RANKL and OPG expression: Jiggling force affects root resorption in rats
Objective: To immunohistochemically investigate the longitudinal changes in root resorption by jiggling force in experimental animal models.
Materials and Methods: Fifty-six 12-week-old male Wistar rats were used. The maxillary first molars were alternately moved in the buccal and lingual direction in 28 rats (experimental group) using an experimental appliance to produce jiggling forces of 10 g. In another 28 rats (control group), the maxillary first molars were moved in only the lingual direction with a force of 10 g. After 1, 3, 7, 10, 14, 17, and 21 days, the maxillae were resected and subjected to immunohistochemical analysis. The resorption area was quantified histomorphometrically and the number of odontoclasts on the root surface was counted. Expression of RANKL and OPG was also examined by immunohistochemical staining.
Results: The root resorption area and the number of odontoclasts were significantly greater in the experimental group than in controls. Odontoclasts were detected in the resorption lacunae and PDL in the experimental group, whereas osteoclasts were located only along the alveolar bone in controls. OPG was detected on the alveolar bone in the experimental group and on the root surfaces of the controls.
Conclusions: Jiggling force is a critical factor in severe root resorption, affecting RANKL and OPG expression, which accelerates and inhibits odontoclastic induction, respectively.ABSTRACT
INTRODUCTION
Root resorption during orthodontic treatment is a critical problem that is difficult to prevent or predict. Risk factors for root resorption include root shape,1,2 patient age,3–5 treatment duration,1,6 type of tooth movement,3 type of appliance,4,7 and force magnitude.8
Jiggling force, which is a repeated force application in opposite directions, has been considered to contribute to root resorption,2,3,5 and this occurs during (1) retraction of maxillary incisors, when tongue pushing forces are exerted against these teeth while they are being lingually displaced; (2) “round-tripping” when the maxillary incisors flare out during the leveling stage (with nickel titanium wires) and are then moved in the opposite direction during space closure; (3) the use of intermaxillary elastics, for example, when using multiloop edgewise archwire to intrude molars, incisor extrusion and intrusion can also occur, depending on patient compliance in wearing elastics; (4) occlusal interference during the leveling and aligning stage of treatment; and (5) the use of active removable appliances.2,3,5,9 However, there have been no reports on the influence of jiggling force on root resorption in experimental animal models.
Odontoclasts are multinucleated cells responsible for the resorption of dental hard tissues. These cells are morphologically and functionally similar to osteoclasts.10,11 However, osteoclasts are part of the normal bone structure, whereas odontoclasts are rarely seen on cementum or the root surface under physiological conditions12; they exist on the root resorption surface.13
Receptor activator of nuclear factor kappa-B ligand (RANKL) is a tumor necrosis factor family member that stimulates the fusion of preosteoclasts, attachment of osteoclasts to bone, osteoclast activation, enhanced resorption, and osteoclast survival. RANKL is expressed by osteoblastic, stromal, dendritic cells, and activated lymphocytes, and is localized in odontoblasts, pulp fibroblasts, periodontal ligament fibroblasts, and odontoclasts. Moreover, the differentiation of odontoclasts is critically regulated by RANKL in response to mechanical stress.14,15 Binding of RANKL to RANK is an essential step in the promotion of osteoclast differentiation and resorption.16
Osteoprotegerin (OPG) is a soluble decoy receptor produced by osteoblastic and periodontal ligament (PDL) cells that inhibits RANKL-mediated osteoclastogenesis.17 OPG binds to RANKL and prevents it from activating RANK. It also inhibits osteoclast formation, attachment to bone and root surfaces, activation and survival of osteoclasts; it rapidly reduces osteoclast numbers and increases its apoptosis.17–19 Moreover, application of tensile stretching to osteoblasts and PDL cells induces OPG expression,20,21 and applying compressive force to PDL cells reduces OPG expression.22 Therefore, RANKL and OPG in periodontal tissue are important determinants for regulating bone remodeling during orthodontic tooth movement as well as root resorption.14,23,24
The purpose of this study was to clarify whether jiggling force is one of the key determinants of root resorption during orthodontic tooth movement.
MATERIALS AND METHODS
Animals
Twelve-week-old male Wistar rats (Charles River, Osaka, Japan) with an average weight of 390 g were used as the experimental animals. All rats were fed powdered fodder (rodent diet CE-2; CLEA Japan, Inc, Tokyo, Japan) and tap water ad libitum and were acclimatized for 1 week under experimental conditions with a standard 12-hour, light-dark cycle at a constant temperature of 23°C and a humidity of 50%. Animals were treated in accordance with the ethical guidelines established by the Animal Care and Use Committee of Hiroshima University. All procedures were carried out while the animals were under anesthesia with pentobarbital (Somnopentyl; Kyoritsu Seiyaku, Tokyo, Japan) at 0.1 mL/100 g body weight and the same amount of atropine (Fuso Pharmaceutical Industries, Ltd, Osaka, Japan).
Experimental Tooth Movement
Fifty-six 12-week-old male Wistar rats were used. In all rats, a quad helix-type appliance was set on the maxilla to deliver 10 g of force to the first molars. The appliance consisted of a 0.5-mm Co-Cr wire as the main wire (Dentsply-Sankin KK, Tokyo, Japan), and 0.016-inch round Co-Cr wires (Rocky Mountain Morita Corp, Tokyo, Japan) were attached to the cervical aspect of the molars. Composite resin was then placed on the incisors to fix the appliance (Figure 1). Force magnitude was quantified using a round, spring-type tension gauge (Oba Keiki Seisakusho, Ltd, Tokyo, Japan) with 0–50-g range of force (sensitivity, 1g) every week. The maxillary first molars were moved in the buccal or lingual direction alternately once a week in the 28 rats of the experimental group using an experimental appliance to produce jiggling forces of 10 g (first week, lingual direction; second week, buccal direction; third week, lingual direction). In another 28 rats (control group), the maxillary first molars were moved in only the lingual direction with a force of 10 g using the same appliance activated once a week. The experimental period was 21 days. A caliper was used to measure the distance between the maxillary right and left first molars in the experimental and control groups. The distances in both groups were corrected by subtracting the number of rats without appliances to eliminate growth and divided by two to obtain the unilateral result.
Citation: The Angle Orthodontist 87, 1; 10.2319/102515-718.1
Tissue Preparation
Experimental periods were set at 1, 3, 7, 10, 14, 17, and 21 days after tooth movement. Rats were deeply anesthetized and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) transcardially. The maxillae were resected and trimmed around the molars. The trimmed tissue blocks were decalcified by 14% ethylenediaminetetraacetic acid (pH 7.4, 4°C) for 4 weeks. After washing overnight with tap water, sections were dehydrated in a graded ethanol series and were embedded in paraffin (Sakura Finetek Japan Co, Ltd, Tokyo, Japan). Embedded tissue blocks were sectioned parallel to the coronal axis of the first molars at 5-μm thickness with a microtome (Microm315; Carl Zeiss, Stuttgart, Germany) and were mounted on slides.
Histomorphometry
The distobuccal and distolingual roots were observed, as these roots have stable conformation. Each section contained the crown, root, and alveolar bone. Sections that included the root canal were stained with hematoxylin and eosin to examine root resorption. Odontoclasts were detected by staining with tartrate-resistant acid phosphatase (TRAP). TRAP-positive cells around the roots with three or more nuclei were counted as odontoclasts. To calculate the percentage of root resorption, two reference points were selected that could be reliably identified on all sections: the cementoenamel junction and the root bifurcation point. The whole root was traced, and areas were calculated. All measurements were performed using ImageJ (National Institutes of Health, Bethesda, Md). To determine the percentage root resorption for each root, the following formula was used:
Immunohistochemical Staining
Immunohistochemical staining was performed as follows: Sections were deparaffinized and endogenous peroxidase activity was quenched by treatment with 0.3% H2O2 in methanol for 30 minutes at room temperature. After the sections were washed in phosphate buffered saline, blocking with 10% bovine serum albumin was performed. Sections were reacted with primary antibodies, polyclonal antirabbit RANKL (working dilution, 1:100; Abcam, Tokyo, Japan), and polyclonal antirabbit OPG (working dilution, 1:50; Santa Cruz Biotechnology, Santa Cruz, Calif) overnight at 4°C. Both RANKL and OPG were stained with a Histofine SAB-PO(R) kit (Nichirei, Tokyo, Japan) in accordance with the manufacturer's protocol. To visualize immunoreactivity, final color reactions were performed using the substrate reagent 3,3′-diaminobenzidine (Nichirei), followed by counterstaining with hematoxylin.
Statistical Analysis
Resorption lacunae areas are given as means ± standard error of the mean. Statistical analysis of the results was performed by ANOVA and Fisher's multiple-comparison test. Probability levels of P < .05 were considered to be statistically significant. The same investigator performed all the measurements.
RESULTS
Animals and Tooth Movement
The experimental animals' weight did not decrease during the weeks after setting the appliance. In addition, there were no significant differences in weight between the control and experimental groups over the whole experimental period. During this period, teeth were moved for 3 weeks at 0.4 ± 0.14 mm per week in the experimental group and 0.3 ± 0.06 mm in the control group.
Histological Evaluation
After force application, osteoclasts were detected on the alveolar bone surface on day 3 in both groups. The number of odontoclasts was significantly greater in the experimental group than in the controls after day 10, and the number of odontoclasts in the experimental group increased even after day 17, although these changes were not seen in the control group (Figure 2a,b). Until day 7, no statistical difference in the number of odontoclasts was observed between the control and experimental groups. Moreover, osteoclasts were seen around the alveolar bone in controls. The amount of root resorption increased gradually by day 7 in both the experimental and control groups (Figure 3). Furthermore, in the experimental group, continuous increases were seen, and on day 21, the root resorption area was significantly greater than in the control group. However, slight increases were observed in the controls after day 10.
Citation: The Angle Orthodontist 87, 1; 10.2319/102515-718.1
Citation: The Angle Orthodontist 87, 1; 10.2319/102515-718.1
Immunohistochemical Staining
RANKL-positive cells were detected in the resorption lacunae and in the periodontal ligament after 10 days in the experimental group, whereas in controls, these cells were mostly located along the alveolar bone (Figure 4). Meanwhile, OPG was detected on the alveolar bone in the experimental group and on the root surfaces in the controls (Figure 5).
Citation: The Angle Orthodontist 87, 1; 10.2319/102515-718.1
Citation: The Angle Orthodontist 87, 1; 10.2319/102515-718.1
DISCUSSION
Through the experimental period, the weight of rats in both groups did not decrease. Therefore, it is clear that the appliance used in this study had no ill effects on rat growth. In previous studies, different types of appliances were used to clarify the factors affecting root resorption in rats; however, these appliances exerted forces in only one direction, that is, either mesially or buccally.25–27 In this study, a new, experimental appliance was used to evaluate the relationship between jiggling force and root resorption by alternately exerting lingual and buccal forces in order to reproduce jiggling force. The results of this investigation have demonstrated that jiggling forces applied alternately every week enhance the rate of root resorption in terms of resorption area.
Many investigators have reported associations between root resorption and intervals of appliance reactivation or desirable inactivated periods of tooth movement.25,28 Previous studies dealing with the relationship between force magnitude and tooth movement have reported that optimal force for human molars is 100–500 cN.29 The root surface area of the human maxillary first molar and canine are 20 and 10 times larger, respectively, than in rat molars.28,30 Moreover, Gonzales et al. reported that a force application of 10 g on rat molars produced significantly more tooth movement and less root resorption.8 If we extrapolate our results with rat molar tooth movement at 10 gf to human tooth movement, this force magnitude in rats seems optimal.
In the control group, expression of mononuclear TRAP-positive cells were seen on root surfaces on days 3 and 7, and the number of multinuclear TRAP-positive odontoclasts peaked on day 7. These changes in number of odontoclasts appearing after experimental tooth movement were similar to the findings of Sakai et al.31 Moreover, after day 10, a small number of odontoclasts appeared once a week after activation. The number of odontoclasts in the experimental group was significantly higher than in the control group after days 10 and 21. Thus, new odontoclasts appeared by switching the tension side to the compression side via repetitive orthodontic force, and odontoclasts seen until day 7 survived and maintained their resorption function. In addition, the odontoclasts on the tension side survived, and more odontoclasts appeared again when compressive force was applied; therefore, the total number of odontoclasts increased on day 21. Moreover, the amount of root resorption in the control group was significantly less than in the experimental group. For this reason, previous studies have reported that optimal force application induced less root resorption than did excessive force.8,32
In the present study, RANKL-positive cells were detected on the alveolar bone surface and PDL in controls; however, odontoclasts were seen in the resorption lacunae outside the alveolar bone surface and PDL in the experimental group. These results are consistent with Nakano's report, in which expression of RANKL was seen when excessive force was applied to teeth.32 Based on this result, we conclude that root resorption initiated by jiggling forces involves activation of RANKL. RANKL-positive cells and cementoblasts were present on the root surfaces in the experimental group. Rego et al. reported that the expression of RANKL in cementoblasts is enhanced by compressive force.33 Therefore, alternately applying compressive and tensile forces on both the buccal and lingual sides of roots induced RANKL expression in the cementoblasts, thereby increasing odontoclastic differentiation and activation.
In the control group, little OPG was detected on the root surfaces and alveolar bone after day 10. However, in the experimental group, OPG was detected on the alveolar bone surface after day 10 and was not present on the root surfaces. As Hartsfield reported, orthodontically induced root resorption may be associated with defective alveolar resorption or turnover, along with tooth movement or force application.11 Moreover, jiggling forces interfere with osteoclast function and may actually contribute to more root resorption by OPG on the alveolar bone surface. OPG on the alveolar bone surface was increased by tensile force in the experimental group, and that contributed to the inhibition of alveolar bone resorption when jiggling force was applied.
This study suggests that when jiggling force is applied, OPG contributes to the inhibition of alveolar bone resorption, and RANKL binds to RANK on the root surface, thus activating the odontoclasts and enhancing resorption. Thus, root resorption is accelerated. Preventing jiggling forces by delivering smooth orthodontic tooth movement should contribute to preventing root resorption.
CONCLUSIONS
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Jiggling force is a critical factor in severe root resorption in terms of RANKL and OPG expression, which accelerates and inhibits odontoclast induction, respectively.
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The present results also suggest that odontoclastic root resorption is enhanced when osteoclastic bone resorption is inhibited or reduced.
Jiggling force appliance. Appliance was attached to rat first molars, and force magnitude was maintained by a hook between the loops.
(a) Number of odontoclasts in experimental and control groups. **P < .01 between the two groups. Odontoclasts areTRAP-positive cells with three or more nuclei on root surface. (b) Odontoclasts in experimental and control group. Bar = 100 μm. T: tension side. C: compression side.
Resorption area (%) in experimental and control groups. *P < .05 between the two groups.
RANKL-positive cells in experimental and control groups. Bar = 100 μm.
OPG-positive cells in experimental and control groups. Bar = 100 μm.
Contributor Notes