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1 Correlation of bone resorption induced by orthodontic tooth movement and expression of RANKL in rats 1 Seifi M. DDS. MS., 2Jessri M. DDS. MS 1Associate Professor, Dept. of Orthodontics, Dental School, Shahid Beheshti University of Medical Sciences, Tehran- Iran 2 Dentist Abstract Background and Aim: The product of RANKL gene expression has a fundamental role in bone resorption and activates the chain of osteoclast differentiation from its predecessor cells. Reports of this gene participation and role in physiologic and pathologic bone resorption led to hypothesis that it may play a role in bone resorption induced by orthodontic tooth movement. Materials & Methods: A closed coil spring (Dentaurum Ispringen, Germany) was fixed by two pieces of ligature wires to mesial side of the maxillary right first molar and anteriorly to both incisors of fifteen 7 week old Wistar male rats to tip the first molars mesially. The maxillary left first molar of each animal used as an internal control group for the contralateral tooth. The animals were sacrificed on the 21st day of the experiment and the tissue from the mesial side of maxillary molars was used for PCR test. Wilcoxon Sign Rank test was used for densitometric analysis of RANKL mRNA on the electrophoresis gel. Results: Densitometric analysis showed a significant increase in expression of gene in the experimental group compared to the control one (P<0.002). Genuineness of the PCR tests was verified by household gene of GAPDH. Conclusion: Based on the data obtained in controlled environment of the study, RANKL expression increased significantly following bone resorption induced by orthodontic tooth movement. ______________________________________________________________________________ Key words: Orthodontic tooth movement, RANKL, Root resorption. Received on:October 2008 Final revision :November2008 2 Accepted on :December2008 Introduction During implementation of orthodontic forces, significant changes occur in periodontal ligaments(1). Orthodontic forces remodel alveolar bone(2); this phenomenon induces tooth movement. Osteoclasts play an important role in bone resorption(3,4), hence studying their molecular characteristics assist in understanding their role in clinical phenomena. Receptor Activator of Nuclear factor kappa β Ligand (RANKL) which is one of the Tumor Necrosis Factors, is consisted of almost 300 amino acids and its major role in bone structure, has been observed to be in osteoclasts differentiation and action(5). A previous study demonstrated resorbing deciduous teeth pulp to have higher amounts of RANKL when compared to a non-resorbing control group(6). This finding, considering active bone and tooth resorption upon permanent teeth eruption, could suggest an important role for RANKL in physiologic bone and root resorption. RANKL has been also shown to express in fibroblasts and osteoclasts of in a resorbing PDL by histochemical analysis(7). Previous studies suggest that RANKL expression in correlation with inflammatory bone resorption due to lipopolysaccharide injection in rats’ gingival tissues increases in osteoclasts of the region(8). This is clearly indicating that RANKL expression in endothelial, inflammatory and periodontium cells increases during bone resorption. Also a close association between occlusal trauma and RANKL expression in bone resorption site has been demonstrated8. When RANKL function is restricted by OPG, periodontitis induced bone resorption is significantly lower than the normal situation(9). This finding is also strongly suggesting a close interaction between bone resorption and RANKL expression. These findings demonstrate RANKL expression in bone resorption. Therefore, the aim of this study was to investigate the role of RANKL in bone resorption induced by orthodontic tooth movement. 3 Method and Materials: Fifteen albino male Wistar rats aged seven weeks, with the mean weight of 200 gr were chosen randomly. In each animal, the maxillary first molar, received orthodontic treatment and considered control group, while the contralateral teeth which did not receive orthodontic treatment, served as internal group. During the experiment period the manipulation and treatment of the animals was performed according to the approved protocol of Institutional Animal Care and Usage Committee and the approval of ethical committee of the dental School of Shahid Beheshti University of Medical Sciences. To minimize the risk of trauma or discomfort to the rats and displacement of the appliances, the animals were fed with ground laboratory chow and drink water ad libitum. The animals were weighed before and after treatment to assess the whole body effects of the intervention. Orthodontic appliance placement: On the first day of the experiments the animals were anesthesized with a Ketamine hydrochloride 50 mg/kgbw (Ketamine Hydrochloride, Alfasan, Holland) injection containing 6mg/kgbw Xylazine HCL (Xylazine hydrochloride, Rompoun, Bayer, Leverkusen, Germany). Phosphoric acid gel 37.4% (Dentaurum® Group, Ispringen, Germany) was applied for 30 seconds to etch the mesial, palatal and buccal surfaces of the maxillary left first molars. The teeth were then wiped with disposable brush (Dentaurum® Group, Ispringen, Germany) soaked in distilled water and dried with air syringe. A closed Ni-Ti coil spring (0.002 × 0.008 inch 12 mm Dentaurum ®- Ispringeu Germany) was used to drift the first molar mesially. By the means of a force measuring gauge (Dentaurum® Group, Ispringen, Germany), it was proved that each spring was capable of delivering 60gf. The springs were tightened to the cervical third of the first maxillary right molars and then fixed anteriorly by using ligature-wire loops (Dentaurum® Steel 4 ligature wire0.010 inch, Dentaurum® Group, Ispringen, Germany) around the cervical third of the maxillary incisors. Anatomic position and structural development of both upper incisors, suggested these teeth as appropriate anchorage units for protraction of first molars. In order to prevent mucosal trauma the ligature wire ends were covered and secured with composite resin (Self cure Degufill, Degussa AG, AG, Frankfurt, Germany). Since tooth movement is directly dependant upon bone resorption, bone resorption was considered equal to bone resorption. Mesial side of alveolar bone was scratched by fine burs and discs and sent for PCR examinations. The obtained materials were then Snap-Frozen in liquid nitrogen and stored at -20ºC for later extraction of mRNA. Specimens were kept in 5% EDTA solution for 24 hours and afterwards they were sonicated for 15 minutes. After primary denaturation, to complete the strands, a final fixation was performed at 72º C for 5 minutes. By using reverse transcriptase enzyme (Super-Script II; Bibco, Wickliffe, OH, USA) and 2 μlit of hexamer primers, cDNA was synthesized (at 42ºC for 1 h). Afterwards cDNA was amplified using taq DNA polymerase (Amplitaq® DNA polymerase applied biosystems,CA, USA), PCR buffer (Applied biosystems,CA,USA), specific primers for RANKL and GAPDH (Sigma Aldrich, India), MgCl2 and according to Table 1 condition. Table 1 - cDNA extension stages Stage Denaturation Annealing Extension Temperature 94 °C 55°C 72°C Duration 30 sec 30 sec 30 sec In order to visualize DNA strands in UV transilluminator, PCR product was electrophoresed on 2% agarose gel and stained by ethidium bromide. PCR product length for RANKL measured 355 5 base pairs and GAPDH was used as control gene. PCR expression was measured defining units as 0,1,2,3 respectively representing none, low, medium, high and performed by a single practitioner and assistant observer. The obtained results were analyzed by Wilcoxon signed-rank test in the SPSS version 11.5 statistical package. Results: The animals had an 8-11% of weight gain during the experiment and this indicates that the orthodontic appliances have not interfered with animals' nourishment. Statistical analysis of tooth movement shows a significant change in control group. (Table 2) Table 2- Orthodontic tooth movement(OTM) in study groups Amount of OTM Mean 0.8942mm SD 0.063mm Minimum 0.85mm Maximum 0.98mm According to the RT-PCR findings, RANKL mRNA was detectable in all experimental groups and only 3 cases of control group. Statistical analysis i.e. Wilcoxon signed-rank of agar gel after electrophoresis for detection of RANKL mRNA showed significant difference (p=0.002) (Table3). 6 Table 3-Densitometric findings of mRNA in control & experimental groups 0 1 2 3 RANKL (Control) - 3 7 5 RANKL (Experiment) 12 1 2 - RANKL (Control) - - 9 6 RANKL (Experiment) - - 8 7 Discussion: The results of comparison between inter-molars distance before and after the treatment signifies the orthodontic treatment, which was a modification of Kobayashi (2000)(10) and Ashizawa (1998)(11) method, to be effective in inducing tooth movement. This finding is in accordance with Low(12) and Seifi (2003)(13) results. Considering the fact that this is the first study investigating the association of RANKL expression with orthodontic bone resorption, we do not have any peer study to compare our findings with. However the results of other investigations which focused on this protein’s role on bone resorption is in accordance with ours. Although Ashizawa(1998)(11) designed his model to induce tooth movement, there was no reports of the tooth movements which he made by his model and only reported the amount of bone appositioned in alveolar sockets’ walls; which he had not observed a significant difference between case and control groups. One may conclude that in Ashizawa’s model there was no significant difference between case and control tooth movement. In his model, Ashizawa used a metal plate over the occlusal surface of the teeth in order to attach the spring to them; this metal plate could induce occlusal trauma and in the same time, the duration of his study, 6 days, in comparison to ours, 21 days, seems insufficient to induce significant tooth movement. 7 The method used to extract RANL mRNA was verified using GAPDH as control gene. RANKL mRNA showed a significant increase in case groups which is consistent with Silva’s (2008)findings(14) who has detected RANKL in periodontium of 65 patients suffering fro active and aggressive periodontitis. Garlet et al(2007)(15), studied the periodontium cytokines, they found out an increase in RANKL expression in tension sides comparing to control group. Nishijima (2006) studied RANKL in gingival sulcular fluid of 10 adloescents receiving orthodontic treatment(16). He found out RANKL expression increases with force magnitude and is also dependant upon time. RANKL was also observed to be of importance in differentiation and maturation of osteoclasts (17). Since RANKL is essential in bone resorption process this finding is in accordance with previously mentioned studies. The mentioned studies results are consistent with the present study; however further studies are needed for valid approval of the present study's findings. Conclusion: According to the results of the present study, the applied method is effective in inducing bone resorption and tooth movement and could be used as a valid method to evaluate orthodontic tooth movement and associated bone resorption. On the other hand, PCR examination results suggested RANKL to be significantly increased on pressure side in case group. This could be concluded that RANKL gene in controlled laboratory environment is associated with orthodontic bone resorption. References 1. Ai H, Xu QF, Lu HF, Mai ZH, An AQ, Liu GP: Rapid tooth movement through distraction osteogenesis of the periodontal ligament in dogs. Chin Med J (Engl) 2008;121:455-462. 2. Sidiropoulou-Chatzigiannis S, Kourtidou M, Tsalikis L: The effect of osteoporosis on periodontal status, alveolar bone and orthodontic tooth movement. A literature review. J Int Acad Periodontol 2007;9:77-84. 3. Zhao H, Patrick Ross F: Mechanisms of osteoclastic secretion. Ann N Y Acad Sci 2007;1116:238-244. 8 4. 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