The effect of IL-1 receptor antagonist on orthodontic toothmovement in mice

Juliana Tito Salla a, Silvana Rodrigues de Albuquerque Taddei a,b,Celso Martins Queiroz-Junior a,b, Ildeu Andrade Junior b,c,Mauro Martins Teixeira b, Tarcılia Aparecida Silva a,b,*aDepartment of Oral Surgery and Pathology, Faculdade de Odontologia, Universidade Federal de Minas Gerais, Minas Gerais, Brazilb Laboratory of Immunopharmacology, Department of Biochemistry and Immunology, Instituto de Ciencias Biologicas, Universidade Federal de

Minas Gerais, Belo Horizonte, Minas Gerais, BrazilcDepartment of Orthodontics, Faculty of Dentistry, Pontifıcia Universidade Catolica de Minas Gerais (PUC Minas), Belo Horizonte, Minas

Gerais, Brazil

1. Introduction

Orthodontic tooth movement (OTM) occurs through remodel-ling of alveolar bone after mechanical stimuli. The orthodonticforces generate and propagate signalling cascades through allparadental tissue cells, triggering important changes in thehomeostatic periodontal environment.1,2

The orthodontic loading leads to a focal tissue injury and,consequently, an aseptic inflammatory response charac-terised by the release of several important inflammatorymediators on periodontal tissues,2,3 such as the cytokineinterleukin-1 (IL-1).4 IL-1 is directly involved in bone resorptionby taking part in the survival, fusion and activation ofosteoclasts and it exerts its activities by binding to two typesof receptors, IL-1-RI and IL-1-RII.5 Whilst the latter has no

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a r t i c l e i n f o

Article history:

Accepted 30 September 2011

Keywords:

Interleukin-1 receptor antagonist

(IL-1Ra)

Interleukin-1 (IL-1)

Mechanical loading

Orthodontic tooth movement

a b s t r a c t

Objective: Orthodontic tooth movement (OTM) is achieved by alveolar bone remodelling

induced by mechanical loading. Whilst interleukin-1 (IL-1) is directly involved in OTM, the

role of interleukin-1 receptor antagonist (IL-1Ra), a naturally occurring IL-1 antagonist, is not

completely defined. Therefore, the aim of this study was to investigate the effects of IL-1Ra

on OTM.

Methods: An orthodontic appliance was placed in C57BL6 mice treated with vehicle or IL-1Ra

(10 mg/kg/day). OTM and TRAP-positive osteoclasts were evaluated after 12 days of me-

chanical loading and the levels of cytokines on periodontal tissues were analysed by ELISA

after 12 and 72 h.

Results: Mice treated with IL-1Ra showed diminished OTM and decreased numbers of TRAP-

positive osteoclasts. In line with this, lower levels of IL-1b and TNF-a, and higher levels of IL-

10, were observed on periodontal tissues of IL-1Ra-treated mice in relation to the vehicle-

treated group.

Conclusion: The present study suggests that IL-1Ra downregulates OTM, probably by its anti-

inflammatory actions.

# 2011 Elsevier Ltd. All rights reserved.

* Corresponding author at: Departamento de Clınica, Patologia e Cirurgia Odontologicas, Faculdade de Odontologia, Universidade Federalde Minas Gerais, Av. Presidente Anto nio Carlos 6627, CEP 31.270-901, Belo Horizonte, Minas Gerais, Brazil. Tel.: +55 31 3409 2478;fax: +55 31 3409 2430.

E-mail addresses: tarcilia@ufmg.br, silva.tarcilia@gmail.com (T.A. Silva).

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.archoralbio.2011.09.018

described signalling properties and acts as a ‘‘decoy’’ target forIL-1, the former develops pro-inflammatory functions, such ascell recruitment and release of other cytokines, which also areinvolved in bone resorption.6 However, IL-1 functions arephysiologically controlled by the naturally occurring interleu-kin-1 receptor antagonist (IL-1Ra), which competitively blocksthe interactions of IL-1 with its receptors and inhibits itsactivity.7,8

IL-1Ra has long been studied in clinical and experimentalsurveys as a physiological and therapeutic target in inflam-matory conditions related to bone resorption, such asrheumatoid arthritis9,10 and periodontal disease.11,12 Thesestudies reported that administration of exogenous IL-1Ra maybe a useful strategy to control bone resorption, mainly for itsanti-inflammatory properties related to the antagonism of IL-1.9–11,13 However, only a few studies have investigated theeffect of IL-1Ra on OTM, showing a positive correlationbetween decreased IL-1Ra gingival expression and fasterOTM in humans.14–17

Despite these findings, there is a lack of evidence describingthe effects of IL-1Ra therapy on bone remodelling aftermechanical loading. Therefore, the aim of this study was toinvestigate the effects of IL-1Ra administration on OTM in amouse model.

2. Materials and methods

2.1. Experimental animals

Thirty five ten-week-old wild-type mice (WT) (C57BL6/J) wereused in this study. For histomorphometric analysis, 10 micewith orthodontic appliance were used. In this set of experi-ments, the left side of maxillae (without orthodontic appli-ance) was used as control. For biochemical analysis, 20 micereceived the orthodontic appliance and treatments describedbelow, and 5 mice did not receive appliance, being used as thecontrol group. All animals were treated under ethical regula-tions for animal experiments, defined by the InstitutionalEthics Committee. Each animal’s weight was recordedthroughout the experimental period and there was nosignificant loss of weight.

2.2. Experimental protocol

The experimental protocol was based on a previous study.18

Briefly, mice were anaesthetized and a Ni–Ti0.25 mm ! 0.76 mm coil spring (Lancer Orthodontics, SanMarcos, CA, USA) was bonded by a light-cured resin(Transbond, Unitek/3M, Monrovia, CA, USA) between themaxillary right first molar and the incisors. The forcemagnitude was calibrated by a tension gauge (ShimpoInstruments, Itasca, IL, USA) to exert a force of 0.35 N appliedin the mesial direction. There was no reactivation during theexperimental period. Thereafter, mice were randomly dividedin two groups for histomorphometric analysis: mice treatedwith vehicle (PBS) (vehicle group) or with IL-1Ra (dailyadministration [s.c.] of 10 mg/kg/day IL-1Ra [Biogen INC;Geneva, Switzerland]) (IL-1Ra group). For biochemical assays,three groups were created: mice without appliance (control

group) and mice with activated coil spring (experimentalgroup) treated with PBS (vehicle group) or with IL-1Ra (IL-1Ragroup). At the end of the experiments, mice were euthanizedwith an overdose of anaesthetic at the following times: 12 daysafter orthodontic appliance placement for histological mea-surements, and 12 h and 72 h for biochemical analysis. Forevery set of experiments, 5 mice/group were used for eachtime-point.

2.3. Histopathological analysis

The right and left halves of maxillae, including first, second, andthird molars, were dissected, fixed in 10% buffered formalin (pH7.4) and rinsed in distilled water. Thereafter, each hemi-maxillawas decalcified in 14% EDTA (pH 7.4) for 14 days and embeddedin paraffin. Samples were cut into sagittal sections of 5 mmthickness. Sections were stained for tartrate-resistant acidphosphatase (TRAP; Sigma–Aldrich, St. Louis, MO, USA),counterstained with haematoxylin, and used for histologicalexamination. The first molar distobuccal root, on the coronaltwo-thirds of the mesial periodontal site, was used for osteoclastcounting on 5 non-consecutive sections (40 mm apart one fromthe other) per mouse. Osteoclasts were identified as TRAP-positive, multinucleated cells on the bone resorption lacunae.

2.4. Measurement of tooth movement

Image J software (National Institutes of Health) was used toquantify the amount of tooth movement, as previouslydescribed.18 Tooth movement was obtained through thedifference between the distance of the cementum-enamel-junction’s (CEJ’s) of the first molar and the second molar (1st and2nd molar distance) of the experimental side (right hemi-maxila) in relation to the control side (left hemi-maxila) of thesame animal. Five sections per mouse were evaluated under amicroscope Axioskop 40 (Carl Zeiss, Gottingen, Niedersachsen,Germany) adapted to a digital camera (PowerShot A620, Canon,Tokyo, Honshu, Japan). Three measurements were conductedfor each evaluation and the variability was below 5%.

2.5. Measurement of cytokine levels

Periodontal ligament and surrounding alveolar bone samplesfrom the areas adjacent to the upper first molars were obtainedusing a stereomicroscope. Samples were weighed and homoge-nized in PBS (0.4 mM NaCl and 10 mM NaPO4) containingprotease inhibitors (0.1 mM PMSF, 0.1 mM benzethoniumchloride, 10 mM EDTA, and 0.01 mg/mL aprotinin A) and 0.05%Tween-20 at 1 mg/mL. The homogenate was centrifuged(8946 ! g) at 4 8C for 10 min. The supernatant was then collectedand stored at "70 8C until further analysis. The levels of IL-1b,TNF-aandIL-10wereevaluatedbydouble-ligandenzyme-linkedimmunosorbent assay (ELISA), according to the manufacturer’sprotocol (R&DSystems,Minneapolis,MN,USA). Theresults wereexpressed as picograms of cytokine/100 mg of tissue.

2.6. Statistical analysis

The results were expressed as the mean # standard error ofthe mean (SEM). Comparison amongst the groups was

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statistically analysed by one-way analysis of variance(ANOVA), followed by the Newman–Keuls multiple compari-son test. P < 0.05 was considered statistically significant.

3. Results

3.1. Tooth movement and TRAP-positive cells

The amount of OTM was significantly less in mice treated withIL-1Ra (Fig. 1A), as well as the number of TRAP-positiveosteoclasts (Fig. 1B), when compared to the vehicle group after12 days of mechanical loading.

Histological characterisation of periodontal tissues alsorevealed that IL-1Ra treated mice demonstrated a decreasedTRAP activity and a smaller number of osteoclasts in thepressure side of the periodontium (Fig. 2E and F), whencompared to the experimental tooth of vehicle treated mice(Fig. 2C and D).

3.2. Levels of cytokines in periodontal tissues

The smaller amount of OTM observed in IL-1Ra treated miceled us to investigate the effects of such therapy on theexpression of cytokines involved in bone remodelling.Mechanical loading applied to tooth triggered a significantrelease of pro-inflammatory and bone resorptive cytokines inperiodontal tissues just after 12 h of stimulation. Whilst thelevels of IL-1b (Fig. 3A) and TNF-a (Fig. 3B) increasedapproximately 6 and 5.5 fold, respectively, IL-10 levels(Fig. 3C) were not altered when compared to control mice.After 72 h of mechanical loading, IL-1b levels were almost 10times higher than control (Fig. 3A), and the levels of TNF-a

(Fig. 3B) and IL-10 (Fig. 3C) were similar to the basal condition.In contrast, treatment of mice with IL-1Ra reduced theinflammatory milieu observed in periodontal tissues afterstimuli. IL-1Ra therapy induced a decrease of 66% and 76% inthe levels of IL-1b (Fig. 3A) and TNF-a (Fig. 3B), respectively,when compared to vehicle-treated mice, whilst the levels ofIL-10 (Fig. 3C) enhanced approximately 2 fold either at 12 or at72 h after mechanical loading.

4. Discussion

Interleukin-1 (IL-1) has been one of the most studied cytokinesand it is one of the major soluble proteins related to osteoclastactivation and bone resorption.5,6 Evidence for the existence ofIL-1 controlling pathways indicates that the IL-1 receptorantagonist (IL-1Ra) can be important for the in vivo regulationof IL-1b activity in bone resorptive conditions.9–11 However,the effect of IL-1Ra on bone remodelling after mechanicalloading is not well described. In the present study, adminis-tration of IL-1Ra diminished OTM by reducing the expressionof the pro-inflammatory cytokines IL-1b and TNF-a, and byincreasing the levels of IL-10, a negative regulator of boneresorption.

When an orthodontic force is applied on teeth, it leads to atransient aseptic inflammation of the periodontium thatculminates in bone remodelling.1 In this context, boneresorption is a fundamental step and several cytokinesassociated to osteoclast differentiation and activation, suchas TNF-a and IL-1b, are early released in the periodontiumafter mechanical loading.3,4,18–20 Accordingly, the levels ofthese cytokines were increased in our experimental condi-tions, whilst the levels of IL-10, a cytokine known to controlbone resorption and osteoclast activation,21 were not affected.

In view of the importance of this inflammatory milieu tobone resorption, it has been suggested that the control of suchinflammation could affect OTM. A previous study showed thatan interference with TNF-a activity might decrease osteoclastmigration and, consequently, diminish OTM.18 In this regard,administration of IL-1Ra to interfere with IL-1b activity couldalso alter mechanically induced bone remodelling. IL-1Ra, firstcalled IL-1 inhibitor, was cloned and identified as an IL-1receptor antagonist after being noticed to bind to IL-1receptors but not to transduce the same signals that IL-1b

did.22,23 Thus, IL-1Ra acts by competitively blocking theinteractions of IL-1 to their receptors, inhibiting its activi-ties.7,8 Indeed, the administration of exogenous IL-1 receptorantagonist has been shown to be effective in reducing signs ofIL-1-related bone resorptive conditions, such as rheumatoidarthritis10 and periodontal disease,11 concomitantly with a

Fig. 1 – (A) Amount of tooth movement in PBS (vehicle) and IL-1Ra treated mice (IL-1Ra). (B) Number of TRAP-positiveosteoclasts in both groups. *P < 0.05 comparing control group to the respective experimental group. #P < 0.05 comparingvehicle- and IL-1Ra-treated experimental groups. One-way ANOVA and Newman–Keuls multiple comparison test.

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reduction of pro-inflammatory cytokines.9,11 In this regard, adecreased physiological IL-1Ra expression in gingival crevi-cular fluid has been shown to correlate with faster OTM inhumans.14–17

In the present study, mice treated with IL-1Ra showedsignificantly diminished OTM and osteoclast numbers thanvehicle-treated animals. This phenotype was associated withreduced early release of TNF-a and IL-1b, concomitantly toincreased expression of IL-10 on periodontal tissues. Thepresent results give support to previous findings showing thatadministration of soluble IL-1 receptors reduces the amount ofOTM in rats24 and go further when showing that this effectoccurs by controlling the expression of cytokines. The datastrongly indicates that interfering with the IL-1b pathway may

impair the pro-inflammatory and pro-osteoclastic milieu onperiodontal tissues, and, consequently, decrease OTM. Inaccordance, in vitro studies have shown that IL-1 receptorantagonists can inhibit the compressive force-induced ex-pression of RANKL (receptor activator of nuclear factor kappaB ligand), a positive regulator of osteoclast differentiation andactivation, by periodontal ligament cells.25 Similarly, theexpression of TNF-a after application of compressive forcesin vitro was decreased with the addition of IL-1Ra to cellcultures.26

In conclusion, the present study suggests that IL-1Ra mightaffect bone remodelling after mechanical loading probably byits anti-inflammatory actions, such as the reduction of pro-inflammatory and bone resorptive cytokines and the increase

Fig. 2 – Histological changes related to OTM in vehicle- and IL-1Ra-treated mice. (A and B) Control group (without mechanicalloading). (C and D) Experimental vehicle-treated and (E and F) experimental IL-1Ra treated groups (12 days after mechanicalloading). Panels (B), (D) and (F) represent the higher view of the identified area in (A), (C) and (E), respectively. Large arrowsindicate TRAP-positive osteoclasts. MB, mesial alveolar bone; DB, distal alveolar bone; PL, periodontal ligament; R, root.Small arrows indicate the direction of tooth movement. Bar = 100 mm.

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of anti-inflammatory cytokine. Furthermore, analysis of ourdata provides new insights into the development of futuretherapeutic interventions with IL-1Ra, which could modulatethe amount of OTM and restrain the relapse of the finalorthodontic result.

Funding: This work was supported by Fundacao de Amparo aPesquisas do Estado de Minas Gerais (FAPEMIG, Brazil), Coor-denacao de Aperfeicoamento de Pessoal de Nıvel Superior(CAPES, Brazil) and Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico (CNPq, Brazil).

Competing interests: The authors state no conflict of interest.

Ethical approval: Mice were treated under ethical regulations foranimal experiments, defined by the Institutional Ethics Com-mittee (Universidade Federal de Minas Gerais), which ap-proved the experimental procedures adopted in the study(protocol number 135/08).

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