Human pulp responses to in-office tooth bleaching

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    completely understood. It has been reported that thelow molecular weight of H2O2 molecules makes themcapable of diffusing across enamel and dentin to reachthe pulpal space.1 Consequently, H O and its degra-daingodthaco






    trayoyears. The parents/guardians as well as the volunteers,after reading and receiving all necessary explanationsincluding the experimental rationale, the clinical pro-cedures, and possible risks, were asked to sign a con-


    107 2doi2 2tion products, which play a role in the tooth bleach-, may cause damage to the pulp cells, especially to

    ontoblasts that underlie dentin.7 It has been reportedt 70% of patients submitted to bleaching have

    mplained about postoperative sensitivity, particularly

    sent form explaining the research protocol, which waspreviously approved by the Ethics Committee.

    Sixteen sound teeth were selected for the study anddivided into 4 groups: bleached premolars (G1; n 6),bleached incisors (G2; n 4), nonbleached premolars(G3; n 3), and nonbleached incisors (G4; n 3). Thelatter 2 groups served as control groups and receivedonly rubber/pumice prophylaxis.

    The teeth were cleaned by rubber/pumice prophy-laxis, thoroughly washed, and dried with an oil-freeair stream. Then, for groups G1 and G2, a light-curedresin-based gingival barrier (Opal Dam; UltradentProducts, South Jordan, UT) was used to protect thesoft tissues circumjacent to teeth being whitened andto protect the adjacent teeth. A 38% H2O2 bleaching

    ported in part by the Fundao de Amparo Pesquisa do EstadoSo PauloFAPESP (grant nos. 2007/50646-3 and 2008/05890-6)Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico

    Pq (grant no. 301029/2007-5).partment of Physiology and Pathology.ivate practice.partment of Restorative Dentistry.partment of Orthodontics and Pediatric Dentistry.

    9-2104/$ - see front matteruman pulp responses to in-office tooCarlos Alberto de Souza Costa,a Heraldo Riehl,Nancy Tomoko Sacono,d and Josimeri Hebling,ARARAQUARA SCHOOL OF DENTISTRY, SO PAUL

    jective. To evaluate and compare the responses of humdy design. A bleaching agent with 38% hydrogen peroxer teeth (G1: 6 premolars; G2: 4 incisors) for 45 minute

    mice prophylaxis were used as control groups G3 and Gteeth were extracted and processed for histologic evalu

    sults. Only in G2 (4 incisors) were any changes in the pagulation necrosis. The radicular pulp showed mild inflanonuclear cells around congested and dilated blood vesups (G3 and G4) or in group G1.nclusion. Bleaching with 38% H2O2 for 45 minutes caumolars. (Oral Surg Oral Med Oral Pathol Oral Radiol E

    drogen peroxide (H2O2) is a thermally unstable ox-en-derived free radical frequently found within thells as the result of a series of intracellular reactionst occur specifically in the mitochondria.1 High con-

    ntrations of this chemical agent, which present oxi-tive power,2-4 have been used to treat discoloredth.5 The oxidative reactions and consequent cellmage caused by free radicals are the main mecha-ms responsible for the toxicity of peroxide-contain-compounds.6 Although it is known that free radicalscapable of degrading complex organic molecules

    t are responsible for tooth coloration,4 the exactchanisms by which the teeth are bleached are notgeac

    010 Mosby, Inc. All rights reserved.:10.1016/j.tripleo.2009.12.002leachingFernando Kina,c

    aquara, BrazilTE UNIVERSITY

    isor and premolar pulps after bleaching.2O2) was applied on the buccal surface of 10 soundee premolars and 3 incisors that received only rubber/ectively. Two days after the bleaching procedure,

    tected. In the coronal pulp there was a large zone ofry changes manifested as an accumulation ofo pulpal damage was seen in either of the control

    eversible pulp damage in lower incisors but not in2010;109:e59-e64)

    their anterior teeth.10 However, although several inro studies have evaluated the cytotoxicity of bleachingents to culture of cells,8,9 few data are available con-ning the effects of bleaching agents on human pulps.erefore, the aim of the present study was to evaluated compare the responses of pulps of anterior (incisors)d posterior (premolars) sound human teeth submitted tooffice bleaching using a gel with 38% H2O2.

    ATERIALS AND METHODSSixteen caries-free human teeth scheduled to be ex-cted for orthodontic reasons were selected fromung patients. The mean age of the patients was 16.2l (Opalescence Xtra Boost; Ultradent) was handledcording to the manufacturers instructions and was


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    OOOOEe60 de Souza Costa et al. April 2010plied to the buccal surface of the teeth in such a wayt a uniform 1-mm layer of material was formed andt undisturbed for 15 min. The bleaching gel wasoved from the tooth surface using a saliva ejector

    th high-power suction, and the bleaching cycle waseated 2 more times, totaling 3 applications of 15nutes each. After the third application, the bleachingl was removed from the enamel surface and the tooths copiously rinsed with an air/water spray and gentlyed with sterile gauze.Two days after the bleaching procedure, the teethre extracted under local anesthesia. The roots weremediately sectioned midway between the cement/amel junction and the root apex with a high-speedndpiece under water spray. The teeth were stored forhours in formalin fixative solution at pH 7.2, decal-ed in buffered Morse solution (equal volumes of

    % acid formic and 20% sodium citrate), under agi-ion, cleared in xylol, vacuum infiltrated with waxraffin, and finally embedded in paraffin. Six-micro-ter-thick serial sections were cut (820; Spencer Mi-tome, Carson, CA), mounted on glass slides, andined with hematoxylin and eosin (H/E) and Massonchrome. Based upon previous in vivo studies,11,12 alltions were evaluated by a calibrated examinernded to the groups. Using a light microscope (62774;rl Zeiss, Oberkchen, Germany) the following his-athologic events were evaluated and classified by a

    ble I. Inflammatory cell responsere Characterization

    None or a few scattered inflammatory cells present in thepulp area corresponding to the buccal surface of thetooth in which the bleaching gel was applied

    Slight inflammatory cell infiltrate withpolymorphonuclear or mononuclear leukocytes

    Moderate inflammatory cell infiltrateSevere inflammatory cell infiltrate

    ble II. Tissue disorganizationre Characterization

    Normal tissueOdontoblastic layer disorganized but central pulp normalTotal disorganization of the pulp tissue morphologyPulp necrosis associated or not with dystrophic calcification

    ble III. Reactionary dentin formationre Characterization

    AbsenceModest hard tissue deposition beneath the buccal surface of

    the tooth in which the bleaching gel was appliedModerate hard tissue deposition beneath the buccal surface

    of the tooth in which the bleaching gel was appliedIntense hard tissue deposition in the pulp tissuescriptive analysis according to the criteria presentedTables I-III: inflammatory cell response, pulp tissueorganization, and reactionary dentin formation.Using a light microscope (Diastar; Cambridge Instru-nts, Buffalo, NY) adapted to a video camera (DXC-7A/107AP; Sony Electronics, Tokyo, Japan), 3 linearasures from the enamel/dentin junction to the pulp

    amber were carried out to determine the dentin thick-ss (DT). The video images were loaded into a com-ter and processed using standard software (Mocha;ndel Scientific, San Rafael, CA). The methodologyed in the present study to measure the DT was similarthat previously used to determine the remaining

    ntin thickness between the cavity floor and the sub-ent pulp tissue.11,12 The 3 readings were averaged totain the mean DT for each tooth. The data regarding

    DT were submitted to nonparametric Kruskal-Wal-test complemented by Mann-Whitney tests at a

    nificance level of 5% (SPSS, Chicago, IL).

    SULTSThe scores for each criterion determined by the his-ogic assessment of the specimens according toups are shown in Table IV. The individual anddian DT values are presented in Table V. RegardingDT, a significant statistical difference was observed

    tween incisors (G1 and G3) and premolars (G2 and), with incisors presenting a smaller DT than pre-lars (P .05). No statistical difference was ob-ved when groups G1 (bleached premolars) and G3nbleached premolars) were compared (P .05).e same lack of difference was seen between G2eached incisors) and G4 (nonbleached incisors).The analysis of the radiographs which were takenmediately before the extractions of all of the teeth

    ble IV. Absolute frequency (n) observed for eachtopathologic event according to the groups


    Histopathologic event Group 0 1 2 3ammatory cell response G1 6 0 0 0

    G2 0 1 3 0G3 3 0 0 0G4 3 0 0 0

    sue disorganization G1 6 0 0 0G2 0 0 1 3G3 3 0 0 0G4 3 0 0 0

    actionary dentin formation G1 6 0 0 0G2 0 0 1 3G3 3 0 0 0G4 3 0 0 0

    , Bleached premolars (n 6); G2, bleached incisors (n 4); G3,bleached premolars (n 3); G4, nonbleached incisors (n 3).

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    OOOOEVolume 109, Number 4 de Souza Costa et al. e61ed in the present in vivo study demonstrated noriapical pathology. The volunteers who had theirerior incisors bleached (G2) reported postoperativein. Therefore, based upon the IRB recommendations,

    teeth were extracted. Conversely, the volunteerso had their premolars bleached (G1) did not report

    y particular postoperative sensitivity. However, tompare the pulp responses occurring in premolars andisors, all premolars also were extracted 2 days after

    ing bleached.Overall, the histologic features showed that the pulpsue observed in G1 (bleached premolars) and bothntrol groups, G3 (nonbleached premolars) and G4nbleached incisors), were quite similar. The pulpponses in G2 (bleached incisors) were different fromse observed in G1, G2, and G3. In G1, all premolarssented pulp tissue with normal histologic character-

    ics, as observed in G3 and G4. Consequently, lack ofammatory response and no tissue disorganizations observed for all specimens (Fig. 1, A and B). Thedian of the dentin thickness for this experimentalup was 3.10 mm (mean SD 3.10 0.11 mm).G2, the coronary pulp tissue of the 3 bleachedisors exhibited a wide zone of coagulation necrosis

    ig. 1, C and D). Deposition of reactionary dentin wasn in the root pulp of these incisors, which showed aderate inflammatory response mediated by mononu-ar cells among dilated and congested blood vessels

    ig. 1, E and F). In only 1 incisor, intense depositionreactionary dentin in the coronal and root pulp tissues observed. In that tooth, the root pulp exhibited mildammatory response characterized by a few inflam-tory mononuclear cells. The median of the dentinckness for this experimental group was 1.83 mmean SD 1.82 0.08 mm). In the nonbleachedntrol groups G3 and G4, all premolars and incisorshibited normal pulp tissue. The pulp-dentin complexhibited tubular dentin and predentin, intact odonto-

    ble V. Dentin thickness (mm) for bleached and nonbl

    ecimen (tooth) G11 2.992 3.213 3.104 2.985 3.106 3.24

    an (SD) 3.10 (0.11) 1.82 (0dian (P25-P75) 3.10 (2.99-3.22)a 1.83 (1roup designations as in Table IV.ach value represents the mean of 3 measurements taken in the sameedians identified with the same letter represent groups that do not dst layer, cell-free zone, cell-rich zone, and normal cen-l part of the pulp tissue (Fig. 2). The medians of thentin thickness for G3 and G4 were 3.02 mm (mean 3.01 0.21) and 1.80 mm (mean SD 1.84

    7 mm), respectively.

    SCUSSIONAccording to Wataha et al.,13 the risk caused byntal materials to the dentin-pulp complex depends on

    capability of the components of these products tofuse across enamel and dentin to reach the pulpsue. Several studies have demonstrated the diffusionH2O2 through enamel and dentin,8,14 which is facil-ted by its low molecular weight as well as its capac-to denature tissue proteins.15 In addition, it has beenorted that the diffusion of H2O2 through enamel and

    ntin increases as the concentration of this unstablee radical in the bleaching gel also increases.6 In thesent in vivo study, the authors did not quantify orntify the products released from the 38% H2O2aching gel applied on the enamel in the pulp tissue.wever, it was demonstrated that the coronal pulp ofst (3 out of 4) of the bleached incisors exhibited

    agulation necrosis (Fig. 1, C and D). In this experi-ntal group, the mean enamel-dentin junctionpulptance was 1.82 0.08 mm. On the other hand, in thentrol group G4, in which the incisors were notached, the pulp tissue exhibited normal histologic

    aracteristics (Fig. 2, C-E). Therefore, it may be sug-sted that toxic components released from the bleach-

    agent were capable of diffusing through the thinamel and dentin present in the inferior incisors touse notable damage to the pulp tissue.It has been demonstrated that the application ofaching agents on enamel increases the porosity ofs highly mineralized dental tissue owing to the dis-tion of matrix protein, which causes loss of struc-al components by free radical oxidation.16 In addi-

    d (control) human premolars and incisorsGroup*

    G3 G42.80 1.693.22 2.023.02 1.80

    3.01 (0.21) 1.84 (0.17))b 3.02 (2.80-3.18)a 1.80 (1.69-1.94)b

    en/tooth.atistically (Mann-Whitney: P .05).disco




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    OOOOEe62 de Souza Costa et al. April 2010n, the contact time of the bleaching agent with thental tissue influences the transenamel and transden-al diffusion of bleaching components to reach thelpal space.3 The increase in hard dental tissue per-ability may also play a role in postoperative sensi-ity or pain after bleaching therapies, which is the

    . 1. Pulp-dentin complex of sound lower human premolar (te that both pulp horns of the premolar present normal histolorn in A related to the tooth surface in which the bleaching gontoblast layer (horizontal arrow), cell-free zone (vertical aponse, necrosis, or pulpal calcification can be seen. H/E,12serve the thickness of the dentin (mean 1.793 m) below thrn exhibits a large zone of coagulation necrosis (N). H/E, 32ween the tubular dentin (TD) and the necrotic pulp tissue (N).ibits notable deposition of reactionary dentin matrix. H/E, DM), which is poorly calcified, the defined predentin (P), aa few congested blood vessels (arrows). H/E, frequent adverse effect claimed by the patientsbmitted to in-office vital tooth bleaching.10 There-e, it may be speculated that the pain claimed by thelunteers in the present in vivo study after bleachingincisors was caused, at least in part, by the increase

    the permeability in the enamel and dentin. However,

    B) and incisors (C-F) submitted to in-office bleaching. A,racteristics. H/E,32. B, High magnification of the pulpalapplied. Observe the tubular primary dentin, the definedand the cell-rich zone (oblique arrow). No inflammatoryentin-pulp complex of a bleached lower incisor (tooth #3).al surface where the bleaching gel was applied. The pulpigh magnification of C. Note the transition border (arrows)125. E, Root pulp tissue of a bleached lower incisor which

    F, Detail of E. Note the tubular reactionary dentin matrixremaining pulp tissue (PT) with some mononuclear cellsmo




    A andgic chael wasrrow),5. C, De bucc. D, HH/E,125.

    nd the

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    OOOOEVolume 109, Number 4 de Souza Costa et al. e63diffusion of H2O2 to the pulp tissue may also beolved in postoperative sensitivity or pain caused bytoxic effects of this chemical agent to the pulp

    lls.17 It is known that H2O2 and its degradation prod-ts, such as hydroxyl (OH) ions, are reactive speciesrived from oxygen, which may cause mutagenesis,rcinogenesis, cell membrane damage by lipid peroxi-tion, and protein fragmentation.18,19 Therefore, therease in the exogenous levels of these highly reac-e free radicals1,18 in contact with cells, as occursring tooth bleaching, may result in cell death anduced cell proliferation.20-23 These data may explainnecrosis occurring in the pulp tissue after bleachingincisors as well as the postoperative pain reportedthe patients. However, future in vivo studies are

    eded to clarify these histologic and clinical observa-ns.Vital teeth present dentinal fluid flow produced byrapulpal pressure and cytoplasmatic prolongations ofontoblasts as well as other intratubular components,ich may impede the diffusion of the bleaching gel

    mponents through the dentinal tubules. In addition,pulp tissue presents lymphatic drainage which is

    pable of moving away toxic products that reach thisnnective tissue. Furthermore, owing to the oxidativeess generated by the presence of free radicals, the

    . 2. Control groups. Dentin-pulp complex of unbleached lowlps tissue exhibits normal histologic features. Masson trichro

    pulp tissue with all defined histological zones. Masson trichhrome, 32. D, High magnification of C. No pulp necrosis,this specific connective tissue. Masson trichrome, 64. Eontoblast layer (arrows) and the subjacent pulp tissue with pfense system of the pulp cells is activated, releasingeral endogenous antioxidant agents, such as peroxi-

    ses and catalases, which promote an enzymatic deg-ation of H2O2 to avoid excessive tissue damage.24wever, it seems that the outward dentin fluid move-nt, the complex network of lymphatic vesselssent in the pulp tissue, as well as the protective rolethe defense system of the pulp were not enough tovent coronal pulp necrosis when the bleaching agent

    th 38% H2O2 was applied on the buccal surface ofer sound incisors. Perhaps, the different pulpal re-

    onses observed in premolars (Fig. 1, A and B) andisors (Fig. 1, C-F) occurred because of the variableckness of enamel and dentin. It has been reportedt the dentin thickness plays an important role in thetection of the pulp tissue against toxic products

    eased from dental materials.12,14 In the present ino study, the decalcification of the teeth during theoratory process resulted in complete enamel loss.nsequently, only the dentin thickness was micro-pically measured. The bleached premolars (G1) andisors (G2) presented mean DTs of 3.10 0.11 mm

    d 1.82 0.08 mm, respectively. Therefore, it may beggested that the protective role of dentin againstfusion of toxic components leached from the bleach-

    agent with 38% H2O2 used in the present study

    nd premolar (A and B) and incisor (C-E). A, The coronal32. B, High magnification of the area indicated in A. Note

    250. C, General view of the coronal pulp tissue. Massonmatory pulpal response, or significant change is observedil of the coronal pulp tissue in D. Note the continuouslls and blood vessels. Masson trichrome, 125.sev






  • increased as a function of the thickness of this tubularhard tissue. Considering the tooth bleaching proceduresco


















    8. Benetti AR, Valera MC, Mancini MNG, Miranda CB, Baldicci I.In vitro penetration of bleaching agents into the pulp chamber.Int Endod J 2004;37:120-4.



















    OOOOEe64 de Souza Costa et al. April 2010mmonly used in esthetic dentistry, it may also beggested that the intensity of pulp responses is in-rsely related to the thickness of the enamel. Because

    enamel is thicker in premolars than in incisors, ity be speculated in the present investigation that theckness of enamel plays also an important role inventing H2O2 and other products leached by theaching gel from reaching the pulp tissue. However,s hypothesis needs further investigations. No pulpmage in bleached premolars was reported by Rob-son and Melfi.25 Those authors demonstrated thatastrous pulp response in bleached premolars wasserved only when the bleaching agent was heated.It is known that the continuous deposition of second-

    and intratubular dentin over time increases theckness of this hard tissue and reduces the innermeter of the dentinal tubules. Both physiologicchanisms, which decrease the dentin permeability,y impede the diffusion of bleaching agent compo-

    nts across dentin to reach the pulp tissue. Therefore,ther in vivo studies are needed to evaluate if similarlpal damage, as demonstrated in the present investi-tion for sound bleached incisors of young patients,ght also take place in teeth of older patients submit-to in-office bleaching.

    NCLUSIONBased on the methodology used in this in vivo study,may be concluded that tooth bleaching with 38%O2 for 45 minutes causes irreversible pulp damage iner incisors but not in premolars.

    FERENCESShackelford RE, Kaufmann WK, Paules RS. Oxidative stress andcell cycle checkpoint function. Free Radic Biol Med 2000;28:1387-404.Arwill T, Myrberg N, Soremark R. Penetration of radioactive iso-topes through enamel and dentin. II. Transfer of 22Na in fresh andchemically treated dental tissues. Odontol Revy 1969;20:47-54.Hanks CT, Fat JC, Wataha JC, Corcoran JF. Cytotoxicity anddentin permeability of carbamide peroxide and hydrogen-perox-ide vital bleaching materials, in vitro. J Dent Res 1993;72:931-8.Tredwin CJ, Naik S, Lewis NJ, Scully C. Hydrogen peroxidetooth-whitening (bleaching) products: review of adverse effectsand safety issues. Br Dent J 2006;200:371-6.Zantner C, Beheim-Schwarzbach N, Neumann K, Kielbassa AM.Surface microhardness of enamel after different home bleachingprocedures. Dent Mater 2007;2:243-50.Li Y. Biological properties of peroxide-containing tooth whiten-ers. Food Chem Toxicol 1996;34:887-904.Goldberg M, Smith AJ. Cells and extracellular matrices of dentinand pulp: a biological basis for repair and tissue engineering. CritRev Oral Biol Med 2004;15:13-27.Camargo SEA, Valera MC, Camargo CHR, Mancini MNG,Menezes MM. Penetration of 38% hydrogen peroxide into thepulp chamber in bovine and human teeth submitted to officebleach technique. J Endod 2007;33:1074-7.Buchalla W, Attin T. External bleaching therapy with activationby heat, light or lasera systematic review. Dent Mater2007;23:586-96.Costa CAS, Teixeira HM, Nascimento ABL, Hebling J. Biocom-patibility of resin-based materials applied as liners in deep cav-ities prepared in human teeth. J Biomed Mater Res B ApplBiomater 2007; Souza Costa CA, Hebling J, Randall RC. Human pulp re-sponse to resin cements used to bond inlay restorations. DentMater 2006;22:954-62.Wataha JC, Hanks CT, Strawn SE, Fat JC. Cytotoxicity ofcomponents of resins and other dental restorative materials.J Oral Rehabil 1994;21:453-62.Gkay O, Mjdeci A, Algin E. In vitro peroxide penetration intopulp chamber from newer bleaching products. Int Endod J2005;38:516-20.Goldstein CE, Goldstein RE, Feinman RA, Garber DA. Bleach-ing vital teeth: state of the art. Quintessence Int 1989;20:729-37.Kwon YH, Huo MS, Kim KH, Kim SK, Kim YJ. Effects ofhydrogen peroxide on the light reflectance and morphology ofbovine enamel. J Oral Rehabil 2002;29:473-77.Haywood VB. Current status of nightguard vital bleaching. Com-pend Contin Educ Dent 2000;28:S10-7.Martindale JL, Holbrook NJ. Cellular response to oxidative stress:signaling for suicide and survival. J Cell Physiol 2002;192:1-15.Sies H. Strategies of antioxidant defense. Eur J Biochem1993;215:213-9.Allen RG, Tresini M. Oxidative stress and gene regulation. FreeRadic Biol Med 2000;28:463-99.Slater AF, Stefan C, Nobel I, van den Dobbelsteen DJ, OrreniusS. Signalling mechanisms and oxidative stress in apoptosis.Toxicol Lett 1995;82:149-53.Trindade FZ, Ribeiro APD, Sacono NT, Oliveira CF, Lessa FCR,Hebling J, Costa CAS. Trans-enamel and trans-dentinal cytotoxiceffects of a 35% H2O2 bleaching agent on an odontoblast cell lineafter consecutive applications. Int Endod J 2009;42:516-24.Dias Ribeiro AP, Sacono NT, Lessa FCR, Nogueira I, ColdebellaCR, Hebling J, de Souza Costa CA. Cytotoxic effect of a 35%hydrogen peroxide bleaching gel on odontoblast-like MDPC-23cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod2009;108:458-64.Esposito P, Varvara G, Murmura G, Terlizzi A, Caputi S. Abilityof healthy and inflamed human dental pulp to reduce hydrogenperoxide. Eur J Oral Sci 2003;111:454-6.Robertson WD, Melfi RC. Pulp response to vital bleachingprocedures. J Endod 1908;6:645-9.

    rint requests:f. Dr. Carlos Alberto de Souza Costapartamento de Fisiologia e Patologiauldade de Odontologia de AraraquaraUNESP

    a Humait, 1680CentroP: 14801-903raquara,



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