ORIGINAL ARTICLE

Kinetics of interleukin-6 and chemokine ligands 2 and 3 expression of periodontal tissues during orthodontic tooth movement
 is Madureira,a Silvana de Albuquerque Taddei,b Mauro Henrique Nogueira Guimara ~es Abreu,c Davidson Fro d d e Henrique Pretti, Elizabeth Maria Bastos Lages, and Tarcilia Aparecida da Silva Belo Horizonte, Minas Gerais, Brazil

Introduction: Mechanical loading induces remodeling of the periodontal ligament and the alveolar bone and is mediated by cytokines and chemokines. In this study, we investigated the kinetics of interleukin-6 and chemokine ligands 2 and 3 levels in periodontal ligaments subjected to orthodontic forces. Methods: We used 64 premolars in this split-mouth design study. The experimental group consisted of premolars subjected to a force of 0.980 N in the apical direction for 3 hours, 15 hours, 3 days, 12 days, or 21 days with a 0.017 3 0.025-in beta-titanium alloy cantilever. The contralateral teeth, without orthodontic appliances, were used as controls. The premolars were extracted for orthodontic reasons, and the periodontal ligaments were scraped for analysis of cytokine levels by ELISA. Results: Compared with the control group, an increase in chemokine ligand 2 was observed on days 3 and 12, and increases in interleukin-6 and chemokine ligand 3 were observed on day 12 in the experimental group. Conclusions: Our data demonstrated differential expressions of interleukin-6 and chemokine ligands 2 and 3 in periodontal ligaments after mechanical loading; this might reect the distinct roles of these molecules in the bone remodeling process. (Am J Orthod Dentofacial Orthop 2012;142:494-500)

rthodontic tooth movement is a combination of force-induced periodontal ligament (PDL) and alveolar bone remodeling.1-8 Mechanical stimuli exerted on a tooth cause vascular changes that lead to an aseptic and transient inammatory response
From the Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. a Postgraduate student, Department of Pediatric Dentistry and Orthodontics, Faculty of Dentistry. b Postgraduate student, Department of Morphology, Instituto de Ci^ encias Biol ogicas. c Associate professor, Department of Community and Preventive Dentistry, Faculty of Dentistry. d Associate professor, Department of Pediatric Dentistry and Orthodontics, Faculty of Dentistry. e Associate professor, Department of Oral Surgery and Pathology, Faculty of Dentistry. The authors report no commercial, proprietary, or nancial interest in the products or companies described in this article. Supported by the Fundac ao de Amparo a Pesquisas do Estado de Minas Gerais, ~ gico, and Pro the Conselho Nacional de Desenvolvimento Cient co e Tecnolo Reitoria de Pesquisa da Universidade Federal de Minas Gerais. Reprint requests to: Tarc lia Aparecida da Silva, Departamento de Cl nica, Patologia e Cirurgia Odontol ogicas, Faculdade de Odontologia, Universidade Federal ^nio Carlos 6627, CEP 31.270-901, Belo Horizonte, de Minas Gerais, Av Anto Minas Gerais, Brazil; e-mail, tarcilia@ufmg.br. Submitted, January 2012; revised and accepted, May 2012. 0889-5406/$36.00 Copyright 2012 by the American Association of Orthodontists. http://dx.doi.org/10.1016/j.ajodo.2012.05.012

in the periodontal tissues. Inammatory mediators are released and trigger biologic processes associated with alveolar bone remodeling such as bone resorption and new bone deposition.1-14 Cytokines are key mediators involved in bone remodeling under physiologic5,6,15,16 and mechanical loading-induced conditions.5-7,14,15,17,18 Interleukin-6 (IL-6) regulates the remodeling process by directly interacting with bone cells.7,10,11,15,16 Orthodontic forces result in an increase of IL-6 expression in periodontal tissues.8,10,16,18-23 Moreover, chemotactic cytokines (chemokines) are important signals for the trafcking, development, activity, and survival of bone cells.24-26 These molecules are expressed in periodontal tissues subjected to orthodontic forces.17,18,26-30 Studies regarding the patterns of cytokine and chemokine expression during orthodontic tooth movement have shown heterogeneity in their methods. In animal studies, periodontal tissue samples were analyzed under varying conditions.11,13,14,17,19,27,30 In studies with human subjects, samples from gingival crevicular uid10,12,20-22,31,32 and periodontal tissues23,28,29,33-35 have been obtained at different times by using distinct experimental protocols. PDL samples have been used to quantify mRNA levels of inammatory molecules

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after palatal expansion28,29 or have been tested in vitro under hypoxic treatment,34 loading of static compressive force,33 stretching-induced mechanical stress,35,36 or the inuence of proinammatory cytokines.37,38 Although the analysis of gingival crevicular uid is noninvasive,7,12,22,31 it provides results that represent indirect measurements of changes in the PDL.12,21,32 Thus, it might not be a specic indicator of periodontal remodeling in pressure or tension areas.31 The side independency of cytokine levels in gingival crevicular uid is probably a result of continuous circulation of the gingival crevicular uid in the PDL.31 Otherwise, we believe that the use of the PDL is a better representation of its environment. In this setting, PDL evaluation during orthodontic tooth movement is an important tool for clarifying the cellular and molecular responses to mechanical loading. This knowledge would be useful for orthodontic treatment because these molecules could be used as diagnostic markers and potential targets for therapeutic intervention.22,39,40 To our knowledge, no study has demonstrated the kinetics of inammatory mediators during orthodontic tooth movement with PDL samples in a split-mouth design. The aim of this study was to determine the kinetics of IL-6 and the chemokine ligands 2 and 3 (CCL2 and CCL3, formerly known as monocyte chemotactic protein-1 and macrophage inammatory protein 1-alpha, respectively) expression in the PDL during orthodontic treatment.
MATERIAL AND METHODS

Eighteen patients (9 male, 9 female), aged 11 to 40 years (median, 13.5 6 6.96 years), seen in the Department of Pediatric Dentistry and Orthodontics, Faculty of Dentistry, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil, were selected to participate in this study. Based on their clinical examinations and orthodontic records, these patients required extraction of the rst or second premolars for orthodontic reasons. The inclusion criteria were as follows: (1) healthy patients with no evidence of type 1 or type 2 diabetes mellitus or osteoporosis; (2) patients who had not taken systemic antibiotics, or anti-inammatory or hormonal drugs for 6 months before the study; (3) patients who required tooth extractions before treatment with xed appliances; and (4) patients with good periodontal health and no radiographic evidence of periodontal bone loss. This study was approved by the institutional ethics committee (protocol number 372/07). Informed consent was obtained from each participant and their guardians when the subject was less than 18 years of age. The experimental group consisted of extracted mandibular or maxillary premolars that had previously

received orthodontic mechanical loading. The contralateral teeth from the same arch without orthodontic appliances were used as the controls. In the experimental group, an orthodontic appliance consisting of 0.022 3 0.028-in light Roth tubes and brackets (Morelli Orthodontics, Sorocaba, S~ ao Paulo, Brazil) was bonded with Transbond XT (3M Unitek, Monrovia, Calif). A 0.017 3 0.025-in beta-titanium alloy cantilever and a 0.010-in metallic ligature (Morelli Orthodontics) were placed between the premolar and the rst molar on the same side (Fig 1, A) by an orthodontist (D.F.M.). A force in the apical direction was applied to the premolar. The force magnitude was 0.980 N, measured by a digital tensiometer (model FGV-1X; Nidec-Shimpo, Itasca, Ill) that was perpendicular to the cantilever (Fig 1, B). No other forces were applied to the teeth before or during this phase. The experimental teeth were randomly selected. If a patient had 4 premolars to be extracted, the pairs of teeth were allocated to 2 time points. The patients were instructed about proper oral hygiene. The teeth were extracted at the following times: 3 hours, 15 hours, 3 days, 12 days, or 21 days. The PDL of each extracted tooth was taken from the whole root surface. Before the extraction, the force was measured again. The PDL of an extracted tooth was immediately scraped by using a 13/14 Gracey curette (Maximus, Contagem, Minas Gerais, Brazil). The sample was placed in a sterile tube and kept frozen at 80 C for further analysis. Afterward, the PDL samples were weighed and homogenized in phosphate-buffered saline solution (0.4 mmol/L of sodium chloride and 10 mmol/L of sodium phosphate [NaPO4]) containing protease inhibitors (0.1 mmol/L of phenylmethylsulfonyl uoride [PMSF], 0.1 mmol/L of benzethonium chloride, 10 mmol/L of ethylenediamine tetraacetic acid [EDTA], and 0.01 mg/ mL of aprotinin A) and 0.05% Tween-20 at 1 mg/mL. The mixture was centrifuged (10,000 rpm) for 10 minutes at 4 C. The supernatant was then collected and assayed with an enzyme-linked immunosorbent assay (ELISA). The concentrations of IL-6, CCL2, and CCL3 were evaluated by using commercially available kits according to the manufacturers instructions (R&D Systems, Minneapolis, Minn). The results were expressed as picograms of cytokine per 100 mg of tissue.
Statistical analysis

The Shapiro-Wilks test was used to assess the quantitative variables. There was no normality of cytokines (P \0.05); thus, nonparametric tests were used. The Mann-Whitney test was performed to verify the inuence of sex on the cytokines. The Kruskal-Wallis test was used to compare cytokine levels and types of teeth.

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2 mandibular second premolars). A mean of 6.4 pairs of teeth was allocated at each time point. The demographic description of the participants is described in the Table. The appliances were well tolerated. The initially applied force magnitude of 0.980 N was gradually reduced to the median of 0.892 6 0.097 N before the extraction of the experimental teeth. Sex, type of tooth, age of the participants, and experimental force had no inuence on IL-6, CCL-2, or CCL-3 concentrations at any time point (P .0.05). The concentrations of IL-6, CCL2, and CCL3 are shown in Figure 2. After 3 hours of force application, there were no signicant differences between the experimental and control groups for any of the evaluated molecules (P .0.05). Although there was no signicant difference at 15 hours, the results showed a tendency toward an increase of IL-6 levels (P 5 0.068). On day 3, the expression of CCL2 was greater in the experimental group than in its control group (P 5 0.028). On day 12, IL-6 (P 5 0.046), CCL2 (P 5 0.028), and CCL3 (P 5 0.046) levels were augmented in the experimental group. On day 21, a reduction of these inammatory mediators was observed; therefore, no difference was detected (P .0.05). In the experimental group, correlations of IL-6 with CCL2 and CCL3 (Spearman's correlation coefcient/Rs 5 0.405, P 5 0.021; Spearman's correlation coefcient/Rs 5 0.382, P 5 0.031, respectively), and CCL2 with CCL3 (Spearman's correlation coefcient/Rs 5 0.426, P 5 0.015), were observed.
DISCUSSION

Fig 1. A, An activated orthodontic appliance consisting of a 0.022 3 0.028-in bracket and tube bonded with light cure adhesive and a 0.017 3 0.025-in beta-titanium alloy cantilever; B, the force was set at 0.980 N in the apical direction, and the force magnitude was measured with a digital tensiometer.

The Spearman correlation was used to assess the association between age and cytokines. The Wilcoxon test was used to assess the inuence of cytokines on the experiment at each time point. The analysis was performed for each time point separately. Thus, although there was more than 1 pair of tooth per patient, it is possible to consider the sample units independent. The level of statistical signicance was set at P \0.05. All statistical evaluations were performed with SPSS software (version 19.0; SPSS, Chicago, Ill).
RESULTS

A total of 64 premolars were obtained (34 maxillary rst premolars, 28 mandibular rst premolars, and

Orthodontic tooth movement is achieved through remodeling of the PDL and the alveolar bone, triggered by the force-induced biologic response of the periodontium.1-3,6,7,14,15 Cytokines and chemokines are the key players in the PDL's response to mechanical loadinginduced conditions.2,3,7,14,15 This is the rst study of the kinetics of cytokine and chemokine expression in the PDL induced by orthodontic mechanical loading in a split-mouth design. We found different patterns of expression of IL-6, CCL2, and CCL3 at the various time points after applying an orthodontic force. As observed in experimental studies, mechanical loading with orthodontic appliances results in the production of signaling molecules (eg, cytokines, chemokines, growth factors, and others) in the periodontal tissues11,17,19,27-30 and gingival crevicular uid.7,10,20-22,31,32,41 Of these, the cytokine IL-6 regulates immune responses in inammation sites10,37 and has an autocrine/paracrine activity that stimulates osteoclast formation and bone-resorbing activity.10,16 It plays an important role in local regulation of bone

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Table. Demographic distribution of the participants (n 5 18) at the time points and the initial and nal forces
Time point 3 hours Patient 1 2 3 4 5 6 7 n57 6 7 8 9 10 11 n56 3 10 12 13 15 16 n56 9 13 14 15 17 18 n56 1 2 4 8 11 12 17 n56 n 5 18 Sex F F M F F M F M F M M F M M F F M F M M M M F F M F F F M M F F M59 F59 Age (y) 12 14 23 17 22 14 12 Median 14 6 4.57 4 12 16 12 12 17 Median 13 6 2.22 23 12 13 18 40 11 Median 15.5 6 11.04 12 18 13 40 11 12 Median 12.5 6 11.21 12 14 17 16 17 13 11 Median 14 6 2.42 Median 13.5 6 6.96 Control tooth 14 14 24 14 14 24 44 34 24 14 44 34 14 34 14 24 34 24 24 14 24 14 34 24 34 44 45 44 34 44 44 34 Experimental tooth 24 24 14 14 24 14 34 44 14 24 34 44 24 44 24 14 44 14 14 24 14 24 44 14 44 34 35 34 44 34 34 44 Initial force (N) 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 0.980 Final force(N) 0.980 0.980 0.980 0.980 0.980 0.980 0.980 Median 0.980 6 0 0.921 0.980 0.892 0.976 0.980 0.967 Median 0.972 6 0.037 0.961 0.686 0.960 0.860 0.872 0.787 Median 0.866 6 0.105 0.768 0.885 0.785 0.892 0.902 0.762 Median 0.835 6 0.067 0.790 0.690 0.835 0.778 0.760 0.745 0.877 Median 0.778 6 0.061 Median 0.892 6 0.097

15 hours

3 days

12 days

21 days

Total

M, Male; F, female; 14, maxillary right rst premolar; 24, maxillary left rst premolar; 34, mandibular left rst premolar; 35, mandibular left second premolar 44, mandibular left rst premolar; 45, mandibular right second premolar.

remodeling and the acute inammation at the beginning of orthodontic tooth movement.10,37 IL-6 is detected in gingival crevicular uid10,12,20-22,31 and PDLs under orthodontic force.11,33,37,38 In-vitro studies have demonstrated that IL-6 is induced after 12 hours of static compressive force by PDL cells33 and is enhanced by proinammatory cytokines such as IL1b,37,38 IL-1a, and TNF-a.37 In rats, mechanical loading induced the production of IL-6 on day 3, followed by a decrease on day 7, and reaching control levels on day 10.11 Human studies with gingival crevicular uid demonstrated, in the experimental group, an increase of IL-6 at 24 hours,10,21 and no signicant levels on days 7 and 21,12 months 2 and 3,21 or months 612 and

12.22 Our results demonstrate increases of IL-6 after 15 hours and 12 days of force application. On day 21, IL-6 reached control levels. Our data partially agree with the literature10,21; however, comparison was sometimes difcult because of different experimental protocols.11,12,21,22 Taken together, we can consider that IL-6 is produced at the beginning of orthodontic tooth movement, and its expression decreases over time. A physiologic homeostasis is probably reached through down regulation via a feedback mechanism.21 At later stages, other mediators, such as chemokines, might govern bone remodeling process.17,27,29,30 Interestingly, IL-6 can induce CCL227 and enhance the effects of CCL3 on osteoclast formation.24

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Fig 2. Median levels of: A, IL-6; B, CCL2; and C, CCL3 in the PDLs after 3 hours, 15 hours, 3 days, 12 days, and 21 days of mechanical loading. The experimental teeth were submitted to 0.980 N of mechanical loading, and the contralateral teeth were used as the controls. PDL samples of 64 teeth were included in this study. The data were expressed as the medians 6 standard deviations. *P \0.05 comparing the groups at the same time point with the Wilcoxon test.

The chemokines CCL2 and CCL3 guide the migration of osteoclasts to bone tissues through interactions with CC chemokine receptors (CCR) such as CCR2 and CCR5/ CCR1, respectively, expressed on the surfaces of osteoclasts.13,39,42,43 Furthermore, CCL2 and CCL3 induce

osteoclast differentiation, activation, and resorbing activity.25,39,43 An in-vitro study with PDL cells demonstrated that intermittent stretching-induced mechanical stress up-regulated the expression of CCL2 and CCL3.35 In mouse models, mechanical loading signicantly increased the levels of chemokines after 12 hours (CCL217), 3 days (CCL227,30 and CCL330), and 7 days (CCL2 and CCL330), reaching control levels on day 10.27 We found no reports regarding CCL2 and CCL3 expression in gingival crevicular uid after orthodontic stimuli. Moreover, after palatal expansion, the compression side of the PDL showed higher expression levels of CCL2 and CCL3.29 Although we used light forces and did not compare compression vs tension sides, our results also demonstrate that the experimental groups showed elevations in CCL2 (days 3 and 12) and CCL3 (day 12) levels in the PDLs. Therefore, mechanical transduction might be responsible for the early release of IL-6 at 15 hours, which might be associated with the later induction of CCL2 on day 3. Both molecules might activate and recruit cells from monocyte or macrophage lineage to the pressure side and contribute to osteoclast formation.10,16,25,27,39,43 A signicant number of positive preosteoclasts was observed in the PDL and the bone surface on day 3.13 On day 7, histologic analysis demonstrated no frontal resorption or tooth movement caused by PDL compression and alveolar bone bending.1 In contrast, on day 14, histologic ndings have shown widened PDL spaces with active frontal alveolar bone resorption and tooth movement.1 These ndings might be associated with the induction of IL-6, CCL2, and CCL3 that we observed on day 12. On day 21, there was little or no evidence of osteoclastic activity, either in frontal or undermining resorption.1 This scenario might explain the low levels of IL-6, CCL2, and CCL3 reached on day 21, as veried in our study. After force application, both matrix strain and uid ow in the PDL and bone cause deformation of cells.8 Through integrin signaling and other transduction pathways, mediators are produced to activate cells (eg, broblasts, osteoblasts, osteocytes, and osteoclasts) involved in bone and PDL remodeling processes8,26,36 during orthodontic tooth movement.8 Direct resorption is associated with light force applications (0.490-0.890 N), tissue cell preservation, and vascular patency. Undermining resorption and hyalinization are associated with heavy or necrotizing forces causing crushing injuries to PDL tissues, cell death, hemostasis, and cell-free PDLs and adjacent alveolar bone zones.44 Unfortunately, during orthodontic tooth movement, some hyalinization appeared to be inevitable,1,15,45 even with forces as low as 0.294 N.15 These hyalinized areas can last from 4 to 49 days.1,7,15,45,46 An inevitable delay of tooth

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movement occurs because of a delay in induction of cell differentiation in the marrow spaces. In addition, a considerable thickness of bone needs to be removed from the underside before any tooth movement can occur.1,6-8,46 It results in activation of the cells participating in the resorption of the hyaline zone and alveolar bone, leading to the remodeling of the compressed periodontium.1,6,7,15,27 Osteoclasts must be formed to remove bone from the compressed area of the PDL from adjacent teeth and hyalinized areas, whereas osteoblasts are needed to form new bone on the tension side.1,7,15,47 Therefore, during the different phases of tooth movement, structural changes in the bone and periodontal tissues occur, altering the local biomechanical environment1,47; this leads to modulation of the biologic response.47 This event might explain the different patterns of expression of cytokines and chemokines at the different time points in this study. Studies concerning orthodontic tooth movement have some limitations: (1) histologic analysis is limited since the teeth moved with orthodontic appliances must be extracted, disrupting the PDL, and the surrounding bone cannot be analyzed47; (2) interindividual variations in mechanobiologic responses are most likely due to differences in bone and PDL cell populations, genomes, and protein expression patterns44; and (3) there are few studies (with different experimental protocols) showing cytokine and chemokine expression in PDL samples during orthodontic tooth movement.28,29 These difculties challenge researchers to clarify these issues.
CONCLUSIONS

1.

2.

This is the rst study demonstrating the kinetics of cytokine and chemokine expression during orthodontic tooth movement with PDL samples in a split-mouth design. Further studies are required to clarify differential cellular and molecular responses to mechanical loading at different times during orthodontic tooth movement. We found elevated levels of IL-6, CCL2, and CCL3 at distinct time points after mechanical loading. These ndings might indicate distinct roles of these molecules in the bone remodeling process.

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