Mast Cells Act as Phagocytes Againstthe Periodontopathogen AggregatibacterActinomycetemcomitansHeliton G. Lima,* Karen H. Pinke,* Taiane P. Gardizani,* Devandir A. Souza-Junior,†

Daniela Carlos,‡ Mario J. Avila-Campos,§ and Vanessa S. Lara*

Background: Evidence to date shows that mast cells playa critical role in immune defenses against infectious agents,but there have been no reports about involvement of these cellsin eliminating periodontopathogens. In this study, the phago-cytic ability of mast cells against Aggregatibacter actinomy-cetemcomitans compared with macrophages is evaluated.

Methods: In vitro phagocytic assays were conducted usingmurine mast cells and macrophages, incubated with A. actinomy-cetemcomitans, either opsonized or not, with different bacterialload ratios. After 1 hour, cells were stainedwith acridine orangeand assessed by confocal laser-scanning electronmicroscopy.

Results: Phagocytic ability of murine mast cells against A.actinomycetemcomitans was confirmed. In addition, the per-centage of mast cells with internalized bacteria was higher inthe absence of opsonization than in the presence of opsoniza-tion. Both cell types showed significant phagocytic activityagainst A. actinomycetemcomitans. However, the percent-age of mast cells with non-opsonized bacteria was higherthan that of macrophages with opsonized bacteria in one ofthe ratios (1:10).

Conclusions: This is the first report about the participationof murine mast cells as phagocytes against A. actinomy-cetemcomitans, mainly in the absence of opsonization withhuman serum. Our results may indicate that mast cells actas professional phagocytes in the pathogenesis of biofilm-associated periodontal disease. J Periodontol 2013;84:265-272.

KEY WORDS

Gram-negative bacteria; macrophages; mast cells;periodontal disease; phagocytosis.

Periodontal biofilm hosts a widediversity of potentially harmfulbacterial species. Aggregatibacter

actinomycetemcomitans has been de-scribed as a member of the indigenous oralmicrobiota in many healthy individualsbut is also primarily involved in the pa-thology of periodontitis,1,2 the majorcause of tooth loss in the world,3 andvarious non-oral infections.4,5 A. acti-nomycetemcomitans is an oral Gram-negative bacterium and has shown theability to invade periodontal tissues, es-tablishing an infection.6

Several studies7-11 have demonstratedthat the evolution of biofilm-associatedperiodontal diseases is influenced by theinflammatory and immune responses ofthe host against pathogens, resulting inthe destruction of soft and mineralizedperiodontal tissues, and involves theparticipation of different immune cellsand their chemical mediators. Currently,mast cells (MCs) have been associatedwith inflammatory periodontal diseasesthrough release of proinflammatory andimmunoregulatory cytokines, which acti-vate other immune cells, such as neutro-phils and lymphocytes, thus contributingto the development and amplificationof the defense response of the host.12,13

MCs are tissue-dwelling granule-con-taining immune cells that play a pivotalrole in inflammatory and other processes,such as the allergic immune response.14,15

MCs are commonly found in tissues that

* Department of Stomatology, Bauru School of Dentistry, Sao Paulo University, Bauru, SP,Brazil.

† Department of Cell and Molecular Biology and Pathogenic Bioagents, School of Medicineof Ribeirao Preto, Sao Paulo University, Ribeirao Preto, SP, Brazil.

‡ Department of Biochemistry and Immunology, School of Medicine of Ribeirao Preto, SaoPaulo University.

§ Department of Microbiology, Institute of Biomedical Sciences, Sao Paulo University, SaoPaulo, SP, Brazil.

doi: 10.1902/jop.2012.120087

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interface with the external environment, such as theskin and mucosa of the respiratory and gastroin-testinal tracts.16 Because these sites are also com-mon portals of infection, MCs are likely to be amongthe first inflammatory cells to interact with invadingpathogens.17

Studies have shown that MCs have the capacityto modulate the innate immune response of the hostagainst bacteria by their ability to phagocytose andkill bacteria, to process and present bacterial an-tigens to T cells, and to recruit phagocytic helpthrough the release of physiologic amounts of pro-inflammatory mediators.18-21 Moreover, MCs displaya phagocytosis-independent antimicrobial activityagainst extracellular bacteria, producing extracel-lular traps and antimicrobial compounds.17,22,23

Although phagocytic and microbicidal activityagainst bacteria has been described in MCs, the in-volvement of these cells in the elimination of peri-odontopathogens via phagocytosis remains unclear.Because A. actinomycetemcomitans is one of themajor periodontal pathogens, it is important to obtainmore knowledge about the protective role of MCsagainst this microorganism. In this study, the phago-cytic ability of MCs against A. actinomycetemcomitansis determined and compared with the phagocyticcapacity of macrophages.

MATERIALS AND METHODS

Isolation and Expansion of Bone Marrow–DerivedMCsPrimary MC lines were derived from femoral bonemarrow of male C57BL/6 mice, 4 to 6 weeks old.These animals were bred and maintained in thecentral animal facility of the Bauru School of Den-tistry, University of Sao Paulo, Bauru, SP, Brazil. Theexperimental protocol was approved by the local In-stitutional Committee for Animal Care and Use (no.028/2010). Mouse femoral bone marrow was re-moved by washing the medullary cavity with cellculture mediumi supplemented with 10% heat-inac-tivated fetal bovine serum,¶ 100 U/mL penicillin,# 100mg/mL streptomycin,** 0.05 mM b-mercaptoethanol,††

and 36 mM sodium bicarbonate.‡‡ Bone marrow cellswere dissociated by aspiration with a Pasteur pipette,and the cells were then rinsed twice by centrifugation(400 · g for 10 minutes) in cell culture medium. Cellswere counted, and the viability was assessed bytrypan blue in a hemocytometer. After this, cellswere cultured, 0.5 · 106 cells/mL, in cell culturemedium supplemented with 100 ng/mL recombi-nant stem cell factor§§ and 20 ng/mL recombinantinterleukin-3ii in a humidified atmosphere contain-ing 5% CO2 at 37�C.24 By the end of 2 to 3 weeks,a non-adherent population of large granular cellswas observed. Over the course of 21 days, the

bone marrow cells differentiated to MCs (bonemarrow–derived MCs [BMMCs]).

By staining with toluidine blue, the isolated cellswere shown to be homogeneous with typical granularappearance. Moreover, to further confirm their differ-entiation into MCs, the cells were immunostained withantibodies against specific surface molecules of mu-rine MCs: antimouse CD117 (c-kit receptor) conju-gated phycoerythrin–cyanine 5,¶¶ antimouse Fcereceptor (FceRI; high-affinity receptor for immuno-globulin E) conjugated phycoerythrin,## and anti-AA4conjugated to fluorescein isothiocyanate (ganglio-sides derived from GD1b, present only on the surfaceof rodent MCs).24-26 After this, cells were analyzed byflow cytometry,*** according to parameters of size(forward-scattered light), granularity (side-scatteredlight), and fluorescence intensity. Thus, the homo-geneity of this cell population also was determinedby flow cytometric analysis, revealing a BMMC pu-rity >98%.

Bacterial GrowthPrevious studies conducted by Malaviya et al.18

demonstrated that different bacterial strains, in-cluding Escherichia coli, bind avidly to mouse MCs.This bacteria/MC interaction causes phagocytosisand bacterial killing. Therefore, to ensure that theBMMCs were able to cause phagocytosis, the sameassay was performed with E. coli as control.

The enteroinvasive E. coli 0:124 were initiallygrown on brain heart infusion agar††† and incubatedat 37�C for 24 hours. A. actinomycetemcomitansATCC 29523 was grown on tryptic soy agar plus 0.6%yeast extract,‡‡‡ and the plates were incubated in10% CO2 at 37�C for 5 days. The bacterial concen-tration of A. actinomycetemcomitans was standard-ized at 1 · 109 colony-forming units (CFU)/mL andE. coli at 1 · 107 CFU/mL by spectrophotometer§§§

(A660 nm).

Phagocytosis AssayThe phagocytosis assay was performed followingthe protocols of previous studies,17,18,22,27 andit was adjusted in accordance with the conditionsof our laboratory. Initially, bacteria were opsonizedvia complement with a pool of human serum under

i RPMI 1640 Medium, Invitrogen, Grand Island, NY.¶ Invitrogen.# Invitrogen.** Invitrogen.†† Sigma-Aldrich, St. Louis, MO.‡‡ Merck, Rio de Janeiro, RJ, Brazil.§§ Peprotech, Rocky Hill, NJ.ii Peprotech.¶¶ eBioscience, San Diego, CA.## eBioscience.*** FACSCalibur/CellQuest, BD Biosciences, San Jose, CA.††† Acumedia, Neogen, Lansing, MI.‡‡‡ Difco, BD Biosciences.§§§ Biotek, Winooski, VT.

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shaking at 37�C for 30 minutes, and non-opsonizedbacteria were used as control. BMMCs were washedtwice with cell culture medium without antibioticsand incubated with A. actinomycetemcomitans at1:5, 1:10, and 1:25 ratios of cell/bacteria or with E. coliat 1:10, for 1 hour at 37�C under gentle agitation.Then, cells were centrifuged at 218 · g, at 24�C, for10 minutes, and the pellet was resuspended in200 mL of cell culture medium. The cell suspensionswere transferred to 24-well plates, containing sterilespherical glass coverslips (13 mm in diameter), andpretreated with tissue adhesive,iii allowing cell adhe-sion. The wells were washed with phosphate-bufferedsaline (PBS) to remove the extracellular bacteria. Thecoverslips with cells attached were fixed with 2%paraformaldehyde in PBS for 20minutes and washedagain with PBS. Then they were stained with 0.05mg/mL acridine orange¶¶¶ for 15 minutes in a darkroom. After this, coverslips were carefully removedand placed on glass slides with mounting medium.###

The slides were qualitatively evaluated by confocallaser-scanning electron microscopy,**** which ini-tially allowed entrapped bacteria to be differentiatedfrom bacteria only bound to the MC surface mole-cules. Thereafter, quantitative analysis was per-formed at ·100 magnification by counting the MCswith internalized bacteria in 20 randomly capturedfields.†††† Phagocytosis was assessed by consideringthe number of internalized bacteria per cell (£5 and>5). Cells with absence of internalized bacteria alsowere counted. The results were expressed in per-centages and were obtained from the average ofthree independent experiments, considering the totalnumber of cells counted per coverslip (20 fields) ineach experiment.

Simultaneously to BMMCs, phagocytosis assaysagainst A. actinomycetemcomitans were performedby using mouse peritoneal macrophages (MPMs), al-lowing the phagocytic capacity of cell types, MCs, andmacrophages to be compared. MPMs were obtainedaccording to the methodology of Murciano et al.28

Statistical AnalysesThe results are expressed as the mean – SD fromthree independent experiments (one mouse in eachexperiment for testing MCs and four mice for mac-rophages). Data were analyzed by using a two-wayanalysis of variance and Tukey test, followed byStudent t test.‡‡‡‡ Values of P <0.05 were consideredstatistically significant.

RESULTS

BMMCs as Phagocytes Against A.actinomycetemcomitansIn this study, by using a ratio of 1:25 (BMMCs/bac-teria), the percentage of BMMCs with opsonized A.

actinomycetemcomitans is higher when comparedwith that of BMMCs without bacteria (P = 0.011).Most of the cells displayed >5 internalized bacteria.

Interestingly, in the absence of opsonization,BMMCs presenting ingested bacteria were morenumerous than cells without bacteria in all BMMCs/bacterial ratios used. In addition, a higher percentageof BMMCs with non-opsonized bacteria was detectedcompared with that of BMMCs with bacteria opson-ized, in the ratios 1:5 and 1:10 (P = 0.02 and P = 0.004,respectively).

Considering BMMCs with >5 internalized bacteria,the cells incubated with non-opsonized bacteria weremore numerous than the BMMCs with opsonizedbacteria only in the ratio of 1:10 (P =0.0008) (Figs. 1and 2).

Phagocytic Activity of BMMCs Versus MPMsWhen comparing the phagocytic activity of BMMCswith professional phagocytes (MPMs) against opson-ized A. actinomycetemcomitans, the percentage ofMPMs with £5 internalized bacteria was higher thanthat of BMMCs in 1:5 and 1:25 ratios (P = 0.03 and P =0.02, respectively). However, the percentage ofBMMCs that had >5 internalized bacteria was higherthan that of MPMs, in the ratio of 1:5 (P = 0.04).

In the absence of opsonization, MPMs without in-ternalized bacteria were more numerous than BMMCsin all the ratios evaluated. Moreover, there were alsohigher percentages of BMMCs with >5 internalizedbacteria than those observed for MPMs in all ratios(Fig. 3).

Considering only the higher percentages of cellswith A. actinomycetemcomitans, the values werehigher for BMMCs with non-opsonized bacteriacompared with MPMs with opsonized bacteria in theratio 1:10 (P = 0.0008) (Fig. 4).

Phagocytosis by BMMCs and MPMs Against A.actinomycetemcomitans and E. coliWhen comparing the two cell types with opsonizedE. coli (1:10), the percentage of MPMs with >5 in-ternalized bacteria was higher than that of BMMCs.Moreover, the percentage of MPMs without opson-ized bacteria was lower than that of BMMCs. In theabsence of opsonization, the opposite was observed;the percentage of BMMCs with >5 internalized bac-teria was higher compared with that of MPMs.

Similar to the phagocytic assays with A. actino-mycetemcomitans, percentages of BMMCs withoutinternalized E. coli ranged according to the presenceor absence of previous bacterial opsonization. BMMCs

iii Biobond, Electron Microscopy Sciences, Hatfield, PA.¶¶¶ Merck, Darmstadt, Germany.### Permount, Thermo Fisher Scientific, Morris Plains, NJ.**** TCS-SPE, Leica, Wetzlar, Germany.†††† AxioVision release 4.6, Carl Zeiss, Goettingen, Germany.‡‡‡‡ Prism, GraphPad Software, San Diego, CA.

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without opsonized E. coli were more numerous thanBMMCs with absence of non-opsonized E. coli. Con-versely, MPMs without non-opsonized E. coli dis-played higher percentages compared with MPMswithout internalization of opsonized bacteria, sug-gesting that serum opsonization amplified bacterialinternalization by MPMs but not by BMMCs (Fig. 5) asobserved for A. actinomycetemcomitans.

DISCUSSION

The contribution of MCs to the host defense has be-come increasingly recognized in recent years. It isknown that MCs have the ability to recognize andphagocytose infectious agents through specific re-ceptors present on their surfaces.29-31 From in vitroassays, we are able to demonstrate for the first timethe participation of murine MCs as phagocytes againstthe periodontopathogen A. actinomycetemcomitans,especially in the absence of opsonization with humanserum. Considering that the Fc portion of immuno-globulin is variable between species,32 the bacterialopsonization by human serum supplied only comple-ment system proteins for opsonin-dependent phago-cytosis by murine cells.

In the present study, opsonized A. actinomy-cetemcomitans with complement was not phagocytosedby BMMCs as efficiently as it was without opsonization.Probably, the recognition of A. actinomycetemcomitansby murine BMMCs, when independent of complementreceptor, seems to have remarkable implicationsbecause percentages of BMMCs with non-opsonized

Figure 1.BMMCs act as phagocytes against A. actinomycetemcomitans,especially in the absence of serum opsonization. Bars show thepercentage of BMMCs with A. actinomycetemcomitans internalizedor not. Results are expressed as the mean – SD from three independentexperiments. Data were analyzed by Tukey test and Student t test.*P <0.05; †P <0.01; ‡P <0.001.

Figure 2.Confocal laser-scanning electron microscopy showing phagocytosisof BMMCs against A. actinomycetemcomitans and E. coli stainedwith acridine orange. A through C) BMMCs stained green, with A.actinomycetemcomitans inside, either red or green. D) BMMCs withE. coli, either green or red, internalized or only adhered to their surfaces.The cell/bacteria ratios are indicated in the top right side of thephotomicrographs (Original magnification ·630).

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bacteria were higher than BMMCs without thesebacteria or than BMMCs with opsonized bacteria,after comparisons in the same ratios. In addition,high percentages of BMMCs had >5 internalizedA. actinomycetemcomitans, especially in the ab-sence of opsonization. Similar results were observedwith BMMCs challenged with E. coli by comparingthe presence or absence of opsonization.

It is now known that the MCs contain numeroussurface receptors, including those that promoterecognition and bacterial binding. MCs exhibit twobasic mechanisms of microbial recognition: opso-nin-dependent (via Fc and C3 receptors) and op-sonin-independent (via integrins, CD48 molecule,and Toll-like receptors [TLRs]).33-36 According toour results, the recognition and ingestion of A. actino-mycetemcomitans by murine BMMCs involve eitheropsonin-dependent or opsonin-independent recep-tors, in agreement with Malaviya et al.,35 who dem-onstrated the recognition of E. coli by MCs.

Because A. actinomycetemcomitans producesdiverse bacterial products, such as lipopolysac-charide and peptidoglycan mediated by TLR2 andTLR4 receptors,31,37-39 the TLR pathway may repre-sent another route of recognition and internalizationof this periodontopathogen by the MCs studied herebecause MCs express TLR1, TLR2, TLR3, TLR4, TLR6,and TLR9.36,40,41 However, additional studies areneeded to unravel which extracellular receptors onthese cells are involved more frequently and effi-ciently in the recognition and ingestion of A. acti-nomycetemcomitans.

In the present study, unlike BMMCs, macrophageswith opsonized bacteria were more frequent thanmacrophages with non-opsonized bacteria, sug-gesting that complement system proteins resulted

in more efficient internalization by these cells thanBMMCs for both A. actinomycetemcomitans andE. coli. Furthermore, in a ratio of 1:10, BMMCs withnon-opsonized A. actinomycetemcomitans weremore numerous than macrophages with opsonizedA. actinomycetemcomitans, suggesting that BMMCsphagocytosed more efficiently non-opsonized peri-odontopathogens than macrophages against opsonizedbacteria with complement. However, we cannot ruleout the possibility that these opsonized bacteria werekilled by macrophages after internalization, sup-porting some findings42-44 that observed phagocy-tosis by macrophages with bacterial death in anopsonin-rich environment. After comparison ofphagocytosis by both cell types, our results showed

Figure 3.A) Phagocytosis ofA. actinomycetemcomitans (Aa) with opsonization.B) Phagocytosis ofAawithout opsonization. In the absence of serum opsonization,BMMCs presented more ingested Aa than MPMs. Bars show the percentage of BMMCs and MPMs with Aa internalized or not. Both cell types wereclassified as follows: absence of bacteria (a) or with £5 (b) or >5 (c) internalized bacteria. Results are expressed as the mean – SD from threeindependent experiments. Data were analyzed by Tukey test and Student t test. *P <0.05; †P <0.01; ‡P <0.001.

Figure 4.Highest percentages of both cell types with internalized A.actinomycetemcomitans. This figure shows the mean of the highestpercentages of MCs and macrophages with internalized A.actinomycetemcomitans from three independent experiments. Thesehigher meanswere obtained fromMCs in the absence of opsonization andfrom macrophages in the presence of opsonization. Data were analyzedby Tukey test. *P <0.05 when comparing both cell types in 1:10 ratio.

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that the BMMCs could be considered professionalphagocytes against A. actinomycetemcomitans inthe same way as macrophages are, irrespective ofthe bacterial killing. It is important to emphasize thatthe bacterial phagocytosis by both cell populationsoccurred in a bacterial load-independent manner.

Based on the methodology used, it was not pos-sible to verify the viability of internalized bacteria.Malaviya et al.18 demonstrated that murine MCs wereable to phagocytose and kill Gram-negative bacteriain a medium containing serum components by meansof non-oxidative and oxidative mechanisms. How-ever, several bacteria can affect this phagocyticability of MCs to their advantage when in opsonin-poor microenvironments,45 thus inducing the cell tobecome an intracellular environment in which bac-teria could proliferate and contribute to bacterialspreading. Under these conditions, MCs would thenserve as reservoirs of viable bacteria, resulting inharmful effects to the host.46 In fact, our results re-vealed high percentages of MCs with >5 internalizedA. actinomycetemcomitans, mainly in the absence ofopsonization through complement. Accordingly, C3-deficient mice presented functional impairment ofMCs with delayed elimination of bacteria and re-duction in their degranulation and production oftumor necrosis factor-alpha, suggesting that productsof the complement system are essential for the ac-tivation and microbicidal activity of MCs after bac-terial challenge.47 Moreover, E. coli phagocytosis byMCs through CD48 molecule (anchored glyco-

sylphosphatidylinositol) promoted bacterial deathmore efficiently than through CR3.35

When MCs interact with opsonized bacteria andperform their phagocytic and microbicidal functions,they simultaneously release considerable amounts ofproinflammatory mediators, thereby leading to themigration of more inflammatory cells at the infectiousfocus.48-50 When there is excessive release of media-tors, it can result in exacerbation of the inflammatoryprocess, causing serious cytotoxic effects on tissues.However, several internalized bacterial pathogensexpress toxins that can inhibit the release of chemicalmediators by MCs.45 Thus, we are tempted to thinkthat the phagocytic response of MCs against A. acti-nomycetemcomitans can be ambiguous in biofilm-associated periodontal diseases, either protectingthe individual by phagocytosis and bacterial killingor leading to additional destruction and progression ofthe disease by exacerbated release of mediators.Moreover, MCs could be an intracellular refuge forperiodontopathogens. With regard to these consid-erations, there continue to be conflicting views onwhether phagocytosis by MCs plays a protective orharmful role in relation to clinical progression ofperiodontal disease. Although there is broad scientificknowledge about the pathogenesis of periodontaldiseases, additional studies are needed for a betterunderstanding of the biologic events involved in thispathology, especially those related to MCs.

CONCLUSIONS

This early in vitro investigation proved the phago-cytic capacity of MCs against A. actinomyce-temcomitans. Although this is an in vitro study and onlyconfocal scanning electron microscopy was used asa method to analyze phagocytosis, the results ob-tained here lead to the hypothesis of a possible roleof MCs as professional phagocytes in a manner simi-lar to that of macrophages. In an additional study, weintend to investigate the microbicidal activity ofMCs against internalized A. actinomycetemcomitans,whether opsonized or not, thus contributing to a betterunderstanding of the real importance of bacterialphagocytosis by MCs in inflammatory PD. In thecontext of infectious diseases, especially PD, it islikely that this will not only expand the scope of ourknowledge of the role of MCs in innate immunity withregard to these diseases but may provide new thera-peutic targets to control the inflammatory responsein gingival and periodontal tissues against the peri-odontopathogens.

ACKNOWLEDGMENTS

This study was supported by Coordenacxao de Aper-feicxoamento de Pessoal de Nıvel Superior and Funda-cxao de Amparo a Pesquisa do Estado de Sao Paulo

Figure 5.Analysis of the phagocytes against E. coli. Bars show the percentage ofBMMCs and MPMs with A. actinomycetemcomitans internalized ornot. Macrophages and MCs were classified as follows: absence ofbacteria (a) or with £5 (b) or >5 (c) internalized bacteria, only theproportion of one cell to 10 bacteria (1:10). Results are expressed as themean– SD from three independent experiments. Datawere analyzed byTukey test and Student t test. *P <0.05; †P <0.01; ‡P <0.001.

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Grant 2009/14152-1.The authors thank Dr. Fernandode Queiroz Cunha (Department of Pharmacology)and Dr. Maria Celia Jamur (Department of Cell andMolecular Biology, School of Medicine of RibeiraoPreto, Sao Paulo University, Ribeirao Preto, SP, Brazil)for providing antibodies. We also thank Dr. MauraRosane Valerio Ikoma, Marcimara Penitenti (bothfrom Laboratory of Hematology, Amaral CarvalhoHospital, Jau, SP, Brazil), Dr. Camila Peres Buzalaf,Marcia Sirlene Zardin Graeff, and Marcelo MilandaRibeiro Lopes (all from Integrated Research Center,Bauru Dental School, University of Sao Paulo, Bauru,SP, Brazil) for their technical support, and ProfessorsJose Roberto Pereira Lauris and Heitor MarquesHonorio (both from Department of Pediatric Denti-stry, Orthodontics, and Public Health, Bauru DentalSchool, University of Sao Paulo) for the statisticalanalysis. The authors report no conflicts of interestrelated to this study.

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Correspondence: Dr. Vanessa Soares Lara, Bauru Schoolof Dentistry, University of Sao Paulo, Al. Dr. OtavioPinheiro Brisolla 9-75, CEP 17.012-901, Bauru, SP, Brazil.Fax: 55-14-3235-8251; e-mail: vanessa@fob.usp.br.

Submitted February 3, 2012; accepted for publicationMarch 30, 2012.

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