anesthetic. Maxillary segments were dissected, rinsed, and fixed in 4% paraformaldehyde solution containing diethyl pyrocarbonate for 24 hours. After decalcification in 10% ethylenediaminetetraacetic acid (pH 7.4) at room temperature for 4 weeks, the samples were dehydrated in a graded ethanol series and then fixed in paraffin. Mesial and distal serial sections, 4 μm thick, were cut along the longitudinal axis of the experimental teeth and then mounted on polylysine-coated glass slides. Immunohistochemistry was performed according to the manual of the immunohistochemical assay kit (West China Hospital of Stomatology) (streptavidin/peroxidase method). The paraffin-embedded sections were dewaxed with xylene and dehydrated with a graded series of ethanol. Endogenous peroxidase activity was blocked and inactivated with 3% hydrogen peroxide at 37°C for 30 minutes, and 0.1% trypsin (HyClone Laboratories, Logan, Utah) was used to retrieve antigen for 20 minutes at 37°C. Sections were incubated with goat serum at 37°C for 20 minutes to seal the nonspecific site and then incubated with primary antibodies—TRAP, Jagged1, Notch2, IL-6, and RANKL polyclonal antibodies (Abcam, Cambridge, United Kingdom)—overnight at 4°C. Negative controls were incubated with phosphate-buffered saline solution (supplementation). Secondary goat antirabbit antibody was added for 30 minutes at room temperature, followed by horseradish peroxidase for 15 minutes. Diaminobenzidine tetrahydrochloride was used to visualize the staining. Sections were counterstained with hematoxylin. Finally, the sections were mounted with neutral balsam and observed under light microscopy. No primary antibody was applied in the negative controls ( Supplementary Fig 1 ).
The inverted microscope (IX71; Olympus, Tokyo, Japan) was applied for observing immunohistochemistry staining. Two visual fields (100× magnification) were selected at the mesial and distal sides of the tooth roots, and images were collected with a cell image analysis system (ACT-1; Nikon, Tokyo, Japan). Positive immunohistochemistry staining in each group was semiquantitatively determined using the Image Pro Plus software (version 6.0; Media Cybernetics, Silver Spring, Md), and the mean optical density was calculated.
To further investigate the mechanism, real-time polymerase chain reaction (qPCR) was performed. The 10 rats in the immature tooth group and the 10 rats in the mature tooth group were randomly divided into group a (n = 5) and group b (n = 5). The orthodontic force was applied as above. After 3 weeks of treatment, the rats were killed, and the tissues were harvested for analysis. Approximately 6 × 3 × 2 mm 3 volumes of bony and root tissues of the maxillary left first molars were dissected, and dental pulps were removed with a microscope ( Supplementary Fig 2 ). Total RNA was extracted, and aliquots containing equal amounts of mRNA were subjected to real-time PCR. The mRNA was reverse transcribed to cDNA using the Prime Script RT Reagent Kit (Takara Bio, Shiga, Japan). Real-time PCR amplification was performed using TB Green Premix Ex Taq Ⅱ (Takara) in a thermal cycler (PIKORed 96; Thermo Fisher Scientific, Waltham, Mass). The PCR primers for Jagged1, Notch2, IL-6, RANKL, and β-actin were purchased from Takara and designed with reference to the respective cDNA sequences, are as follows:
Jagged1
Forward: 5 ′ -CGCTGTATCTGTCCACCTGGCTATGC-3 ′
Reverse: 5 ′ -GGCAAGGGTTGGGCTCGCAGTAATC-3 ′
Notch2
Forward: 5 ′ -TCTGCTTGCGGTTGCTGTGGTCATT-3 ′
Reverse: 5 ′ -TTGTGATTGCTGGAGTCTCGGCGAAG-3 ′
IL-6
Forward: 5 ′ -TTGTGATTGCTGGAGTCTCGGCGAAG-3 ′
Reverse: 5 ′ -CGGAACTCCAGAAGACCAGAGCAGAT-3 ′
RANKL
Forward: 5 ′ -GACTTCACCGAGCCTCCAAGCAGAAC-3 ′
Reverse: 5 ′ -TGCCTGTGTAGCCATCCGTTGAGTTG-3 ′
β-actin
Forward: 5 ′ -GAAGATCAAGATCATTGCTCC-3 ′
Reverse: 5 ′ -TACTCCTGCTTGCTGATCCA-3 ′
Statistical analysis
Data were expressed as means and standard deviations. Intergroup comparisons of immature tooth group and mature tooth group were achieved with 1-way analysis of variance. Values of P <0.05/n were considered to have statistical significance (n means the number of groups brought into the Bonferroni test). For example, if 3 groups are included in the statistical analysis, P <0.017 means significant between-group differences in the comparison. All statistical testing was performed with SPSS software (version 20.0; IBM, Armonk, NY).
Results
The intraobserver reproducibility of measurements was all high (all intraclass correlation coefficients >0.8). The reconstruction of microCT image and hematoxylin and eosin staining for the maxillary left first molars in rats aged from 4 weeks to 13 weeks were shown in Figure 1 , A and B and in Supplementary Figures 3 and 4 , indicating the tooth development process of rats. Figure 1 , C presented the mesial root volume change of the maxillary left first molars, revealing that the root volume increased obviously at the beginning and then gradually stabilized. Finally, 4-week-old rats and 10-week-old rats were determined to be used in the immature tooth group and mature tooth group, respectively.
After moving the maxillary left first molar for 3 weeks with nickel-titanium coil springs ( Fig 1 , D ), the mesial root volume of the maxillary left first molars was measured ( Fig 1 , E ). Figure 1 , F showed the amount of mesial root volume in 2 groups, indicating that the experimental group has less root volume than the sham and blank control group either in the immature tooth group ( P <0.001) or mature tooth group ( P <0.001), which were both statistically significant. Moreover, in this study, the real root resorption volume was obtained by adding the growth volume to root volume change for 3 weeks ( Fig 1 , C ). Equivalently, the real root resorption volume = root volume before the interventions − root volume 3 weeks later + the growth volume for 3 weeks. Therefore, the volume of root resorption in group a of the immature tooth group was 0.0869 ± 0.0244 mm 3 , which was less than that in the mature tooth group (0.1218 ± 0.0123 mm 3 ) ( P <0.001) ( Fig 1 , G ). In addition, the ratio of the root resorption volume to root volume was obtained by dividing real root resorption volume by real root volume 3 weeks without interventions. Equivalently, root resorption volume/root volume = real root resorption volume/(root volume before the interventions + the growth volume during 3 weeks). Thus, the ratio of the immature tooth group was 6.78%, which was less than that in the mature tooth group (7.34%).
Then, the crown movement distance, neck movement distance, root inclination, and apical distance were measured, as shown in Supplementary Figure 5 . Trabecular bone cubes on the mesial and distal side of the mesial root were also selected for analysis of BV/TV, Tb.Th, Tb.N, and Tb.Sp.
Statistical analysis indicated no significant intergroup difference in the apical distance between groups a, b, and c. However, there were significant differences in crown movement distance, neck movement distance, and root inclination between groups a, b, and c (all P <0.001). Significant differences were all in crown movement distance, neck movement distance, root inclination, and apical distance between immature tooth group and mature tooth group under orthodontic force (all P <0.001) ( Fig 2 , A ). On the compression side, Tb.Sp increased and Tb.N decreased in the immature tooth group, and Tb.N decreased in the mature tooth group ( Supplementary Fig 6 , A ). On the tension side, Tb.N increased as well as BV/TV and Tb.Th decreased in immature tooth group, and Tb.Sp increased and BV/TV decreased in the mature tooth group ( Supplementary Fig 6 , B ).
