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INTRODUCTION
The etiology of undesirable root resorption is considered to be multifactorial. The factors responsible for root resorption are now investigated at the molecular level.12 Therefore, a root resorption-inducing model should be established to facilitate studies at morphological, cellular, and molecular levels. In the past, a number of etiological studies concerning root resorption have been conducted, and various root resorption models have been developed to test several hypotheses. The replantation model,3 internal resorption model,4 luxation model,5 surgical periodontal injury model,6 orthodontic injury model,78910 and freezing injury model11 have all provided novel information about the initial phases of the resorption process. An in-vitro model has also been developed.12 However, orthodontic root resorption differs from other types of root resorption because it is primarily caused by mechanical stress to the periodontium, not by bacterial infection or systemic/metabolic diseases.12 To date, the maxillary first molar has been moved and investigated in most orthodontic root resorption models in rats.12 However, the maxillary first molar in rats has five roots, including a thick mesial root and a narrow mesiobuccal root, that are quite different from their human counterparts in shape and composition. On the other hand, the mandibular first molar in rats has two thick roots that are quite similar to those of the human mandibular first molar. In order to clarify the characteristics of apical orthodontic root resorption that are different from those of other types of root resorption, we tried to establish an apical orthodontic root resorption model in the rat mandibular first molar for use in experiments examining factors such as the effects of differences in periodontal conditions and the effects of mechanical stimuli on periodontal and cellular responses. The purpose of this study was to clarify the effects of continuous force application for extrusive tipping movement and occlusal interference on periapical root resorption in the rat mandibular first molar.
MATERIALS AND METHODS
Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (approval No. 0150192A, 0160246A). Twenty-five 8-week-old male Sprague Dawley rats were randomly divided into a non-treated control group (n = 5) and an experimental group (n = 20) to examine the body effect of an appliance. The rats in the experimental group were further randomized into an 8-day movement group (8d group, n = 10) and a 15-day movement group (15d group, n = 10). The animals were kept in separate cages in a 12-hour light/dark environment at a constant temperature of 23℃ and were provided food and water ad libitum. All procedures were conducted under general anesthesia with ketamine hydrochloride (KETALAR 50; Sankyo Co., Ltd., Tokyo, Japan) and 20% xylazine hydrochloride (Celactal 2% injections; BAYER-Japan Co., Ltd., Tokyo, Japan). During the study period, the rats were weighed every week.
Experimental tooth movement
When the rats in the experimental groups were 8 weeks old, the mandibular left first molars were mesioocclusally moved for 8 or 15 days. The necessary anchorage was provided by a 3.5-mm-long titanium screw implant with a 1.0-mm diameter (Shioda Co, Ltd., Tochigi, Japan)13 that was fixed to the left body of the mandible and a cobalt-chromium (Co-Cr) alloy wire with a 1.2-mm diameter extending along the incisal axis as a post (Figure 1A and 1B). A 2-mm-long, 50-cN, superelastic nickel-titanium (Ni-Ti) alloy closed coil spring (Tomy International Co., Ltd., Tokyo, Japan) extended from the tip of the Co-Cr alloy post to a clamp at the furcation of the mandibular left first molar (Figure 1C)14 to facilitate mesio-occlusal tipping movement. The antagonistic teeth were retained to produce occlusal interference.
Histological analysis
After the completion of tooth movement in the experimental groups, the animals were anesthetized using diethyl ether and sacrificed by cervical dislocation. The left half of the mandible was dissected and immersed overnight in 10% neutral buffered formalin (pH 7.4) at 4℃. Before decalcification, the horizontal distance between the first and second molars was recorded from the center of the distal contact area of the first molar to the center of the mesial contact area of the second molar on the horizontal stage of a noncontact digital microscopic gauge (MS-214; FUSOH Co., Ltd, Tokyo, Japan; Figure 1D).15 The specimens were then decalcified in 10% (W/V) ethylene diamine tetra-acetic acid for 4 weeks at 4℃, dehydrated, and embedded in paraffin. Serial sections with a 5.0-µm thickness were cut along the sagittal axis. The sections that included the root canal were stained with hematoxylin and eosin to examine root resorption in the compression zone. Active resorption lacunae were identified by the presence of odontoclasts on the tooth surface. These multinucleated odontoclasts were detected by staining with tartrate-resistant acid phosphatase (TRAP). According to our previous study,16 specimens were incubated at 37℃ for 10 minutes in 0.1 M acetate-buffered medium (pH 5.4) containing naphthol AS-MX (Sigma, St. Louis, MO, USA) as a substrate, fast red violet (Sigma) for a color reaction, and 10 mM sodium tartrate (Sigma). The reaction was stopped by the addition of distilled water and the specimens were counterstained with hematoxylin. The control specimens were incubated in medium without a substrate under the same conditions.
Quantitative evaluation of root resorption
The distoapical region of the distal root was observed as the compression zone. All sections that included the root canal were examined. Sagittal sections from the buccal, central, and lingual portions of the middle third of the distal root were selected for measurement (Figure 1D).
Because the distal root has a stable conformation, the root outline can be easily estimated using a reference template. The distal root was microphotographed using a digital camera (DXm1200; Nikon, Tokyo, Japan), and the length (RL), depth (RD), and area (RA) of the resorption lacunae were three-dimensionally estimated using an image analysis program (Image-Pro Plus 4.0; Media Cybernetics, Silver Spring, MD, USA). RL and RA represented the lacunar surface area and volume, respectively,17 while RD was the deepest point from the simulated root surface to the resorption surface of the lacuna16 (Figure 1E).
Statistical analysis
The amount of tooth movement and the dimensions of the root resorption lacunae are expressed as means ± standard deviations (n = 10). Comparisons among the control, 8d, and 15d groups were performed using t-tests and Wilcoxon signed-rank tests with a statistical software program (SPSS ver. 15.0; SPSS Inc., Chicago, IL, USA). The level of significance was set at 0.05.
RESULTS
The animals in the control and experimental groups exhibited no significant differences with regard to changes in the body weight during the study period (Figure 2).
Amount of tooth movement
Horizontal tooth movement was observed in both the 8d (0.15 ± 0.09 mm, n = 10) and 15d (0.26 ± 0.13 mm, n = 10) groups, but not in the control group. The amount of tooth movement was not significantly different between the two experimental groups (Figure 3).
Characteristics of root resorption
TRAP-positive multinucleated cells, i.e., odontoclasts, were identified in the root resorption lacunae, indicating newly formed lacunae (Figure 4). The control group did not exhibit any active lacunae. In all specimens from the 8d and 15d groups, resorption facing the alveolar bone close to the root apex had occurred in the distoapical third of the mesial and distal roots; this resorption was more severe in the mesial roots. Moreover, most root resorption areas did not exhibit a hyalinization zone or necrotic periodontal area.
Quantitative analysis
The amount and severity of root resorption exhibited no significant differences between the 8d and 15d groups (Figure 5).
DISCUSSION
In our root resorption model, we applied known root resorption factors during tooth movement, such as continuous force application,8151819 long duration of force application or tooth movement,720 mesio-occlusal tipping for the induction of high stress at the root apex,2122 and movement under occlusal interference from antagonist teeth.2324 Therefore, this experimental model could result in root resorption in the anticipated compression zone.
Because of mesio-occlusal tipping movement, the distoapical third of the mesial and distal roots was defined as the compression zone. Mesial tipping alone may not create adequate stress or stress distribution to induce root resorption in the distoapical third of the root.22
Because tipping movement and occlusal interference were simultaneously applied, resorption was more evident in the mesial root than in the distal root. We speculate that an intruding vector of force for a jiggling effect may have occurred along the mesial root and was focused in the apical region, whereas the extruding vector occurred along the distal root.2225
Intermittent orthodontic forces have often been applied to the rat molar resulting in tooth movement without root resorption; this may be due to a decrease in force toward the end of the experiment.26 In the present study, we utilized a continuous force for the maintenance of mechanical forces acting on the periodontium. This continuous force application stimulates inflammation through the promotion of inflammatory mediators27 secreted from local cells and migrated leukocytes to induce bone and tooth destruction by maintaining the resorption function of osteoclasts and odontoclasts and protecting them from apoptosis.28
Moreover, most of the root resorption areas did not exhibit a hyalinization zone or necrotic periodontal area, probably because of a relatively long duration of tooth movement.29
The increase in the severity of root resorption with time may have resulted from the continuous force applied by the superelastic Ni-Ti closed coil spring, which does not provide a resting period to allow tissue repair.30
Unlike that in previously developed root resorption models such as the replantation model3 and the periodontal ligament injury model induced by surgical procedures,6 root resorption in our model was not caused by damage due to necrosis/infection. In addition, it occurred in the main roots of the rat mandibular first molar, which is convenient for several experimental procedures such as extraction and sectioning. Moreover, the rat mandibular first molar is a suitable representative of the human mandibular first molar because it exhibits a similar root configuration.
CONCLUSION
In conclusion, periapical root resorption was induced by continuous extrusive tipping force and occlusal interference in rat mandibular molars. These data suggest that we orthodontists had better take care not to induce severe and long lasting occlusal interference during our orthodontic treatment.
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