Abstract
Orthodontic movement of non-occluding teeth may result in undesirable apical root resorption. These teeth present with a histologically altered periodontium and are considered to be hypofunctional. The purpose of this study was to compare the amount of root resorption associated with a normal and a hypofunctional periodontium in rats during experimental tooth movement caused by heavy continuous force. The mandibular first molar was induced into a non-occluding condition in the hypofunctional periodontium group. Mesial orthodontic force was applied by means of 50-gram-force closed-coil springs for 15 days in both groups. The active root-resorption lacunae from histological sections, identified by tartrate-resistant acid phosphatase, were measured in terms of length, depth, and area. The results showed that the amount of root resorption was significantly greater in teeth with a hypofunctional periodontium than in those with a normal periodontium (p < 0.05). These results suggest that orthodontic movement of non-occluding teeth should be performed with caution.
INTRODUCTION
The cause of root resorption during orthodontic treatment is considered to be multifactorial. One of the possible etiological factors is hypofunction during normal occlusion (Newman, 1975). This condition includes teeth in malposition, or in vertical or horizontal open bite. Clinical study showed that the root-resorption severity in open-bite teeth was radiographically greater than in deep-bite teeth before and after orthodontic treatment (Harris and Butler, 1992).
Histologically, in the absence of occlusal function, teeth exhibit a hypofunctional periodontium (Kronfeld, 1931; Newman, 1975) characterized by loss of functional structures. The periodontal ligament space becomes narrow (Kronfeld, 1931), and atrophic changes such as the disappearance of the functional arrangement of Sharpey’s fibers and a decrease in fibroblastic proliferation activity (Koike, 1996) occur. Changes in vascularity (Tanaka et al., 1998) and distribution of Ruffini’s nerve endings and proteoglycans (Muramoto et al., 2000; Kaneko et al., 2001) have also been reported. Large marrow spaces and thinner outer shells of alveolar bone were observed in the mandibular teeth after extraction of the opposing teeth in rats (Lee and Nakamura, 1999). Based on these clinical and histological findings, an altered response to orthodontically induced mechanical stress could be expected in hypofunctional non-occluding teeth. Moreover, no histological investigations have been performed to examine the possible etiology of root resorption under hypofunctional conditions.
The aim of the present study was to compare the incidence and extent of root resorption in hypofunctionally non-occluding teeth with those in normally occluding teeth during experimental tooth movement in rats. We established the experimental hypofunctional periodontal condition by relieving the teeth from normal functional stress and measured orthodontically induced root resorption quantitatively. We hypothesized that non-occluding teeth would have a higher risk for orthodontically induced root resorption than would normally occluding teeth.
MATERIALS & METHODS
Animals
Twenty male five-week-old Sprague-Dawley rats (Sankyo Labo Service Corporation, Inc., Tokyo, Japan) were divided into two groups: those with a normal periodontium and those with a hypofunctional periodontium. Orthodontic appliances were placed for 15 days (Fig. 1). The animals were fed with powder-form fodder (Rodent Diet CE-2; Japan Clea Inc., Shizuoka, Japan) and water ad libitum in separate cages, in a 12-hour light/dark environment at a constant temperature of 23°C. During the experimental period, the rats were weighed twice a week. The experimental procedures were approved by the Animal Ethics Committee of Tokyo Medical and Dental University. All procedures were carried out while the animals were under general anesthesia with ketamine hydrochloride (KETALAR 50, Sankyo Co., Ltd., Tokyo, Japan) and 20% xylazine hydrochloride (Celactel 2% injections, BAYER-Japan Co., Ltd., Tokyo, Japan) administered intraperitoneally.
Induction of Hypofunctional Condition
The left mandibular first molar was induced into hypofunctional occlusion by the bite-raising technique. With this technique, the non-occluding teeth were orthodontically moved to a new occluding position with their opposing teeth. When the animals were 5 wks old, composite resin (Clearfil Photo SC; Kuraray Co., Ltd., Osaka, Japan) was applied to the occlusal surface of the right maxillary first to third molars and the left maxillary third molar until the incisal overbite became -1 mm. This procedure was repeated once a week for 3 wks (Fig. 1A). We performed a preliminary experiment to verify the hypofunctional condition. Occlusal records were registered in the closed-jaw position by means of silicone impression material (Dent Silicone-V, regular type, Shofu Inc., Kyoto, Japan) prior to tooth movement. These records showed the contact area between maxillary and mandibular teeth, with a lack of impression material at the area of composite resin. The interocclusal space at the left mandibular first and second molars indicated a non-occluding condition. In addition, we examined paraffin-embedded sagittal sections histologically by staining them with hematoxylin and eosin to verify the hypofunctional periodontium.
Experimental Tooth Movement
When the rats were 8 wks old, the left mandibular first molars were moved mesio-occlusally for 15 days in both groups (Fig. 1A). Necessary anchorage was provided by a 3.5-mm-long titanium-screw implant with a diameter of 1.0 mm (Kameyama et al., 2002), fixed to the left body of the mandible, and a 1.2-mm-diameter cobalt-chromium (Co-Cr) alloy wire extending along the incisal axis. A 2-mm-long 50-gram-force super-elastic titanium-nickel (Ti-Ni) alloy closed-coil spring (Sentalloy® closed coil spring, EX Light, Tomy International Inc., Tokyo, Japan) (Tobiume et al., 2000), which delivered sufficient continuous heavy force to initiate root resorption (King and Fischlschweiger, 1982), extended from the tip of the Co-Cr alloy wire to the left mandibular first molar (Fig. 1B). This spring was attached to the tooth by a clamp at the mesial furcation and was reinforced by composite resin filling the gap between the clamp and the tooth surface.
Histochemical Analysis
After 15 days of tooth movement, the animals were anesthetized by diethyl ether and killed by cervical dislocation. The left half of mandible was dissected and immersed overnight in 10% neutral buffered formalin (pH 7.4) at 4°C. Before decalcification, the amount of horizontal tooth movement was determined from the space between the contact areas of first and second molars (Kohno et al., 2002). The specimens were then decalcified in 10% (W/V) ethylene diamine tetra-acetic acid for 4 wks at 4°C, dehydrated, and embedded in paraffin. Serial sections of 5.0-μm thickness were made along the sagittal axis. The sections that included the root canal were stained with hematoxylin and eosin for examination of root resorption at the compression area. Odontoclasts were detected by being stained with tartrate-resistant acid phosphatase (TRAP) (Domon et al., 1999). In brief, after fixation in citrate-acetone-formaldehyde solution for 30 sec, the sections were incubated in de-ionized water containing naphthol AS-MX phosphate (Sigma, St. Louis, MO, USA) as the substrate and Fast Red Violet LB salt (Sigma, St. Louis, MO, USA) for color reaction at pH 5.4 with 50 mM sodium tartrate. The reaction was stopped with distilled water. The sections were counterstained with hematoxylin and mounted with permanent aqueous mounting medium (Crystal/Mount, Biomeda Corp., Foster City, CA, USA). The newly formed resorption lacunae were identified by the presence of TRAP-positive multinucleated cells.
Quantitative Evaluation of Root Resorption
The disto-apical region of the distal root was observed as the compression area. All sections that included the root canal were examined. In both normal and hypofunctional periodontium groups, root resorption was approximately equal in dimension bucco-lingually, but differed occluso-apically. Hence, the buccal, middle, and lingual one-third sections were measured, and the mean value for each animal was calculated for statistical analysis.
Since the distal root has a stable conformation, the root-shape outline can be easily estimated by means of a template reference. The distal root was photographed by a digital camera (DXm1200, Nikon, Tokyo, Japan), and the resorption lacunar length, depth, and area were estimated three-dimensionally by image analysis software (Image-Pro Plus 4.0, Media Cybernetics, Silver Spring, MD, USA). Resorption lacunar length and area represent the lacunar surface area and volume, respectively (Kameyama et al., 1994), while resorption lacunar depth is the length of the deepest point from the simulated root surface to the resorption surface of the lacuna (Owman-Moll et al., 1995).
Statistical Analysis
The amount of tooth movement and dimensions of root-resorption lacuna were represented as the mean ± standard error of the mean (SEM) (n = 10). Comparisons between normal and hypofunctional periodontium groups were performed with the Mann-Whitney U test and the use of statistical analysis software (Statview 5.0, SAS Institute, Cary, NC, USA). The level of significance was set at 0.05.
RESULTS
The animals in both normal and hypofunctional periodontium groups exhibited normal growth during the experiment, according to the monitored changes in body weight.
Histological Examination of Hypofunctional Periodontium
When compared with normal periodontium, the hematoxylin/eosin-stained sagittal sections of the hypofunctional periodontium group demonstrated a narrow periodontal width, atrophic changes, and derangement of principal collagen fibers. Fibroblasts were also relatively few in the hypofunctional periodontium group (Fig. 2).
Amount of Tooth Movement
There was no significant difference in the amount of horizontal tooth movement between the normal periodontium group (0.26 mm ± 0.06) and the hypofunctional periodontium group (0.33 mm ± 0.05) after 15 days.
Characteristics of Root Resorption
In all specimens of both the normal and hypofunctional periodontium groups, root resorption had occurred on the disto-apical third of the distal root facing the alveolar bone in the compression zone (Fig. 3). In the hypofunctional periodontium group, the roots were resorbed extensively into dentin after 15 days of orthodontic movement.
Quantitative Analysis
Root resorption, measured in terms of resorption lacunar length, depth, and area, was significantly greater in the hypofunctional periodontium group (length, 1.09 mm ± 0.18; depth, 0.16 mm ± 0.01; area, 0.06 mm2 ± 0.01) than in the normal periodontium group (length, 0.49 mm ± 0.19; depth, 0.06 mm ± 0.02; area, 0.02 mm2 ± 0.01) (Fig. 4).
DISCUSSION
In the preliminary study, we established an experimental hypofunctional periodontal condition by relieving the teeth from normal functional stress with a bite-raising technique. This technique could establish a hypofunctional periodontium in the rat model by 3 wks. The histological findings indicated that the normal functional periodontal structure was completely lost, as reported previously (Bondevik, 1984; Koike, 1996).
The method used in the present study for quantitative assessment of root resorption has been widely used in histomorphometric studies (Kameyama et al., 1994; Owman-Moll et al., 1995). These procedures are performed to estimate the resorption three-dimensionally. The presence of TRAP-positive cells in the resorption lacunae confirms the resorptive activity. This is a standard method for the detection of active root resorption (Brudvik and Rygh, 1994; Baroukh et al., 2000).
In the present study, heavy and continuous orthodontic forces were applied to both groups. This force would naturally induce root resorption in teeth with normal periodontium (Brezniak and Wasserstein, 1993). However, root resorption was greater in the hypofunctional periodontium group. This implies that factors other than the applied force were responsible for root resorption.
Since the hypofunctional periodontium exhibited progressive atrophic changes in all functional structures, this might have accelerated the root destruction resulting from the mechanical stress of orthodontic force. Due to the narrow periodontal space in the hypofunctional periodontium, the applied force might concentrate in the compression area (Jeon et al., 1999). In addition, the narrow periodontal space and derangement of functional fibers would eliminate the normal cushioning effect of the periodontal ligament (Selliseth and Selvig, 1994), thus resulting in a high concentration of force. This would stimulate inflammation by the promotion of inflammatory mediators (Cooper and Sims, 1989) secreted from local cells to induce destruction of tooth and bone.
Our results suggest that orthodontists should be cautious while applying orthodontic force to non-occluding teeth with a hypofunctional periodontium so as to avoid the occurrence of undesirable root resorption.
Experimental schedule and orthodontic appliance fixed to the left mandible of the rat. Histological comparison of normal and hypofunctional periodontium after bite-raising for 3 wks at the disto-apical third region of the distal root. The reduction in periodontal width, atrophic changes, and derangement of the functional principal collagen fibers in the hypofunctional periodontium group Active root resorption lacunae of normal periodontium Quantitative analysis of active root resorption in normal and hypofunctional periodontium groups. The root resorption lacunar length 
Footnotes
Acknowledgements
This study was financially supported by Grants-in-Aid for Scientific Research (Nos. 10771169, 12771271, 19671989) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The work is based on a thesis submitted to the Graduate School, Faculty of Dentistry, Tokyo Medical and Dental University, in partial fulfillment of the requirements for PhD degree. Part of this study was presented at the 79th General Session & Exhibition of the International Association for Dental Research, Chiba, Japan, June 27-30, 2001.