ABSTRACT
http://dx.doi.org/10.1590/1678-775720150393
Ev alu at ion of an ex p er im en t al r at m od el f or
com parat ive st udies of bleaching agent s
/XFLDQR7DYDUHV$QJHOR&,175$1, Francine BENETTI1, Luciana Lousada FERREIRA19DQHVVD5$+$/2, Edilson
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1- Univ. Estadual Paulista - UNESP, Faculdade de Odontologia de Araçatuba, Departamento de Endodontia, Araçatuba, SP, Brasil.
2- Univ. Estadual Paulista - UNESP, Faculdade de Odontologia de Araçatuba, Departamento de Odontologia Restauradora, Araçatuba, SP, Brasil. 3- Univ. Estadual Paulista - UNESP, Faculdade de Odontologia de Araçatuba, Departamento de Ciências Básicas, Araçatuba, SP, Brasil.
&RUUHVSRQGLQJDGGUHVV Dr. Luciano Tavares Angelo Cintra - Department of Endodontics - Araçatuba Dental School - UNESP - Univ. Estadual Paulista - R. José Bonifácio, 1193 - Araçatuba - São Paulo - Brazil - Phone: + 55 18 3636-2867 - Fax: +55 18 3636-3253 - e-mail: lucianocintra@foa.unesp.br
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ent al m at er ials, in general, ar e t est ed in differ ent anim al m odels pr ior t o t heir clinical use in hum ans, except for bleaching agent s. Obj ect ives: To evaluat e an exper im ent alUDWPRGHOIRUFRPSDUDWLYHVWXGLHVRIEOHDFKLQJDJHQWVE\LQYHVWLJDWLQJWKHLQÀXHQFHRI
differ ent concent rat ions and applicat ion t im es of H2O2JHOLQWKHSXOSWLVVXHGXULQJLQRI¿FH bleaching of rat s’ vit al t eet h. Mat er ial and m et hods: The r ight and left m axillar y m olar s of 50 Wist ar rat s w er e bleached w it h 20% and 35% H2O2 gels, r espect ively, for 5, 10, 15, 30, or 45 m in ( n= 10 rat s/ gr oup) . Ten anim als ( cont r ol) w er e unt r eat ed. The rat s w er e killed
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w er e at t r ibut ed t o t he odont oblast ic layer and t o vascular changes. The t er t iar y dent in ar ea and t he pulp cham ber cent ral ar ea w er e hist om or phom et r ically m easur ed. Dat a w er e com par ed by t he analysis of var iance and t he Kr uskal- Wallis t est ( p< 0.05) . Result s: Aft er
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pulp unt il t he t im e of 15 m in for bot h concent rat ions of bleaching gels. I n 30 and 45 m in
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t he appearance of necr ot ic ar eas. Aft er 30 days, a r educt ion in t he pulp cham ber cent ral ar ea and an enlar gem ent of t er t iar y dent in ar ea w er e obser ved w it hout t he det ect ion of
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adequat e for bleaching pr ot ocols st udies, as it was possible t o obser ve alt erat ions in t he pulp t issues and in t he t oot h st r uct ur e caused by differ ent concent rat ions and per iods of applicat ion of bleaching agent s.
Ke y w or ds: Bleaching agent s. Anim al m odels. Hydr ogen per oxide.
I N TROD UCTI ON
,QRI¿FHEOHDFKLQJZLWK+2O2 gel is consider ed a conser vat ive and affor dable aest het ic t r eat m ent18. I t s effect iveness is due t o t he low m olecular m ass of t he m ain act ive com pound: H2O2, w hich easily diffuses t hr ough enam el and dent in and r eleases r eact iv e ox y g en sp ecies ( ROS) , t h u s ox id izin g or ganic st r uct ur es2.
I m p or t an t ly, H2O2 an d it s b y - p r od u ct s h av e var ying biological effect s on hum an oral t issues30. Th e ROS- i n d u ced ox i d a t i v e st r ess ca n ca u se
m ut at ion, enzym e inact ivat ion, prot ein degradat ion, and fragm ent at ion of pulp cells t hat m ight m anifest as pu lpit is an d t oot h sen sit iv it y3. Th e sev er it y
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bleaching pr ot ocols, and t his pr ocedur e has been incr easingly quest ioned2,6,8.
in cr eased d ep osit ion of r ep ar at iv e d en t in , t h e
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r ev er sed af t er 6 0 d ay s2 5 ,Q KXPDQV LQRI¿FH bleaching of m andibular incisor s using t he above-m ent ioned pr ot ocol caused par t ial necr osis in t he
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t he radicular pulp8. Mor eover, a 45 m in bleaching wit h 35% H2O2 gel result ed in necrosis near t he pulp hor ns in rat s6. On t he ot her hand, t he applicat ion of 38% H2O2 gel on hum an pr em olar s did not cause pat hological changes in t he dent al pulp17. Therefore, it is evident t hat t he anat om ical charact er ist ics of t he t eet h and of t he in vivo m odel analyzed, as w ell as t he bleaching pr ot ocols em ployed, det er m ined differ ent r esult s.
Thus, t he lesser t hickness of enam el and dent in in t eet h of rat s m ight allow a gr eat er penet rat ion of H2O2, consequent ly causing m or e dam ages t o pulp t issues8. Therefore, it is essent ial t o charact erize t he
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prot ocol t o be applied in t his m odel and t o allow t he per for m ance of fur t her st udies on H2O2 dam age t o pulp t issues. This m odel w ill enable t he evaluat ion of new dosages, for m ulat ions, and concent rat ions of bleaching agent s t hat ar ise in t he m ar ket , and also of t he pot ent ial t herapeut ic agent s t hat m ay be used t o m inim ize t he dam age caused by H2O2 in differ ent applicat ion pr ot ocols6,9.
The choice by t he rat s in t his anim al m odel w as d u e t o t h e easin ess in st an d ar d izin g an d cont r olling t hese anim als, and t he possibilit y of per for m ing ot her t est s7,9. Thus, it is possible for us t o st udy differ ent var iables and t o be able t o pr opose, in a second st age and w it h t he r esult s alr eady st andar dized and evaluat ed for anim als, t he validat ion of t he sam e r esult s in sm aller gr oups of h u m an s by f ollow in g t h e et h ical pr in ciples9. Research involving bot h dogs’ and hum an’s t eet h t o st udy bleaching pr ot ocols ar e im pract ical because
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and also because of et hical principles. Furt herm ore,
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of t he num ber of bleaching sessions in pulp t issues, indicat ed t he possibilit y of using t eet h of rat s for t he st udy of bleaching pr ot ocols. Using t he rat m odel for st udying bleaching agent s is r elat ively sim ple and easy t o r epr oduce.
Th e r e f o r e , t h e p u r p o se o f t h i s st u d y w a s t o ch ar act er ize an ex p er im en t al an im al m od el for com parat iv e st udies of bleaching agent s by
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and applicat ion t im es of H2O2JHOGXULQJLQRI¿FH bleaching of rat s’ vit al t eet h. We hypot hesized t hat : ( I ) t he H2O2 bleaching gel is capable of penet rat ing t he pulp t issue and causing gr eat er dam ages w hen t he t im e of applicat ion and t he concent rat ion of
H2O2 ar e incr eased; ( I I ) t he pulp t issue is capable of r ecover ing fr om t he dam ages caused by H2O2 aft er long per iods of t im e.
M ATERI AL AN D M ETH OD S
An im a ls
Si x t y m a l e W i st a r r a t s ( 1 8 0 - 2 0 0 g ) w e r e used in t his st udy. The anim als w er e housed in a t em perat ur e- cont r olled envir onm ent ( 22°C ± 1°C) in a st andar d light - dar k schedule w it h unr est r ict ed access t o food and wat er. The experim ent al prot ocol was approved by t he Et hics Com m it t ee ( CEUA 2013-01253) and conduct ed accor ding t o Guide for t he Car e and Use of Laborat or y Anim als of t he Nat ional I nst it ut es of Healt h ( Bet hesda, Mar yland, USA) .
Toot h ble a ch in g
The rat s w er e anest het ized w it h int ram uscular inj ect ions of ket am ine ( 87 m g/ kg; Francot ar, Virbac do Brasil I ndúst r ia e Com ér cio Lt da., Roseira, São Paulo, Brazil) and xylazine ( 13 m g/ kg; Rom pum , Bayer SA, São Paulo, São Paulo, Brazil) . The r ight and left m olar s in ever y anim al w er e bleached w it h 20% ( Whit eness HP Blue, FGM Dent al Pr oduct s, Joinv ille, Sant a Cat ar ina, Brazil) and 35% H2O2 ( W h i t e n e ss HP Ma x x , FGM D e n t a l Pr o d u ct s, Joinville, Sant a Cat ar ina, Brazil) , r espect ively, for 5, 10, 15, 30, or 45 m in ( n= 10 rat s/ gr oup) . Ten anim als ( cont r ols) did not r eceive any t r eat m ent .
H ist ology
Anim als w er e killed by t he anest het ic solut ion over dose 2 or 30 days aft er t he bleaching sessions. Their bilat eral m axillae w er e separat ed, dissect ed,
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solut ion for t hr ee m ont hs and t hen dehydrat ed in
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hem at oxylin and eosin ( H&E) . The serial hist ological sect ions of each specim en w er e select ed fr om t he
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seen in all it s longit udinal ext ension.
Th e cor on al pu lp w as div ided in t o occlu sal, m iddle, and cer vical t hir ds and t he radicular pulp was divided int o cervical, m iddle, and apical t hirds6.
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cor on al an d r ad i cu l ar t h i r d s of t h e p u l p . Th e
FRXQWLQJRI¿EUREODVWVZDVDOVRSHUIRUPHG7KHFHOO FRXQWLQJZDVSHUIRUPHGLQDNjP2 ¿HOGLQHDFK t hird of t he pulp t issue of each specim en, exam ined u n d er lig h t m icr oscop y ( × 1 0 0 0 m ag n if icat ion ; DM4000 B, Leica Microsyst em s, Wet zlar, Germ any) .
layer ; or 3- disr upt ion of t he odont oblast ic layer. Scor es t o vascular changes w er e also assigned as follow s: 1- nor m alit y; 2- incr ease in t he num ber of blood vessels; or 3- necr osis.
The m ean of cent ral ar ea of t he pulp cham ber was m easured by im age processing soft ware ( Leica QWin V3, Leica Micr osyst em s, Wet zlar, Ger m any) ( Figur e 1) . By t ak ing int o account t he obt ained values it was possible t o calculat e t he r educt ion per cent age in t he cent ral ar ea of t he pulp cham ber in t he t r eat ed gr oups consider ing t he cent ral ar ea of t he cont r ol gr oup.
Aft er t he applicat ion of t he Kolm ogorov- Sm irnov t est of nor m alit y, t he dat a obt ained in t he count ing
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t o t he Two- way analysis of variance and Tukey t est s
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5% ( p< 0.05) . The scor es obt ained in t he analysis of t he odont oblast ic layer and of vascular changes w er e subm it t ed t o t he Kr usk al- Wallis and Dunn t est s ( p< 0.05) . The values obt ained in t he m ean of cent ral area of t he pulp cham ber w ere subm it t ed t o Kolm ogorov- Sm irnov t est of norm alit y and One- way analysis of var iance t est ( p< 0.05) .
RESULTS
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Con t r ol gr ou p
The dent al pulp of cont rol anim als exhibit ed
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odont oblast ic layer and even dist r ibut ion of cells, blood vessels, and ext racellular m at r ix st r uct ur es ( Figur e 2) .
2 0 % - 5 m in gr ou p
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The dent al pulp appeared t o be sim ilar t o t hat in t he cont r ol gr oup. The odont oblast ic layer was int act and blood vessels show ed nor m al charact er ist ics. The cem ent um , periodont al ligam ent , alveolar bone, and ot her suppor t ing st r uct ur es also appear ed t o be nor m al ( Figur e 3A) .
2 0 % - 1 0 m in gr ou p
This group did not exhibit a considerable am ount
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t hir ds of t he cor onal pulp. The odont oblast ic layer w as par t ially disor ganized in t he occlusal t hir d, and t her e was an incr ease in t he num ber of blood vessels in t he occlusal and m iddle t hir ds of t he cor onal pulp ( Figur e 3B) .
Figure 1- Measurement of the central area of the pulp chamber in the experimental groups using the Leica QWin V3 Image
2 0 % - 1 5 m in gr ou p
Th e r e w a s a n i n cr e a se i n t h e a m o u n t o f
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of t he cor onal pulp, a r educt ion in t he am ount
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blood vessels. The odont oblast ic layer was part ially disor ganized in t he occlusal t hir d ( Figur e 3C) .
2 0 % - 3 0 m in gr ou p
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cells was found in t he m iddle t hir d of t he cor onal pulp. Ther e was a lar ge r educt ion in t he am ount of
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disrupt ion of t he odont oblast ic layer. The am ount of blood vessels incr eased in t he occlusal and m iddle t hirds of t he coronal pulp ( Figure 3D) . The radicular pulp appear ed t o be nor m al in all cases.
2 0 % - 4 5 m in gr ou p
Th i s g r o u p s h o w e d i n c r e a s e d n u m b e r o f
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of t h e cor on al pu lp. Th e occlu sal t h ir d sh ow ed n ecr ot ic ar eas. A r ed u ct ion in t h e n u m b er of
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cr ow n. The odont oblast ic layer was absent in t he occlusal t hir d and par t ly disor ganized in t he m iddle
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of t he radicular pulp ( Figur e 3E) .
3 5 % - 5 m in gr ou p
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r e d u ce d i n t h e o ccl u sa l t h i r d o f t h e co r o n a l pulp, w her e t he odont oblast ic layer was par t ially disor ganized. An incr ease in t he num ber of blood vessels was obser ved in all ar eas of t he cor onal pulp ( Figur e 3F) .
3 5 % - 1 0 m in gr ou p
I n t h is gr ou p, t h er e w as an in cr ease in t h e
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m iddle t h ir ds of t h e cor on al pu lp. Th e am ou n t
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odont oblast ic layer was absent in t he occlusal t hir d and was par t ly disor ganized in t he m iddle t hir d of t he cor onal pulp. Ther e was an incr ease in t he num ber of blood vessels t hr oughout t he cor onal pulp ( Figur e 3G) .
3 5 % - 1 5 m in gr ou p
Th e r e w a s a n i n cr e a se i n t h e n u m b e r o f
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r educed. The odont oblast ic layer was absent in t he occlusal t hird, and part ly disorganized in t he m iddle t hird of t he coronary pulp. There was an increase in t he num ber of blood vessels t hroughout t he coronal pulp ( Figur e 3H) .
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3 5 % - 3 0 m in gr ou p
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r educed in t he occlusal t hir d, w her e t her e was t he pr esence of necr ot ic ar eas. Ther e was an incr ease
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and cervical t hirds of t he coronary pulp. The am ount
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cor onar y pulp. The odont oblast ic layer was absent in occlusal and m iddle t hir ds of t he cr ow n. Ther e was an incr ease in t he num ber of blood vessels in t he m iddle and cer vical t hir ds of t he cor onal pulp. The occlusal t hir d w er e charact er ized as necr osis.
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t he cer vical t hir d of t he radicular pulp ( Figur e 3I ) .
3 5 % - 4 5 m in gr ou p
Ther e was necr osis in t he occlusal t hir d of t his g r ou p w it h ab sen ce of in f lam m at or y cells an d f ib r ob last s. Th e n u m b er of in f lam m at or y cells in cr eased in t h e cer v ical an d m id d le t h ir d s of t he cor onal pulp, and in t he cer vical t hir d of t he
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in t hese t hir ds. Ther e was no odont oblast ic layer in t he occlusal and m iddle t hir ds of t he cr ow n, and t his layer was par t ially disor ganized in t he cer vical t hir d. The num ber of blood vessels incr eased in t he
cer vical t hir d of cor onar y and radicular pulps. The r em aining t hir ds seem ed t o be nor m al ( Figur e 3J) .
Re pa r a t iv e D e n t in Ar e a
Th ir t y day s aft er t h e bleach in g session s, all specim ens show ed nor m al dent al pulp. How ever, t he cent ral ar ea of t he pulp cham ber r educed, and t he t er t iar y dent in ar ea incr eased ( Figur e 4) .
I n t e r gr ou p Com pa r ison s
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of all exper im ent al gr oups. The m ost pr edom inant i n f l a m m a t o r y ce l l s f o u n d w e r e m o n o n u cl e a r cells, su ch as ly m ph ocy t es, m acr oph ages, an d
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gradually w it h t he incr easing concent rat ions and t he applicat ion t im e of t he bleaching gel, up t o t he 15 m in applicat ion gr oups of each bleaching gel, in t he occlusal t hir d of t he cor onal pulp. The gr oups t hat r eceived t he applicat ion of 30 and 45 m in of each bleaching agent show ed ar eas of necr osis in t he occlusal t hir d w it h a decr ease in t he am ount o f i n f l am m at o r y cel l s. Si g n i f i can t d i f f er en ces w er e obser ved bet w een t he bleached gr oups and
Figure 3- Representative images of H&E-stained sections showing the coronal pulp 2 days after bleaching. Panels a, b, c,
d, and e represent the groups treated with 20% H2O2 gel and panels f, g, h, i, and j represent those treated with 35% H2O2
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t he cont r ol gr oup in t he occlusal t hir d ( p< 0.05) , except for t he 35% - 45 m in gr oup, w it h absence of
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t he cor onal pulp w er e not ed bet w een t he cont r ol
gr oup and t he 20% - 10 t o 45 m in and t he 35% - 5 t o 45 m in gr oups ( p< 0.05) . I n t he cer vical t hir d, t he differ ence w it h t he cont r ol gr oup r em ained in 20% - 15 t o 45 m in and 35% - 10 t o 45 m in gr oups
Group &RURQDO Radicular
Occlusal Middle &HUYLFDO &HUYLFDO Middle Apical
Control 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a
20% H2O2 gel 5 min 3.6 ±0.5b 2.2 ±0.4ab 1.0 ±0.4ab 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a
10 min 5.6 ±1.1bc 4.6 ±1.1bcd 2.2 ±0.5abc 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a
15 min 7.6 ±1.5c 6.0 ±1.4ce 3.4 ±0.9bd 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a
30 min 5.2 ±0.8bc 8.2±1.3e 5.2 ±1.1d 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a
45 min 3.2 ±0.8b 13.0 ±2.9fg 9.0 ±2.3e 5.6 ±0.9b 0.0 ±0.0a 0.0 ±0.0a
35% H2O2 gel 5 min 4.0 ±0.7b 4 ±1.0bc 2.2 ±0.5abc 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a
10 min 10.6±2.3d 7.4 ±1.1de 4.4 ±1.1cd 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a
15 min 14.6±3.6e 11.8±2.3f 5.2 ±1.3d 0.0 ±0.0a 0.0 ±0.0a 0.0 ±0.0a
30 min 6.0±1.4bc 15.6±1.3g 9.8 ±2.6e 5.8 ±0.8b 0.0 ±0.0a 0.0 ±0.0a
45 min 0.0 ±0.0a 12.0±2.9f 11.0±2.6e 7.6±1.5c 0.0 ±0.0a 0.0 ±0.0a
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the Two-way ANOVA and Tukey test - p<0.05).
Table 1-,QÀDPPDWRU\FHOOFRXQWperȝP2) in the pulp thirds of each group (Mean ±SD)
Figure 4- Representative images of H&E-stained sections showing the coronal pulp 30 days after bleaching. Panels a, b, c,
d, and e represent the groups treated with 20% H2O2 gel and panels f, g, h, i, and j represent those treated with 35% H2O2
JHOIRUDQGPLQUHVSHFWLYHO\îPDJQL¿FDWLRQ3DQHOVDMDUHPDJQL¿HGLPDJHVîRIWKHLQVHWVLQ
( p< 0 . 0 5 ) . I n t h e cer v ical t h ir d of t h e radicu lar
SXOS VLJQL¿FDQW GLIIHUHQFHV ZHUH QRWHG EHWZHHQ
t he 20% - 45 m in and t he 35% - 30 m in gr oups w it h t he ot her gr oups and 35% - 45 m in w it h all gr oups.
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ot her radicular t hir ds ( p> 0.05) .
7DEOHVKRZVWKHDPRXQWRI¿EUREODVWVRIWKH
exper im ent al gr oups. The 20% - 10 t o 45 m in, and
JURXSVVKRZHGDVLJQL¿FDQWGHFUHDVHLQWKH QXPEHURI¿EUREODVWVLQWKHRFFOXVDOWKLUGFRPSDUHG
t o t h e con t r ol g r ou p ( p < 0 . 0 5 ) . Th is d ecr ease r em ained in t he m iddle t hir d of t he cor onal pulp in t he 20% - 15 t o 45 m in, and 35% - 15 t o 45 m in gr oups ( p< 0.05) . I n t he cer v ical t hir d, only t he gr oups t hat r eceived t he bleaching gels for 45 m in
VKRZHGDVLJQL¿FDQWGLIIHUHQFHRIWKHFRQWUROJURXS
S6LJQL¿FDQWGLIIHUHQFHVZHUHQRWREVHUYHG
in t he radicular t hir ds of any gr oup ( p> 0.05) . Tab l e 3 sh o w s t h e sco r es assi g n ed t o t h e odon t oblast lay er of each ex per im en t al gr ou p.
6LJQL¿FDQW GLIIHUHQFHV ZHUH REVHUYHG EHWZHHQ
t h e 2 0 % - 4 5 m in , 3 5 % - 3 0 an d 4 5 m in in t h e occlusal t hird com pared t o t he cont rol and t he 20% -5 m in gr oups ( p< 0.0-5) . I n t he m iddle t hir d of t he cor onal pulp, t he differ ence r em ained bet w een t he 35% - 45 m in gr oup w it h t he cont r ol and 20% - 5 t o
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differ ences in t he cer v ical t hir d and in radicular t hir ds ( p> 0.05) .
Tab l e 4 sh o w s t h e sco r es assi g n ed t o t h e v ascu lar ch an g es of each ex p er im en t al g r ou p.
6LJQL¿FDQWGLIIHUHQFHVZHUHREVHUYHGEHWZHHQWKH
Group &RURQDO Radicular
Occlusal Middle &HUYLFDO &HUYLFDO Middle Apical
Control 65.2 ±8.7a 67.0 ±7.8ab 44.0 ±4.6abc 38.0 ±3.7a 33.8 ±2.9a 37.2 ±3.3a
20% H2O2 gel 5 min 51.8 ±6.6ab 72.2 ±11.9a 52.8 ±8.5bc 39.4 ±4.2a 35.8 ±3.0a 37.0 ±2.3a
10 min 45.2 ±9.2bc 54.0 ±4.2bc 55.6 ±11.2c 36.8 ±2.9a 37.6 ±3.1a 35.0 ±3.2a
15 min 36.2 ±3.6c 46.4 ±2.4cd 43.2 ±8.4ac 41.2 ±0.8a 38.2 ±3.6a 35.2 ±3.3a
30 min 5.8 ±1.6de 32.4±7.5de 34.6 ±5.5ad 39.6 ±1.9a 36.2 ±3.8a 37.2 ±2.2a
45 min 0.0 ±0.0d 26.0 ±1.6efg 29.0 ±8.2def 36.0 ±4.5a 37.6 ±4.2a 38.2 ±2.6a
35% H2O2 gel 5 min 15.2 ±2.8e 64.8 ±11.3ab 40.8 ±8.0abe 35.2 ±2.9a 34.8 ±3.9a 39.0 ±2.3a
10 min 13.2±3.0de 54.6±12.4abc 35.2 ±3.6ae 38.6 ±1.5a 36.2 ±4.3a 37.6 ±3.6a
15 min 10.8±2.9de 41.6±9.7cdf 34.2 ±3.0ae 37.6 ±2.5a 38.8 ±1.6a 37.6 ±4.3a
30 min 5.8±0.8de 23.2±5.0eg 30.4 ±4.0aef 36.0 ±4.2a 37.8 ±2.2a 39.6 ±4.5a
45 min 0.0 ±0.0d 10.2±2.7g 19.6±1.8f 38.0±2.7a 36.8 ±2.6a 40.2 ±4.2a
'LIIHUHQWOHWWHUVLQWKHFROXPQVLQGLFDWHVLJQL¿FDQWGLIIHUHQFHEHWZHHQWKHJURXSV.ROPRJRURY6PLUQRYQRUPDOLW\WHVWDQG
the Two-way ANOVA and Tukey test - p<0.05).
Table 2- Fibroblasts count (per ȝP2) in the pulp thirds of each group (Mean ±SD)
Group &RURQDO Radicular
Occlusal Middle &HUYLFDO &HUYLFDO Middle Apical
Control 1a 1a 1a 1a 1a 1a
20% H2O2 gel 5 min 1a 1a 1a 1a 1a 1a
10 min 2ab 1a 1a 1a 1a 1a
15 min 2ab 1a 1a 1a 1a 1a
30 min 3ab 1a 1a 1a 1a 1a
45 min 3b 2ab 1a 1a 1a 1a
35% H2O2 gel 5 min 2ab 1ab 1a 1a 1a 1a
10 min 3ab 2ab 1a 1a 1a 1a
15 min 3ab 2ab 1a 1a 1a 1a
30 min 3b 3ab 1a 1a 1a 1a
45 min 3b 3b 2a 1a 1a 1a
'LIIHUHQWOHWWHUVLQWKHFROXPQVLQGLFDWHVLJQL¿FDQWGLIIHUHQFHEHWZHHQWKHJURXSV.UXVNDO:DOOLVDQG'XQQWHVWVS
20% - 45 m in and 35% - 45 m in in t he occlusal t hir d com par ed t o t he cont r ol and 20% - 5 m in gr oups ( p< 0.05) . I n t he m iddle t hir d of t he cor onal pulp, t he differ ence r em ained bet w een t he 35% - 45 m in group wit h t he cont rol and 20% - 5 groups ( p< 0.05) .
7KHUHZDVQRVLJQL¿FDQWGLIIHUHQFHLQWKHFHUYLFDO
t hir d and in radicular t hir ds ( p> 0.05) .
Aft er 30 days, t he specim ens show ed a gradual
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differ ences w er e obser ved bet w een t he 35% - 45 m in and t he ot her gr oups, except for t he 35% -30 m in gr oup ( p< 0.05) . The gr oups 20% - 5 m in, 20% - 10 m in, 20% - 15 m in, and 35% - 5 m in did not
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( Table 5) .
D I SCUSSI ON
Toot h bleaching is an aest het ic alt er nat ive for discolored t eet h, but it has pot ent ial adverse effect s t hat ar e not com plet ely under st ood yet28. A single
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r esult s, but longer applicat ion t im es and m ult iple sessions m ay be r equir ed for opt im al out com es, incr easing t he r isk of t oot h sensit ivit y20 and pulp dam age6.
A lar ge num ber of in vit r o st udies have show n t he ROS generat ed by H2O2 of bleaching gels is capable of causing hist ochem ical and m orphological changes in enam el and dent in4,5.
I n vivoVWXGLHVVKRZHGFHOOXODUGDPDJHFODVVL¿HG
as m ild t o sever e. These include st udies per for m ed in dogs25,26, hum an m andibular incisor s8,19, and in
Group Mean (105)* SD (105) % reduction
Control 18.46a 2.38 0.00
20% H2O2 gel 5 min 17.57a 2.30 4.82
10 min 17.18a 1.85 6.93
15 min 15.65ab 1.78 15.22
30 min 13.47bc 1.43 27.03
45 min 11.23cd 1.17 39.16
35% H2O
2 gel 5 min 14.61
ab 0.46 20.85
10 min 13.41bc 0.72 27.36
15 min 12.56bcd 0.71 32.33
30 min 10.01de 0.62 45.77
45 min 6.98e 0.51 62.18
'LIIHUHQWOHWWHUVLQWKHFROXPQLQGLFDWHVLJQL¿FDQWGLIIHUHQFH.ROPRJRURY6PLUQRYQRUPDOLW\WHVWDQGWKH2QHZD\$129$
test - p<0.05).
Table 5-&KDQJHLQ3XOS&KDPEHU&HQWUDO$UHDȝP2)
Group &RURQDO Radicular
Occlusal Middle &HUYLFDO &HUYLFDO Middle Apical
Control 1a 1a 1a 1a 1a 1a
20% H2O2 gel 5 min 1a 1a 1a 1a 1a 1a
10 min 2ab 2ab 1a 1a 1a 1a
15 min 2ab 2ab 1a 1a 1a 1a
30 min 2ab 2ab 1a 1a 1a 1a
45 min 3b 2ab 1a 1a 1a 1a
35% H2O2 gel 5 min 2ab 2ab 2a 1a 1a 1a
10 min 2ab 2ab 2a 1a 1a 1a
15 min 2ab 2ab 2a 1a 1a 1a
30 min 3ab 2ab 2a 1a 1a 1a
45 min 3b 3b 2a 1a 1a 1a
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rat incisor s12,13 and m olar s6. St udies oncell cult ur es also dem onst rat ed cellular dam age not only t o DNA cells23 but also such as apopt osis14LQÀDPPDWLRQ3, cy t ot ox icit y3 0, cell v iab ilit y r ed u ct ion2 7, or t h e ageing of t he dent al pulp1,28. The cy t ot ox icit y of t he bleaching gel was also obser ved in t his st udy.
St udies predom inant ly wit h ex vivo m anipulat ed
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st udies on bleaching agent s. How ev er, in t hose st udies, pulp cells ar e not exam ined as or ganized t issues. Teet h have vit al pulp com ponent s t hat can pr event or hinder t he H2O2 effect s in pulp t issues,
DVGHQWLQDOÀXLGF\WRSODVPDWLFH[WHQVLRQV17,30, and ant ioxidant enzym es as super oxide dism ut ase and cat alase t hat pr om ot e t he enzym at ic degradat ion of H2O211,17. Ther efor e, in vivo exper im ent s ar e t he ones t hat best r epr esent t he r ealit y of bleaching effect s.
Th e ap p licat ion of 3 8 % H2O2 g el in h u m an pr em olar s does not cause pat hological changes in t he dent al pulp17. How ever, t he applicat ion of t he sam e concent rat ion in hum an m andibular incisor s causes necr osis in t he cor onal pulp in a sim ilar w ay t o t hat obser v ed in rat s’ m olar s6, possibly because of t he t hinner enam el and dent in8. These
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pulp dam age. I deally, upper ant er ior hum an t eet h should be used t o det er m ine t he pulp changes accu r at ely. Ev en u n der t h ese con dit ion s, ot h er
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pr esence of r est orat ions, pr evious t raum a, am ong ot her s.
Even t hough var iat ions of t he pulp r esponse have been show n in hum an t eet h, our st udy aim ed t o charact er ize an exper im ent al anim al m odel of ease r epr oduct ion and st andar dizat ion t o t he st udy of new bleaching agent s, posology, concent rat ions, and applicat ion t im e.
I n dogs’ t eet h, dent al bleaching using 35% H2O2 show ed gr eat er changes im m ediat ely beneat h t he r egion w her e t he gel w as applied27, sim ilar ly t o t hat obser ved in gr oups of 35% H2O2 gel applied for 30 or 45 m in, and 20% H2O2 gel for 45 m in in t his st udy. Sever e pulp dam age m ay occur w hen bleaching agent s are applied in t he buccal surface of t eet h w it h t hin enam el and dent in8,25,26. Dog’s t eet h
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Furt herm ore, nowadays, st udies in dogs have been avoided by et hical r easons.
Th e u se of r at s in t h e ex p er im en t al m od el pr esent s advant ages such as st raight for war dness in t h e h an d lin g an d in t h e r ep r od u ct ion , an d m ainly, t he easiness for cont r olling, pr edict ing22, and st andar dizing6. Mor eover, t his m odel is bet t er accept ed regarding et hical and econom ic concerns9.
Despit e t he differ ence in enam el and dent in
t hick ness bet w een hum ans’ and rat s’ t eet h ( 2.5
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t h e sam e p r op or t ion of t h ese st r u ct u r es6 , 9. I n addit ion, rat s’ m olars have anat om ical, hist ological, biological, an d phy siological feat u r es sim ilar t o hum an m olars9,24. Also, rat m olars exhibit t he sam e st r uct ural charact er ist ics of t he pulp cham ber and of t he pulp t issues, w her e t he essent ial biological r eact ions and t he w ound healing of rat s’ m olar t eet h ar e com parable t o t hat of ot her m am m als9. Conver sely, rat s’ incisor s ar e t y pical of r odent s, having per m anent gr ow t h, w it h a w ide- open apex, and cannot be com par ed t o hum an t eet h9.
I n t he present st udy, 35% H2O2 gel applied for 30
RUPLQFDXVHGQHFURVLVRUDVHYHUHLQÀDPPDWRU\
r esponse in t he dent al pulp, especially in t he upper
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w it h high H2O2 concent rat ions for 30- 45 m in in a single session is fr equent ly associat ed w it h a high in ciden ce of t oot h sen sit iv it y8. Con sider in g t h e sim ilar it y of t he r esult s found in t his st udy w it h t he r esult s of Cost a, et al.8 ( 2010) , w e suggest ed t h at r at m olar s can be t ar get ed an d im pr ov ed as an ex per im ent al m odel t o pr edict t he r esult s of pr ocedu r es per for m ed in h u m an m an dibu lar incisor s w it h t he sam e concent rat ion and durat ion of applicat ion6.
The am ount of H2O2 det ect ed in t he pulp cham ber is r elat ed t o t he concent rat ion and applicat ion t im e of t he gel2. The use of 35% H
2O2 gel applied for 30 m in, and 20% and 35% H2O2 gel for 45 m in was r elat ed t o changes in t he vascular per m eabilit y12. Ther efor e, our st udy was car r ied out w it h several applicat ion t im es and t w o concent rat ions, one of w hich m or e com m only em ployed at t he clinic ( H2O2 35% )16, 29, and anot her w it h low er concent rat ion ( H2O2 20% ) . Our r esult s allow us t o choose t he concent rat ion and t he t im e of applicat ion t o analyze
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( af t er t w o day s) as w ell as in t h e su bsequ en t r eparat ive pr ocess ( aft er 30 days) .
I n our evaluat ion by 30 days aft er bleaching, w e ob ser v ed t h at all g r ou p s sh ow ed sig n s of r ep ai r. Ter t i ar y d en t i n w as f or m ed t o p r ot ect t he dent al pulp, r educing pulp cham ber cent ral
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con cen t r at ion / ap p licat ion t im e g r ou p s sh ow ed
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concent rat ions/ applicat ion t im e gr oups. St udies of t he effect s of high concent rat ions of bleaching gels on pulp cell cult ur es have show n t hat pr oduct s r eleased by 3 5 % H2O2 gel can dif f u se t h r ou gh en am el an d d en t i n an d cau se si g n i f i can t cel l dam age10,30.
I n cr e a se d a l k a l i n e p h o sp h a t a se a ct i v i t y a n d ex t racellular m at r ix m ineralizat ion r eveal dent in pr oduct ion28. The m odel in rat s can also be used in long t er m analysis t o det er m ine clinical pr ot ocols of applicat ion t h at pr odu ce less pu lp dam ages along t im e.
The charact er izat ion of t his exper im ent al m odel does not r eplace hum an t r ials, but allow s us t o k n ow t h e act ion m ech an ism s of n ew bleach in g agent s; t o com par e bleaching pr ot ocols; and t o st udy desensit izing and rem ineralizing agent s used befor e and aft er bleaching t o m inim ize it s effect s on pulp t issues.
Th er ef o r e, t h e r a t m o d el o f ex t r a co r o n a l bleaching showed t o be adequat e t o st udy bleaching pr ot ocols, as it was possible t o obser ve alt erat ions in t he pulp t issues and in t oot h st r uct ur e caused by differ ent concent rat ions and per iods of applicat ion
RIEOHDFKLQJDJHQWV,QRI¿FHEOHDFKLQJZLWK+2O2
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t h e ag in g of t h e d en t al p u lp b y in d u cin g t h e deposit ion of t he t er t iar y dent in, and t he degr ee of dam age gr ew w it h t he incr easing concent rat ions and applicat ion t im es of t he bleaching agent .
ACKN OW LED GEM EN TS
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pr oj ect 2011/ 13709- 2 and 2013/ 25429- 0.
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