ABSTRACT
http://dx.doi.org/10.1590/1678-775720150289
Pr ev en t i v e ef f ect o f t o o t h p a st es w i t h MMP
inhibit ors on hum an dent ine erosion and abrasion
in vit ro
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1- Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Ciências Biológicas, Bauru, SP, Brasil. 2- Universidade do Sagrado Coração, Departamento de Odontologia, Bauru, SP, Brasil.
3- Universidade do Sagrado Coração, Departamento de Odontopediatria, Bauru, SP, Brasil.
4- Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Dentística, Endodontia e Materiais Odontológicos, Bauru, SP, Brasil. 5- Oulu University Hospital and University of Oulu, Medical Research Center, Institute of Dentistry, Oulu, Finland.
&RUUHVSRQGLQJDGGUHVV Marília Afonso Rabelo Buzalaf - Universidade de São Paulo Faculdade de Odontologia de Bauru - Departamento de Ciências Biológicas - Al. Octávio Pinheiro Brisolla, 9-75 - Bauru - SP - 17012-901 - Brazil - Phone: +55 -1432358346 - e-mail: mbuzalaf@fob.usp.br
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he use of gels and m out hrinses w it h MMP inhibit ors ( chlorhexidine, and green t ea ext ract ) was show n t o prevent erosive w ear. The aim of t his st udy was t o analyze t he prot ect ive effect of t oot hpast es cont aining MMP inhibit ors on dent ine loss induced by erosionin vit ro. Mat erial and Met hods: Five groups each cont aining 12 specim ens of hum an root dent ine w ere prepared. The specim ens w ere subj ect ed t o 1 m in erosion by im m ersion in DFRODGULQNWLPHVDGD\IRUG(DFKGD\DIWHUWKH¿UVWDQGODVWHURVLYHFKDOOHQJHV t he specim ens w ere brushed for 15 s w it h a slurry of dent ifrice and wat er ( 1: 3) cont aining SODFHERSSPÀXRULGHJUHHQWHDH[WUDFWFKORUKH[LGLQHRU chlorhexidine ( com m ercial t oot hpast e) . Bet w een t he acid challenges, t he specim ens w ere VWRUHGLQDUWL¿FLDOVDOLYDZLWKUHPLQHUDOL]LQJSRWHQWLDOXQWLOWKHQH[WWUHDWPHQW'HQWLQH ORVVZDVGHWHUPLQHGXVLQJSUR¿ORPHWU\'DWDZHUHDQDO\]HGXVLQJRQHZD\$129$DIWHU ORJWUDQVIRUPS5HVXOWV7KHPHDQZHDUYDOXHVNjPZHUHDVIROORZVSODFHER JUHHQWHDH[WUDFWÀXRULGHFKORUKH[LGLQH DQGFKORUKH[LGLQH7KHUHZDVDVLJQL¿FDQWGLIIHUHQFHLQ w ear bet w een placebo and all t he t reat m ent t oot hpast es, w hich did not differ from each ot her. Conclusion: The result s suggest t hat t oot hpast es cont aining MMP inhibit ors are as effect ive as t hose based on NaF in prevent ing dent ine erosion and abrasion.
Ke yw or ds: Erosion. Dent in. Mat rix m et alloprot einases. I nhibit ors. Toot hpast es.
I N TROD UCTI ON
The reduct ion in caries prevalence, associat ed wit h an increase in t oot h- ret ent ion rat es, has posed new challenges r egar ding pr ev ent iv e m easur es in or al h ealt h . Th e au gm en t ed con su m pt ion of acid ic f ood s an d b ev er ag es h as led t o h ig h er
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as t he loss of t oot h subst ance caused by acids ( pH< 4 . 5 ) , under sat urat ed r egar ding apat it e, in t he absence of m icroorganism s15,22. The frequent
in g est ion of acid ic b ev er ag es h as a p r of ou n d effect on dent ine, especially for adult s and for t he
elderly populat ion. Wit h age, gingival recession is a com m on occurrence, leading t o t he subsequent exposure of cem ent and root dent ine t hat are quit e vulnerable t o erosion2,13.
The exact m echanism of t he er osion pr ocess in ex posed dent ine r oot sur faces is not k now n. Alt hough t he m ineral phase of t he dent ine is lost aft er t he erosive challenge, t he fat e of t he organic phase is not com plet ely under st ood2, 11, 29. When
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can be det ect ed. This dem ineralized organic layer is resist ant t o m echanic rem oval by brushing forces up t o 4 N5,8, and it is plausible t hat t his out erm ost layer
of collagenous m at rix m ight prot ect t he rem aining dent ine against m echanical forces, like t oot hbrush abrasion8. I t could also lim it ionic diffusion int o and
out of t he dem ineralized sur face16,19,21. Toget her,
t hese propert ies m ay explain w hy it s m aint enance has been at t ribut ed t o reduce t he progression of dent ine erosion2,3.
I t is not know n t o w hich ext ent t he prot ect ive effect of t he dem ineralized organic m at rix is relevant in clinical realit y. This layer could be degraded by m at rix m et alloprot einases ( MMPs)2,10,28, which have
been suggest ed t o cont ribut e t o t he progression of den t in e er osion2 , 1 1. MMP in h ibit or s, su ch as
chlorhexidine, green t ea polyphenols, and FeSO4, in solut ions or gels, r educe t he dent ine er osive w ear4,17,18,24. How ever, t he use of t oot hpast es as
a vehicle t o deliver MMPs t o dent ine has not been st u died so f ar. Th is v eh icle seem s par t icu lar ly at t ract ive, since t oot hbrushing wit h t oot hpast e is an int egral part of daily oral hygiene m easures, and t he
dem ineralized organic m at rix is not rem oved during t his procedure5,8. Thus, t he present st udy t est ed t he
null hypot hesis t hat t oot hpast es cont aining MMP
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erosion associat ed w it h t oot hbrush abrasion.
M ATERI AL AN D M ETH OD S
Pr e pa r a t ion of de n t in e spe cim e n s
Six t y r ecen t ly ex t r act ed n on - car iou s h u m an t hird m olars w ere used in t his st udy. The prot ocol was approved by t he I nst it ut ional Review Board of Bauru School of Dent ist ry, Universit y of São Paulo ( 011/ 2008) . Prior t o t he ext ract ion, t he pat ient s w er e infor m ed about t he use of t heir t eet h for research purposes and consent was obt ained. Teet h were st ored in 0.9% NaCl w it h 0.2% NaN3 at 4° C. The crow ns w ere sect ioned from t he root s w it h a diam ond saw ( I som et 1000; Buehler, Lake Bluff, I L, USA) . Dent ine slabs ( 4x4 m m ) w ere cut from t he cervical t hird of t he root under wat er cooling,
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wat er- cooled car bor undum discs ( 320, 600 and 1,200 grades of Al2O3 papers; Buehler) . Aft er each
Figure 1- Schematic illustration of the experimental sequence. (A) The crowns were sectioned from the roots with a
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(320, 600, and 1,200 grades of Al2O3 papers). Dentine specimens (n=12) were randomly divided into 5 groups. (E) The
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last erosive challenges, the specimens were brushed for 15 s with slurry of dentifrice and water (1:3) with the following treatment toothpastes: placebo; 1,100 ppm F as NaF; 0.61% green tea extract (EGCG); 0.12% chlorhexidine (CHX);
disc, t he specim ens were cleaned in ult rasound bat h (T7 Thornt on, Unique I nd. e Com . Lt da., São Paulo, SP, Brazil) for 2 m in. Finally, t he specim ens w ere polished w it h felt paper w et by diam ond spray ( 1
P%XHKOHUon a rot at ing polishing m achine. The specim ens wereUDQGRPO\DVVLJQHGLQWR¿YHJURXSV of 12 specim ens each t hat differed according t o t he t ype of t oot hpast e used. Sam ple size was calculat ed based on a previous st udy26. I n order t o est ablish reference surfaces for lesion dept h det erm inat ion, t w o layers of nail varnish (Risqué: Niasi I ndúst ria de Cosm ét icos LTDA, Taboão da Serra, SP, Brazil) w ere applied on each side of t he surface of each specim en, leaving 1 m m of t he m iddle unprot ect ed ( Figure 1) .
Tr e a t m e n t pr ot ocol
For er osiv e dem ineralizat ion, each specim en was im m ersed in 30 m L of Coke® ( Cia de Bebidas
I piranga, Ribeirão Pret o, SP - Brazil, pH 2.6) for 1 m in, 4 t im es a day, dur ing 5 d. Bet w een t he dem ineralizat ion phases, t he sam ples were st ored in
DUWL¿FLDOVDOLYD20 cont aining 0.2 m M glucose, 9.9 m M
NaCl, 1.5 m M CaCl2.2H2O, 3 m M NH4Cl, 17 m M KCl, 2 m M NaSCN, 2.4 m M K2HPO4, 3.3 m M urea, 2.4 m M NaH2PO4, and t races of ascorbic acid ( pH 6.8) at 37° C for 59 m int o allow rem ineralizat ion. Each
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br ushing w as per for m ed for 15 s per specim en using pow ered t oot hbrush w it h 166 oscillat ions/ s
( Oral- B®, Cross Act ion Pow er, Proct er & Gam ble,
Cuaj im alpa, Mex ico) . For st andar dized br ushing force, a cust om - m ade device was used, allow ing for a pressure of approxim at ely 2 N32. The brushing
heads w ere renew ed aft er 2.5 days of t reat m ent . For each specim en, brushing was perform ed w it h 1 m L of a 1: 3 ( w/ v) slurry of t oot hpast e and deionized w at er ( Fig u r e 1 ) . Placeb o t oot h p ast e g el w as prepared wit h t he sam e com posit ion and pH ( 7.0) of t he ot her experim ent al t oot hpast es and w it h no act ive com pound. I t s com posit ion included et hanol, wat er, sorbit ol, glycerine, carboxym et hylcellulose, x a n t h a n g u m , p o l i e t h y l e n e g l y c o l 4 0 0 , m et hy lpar aben , pr opy lpar aben , silicon diox ide, abrasive silicon diox ide, sodium laur y l sulphat e, cocoam idopropylbet ain, sodium saccharine, m int essen ce, an d or t h op h osp h or ic acid . Th is also ser v ed as t h e base f or t h e ex per im en t al gels. Th r ee ex per im en t al t oot h past es w it h a sim ilar com posit ion and pH ( 7 . 0 ) w er e pr epar ed, w it h t he except ion of t he com pound t est ed, as follows:
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Germ any) , 0.61% green t ea ext ract ( OM24, 100%
Cam ellia sinensis- leaf ext ract wit h 30± 3% cat echin; Om n i m ed i ca, Sch l i er en , Sw i t zer l an d ) , 0 . 1 2 % chlor hex idine digluconat e ( Sigm a) . Com m er cial t o o t h p a st e co n t a i n i n g 0 . 0 0 4 % ch l o r h e x i d i n e digluconat e ( Elgydium , Pierre Fabre, Swit zerland, pH 7.0) was also t est ed ( Figure 1) . Aft er com plet ion of t he 5- day brushing, t he specim ens w ere rinsed
Figure 2- Mean loss of dentine (± standard deviation) in groups submitted to erosive and abrasive challenges in vitro,
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unt il analyzed ( Figure 1) .
D e n t in e loss a sse ssm e n t
The nail varnish over t he surfaces was carefully r em ov ed w it h a scalp el. Th e sp ecim en s w er e m a i n t a i n e d w e t t o av o i d sh r i n k a g e . D e n t i n e
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b y a con t act p r of ilom et er ( Mah r Per t h om et er, Mahr, Göt t ingen, Germ any) as described before3. %ULHÀ\WKHVSHFLPHQVZHUHVOLJKWO\GULHGJHQWO\ UHPRYLQJRQO\WKHH[FHVVRIZDWHUZLWK¿OWHUSDSHU
and im m ediat ely analy zed. The diam ond st y lus was m oved fr om one r efer ence ar ea t o anot her across t he exposed area ( 2.5 m m lengt h and 2.0
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program ( Soft ware Mahr Surf XT20, Mahr) . Five p r of ile m easu r em en t s w er e p er f or m ed in t h e cent er of each specim en at int ervals of 0.5 m m . The v er t ical dist ance bet w een t he m idpoint s of regression lines on t he reference and experim ent al
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of t he m et hod, evaluat ed in previous st udies, is
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St a t ist ica l a n a lysis
Aft er check ing for equalit y of var iances and norm al dist ribut ion of errors, dat a w ere analyzed by on e- w ay ANOVA an d Tu k ey ’s t est af t er log t ransform at ion. The soft ware GraphPad I nst at for Window s version 3.0 ( La Jolla, CA, USA) was used.
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RESULTS
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t he specim ens t reat ed w it h t he placebo t oot hpast e
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p= 0.008) t han t he values found for t he specim ens fr om t he t oot hpast es cont aining MMP inhibit or s ( 1.00± 0.21, 1.19± 0.30, and 1.22± 0.46 for 0.61% green t ea ext ract , 0.12% chlorhexidine or 0.004%
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Th e t oot h past es con t ain in g act iv e com pou n ds,
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anot her ( Figure 2) .
D I SCUSSI ON
Th e st u d y d e si g n a i m e d t o si m u l a t e t h e habit s of pat ient s w it h erosive lesions. The t ot al dent ine erosion t im e was 20 m in w it h st orage in rem ineralizing solution between the acidic challenges t o allow clinically r elevant r em ineralizat ion. The t ot al abrasion t im e t hroughout t he st udy was 2.5 m in, which would correspond t o 30 brushings of 5 s each for one t oot h in t he clinical condit ion31. Despit e
t he experim ent al in vit ro condit ions em ployed in
an at t em pt t o resem ble as closely as possible t he clinical condit ion, som e sit uat ions could not be reproduced by our prot ocol, such as t he presence
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t he presence of bact eria, as w ell as variat ions in t em per at u r e. I n addit ion , t h e con cen t r at ion of t he act ives used in t he ex per im ent al dent ifr ices ( 0.61% green t ea ext ract and 0.12% chlorhexidine digluconat e) had a good effect on t he reduct ion in dent ine erosion w hen added t o rinse solut ions in a previous in sit u st udy24.
The rat e of loss in dent ine reduces along t im e if t he dem ineralized organic m at rix is not rem oved3,6. Th e d em i n er al i zed co l l ag en l ay er w o r k s as a diffusion obst acle, which occurs also in root caries27. I t has also been speculat ed t hat t his organic layer m ay present buffering capacit y. During an erosive challenge from t he out er surface, it m ay adsorb H+, t her efor e pr ot ect ing t he inner dent ine fr om pH decrease2,19. The presence and m aint enance of t he organic dent ine m at rix, t hrough t he inhibit ion of collagenolyt ic enzym es present in dent ine and saliva, is required for t he rem ineralizat ion of eroded dent ine12,30 $V IDU DV ZH NQRZ WKLV LV WKH ¿UVW
st udy t o assess t he effect of t oot hpast es w it h MMP inhibit ors on dent ine loss and t he second in vit ro
st udy t o show t hat dent inal enzym es have a role in erosion progression1. Most of t he previous st udies
have been perform ed in sit u, inw hich t he pot ent ial r ole of saliv ar y MMPs can n ot be dif f er en t iat ed f r om t h e d en t in al MMPs. Sin ce d em in er alized dent ine organic m at rix seem s t o be quit e resist ant t o m echanical for ces, even under high br ushing forces7, avoiding it s degradat ion by em ploying MMP
inhibit ors has em erged as a prom ising m et hod t o prevent dent ine erosion. This approach has been t est ed by using rinses and gels as vehicles t o deliver MMP inhibit ors17,18,24+RZHYHUHYHQWKRXJKÀXRULGH
t oot hpast es ar e w idely t est ed t o pr event dent al erosion23, t o our know ledge, t hey have never been
used as vehicles t o deliver MMP inhibit ors t o t he dent al st ruct ure.
Th e r e s u l t s o f t h e p r e s e n t i n v e s t i g a t i o n dem onst rat ed t hat t oot hpast es w it h chlorhexidine or green t ea ext ract are able t o decrease dent ine loss u n d er m ild in v it r o er osiv e an d ab r asiv e con dit ion s. Addit ion ally, t h ey w er e at least as effect ive as convent ional t oot hpast e w it h 1,100 ppm F. Bot h chlorhexidine and green t ea cat echins are MMP inhibit ors4,9,24 wit h unknown rem ineralizing
pot ent ial. As a m at t er of fact , while bot h hesperidin
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t he collagenase- induced loss of m ineral and erosive lesion pr ogr ession, only hesper idin w as able t o induce m ineral upt ak e in a r ecent st udy14. The
fur t her indicat es t he dist inct ive r ole of MMPs in t he progression of dent ine erosion. Thus, t he null hypot hesis was rej ect ed.
I n t he pr esent st udy, t he t oot hpast es t est ed
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been suggested to be dependent on the m aintenance of t he dem ineralized organic m at rix2,7,16,30, it would
be int erest ing t o t est t oot hpast es cont aining bot h
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prevent dent ine erosion seem s t o be dependent on t he vehicle used t o deliver t hese inhibit ors. The single applicat ion of gels17 is m ore effect ive t han
m ult iple r inses24 under sim ilar er osive pr ot ocols.
I n fact , t his applicat ion of gels cont aining EGCG or ch lor h ex idin e com plet ely pr ev en t ed den t in e er osion u n der er osiv e ch allen ges con du ct ed in sit u/ex vivo ( 100 m in of erosion)17. How ever, when
t he er osive challenges w er e conduct ed for 10 d ( 200 m in of erosion) , som e degree of dent ine loss occurred ( Kat o, et al., unpublished observat ions) . Thus it seem s t hat even in t he best case scenario, r epeat ed MMP inhibit or applicat ions ar e needed for per m anent pr event ion. This w ould be easier t o achieve w it h t he daily oral hygiene product s, especially t oot hpast es, w it h MMP- inhibit ing act ion.
CON CLUSI ON
I n conclusion, under t he lim it at ions of t his in vit ro st udy, t oot hpast es cont aining MMP inhibit ors ( 0.12% or 0.004% chlorhexidine, or 0.61 % green t ea ex t ract ) w er e able t o pr event dent ine w ear caused by erosion and abrasion. They perform ed sim ilarly t o t he t oot hpast e cont aining 1,100 ppm
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er o si o n . Si n ce MMP i n h i b i t i o n h a s a l so b een suggest ed as a st rat egy t o reduce dent ine caries pr ogr ession2 8 , 3 0, t oot hpast e w it h MMP inhibit ing DFWLRQ FRPELQHG ZLWK ÀXRULGH PLJKW EH HIIHFWLYH
in t he prot ect ion of t he t w o m ost com m on reasons for t he loss of t oot h st ruct ure, caries and erosion.
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furt her in sit u and clinical st udies.
ACKN OW LED GEM EN TS
Th is st u dy w as f u n ded by CNPq ( Gr an t n o. 557863/ 2008- 2) . The funders had no role in st udy design, dat a collect ion and analysis, decision t o publish, or preparat ion of t he m anuscript .
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