ORIGINAL ARTICLE

Kinetics of interleukin-6 and chemokine ligands2 and 3 expression of periodontal tissues duringorthodontic tooth movement

Davidson Fr�ois Madureira,a Silvana de Albuquerque Taddei,b Mauro Henrique Nogueira Guimar~aes Abreu,c

Henrique Pretti,d Elizabeth Maria Bastos Lages,d and Tarcilia Aparecida da Silvae

Belo Horizonte, Minas Gerais, Brazil

FromBrazilaPostgFaculbPostBiol�ogcAssoFaculdAssoFaculeAssoDentiThe aproduSuppothe CReitorReprinlogiade MMinasSubm0889-Copyrhttp:/

494

Introduction: Mechanical loading induces remodeling of the periodontal ligament and the alveolar bone and ismediated by cytokines and chemokines. In this study, we investigated the kinetics of interleukin-6 andchemokine ligands 2 and 3 levels in periodontal ligaments subjected to orthodontic forces. Methods: Weused 64 premolars in this split-mouth design study. The experimental group consisted of premolars subjectedto a force of 0.980 N in the apical direction for 3 hours, 15 hours, 3 days, 12 days, or 21 days with a 0.0173 0.025-in beta-titanium alloy cantilever. The contralateral teeth, without orthodontic appliances, were usedas controls. The premolars were extracted for orthodontic reasons, and the periodontal ligaments werescraped for analysis of cytokine levels by ELISA. Results:Compared with the control group, an increase in che-mokine ligand 2 was observed on days 3 and 12, and increases in interleukin-6 and chemokine ligand 3 wereobserved on day 12 in the experimental group. Conclusions: Our data demonstrated differential expressionsof interleukin-6 and chemokine ligands 2 and 3 in periodontal ligaments after mechanical loading; this mightreflect the distinct roles of these molecules in the bone remodeling process. (Am J Orthod Dentofacial Orthop2012;142:494-500)

Orthodontic tooth movement is a combination offorce-induced periodontal ligament (PDL) andalveolar bone remodeling.1-8 Mechanical

stimuli exerted on a tooth cause vascular changes thatlead to an aseptic and transient inflammatory response

the Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais,.raduate student, Department of Pediatric Dentistry and Orthodontics,ty of Dentistry.graduate student, Department of Morphology, Instituto de Cienciasicas.ciate professor, Department of Community and Preventive Dentistry,ty of Dentistry.ciate professor, Department of Pediatric Dentistry and Orthodontics,ty of Dentistry.ciate professor, Department of Oral Surgery and Pathology, Faculty ofstry.uthors report no commercial, proprietary, or financial interest in thects or companies described in this article.rted by the Fundac~ao de Amparo a Pesquisas do Estado de Minas Gerais,onselho Nacional de Desenvolvimento Cient�ıfico e Tecnol�ogico, and Pr�o-ia de Pesquisa da Universidade Federal de Minas Gerais.t requests to: Tarc�ılia Aparecida da Silva, Departamento de Cl�ınica, Pato-e Cirurgia Odontol�ogicas, Faculdade de Odontologia, Universidade Federalinas Gerais, Av Antonio Carlos 6627, CEP 31.270-901, Belo Horizonte,Gerais, Brazil; e-mail, tarcilia@ufmg.br.itted, January 2012; revised and accepted, May 2012.5406/$36.00ight � 2012 by the American Association of Orthodontists./dx.doi.org/10.1016/j.ajodo.2012.05.012

in the periodontal tissues. Inflammatory mediators arereleased and trigger biologic processes associated withalveolar bone remodeling such as bone resorption andnew bone deposition.1-14

Cytokines are key mediators involved in boneremodeling under physiologic5,6,15,16 and mechanicalloading-induced conditions.5-7,14,15,17,18 Interleukin-6(IL-6) regulates the remodeling process by directly inter-acting with bone cells.7,10,11,15,16 Orthodontic forcesresult in an increase of IL-6 expression in periodontal tis-sues.8,10,16,18-23 Moreover, chemotactic cytokines(chemokines) are important signals for the trafficking,development, activity, and survival of bone cells.24-26

These molecules are expressed in periodontal tissuessubjected to orthodontic forces.17,18,26-30

Studies regarding the patterns of cytokine and che-mokine expression during orthodontic tooth movementhave shown heterogeneity in their methods. In animalstudies, periodontal tissue samples were analyzed undervarying conditions.11,13,14,17,19,27,30 In studies withhuman subjects, samples from gingival crevicularfluid10,12,20-22,31,32 and periodontal tissues23,28,29,33-35

have been obtained at different times by using distinctexperimental protocols. PDL samples have been usedto quantify mRNA levels of inflammatory molecules

Madureira et al 495

after palatal expansion28,29 or have been tested in vitrounder hypoxic treatment,34 loading of static compres-sive force,33 stretching-induced mechanical stress,35,36

or the influence of proinflammatory cytokines.37,38

Although the analysis of gingival crevicular fluid isnoninvasive,7,12,22,31 it provides results that representindirect measurements of changes in the PDL.12,21,32

Thus, it might not be a specific indicator of periodontalremodeling in pressure or tension areas.31 The side inde-pendency of cytokine levels in gingival crevicular fluid isprobably a result of continuous circulation of the gingi-val crevicular fluid in the PDL.31 Otherwise, we believethat the use of the PDL is a better representation of itsenvironment. In this setting, PDL evaluation during or-thodontic tooth movement is an important tool for clar-ifying the cellular andmolecular responses tomechanicalloading. This knowledge would be useful for orthodontictreatment because these molecules could be used as di-agnostic markers and potential targets for therapeuticintervention.22,39,40 To our knowledge, no study hasdemonstrated the kinetics of inflammatory mediatorsduring orthodontic tooth movement with PDL samplesin a split-mouth design. The aim of this study was todetermine the kinetics of IL-6 and the chemokine ligands2 and 3 (CCL2 and CCL3, formerly known as monocytechemotactic protein-1 and macrophage inflammatoryprotein 1-alpha, respectively) expression in the PDLduring orthodontic treatment.

MATERIAL AND METHODS

Eighteen patients (9 male, 9 female), aged 11 to40 years (median, 13.56 6.96 years), seen in the Depart-ment of Pediatric Dentistry and Orthodontics, Faculty ofDentistry, Universidade Federal de Minas Gerais, BeloHorizonte, Minas Gerais, Brazil, were selected to partici-pate in this study. Based on their clinical examinationsand orthodontic records, these patients required extrac-tion of the first or second premolars for orthodonticreasons. The inclusion criteria were as follows: (1) healthypatients with no evidence of type 1 or type 2 diabetesmellitus or osteoporosis; (2) patients who had not takensystemic antibiotics, or anti-inflammatory or hormonaldrugs for 6 months before the study; (3) patients who re-quired tooth extractions before treatment with fixed ap-pliances; and (4) patients with good periodontal healthand no radiographic evidence of periodontal bone loss.This study was approved by the institutional ethics com-mittee (protocol number 372/07). Informed consent wasobtained from each participant and their guardians whenthe subject was less than 18 years of age.

The experimental group consisted of extractedmandibular or maxillary premolars that had previously

American Journal of Orthodontics and Dentofacial Orthoped

received orthodontic mechanical loading. The contralat-eral teeth from the same arch without orthodontic appli-ances were used as the controls. In the experimentalgroup, an orthodontic appliance consisting of 0.022 30.028-in light Roth tubes and brackets (Morelli Ortho-dontics, Sorocaba, S~ao Paulo, Brazil) was bonded withTransbond XT (3M Unitek, Monrovia, Calif). A 0.017 30.025-in beta-titanium alloy cantilever and a 0.010-inmetallic ligature (Morelli Orthodontics) were placedbetween the premolar and the first molar on the sameside (Fig 1, A) by an orthodontist (D.F.M.). A force inthe apical direction was applied to the premolar. Theforce magnitude was 0.980 N, measured by a digital ten-siometer (model FGV-1X; Nidec-Shimpo, Itasca, Ill) thatwas perpendicular to the cantilever (Fig 1, B). No otherforces were applied to the teeth before or during thisphase. The experimental teeth were randomly selected.If a patient had 4 premolars to be extracted, the pairsof teeth were allocated to 2 time points. The patientswere instructed about proper oral hygiene.

The teeth were extracted at the following times:3 hours, 15 hours, 3 days, 12 days, or 21 days. ThePDL of each extracted tooth was taken from the wholeroot surface. Before the extraction, the force was mea-sured again. The PDL of an extracted tooth was immedi-ately scraped by using a 13/14 Gracey curette (Maximus,Contagem, Minas Gerais, Brazil). The sample was placedin a sterile tube and kept frozen at �80�C for furtheranalysis. Afterward, the PDL samples were weighedand homogenized in phosphate-buffered saline solution(0.4 mmol/L of sodium chloride and 10 mmol/L of so-dium phosphate [NaPO4]) containing protease inhibitors(0.1 mmol/L of phenylmethylsulfonyl fluoride [PMSF],0.1 mmol/L of benzethonium chloride, 10 mmol/L ofethylenediamine 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 min-utes at 4�C. The supernatant was then collected and as-sayed with an enzyme-linked immunosorbent assay(ELISA). The concentrations of IL-6, CCL2, and CCL3were evaluated by using commercially available kits ac-cording to the manufacturer’s instructions (R&D Sys-tems, Minneapolis, Minn). The results were expressedas picograms of cytokine per 100 mg of tissue.

Statistical analysis

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

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Fig 1. A,An activated orthodontic appliance consisting ofa 0.022 3 0.028-in bracket and tube bonded with lightcure adhesive and a 0.0173 0.025-in beta-titanium alloycantilever; B, the force was set at 0.980 N in the apical di-rection, and the forcemagnitudewasmeasuredwith adig-ital tensiometer.

496 Madureira et al

The Spearman correlation was used to assess the associ-ation between age and cytokines. The Wilcoxon test wasused to assess the influence of cytokines on the experi-ment at each time point. The analysis was performed foreach time point separately. Thus, although there wasmore than 1 pair of tooth per patient, it is possible toconsider the sample units independent. The level of sta-tistical significance was set at P \0.05. All statisticalevaluations were performed with SPSS software (version19.0; SPSS, Chicago, Ill).

RESULTS

A total of 64 premolars were obtained (34 maxillaryfirst premolars, 28 mandibular first premolars, and

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2 mandibular second premolars). A mean of 6.4 pairsof teeth was allocated at each time point. The demo-graphic description of the participants is described inthe Table. The appliances were well tolerated. The ini-tially applied force magnitude of 0.980 N was graduallyreduced to the median of 0.892 6 0.097 N before theextraction of the experimental teeth. Sex, type of tooth,age of the participants, and experimental force had noinfluence on IL-6, CCL-2, or CCL-3 concentrations atany time point (P .0.05).

The concentrations of IL-6, CCL2, and CCL3 areshown in Figure 2. After 3 hours of force application,there were no significant differences between the exper-imental and control groups for any of the evaluatedmolecules (P .0.05). Although there was no significantdifference at 15 hours, the results showed a tendencytoward an increase of IL-6 levels (P 5 0.068). On day3, the expression of CCL2 was greater in the experimen-tal group than in its control group (P 5 0.028). Onday 12, IL-6 (P 5 0.046), CCL2 (P 5 0.028), and CCL3(P 5 0.046) levels were augmented in the experimentalgroup. On day 21, a reduction of these inflammatorymediators was observed; therefore, no difference wasdetected (P .0.05). In the experimental group, correla-tions of IL-6 with CCL2 and CCL3 (Spearman's correla-tion coefficient/Rs 5 0.405, P 5 0.021; Spearman'scorrelation coefficient/Rs 5 0.382, P 5 0.031, respec-tively), and CCL2 with CCL3 (Spearman's correlation co-efficient/Rs 5 0.426, P 5 0.015), were observed.

DISCUSSION

Orthodontic tooth movement is achieved throughremodeling of the PDL and the alveolar bone, triggeredby the force-induced biologic response of the periodon-tium.1-3,6,7,14,15 Cytokines and chemokines are the keyplayers in the PDL's response to mechanical loading-induced conditions.2,3,7,14,15 This is the first study ofthe kinetics of cytokine and chemokine expression inthe PDL induced by orthodontic mechanical loading ina split-mouth design. We found different patterns ofexpression of IL-6, CCL2, and CCL3 at the various timepoints after applying an orthodontic force.

As observed in experimental studies, mechanicalloading with orthodontic appliances results in theproduction of signaling molecules (eg, cytokines,chemokines, growth factors, and others) in the peri-odontal tissues11,17,19,27-30 and gingival crevicularfluid.7,10,20-22,31,32,41 Of these, the cytokine IL-6 regu-lates immune responses in inflammation sites10,37 andhas an autocrine/paracrine activity that stimulatesosteoclast formation and bone-resorbing activity.10,16

It plays an important role in local regulation of bone

Journal of Orthodontics and Dentofacial Orthopedics

Table. Demographic distribution of the participants (n 5 18) at the time points and the initial and final forces

Time point Patient Sex Age (y) Control tooth Experimental tooth Initial force (N) Final force(N)3 hours 1 F 12 14 24 0.980 0.980

2 F 14 14 24 0.980 0.9803 M 23 24 14 0.980 0.9804 F 17 14 14 0.980 0.9805 F 22 14 24 0.980 0.9806 M 14 24 14 0.980 0.9807 F 12 44 34 0.980 0.980

n 5 7 Median 14 6 4.57 Median 0.980 6 015 hours 6 M 4 34 44 0.980 0.921

7 F 12 24 14 0.980 0.9808 M 16 14 24 0.980 0.8929 M 12 44 34 0.980 0.976

10 F 12 34 44 0.980 0.98011 M 17 14 24 0.980 0.967

n 5 6 Median 13 6 2.22 Median 0.972 6 0.0373 days 3 M 23 34 44 0.980 0.961

10 F 12 14 24 0.980 0.68612 F 13 24 14 0.980 0.96013 M 18 34 44 0.980 0.86015 F 40 24 14 0.980 0.87216 M 11 24 14 0.980 0.787

n 5 6 Median 15.5 6 11.04 Median 0.866 6 0.10512 days 9 M 12 14 24 0.980 0.768

13 M 18 24 14 0.980 0.88514 M 13 14 24 0.980 0.78515 F 40 34 44 0.980 0.89217 F 11 24 14 0.980 0.90218 M 12 34 44 0.980 0.762

n 5 6 Median 12.5 6 11.21 Median 0.835 6 0.06721 days 1 F 12 44 34 0.980 0.790

2 F 14 45 35 0.980 0.6904 F 17 44 34 0.980 0.8358 M 16 34 44 0.980 0.778

11 M 17 44 34 0.980 0.76012 F 13 44 34 0.980 0.74517 F 11 34 44 0.980 0.877

n 5 6 Median 14 6 2.42 Median 0.778 6 0.061Total n 5 18 M 5 9

F 5 9Median 13.5 6 6.96 Median 0.892 6 0.097

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

Madureira et al 497

remodeling and the acute inflammation at thebeginning of orthodontic tooth movement.10,37 IL-6 isdetected in gingival crevicular fluid10,12,20-22,31 andPDLs under orthodontic force.11,33,37,38 In-vitro studieshave demonstrated that IL-6 is induced after 12 hoursof static compressive force by PDL cells33 and is en-hanced by proinflammatory cytokines such as IL-1b,37,38 IL-1a, and TNF-a.37 In rats, mechanical loadinginduced the production of IL-6 on day 3, followed bya decrease on day 7, and reaching control levels onday 10.11 Human studies with gingival crevicular fluiddemonstrated, in the experimental group, an increaseof IL-6 at 24 hours,10,21 and no significant levels ondays 7 and 21,12 months 2 and 3,21 or months 612 and

American Journal of Orthodontics and Dentofacial Orthoped

12.22 Our results demonstrate increases of IL-6 after15 hours and 12 days of force application. On day 21,IL-6 reached control levels. Our data partially agreewith the literature10,21; however, comparison wassometimes difficult because of different experimentalprotocols.11,12,21,22 Taken together, we can considerthat IL-6 is produced at the beginning of orthodontictooth movement, and its expression decreases overtime. A physiologic homeostasis is probably reachedthrough 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 theeffects of CCL3 on osteoclast formation.24

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Fig 2. Median levels of:A, IL-6;B,CCL2; andC,CCL3 inthe PDLs after 3 hours, 15 hours, 3 days, 12 days, and21 days of mechanical loading. The experimental teethwere submitted to 0.980 N of mechanical loading, andthe contralateral teeth were used as the controls. PDLsamples of 64 teeth were included in this study. Thedata were expressed as the medians 6 standard devia-tions. *P\0.05 comparing the groups at the same timepoint with the Wilcoxon test.

498 Madureira et al

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

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osteoclast differentiation, activation, and resorbingactivity.25,39,43 An in-vitro study with PDL cells demon-strated that intermittent stretching-induced mechanicalstress up-regulated the expression of CCL2 and CCL3.35

In mouse models, mechanical loading significantlyincreased the levels of chemokines after 12 hours(CCL217), 3 days (CCL227,30 and CCL330), and 7 days(CCL2 and CCL330), reaching control levels on day10.27 We found no reports regarding CCL2 and CCL3expression in gingival crevicular fluid after orthodonticstimuli. Moreover, after palatal expansion, the compres-sion side of the PDL showed higher expression levels ofCCL2 and CCL3.29 Although we used light forces anddid not compare compression vs tension sides, our re-sults also demonstrate that the experimental groupsshowed elevations in CCL2 (days 3 and 12) and CCL3(day 12) levels in the PDLs. Therefore, mechanical trans-duction might be responsible for the early release of IL-6at 15 hours, which might be associated with the laterinduction of CCL2 on day 3. Both molecules mightactivate and recruit cells from monocyte or macrophagelineage to the pressure side and contribute to osteoclastformation.10,16,25,27,39,43 A significant number ofpositive preosteoclasts was observed in the PDL andthe bone surface on day 3.13 On day 7, histologic analysisdemonstrated no frontal resorption or tooth movementcaused by PDL compression and alveolar bone bending.1

In contrast, on day 14, histologic findings have shownwidened PDL spaces with active frontal alveolar boneresorption and tooth movement.1 These findings mightbe associated with the induction of IL-6, CCL2, andCCL3 that we observed on day 12. On day 21, therewas little or no evidence of osteoclastic activity, eitherin frontal or undermining resorption.1 This scenariomight explain the low levels of IL-6, CCL2, and CCL3reached on day 21, as verified in our study.

After force application, both matrix strain and fluidflow in the PDL and bone cause deformation of cells.8

Through integrin signaling and other transductionpathways, mediators are produced to activate cells (eg,fibroblasts, osteoblasts, osteocytes, and osteoclasts) in-volved in bone and PDL remodeling processes8,26,36

during orthodontic tooth movement.8 Direct resorptionis associated with light force applications (0.490-0.890N), tissue cell preservation, and vascular patency. Under-mining resorption and hyalinization are associated withheavy or necrotizing forces causing crushing injuries toPDL tissues, cell death, hemostasis, and cell-free PDLsand adjacent alveolar bone zones.44 Unfortunately,during orthodontic tooth movement, some hyalinizationappeared to be inevitable,1,15,45 even with forces as lowas 0.294 N.15 These hyalinized areas can last from 4 to49 days.1,7,15,45,46 An inevitable delay of tooth

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Madureira et al 499

movement occurs because of a delay in induction of celldifferentiation in the marrow spaces. In addition,a considerable thickness of bone needs to be removedfrom the underside before any tooth movement canoccur.1,6-8,46 It results in activation of the cellsparticipating in the resorption of the hyaline zone andalveolar bone, leading to the remodeling of thecompressed periodontium.1,6,7,15,27 Osteoclasts mustbe formed to remove bone from the compressed areaof the PDL from adjacent teeth and hyalinized areas,whereas osteoblasts are needed to form new bone onthe tension side.1,7,15,47 Therefore, during the differentphases of tooth movement, structural changes in thebone and periodontal tissues occur, altering the localbiomechanical environment1,47; this leads tomodulation of the biologic response.47 This event mightexplain the different patterns of expression of cytokinesand chemokines at the different time points in this study.

Studies concerning orthodontic tooth movementhave some limitations: (1) histologic analysis is limitedsince the teeth moved with orthodontic appliancesmust be extracted, disrupting the PDL, and the sur-rounding bone cannot be analyzed47; (2) interindivid-ual variations in mechanobiologic responses are mostlikely due to differences in bone and PDL cell popula-tions, genomes, and protein expression patterns44; and(3) there are few studies (with different experimentalprotocols) showing cytokine and chemokine expressionin PDL samples during orthodontic tooth move-ment.28,29 These difficulties challenge researchers toclarify these issues.

CONCLUSIONS

1. This is the first study demonstrating the kinetics ofcytokine and chemokine expression during ortho-dontic tooth movement with PDL samples ina split-mouth design. Further studies are requiredto clarify differential cellular and molecular re-sponses to mechanical loading at different timesduring orthodontic tooth movement.

2. We found elevated levels of IL-6, CCL2, and CCL3 atdistinct time points after mechanical loading. Thesefindings might indicate distinct roles of these mole-cules in the bone remodeling process.

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