INTRODUCTION

Bone remodeling is a lifelong process, and it is achieved by boneresorption at compression sites and new bone formation on the tension

sides (Thilander et al., 2000; Klein-Nulend et al., 2005). The availability oftransgenic-mutated mice, including those with genes related to bonemetabolism, for investigating the effect of mechanical loading on boneremodeling is of great interest to enhance our understanding of the processof orthodontic tooth movement.

Orthodontic tooth movement is achieved by the process of osteoclasticbone resorption and osteoblastic bone formation. Numerous substances—such as hormones, cytokines, growth factors, and bone matrixconstituents—are involved with osteoclastogenesis (Boyle et al., 2003;Kohno et al., 2003; Teitelbaum, 2007). For example, pro-inflammatorycytokines, such as TNF-!, are thought to play a role in bone remodeling(Horowitz et al., 2001; Wise and Yao, 2003) and osteoclast differentiation(Azuma et al., 2000). In rats (Ogasawara et al., 2004) and in humans(Basaran et al., 2006), orthodontic tooth movement increases the levels ofTNF-! in the periodontal tissues. TNF-! induces several biologicalresponses via 2 cell-surface receptors, namely, TNF-receptor type 1 (p55)and TNF receptor type 2 (p75) (Peschon et al., 1998). TNF-! enhancesbasal osteoclastogenesis, in vitro, via p55 and suppresses it via p75 (Abu-Amer et al., 2000). In addition to modifying processes directly associatedwith bone movement, TNF-! may also induce mediators of theinflammatory process, which will then influence osteoclast recruitment andfunction. Recent studies from our group have shown that TNF-! maymodulate the expression of the chemokines Regulated upon Activaton,Normal T-cell Expressed and Secreted (RANTES/CCL5) (Garlet et al.,2007) and monocyte chemoattractant protein-1 (MCP-1/CCL2) (Barcelos etal., 2005), and, hence, influence the outcome of the inflammatory response.

Chemokines are a large family of chemotactic cytokines that providekey signals for the trafficking and homing of specific subpopulations ofleukocytes and other cells in both physiological and pathological processes(Rossi and Zlotnik, 2000; Gerard and Rollins, 2001). Among the manymembers of the chemokine family, 2 chemokines were evaluated in thepresent study, CCL2 and CCL5. Both CCL2 and CCL5 may be modulatedby TNF-! (Barcelos et al., 2005; Garlet et al., 2007) and play a role inmechanisms underlying osteoclast recruitment and activation (Chae et al.,2002; Graves et al., 2002). CCL5 has been shown to induce osteoclastchemotaxis (Yu et al., 2004). A recent study showed that CCL2 and CCL5are involved in human osteoclast differentiation from monocyte precursorsin vitro (Kim et al., 2006).

A better understanding of cellular and molecular responses tomechanical loading is crucial for future improvements in orthodontictreatment. In the present study, we applied a tooth movement modeldeveloped in mice to investigate the role of TNF receptor signaling in

ABSTRACTOrthodontic tooth movement is dependent onosteoclast activity. Tumor necrosis factor (TNF)-!plays an important role, directly or via chemokinerelease, in osteoclast recruitment and activation.This study aimed to investigate whether the TNFreceptor type 1 (p55) influences these events and,consequently, orthodontic tooth movement. Anorthodontic appliance was placed in wild-typemice (WT) and p55-deficient mice (p55-/-). Levelsof TNF-! and 2 chemokines (MCP-1/CCL2,RANTES/CCL5) were evaluated in periodontaltissues. A significant increase in CCL2 and TNF-!was observed in both groups after 12 hrs ofmechanical loading. However, CCL5 levelsremained unchanged in p55-/- mice at this time-point. The number of TRAP-positive osteoclastsin p55-/- mice was significantly lower than that inWT mice. Also, there was a significantly smallerrate of tooth movement in p55-/- mice. Analysis ofour data suggests that the TNFR-1 plays asignificant role in orthodontic tooth movementthat might be associated with changes in CCL5levels.

KEY WORDS: orthodontic tooth movement,mechanical loading, bone remodeling, TNF-!,chemokines.

Received February 20, 2007; Last revision July 11, 2007;Accepted August 2, 2007

A supplemental appendix to this article is publishedelectronically only at http://www.dentalresearch.org.

The Role of Tumor Necrosis FactorReceptor Type 1 in OrthodonticTooth Movement

I. Andrade, Jr.1, T.A. Silva2*, G.A.B. Silva3,A.L. Teixeira4, and M.M. Teixeira5

1Department of Orthodontics, Pontifícia UniversidadeCatólica de Minas Gerais (PUC-Minas), Faculty ofDentistry, Belo Horizonte/MG, Brazil; 2Department of OralPathology, Universidade Federal de Minas Gerais, Facultyof Dentistry, Av. Antônio Carlos 6627, CEP 31.270-901,Belo Horizonte/MG, Brazil; 3Department of Morphology,Universidade Federal de Minas Gerais, Instituto de CiênciasBiológicas, Belo Horizonte/MG, Brazil; 4Department ofClinical Medicine, Universidade Federal de Minas Gerais,Faculty of Medicine, Belo Horizonte/MG, Brazil; and5Department of Biochemistry and Immunology, Instituto deCiências Biológicas, Universidade Federal de Minas Gerais,Belo Horizonte/MG, Brazil; *corresponding author,tarcilia@odonto.ufmg.br

J Dent Res 86(11):1089-1094, 2007

RESEARCH REPORTSBiological

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osteoclast recruitment. An orthodontic device was placed, andthe phenotype—osteoclast recruitment and tooth movement—of tumor necrosis factor type 1-deficient (p55-/-) and wild-type(WT) mice were compared. To gain insight into themechanisms potentially responsible for the recruitment ofosteoclasts, we also examined the expression of CCL2 andCCL5 in the mouse periodontium under mechanical loading.

MATERIALS & METHODSExperimental AnimalsForty ten-week-old wild-type mice (C57BL6/J) and 40 ten-week-old TNF-RI (p55-/-)-deficient mice were used in this experiment.All animals were treated under ethical regulations for animalexperiments, defined by the Ethics Committee, Federal Universityof Minas Gerais (UFMG). Each animal's weight was recordedthroughout the experimental period, and there was no significantloss of weight.

Experimental ProtocolThe mice were anesthetized i.p. with 0.2 mL of a solutioncontaining xylazine (0.02 mg mL-1), ketamine (50 mg mL-1), andsaline in a proportion of 1:0.5:3, respectively. An orthodonticappliance consisted of a Ni-Ti 0.25 x 0.76 mm (LancerOrthodontics, San Marcos, CA, USA) coil spring, bonded by alight-cured resin (Transbond, Unitek/3M, St. Paul, MN, USA),between the maxillary right first molar and the incisors (Fig. 1).The force magnitude was calibrated by a tension gauge (ShimpoCorp., Tokyo, Japan) to exert a force of 0.1 N applied in the mesialdirection. The amount of force produced by the activation of thecoil was based on results from our previous experiments, whichwere adapted from the method proposed by Pavlin et al. (2000).There was no reactivation during the experimental period. Toavoid traumatic occlusal interference during tooth movement, weextracted the opposing mandibular first molar, using fineanatomical tweezers. The animals were divided into 3 groups:Control (non-operated animals), SHAM (with inactivated coilspring), and experimental group (with activated coil spring). Micewere killed with an overdose of anesthetic at the following times:0, 12 hrs, and 3, 6, and 12 days. For every set of experiments

(histological and biochemical measurements), 5 animals were usedfor each time-point.

Measurement of Cytokine LevelsUsing a stereomicroscope, we extracted periodontal ligament andsurrounding alveolar bone samples from the areas adjacent to theupper first molars. The samples were weighed and homogenized inPBS (0.4 mM NaCl and 10 mM NaPO4) containing proteaseinhibitors (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mMEDTA, and 0.01 mg/mL aprotinin A) and 0.05% Tween-20 at 1mg/mL. The mixture was placed on ice and centrifuged (10.000 g)for 10 min. The supernatant was then collected and stored at -70°Cuntil further analysis. The levels of TNF-!, CCL2, and CCL5 wereevaluated by a double-ligand enzyme-linked immunosorbent assay(ELISA), according to the manufacturer's protocol (R&D Systems,Minneapolis, MN, USA). The results were expressed as picogramsof cytokine/mg tissue. In preliminary experiments, the inter-experimental variability was below 5%.

HistologyThe right and left halves of the maxillae, including first, second,and third molars, were dissected, fixed in 4% paraformaldehyde,and rinsed in distilled water. After fixation, the hemimaxillae weredecalcified in 14% EDTA (pH 7.4) for 14 days and embedded inparaffin. Paraffin-embedded samples were cut into vertical sectionsof 4-"m thickness. The selection was based on morphologicalcriteria, such as the position of the distal root, where it appeared tobe as long as possible. The sections were stained for tartrate-resistant acid phosphatase (TRAP; Sigma-Aldrich, St. Louis, MO,USA), counterstained with hematoxylin, and used for histologicalexamination. The distal-buccal root, on its coronal two-thirds of themesial periodontal site, was used for the osteoclast counts, on 5sections per animal. Osteoclasts were identified as TRAP-positive,multinucleated cells located on the bone surface. To validate theconsistency of the measurement, two examiners measured 20successive slides until an r2 of at least 0.85 was repeatedly obtained.Measurement of Tooth MovementWe evaluated the amount of tooth movement morphometrically bymeasuring the distance between the cementum-enamel junctions(CEJs) from the first molar and the second molar (1st and 2ndmolar distances) in 5 vertical sections per animal under anAxioskop 40 microscope (Carl Zeiss, Göttingen, Germany), linkedto a digital camera (PowerShot A620, Canon, Tokyo, Japan),adapted from a previous study (Mavragani et al., 2005). We usedNational Institutes of Health Image J software. A single examiner(I.A.) conducted observations blinded to the group status. Threemeasurements were conducted for each evaluation, and thevariability was below 5% in all cases.Statistical AnalysisThe evaluation of each group was expressed as the mean ± SEM.Comparison among the groups was statistically analyzed by one-way analysis of variance (ANOVA), followed by the Newman-Keuls’ multiple comparison test. P < 0.05 was consideredstatistically significant.

RESULTSCCL5 Levels in the Periodontal Tissue were Diminished in p55-/- MiceThere was no significant difference in cytokine levels betweencontrol and SHAM-operated groups. In WT mice, there was a

Figure 1. Occlusal view of a Ni-Ti open coil spring placed between theupper right first molar and the upper incisors. The orthodontic applianceapplied a force of 0.1 N.

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significant increase in periodontal tissue levels of TNF-!,CCL2, and CCL5 at 12 hrs after mechanical loading (p < 0.05).Levels of mediators returned to baseline at 72 hrs (Figs. 2A,2B, 2C). In p55-/- mice, there was an increase in TNF-! andCCL2, similar to that found in WT mice (Figs. 2A, 2B).However, there was no significant increase in levels of CCL5after mechanical loading (Fig. 2C).

TRAP Activity and the Number of TRAP-positive Cells inthe Periodontal Tissue was Diminished in p55-/- MiceIn the control and SHAM-operated animals, TRAP activity wasfound on the distal side of the alveolar bone surface, and noactivity was observed in the mesial region of the periodontium(Figs. 3A, 3B). On day 6, TRAP activity appeared to increaseon the mesial periodontium of the distobuccal root, the side ofpressure after mechanical loading, and to decrease on the distalside of this root in WT mice (Fig. 3C). In p55-/- mice, thereappeared to be a similar decrease in the distal side, but asmaller increase of TRAP activity on the mesial side (Fig. 3D).On day 12, TRAP activity appeared to increase moreextensively in WT mice, which presented a greater alveolarbone resorption area than did p55-/- mice (Figs. 3E, 3F).Overall, there was a good correlation between the intensity ofTRAP activity, as evaluated qualitatively, and the number ofTRAP-positive osteoclasts. Indeed, the quantification ofTRAP-positive osteoclasts had a steady increase from days 3 to12 in WT mice subjected to orthodontic force (Fig. 4A). Theincreased number of TRAP-positive osteoclasts was greater inWT than in p55-/- mice (Fig. 4A).

The Amount of Tooth Movement was Reduced in p55-/- MiceAnalysis of the above data demonstrates that production ofCCL5 and recruitment of TRAP-positive osteoclasts inresponse to orthodontic force are diminished in p55-/- mice.Next, we evaluated whether the latter changes were reflected inchanges of tooth movement. After an initial displacement phasethat peaked on day 3, appreciable tooth movement was notobserved between days 3 and 6 in both groups. From days 6 to12, there was another phase of tooth movement (Fig. 4B).Overall, tooth movement in p55-/- mice followed a similarpattern, but the movement was significantly smaller than thatobserved in WT mice (Fig. 4B).

DISCUSSIONThe recruitment of osteoclasts to a site of periodontal ligamentcompression is essential for bone remodeling and consequenttooth movement. Several studies have attempted to elucidatethe role of mediators of the inflammatory process on themechanisms controlling the appearance of osteoclasts atcompression sites and consequent osteoclast-dependent toothmovement (Brezniak and Wasserstein, 2002; Kohno et al.,2003; Jager et al., 2005). Previous studies showed that TNF-!,a pro-inflammatory cytokine, might be associated withosteoclast differentiation (Abu-Amer et al., 2000; Azuma et al.,2000) and bone remodeling (Horowitz et al., 2001; Wise andYao, 2003). The present study aimed to investigate the role ofthe TNFRI in controlling the production of chemokines andosteoclast and tooth movement after the application oforthodontic force in mice.

Initial studies investigated the expression of cytokines in

Figure 2. Mean concentrations of TNF-! (A), CCL2 (B), and CCL5 (C) inthe mouse periodontium (WT and p55-/-) after 12 hrs and 72 hrs ofmechanical loading, respectively. There were 5 animals in each groupon each day. The data were expressed as the mean ± SEM. *P < 0.05compared with control and 72 hrs of orthodontic force (in the sameanimal strain). #P < 0.05 compared with WT groups at the samemoment, by one-way ANOVA and Newman-Keuls’ multiple-comparison test.

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the periodontium of WT mice during tooth movement andrelated histological changes. The results demonstrated thatexpression of TNF-! was detectable at 12 hrs after mechanical

loading. After 3 days, TNF-!returned to basal levels. Levels ofthe chemokines CCL2 and CCL5followed a pattern similar to that ofTNF-!. One limitation of thisapproach is that it evaluates levels ofmediators in both the compressionand the tension sites aftermechanical loading. Nevertheless, itis clear that the local enhancement ofTNF-! and chemokine activitypreceded the enhancement in TRAPactivity and the number of TRAP-positive cells in the mesial alveolarbone. We measured the distancebetween the CEJs to evaluate theeffect of the orthodontic device ontooth movement. The resultsdemonstrated that, under the appliedexperimental conditions, the toothmovement had an initialdisplacement, followed by a plateauphase between days 3 and 6. Inagreement with this finding, recentstudies demonstrated that toothmovement may be delayed by thedevelopment of hyalinized areas andprotracted osteoclast recruitment(van Leeuwen et al., 1999; Rody etal., 2001; von Böhl et al., 2004a,b;Krishnan and Davidovitch, 2006).After day 6, the tooth movement wasre-initiated and increased steadilyuntil day 12. This tooth movementwas preceded and paralleled by asignificant increase in the number ofTRAP-positive osteoclasts, TRAPactivity, and histological changesassociated with osteoclast function.

To evaluate the role of TNFRIon orthodontic tooth movement, weused p55-/- mice, in which there werelevels of both TNF-! and CCL2similar to those found in WT mice.However, the local production ofCCL5 was reduced. We also foundless TRAP activity, significantlyfewer osteoclasts, and diminishedtooth movement on days 6 and 12after the application of orthodonticforce than in WT mice. TNF-!and its TNFRI are well-known fortheir ability to enhance tissueexpression of cell adhesionmolecules (Zhou et al., 2007) and tofacilitate leukocyte influx (Barceloset al, 2005). TNF-! may alsoenhance osteoclast differentiation

and basal osteoclastogenesis, in vitro, via p55 (Azuma et al.,2000; Abu-Amer et al., 2000). A recent study has shown thatpre-osteoblast migration could be induced by CCL5 secreted

Figure 3. Histological changes related to orthodontic tooth movement in WT (A,C,E,G) and p55-/- mice(B,D,F,H). Vertical sections (4-"m thickness) of the periodontium around the distobuccal root of the firstmolar stained with TRAP. (A-B) Controls (before mechanical loading). TRAP activity was found on thedistal alveolar bone, demonstrating physiological distal tooth movement. (C-D) Experimental group (6days after the application of orthodontic force). TRAP activity increased on the mesial alveolar boneand decreased on the distal alveolar bone. In p55-/- mice, there was a smaller increase of TRAPactivity on the mesial alveolar bone. (E-F) Experimental group (12 days after mechanical loading).TRAP activity was less in p55-/- mice, which showed a smaller mesial alveolar bone resorption area.(G-H) Close-up view of the detached area in E and F. TRAP-positive osteoclasts are shown in bluearrows. MB, mesial alveolar bone; DB, distal alveolar bone; PL, periodontal ligament; R, root. Theblack arrows indicate the orthodontic tooth movement. Bar = 100 "m.

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from osteoclasts in a paracrine mode of action, and indicatedCCL5 as being an important molecule for communicationbetween osteoclasts and osteoblasts (Yano et al., 2005).Moreover, CCL5 has been shown to induce osteoclastchemotaxis (Yu et al., 2004), and may be involved in humanosteoclast differentiation in vitro (Kim et al., 2006). Together,the actions of TNF-!, via its TNFRI receptor, and CCL5 couldfacilitate osteoclast chemotaxis, differentiation, and activity. Inthe absence of the TNFRI and the consequent diminishedproduction of CCL5, the latter parameters would be deficient,as would tooth movement. In accordance with this hypothesis,a previous study demonstrated decreased levels of CCL5 andits receptor in p55-/- mice with periodontal disease (Garlet etal., 2007).

Our results are not in agreement with those of a previousstudy (Yoshimatsu et al., 2006) that showed a similar numberof TRAP-positive cells in tissues from WT and p55-/- miceunder orthodontic force, without a significant differencebetween the amounts of tooth movement in both groups. Thereasons for the discrepancy between findings are not known.However, it is possible that the different methodologies used toquantify TRAP-positive osteoclasts and to measure toothmovement in both studies could be an underlying reason. In ourstudy, the use of careful morphometric analysis to quantifyosteoclast numbers and tooth movement clearly showed aninhibition in mice lacking the TNFRI.

In conclusion, our study suggests that TNF signaling viap55 plays a significant role in osteoclast recruitment induced bymechanical loading, and this mechanism is related (andpossibly secondary) to decreased CCL5 production. Furtherstudies are now required for a better understanding of the roleof chemokines, such as CCL2 and CCL5, their receptors, andother cytokines on tooth movement induced by an orthodonticappliance.

ACKNOWLEDGMENTSWe are grateful to Fundação de Amparo a Pesquisas do Estadode Minas Gerais (FAPEMIG, Brazil) and Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq, Brasil) forfinancial support.

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