dental erosion is a common problem in modern societies , owing to the increased 
 consumption of acidic drinks , such as soft drinks , sport drinks , fruit juices , and fruit 
 teas , which in turn have a high potential to provoke dental demineralization . up to now
, most clinical reports are 
 generally related with a later intervention , in which the non - carious lesions as 
 erosion , especially in cervical area of the tooth , present dentin exposure , 
 hypersensitivity , and more complex restorative needs .
erosion is a superficial demineralization process that softens the surface with 
 subsequent wear until reaching dentin .
is normally associated with other 
 non - carious lesions such as abrasion , its sole cause in dental substrates is difficult 
 to establish , since erosion consists of lesions from multiple etiologies , which may 
 result in the need for a restorative procedure . although enamel is considered a simple and safe substrate for bonding , there is lack of research regarding this property to eroded tooth . 

as erosion provokes alterations on this substrate , details of these modifications on 
 bonding turn the investigations necessary .
as 
 these substrates will be subsequently restored , mostly using adhesive materials , this 
 interaction needs to be clarified .
thus , this investigation aimed to test the impact of different coke drinks on adhesion 
 to previouslyeroded enamel surfaces .
the null hypotheses are that ( 1 ) different coke 
 drinks do not provoke differences on adhesion to enamel , and ( 2 ) their association with 
 toothbrushing does not have any impact on the adhesion process either .
this experiment was conducted considering two factors : erosive challenges by soft 
 drinks ( in four levels : none / artificial saliva , rc , lc , and zc ) and toothbrushing 
 effect ( in two levels : none or following erosive challenge ) . the response variable 
 was based on bond strength .
fifty - six enamel specimens ( 4x4x2 mm ) were obtained from freshly - extracted bovine 
 incisors , which were previously stored in 0.1% thymol solution at room temperature . 

one specimen was cut from each crown using an isomet low - speed saw cutting machine 
 ( buehler , lake bluff , il , usa ) and two diamond disks ( extec corp .
, enfield , ct , usa ) , 
 which were separated by a 4 mm thickness spacer .
the enamel surface was flat with 
 watercooled carborundum discs ( # 320 , 600 , and 1200 of al2o3 papers ; buehler , lake 
 bluff , il , usa ) , resulting in enamel removal of about 100 m depth .
this series was 
 completed with polishing using felt paper made wet by diamond spray ( 1 m ; buehler , 
 lake bluff , il , usa ) . in order to standardize the enamel surfaces ,
they were selected 
 using a micro - hardness test by performing five indentations in different regions of 
 the block ( knoop diamond , 25 g , 5 s , hmv-2000 ; shimadzu corporation , tokyo , japan ) . 

enamel blocks with a knoop hardness number ranging from 320 to 385 khn were 
 selected .
selected specimens were randomly 
 assigned into seven groups ( n=8 ) according to immersion media and toothbrushing 
 association or not as presented in figure 
 2 .
composition of artificial saliva and coke beverages based on manufacturer information , except for ph and tritability , which were 
 assessed by the authors groups tested according to erosive / abrasive challenges after the erosive / abrasive challenge , each specimen was carefully cleaned under a 
 deionized water flow for 2 minutes .
ltda , petrpolis , rj , brazil ) for 15 s , which was washed out 
 for a 30 s. a gentle air - stream was used to promote water evaporation , 
 which was completed with absorbent paper .
two thin coats of an etch - and - rinse dentin 
 bonding system ( adper single bond 2- 3 m espe , st .
paul , mn , usa ) were subsequently 
 dispensed with a disposable microbrush and gently air - dried for 2 - 5 s to allow 
 solvent evaporation and followed by light curing for 10 s with a 1,000 
 mw / cm power density led unit ( radi cal - sdi , bayswater , victoria , 
 australia ) .
thus , the enamel surface was restored with two layers of 2 mm 
 thickness increments of a nano - filled a2 shade resin composite ( filtek z350 - 3 m espe , 
 st .
paul , mn , usa ) and light - activated for 20 s. after 24 hours of water immersion in 37c , each restored enamel specimen was 
 longitudinally sectioned in directions across the bonded interface using an isomet 
 1000 digital saw ( buehler ltd .
, lake bluff , il , usa ) to obtain specimens with an 
 interface area of approximately 1 mm .
each beam was attached to a modified bencor multi - t testing apparatus 
 ( danville engineering co. , danville , ca , usa ) with a cyanoacrylate resin ( super 
 bonder flex gel - loctite , henckel ltda , itapevi , sp , brazil ) and submitted to test 
 under tension in a universal testing machine ( emic , so jos dos pinhais , pr , brazil ) 
 operating at a crosshead speed of 0.5 mm/ min . after testing , the cross - sectional 
 area at the site of fracture
was measured with a digital caliper ( mitutoyo digimatic 
 caliper series / code 500 - 144 , mitutoyo sul americana , rj , brazil ) to calculate bond 
 strength in mega pascal ( mpa ) .
data analysis was accomplished by the graph pad / prisma statistical package ( graphpad 
 instat for windows version 4.0 , san diego , ca , usa ) .
the assumptions of equality of 
 variances and normal distribution of errors were checked for all the variables 
 tested . since the assumptions were satisfied , two - way analysis of variance ( anova ) 
 and
bonferroni post hoc tests were carried out for statistical 
 comparisons and the significance was preset to 5% . after bonding tests , each interface was analyzed with a stereomicroscopy 40x and was 
 categorized according to failure as : adhesive failure ( failure between the enamel and 
 bonding layer ) , cohesive failure in enamel ( when failure occurred predominantly in 
 enamel ) or in resin ( when failure occurred predominantly in resin ) or mixed failure 
 ( when two or more types were observed simultaneously ) .
for each group , two additional specimens were prepared with half of the surface 
 protected with nail varnish in order to maintain a control surface .
after the 
 challenges , nail varnish was removed with acetone and the specimens were restored in 
 similar conditions as described above .
however , rhodamine b ( sigma - aldrich brasil , 
 so paulo , sp , brazil ) was added to adper single bond 2 in 0.16 mg / ml as a 
 fluorescent ingredient to be detected in clsm .
following , the specimens were cut in the middle to obtain two 
 halves containing control and exposed specimens to analyze the interfaces with the 
 confocal microscope ( leica tcs spe , leica microsystems cms , mannheim , germany ) using 
 the microscope 's software ( leica application suite advanced fluorescence , leica 
 microsystems cms , mannheim , germany ) .
the quality of the interfaces was analyzed by 
 examining both halves of each specimen with 40x ( each 1.0- 1.0 mm , 1,024 pixels and 
 0.976 m in resolution ) .
this experiment was conducted considering two factors : erosive challenges by soft 
 drinks ( in four levels : none / artificial saliva , rc , lc , and zc ) and toothbrushing 
 effect ( in two levels : none or following erosive challenge ) . the response variable 
 was based on bond strength .
fifty - six enamel specimens ( 4x4x2 mm ) were obtained from freshly - extracted bovine 
 incisors , which were previously stored in 0.1% thymol solution at room temperature . 

one specimen was cut from each crown using an isomet low - speed saw cutting machine 
 ( buehler , lake bluff , il , usa ) and two diamond disks ( extec corp .
, enfield , ct , usa ) , 
 which were separated by a 4 mm thickness spacer .
the enamel surface was flat with 
 watercooled carborundum discs ( # 320 , 600 , and 1200 of al2o3 papers ; buehler , lake 
 bluff , il , usa ) , resulting in enamel removal of about 100 m depth .
this series was 
 completed with polishing using felt paper made wet by diamond spray ( 1 m ; buehler , 
 lake bluff , il , usa ) . in order to standardize the enamel surfaces ,
they were selected 
 using a micro - hardness test by performing five indentations in different regions of 
 the block ( knoop diamond , 25 g , 5 s , hmv-2000 ; shimadzu corporation , tokyo , japan ) . 

enamel blocks with a knoop hardness number ranging from 320 to 385 khn were 
 selected .
selected specimens were randomly 
 assigned into seven groups ( n=8 ) according to immersion media and toothbrushing 
 association or not as presented in figure 
 2 .
composition of artificial saliva and coke beverages based on manufacturer information , except for ph and tritability , which were 
 assessed by the authors groups tested according to erosive / abrasive challenges
after the erosive / abrasive challenge , each specimen was carefully cleaned under a 
 deionized water flow for 2 minutes .
ltda , petrpolis , rj , brazil ) for 15 s , which was washed out 
 for a 30 s. a gentle air - stream was used to promote water evaporation , 
 which was completed with absorbent paper .
two thin coats of an etch - and - rinse dentin 
 bonding system ( adper single bond 2- 3 m espe , st .
paul , mn , usa ) were subsequently 
 dispensed with a disposable microbrush and gently air - dried for 2 - 5 s to allow 
 solvent evaporation and followed by light curing for 10 s with a 1,000 
 mw / cm power density led unit ( radi cal - sdi , bayswater , victoria , 
 australia ) .
thus , the enamel surface was restored with two layers of 2 mm 
 thickness increments of a nano - filled a2 shade resin composite ( filtek z350 - 3 m espe , 
 st .
after 24 hours of water immersion in 37c , each restored enamel specimen was 
 longitudinally sectioned in directions across the bonded interface using an isomet 
 1000 digital saw ( buehler ltd . , lake bluff , il , usa ) to obtain specimens with an 
 interface area of approximately 1 mm .
each beam was attached to a modified bencor multi - t testing apparatus 
 ( danville engineering co. , danville , ca , usa ) with a cyanoacrylate resin ( super 
 bonder flex gel - loctite , henckel ltda , itapevi , sp , brazil ) and submitted to test 
 under tension in a universal testing machine ( emic , so jos dos pinhais , pr , brazil ) 
 operating at a crosshead speed of 0.5 mm/ min . after testing , the cross - sectional 
 area at the site of fracture
was measured with a digital caliper ( mitutoyo digimatic 
 caliper series / code 500 - 144 , mitutoyo sul americana , rj , brazil ) to calculate bond 
 strength in mega pascal ( mpa ) .
data analysis was accomplished by the graph pad / prisma statistical package ( graphpad 
 instat for windows version 4.0 , san diego , ca , usa ) .
the assumptions of equality of 
 variances and normal distribution of errors were checked for all the variables 
 tested . since the assumptions were satisfied , two - way analysis of variance ( anova ) 
 and
bonferroni post hoc tests were carried out for statistical 
 comparisons and the significance was preset to 5% .
after bonding tests , each interface was analyzed with a stereomicroscopy 40x and was 
 categorized according to failure as : adhesive failure ( failure between the enamel and 
 bonding layer ) , cohesive failure in enamel ( when failure occurred predominantly in 
 enamel ) or in resin ( when failure occurred predominantly in resin ) or mixed failure 
 ( when two or more types were observed simultaneously ) .
for each group , two additional specimens were prepared with half of the surface 
 protected with nail varnish in order to maintain a control surface . after the 
 challenges , nail varnish was removed with acetone and the specimens were restored in 
 similar conditions as described above .
however , rhodamine b ( sigma - aldrich brasil , 
 so paulo , sp , brazil ) was added to adper single bond 2 in 0.16 mg / ml as a 
 fluorescent ingredient to be detected in clsm .
following , the specimens were cut in the middle to obtain two 
 halves containing control and exposed specimens to analyze the interfaces with the 
 confocal microscope ( leica tcs spe , leica microsystems cms , mannheim , germany ) using 
 the microscope 's software ( leica application suite advanced fluorescence , leica 
 microsystems cms , mannheim , germany ) .
the quality of the interfaces was analyzed by 
 examining both halves of each specimen with 40x ( each 1.0- 1.0 mm , 1,024 pixels and 
 0.976 m in resolution ) .
bond strength means and standard deviations are summarized in table 1 and figure 3 . means and standard deviations ( mpa ) of eroded / abraded enamel specimens restored 
 with resin composite * uppercase letters show significant differences among the erosive challenges 
 for each abrasion condition ( columns ) ( p<0.05 ) .
lower case letters show 
 significant differences between the association with or without abrasive 
 challenge for each erosive condition ( rows ) ero - rc = eroded with regular coke ; ero - lc = eroded with light coke ; ero - zc = eroded 
 with zero coke ; no = not abraded and ab = abraded bond strengths ( mpa ) for all tested conditions ; comparing each eroded challenge in 
 abraded and not abraded conditions the factor erosion ( type of coke drink ) revealed statistical significance ( p<0.05 ) . 

in contrast , no significance was attributed to abrasion performed by toothbrushing 
 ( p>0.05 ) .
the control group ( neither eroded nor abraded ) showed significantly greater bond 
 strength compared to all other situations ( p<0.05 ) . for the eroded groups , all coke 
 drinks resulted in a significant decrease in bond strength compared to the control group 
 ( p<0.05 ) ; however , no differences were found among the bonding to eroded groups 
 previously treated with any coke drinks ( p>0.05 ) . in the comparison of bond strength 
 of specimens submitted to erosion to each coke drink to their respective association 
 with abrasion ,
description of distribution of the failure modes for each tested group is presented in 
 table 2 .
it could be observed that the sum of 
 mixed and adhesive failures was evident in all conditions .
cohesive failure in resin was 
 only present in groups eroded by zc ( 3.70% ) .
cohesive failure in enamel was also present 
 for all conditions , except for rc associated with abrasion .
failure mode distribution according to challenge ( % ) ero - rc = eroded with regular coke ; ero - lc = eroded with light coke ; ero - zc = eroded 
 with zero coke ; no = not abraded and ab = abraded ; rc = regular coke ;
lc = light coke ; 
 zc = zero coke under clsm observation , a control pattern in specimens ( not challenged ) is illustrated 
 in figure 4 , which showed a uniform and regular 
 tag formation , with homogeneous thickness and regular extension into enamel .
on the 
 other hand , distinct performance was revealed comparatively regarding each coke drink 
 that was used .
tags are presented 
 in homogeneous extent rc ( ero - rc and eroab - rc specimens ) caused superficial and heterogeneous tag formation 
 ( figures 5a , b ) .
however , for eroab - rc specimens , even also poorly visible , tags were more 
 regular than for ero - rc specimens .
a similar result was shown for ero - lc , in which the 
 adhesive impregnation was more superficial compared to control ( figures 6a , b ) .
for eroab - lc , 
 the adhesive layer showed to be more regular than only eroded specimens .
interface aspect of bonding system to enamel surface eroded by regular coke ( rc ) . 

hybrid layer is based on superficial tags 
 formation interface aspect of bonding system to enamel surface eroded by regular coke ( rc ) 
 followed by toothbrushing .
hybrid layer is irregular and superficial , but it is 
 more evident than only eroded surface interface aspect of bonding system to enamel surface eroded by light coke ( lc ) . 

tags are thin and irregular interface aspect of bonding system to enamel surface eroded by light coke ( lc ) 
 followed by toothbrushing .
hybrid layer is also superficial , but much more regular 
 than only eroded surface only zc allowed similar tag formation compared to the control group , regardless of the 
 abrasion .
the interface characteristics of these groups ( ero - zc and eroab - zc ) can be 
 observed in figure 7a , b. interface aspect of bonding system to enamel surface eroded by zero coke ( zc ) . 

tags present homogeneous pattern interface aspect of bonding system to enamel surface eroded by zero coke ( zc ) 
 followed by toothbrushing .
poor evidence is reported on the adhesion to enamel previously eroded by different soft 
 drinks .
while enamel is a dental substrate that allows the formation of regular and 
 strong adhesion , changes in this 
 substrate might affect the bond strength , failure mode , and the tag formation .
based on the results of this study , the first null hypothesis was rejected while the 
 second null hypothesis was accepted .
data attested the potential of cola - based drinks , 
 as coke types , to reduce bond strength in enamel compared with the control condition .
as 
 coke drinks are based on phosphoric acid content ( ph 2.6 - 3.0 ) , they showed to be 
 potentially erosive , which was previously stated . according to 
 figure 1 , all tested drinks presented similar 
 ph .
rc and lc present the same titratable acidity , which were higher than presented by 
 zc .
titratable acidity is related to the amount of base required to allow a solution 
 with neutral ph , which exhibits relevant influence on demineralization .
thus , it can be expected that there will be higher compromising by 
 rc and lc . by means of bond strength , this performance was not confirmed as all drinks 
 negatively influenced adhesion with no difference among them . however , in light of the 
 failure mode interpretation , we can observe a similar pattern of failure mode between rc 
 and lc , with a predominant occurrence of mixed and adhesive failures , which differed 
 from zc ( table 2 ) .
this less - erosive potential was attributed to the presence of the amino acid 
 phenylalanine , which is provided by the hydrolysis of aspartame in the presence of 
 saliva . as the present study was conducted under the in vitro 
 experimental model
, there was no influence of saliva , which in turn may be responsible 
 for the lack of differences between rc and lc .
the performance of zc reveals that it was the only group that presented cohesive failure 
 in resin .
likely , this beverage might provoke irregularities of surface , which intensity 
 was favorable to bond .
a rougher surface is attributed to playing a relevant role in the 
 adhesion mechanism , as it contributes to promoting more intense interlocking to 
 enamel .
however , the fragile enamel 
 surface can be lost if the erosive challenge continues .
this softened zone is also more 
 susceptible to mechanical forces , such as abrasion.control group , associated with 
 toothbrushing , presented no difference in relation to respective solely eroded group . 

when rc was associated with toothbrushing ( eroab - rc ) , cohesive failure in enamel was not 
 evident , suggesting that the eroded surface was removed by abrasion .
for lc and zc , it 
 might be prudent to speculate that the toothbrushing has a minor impact on the enamel 
 loss , due to the low erosive demineralization provoked by these drinks .
despite the fact that some studies have 
 shown that toothbrushing seems to have some effects on acid - softened hard 
 tissues , the abrasion of the eroded enamel surface did not have 
 a major impact on bond strength and failure mode in the present study .
clsm images are in accordance with the speculated interpretation of overall results 
 ( figures 4 to 7b ) . except for zc ,
however , enamel eroded with 
 rc showed more irregular interface than the lc - eroded surface .
for eroded/ abraded 
 enamel specimens , the hybrid layer was more evident than for only eroded enamel , except 
 for zc , which was similar to the control group .
we suggest that the toothbrushing 
 abrasion partially removed the fragile enamel layer , especially in the case of rc , 
 allowing better hybrid layer formation , even though the bond strength remained as low as 
 that for only the eroded enamel .
practitioners should be aware when they restore enamel erosion lesions of patients with 
 resin - based materials , as this property seems to be somehow negatively affected . 
 however , once more it is important to state that this investigated potential did not 
 consider the influence of saliva and its components present in the oral environment , 
 which could alter the results .
clinical investigations have shown some concern about eroded - tooth restoration ; however , 
 most of them regard the adhesion failures in dentin . in the present study
, 
 we focused on early treatment strategies for dental erosion which has not reached 
 dentin .
clinically , the findings of the present study are relevant for restoring enamel 
 erosive lesions in a facial surface of anterior teeth , which in turn might compromise 
 the aesthetic
. the early adhesive 
 restoration could also prevent the progression of enamel erosion in anterior teeth . 

thus , the results of the present study highlight the bonding mechanism when enamel is 
 involved in early stages of erosive demineralization .
furthermore , the study also showed 
 the importance of combining different analyses to better understand the adhesion process 
 in enamel .
based on the results of this study , we can conclude that all coke drinks reduced the 
 bond strength no matter the type .
qualitative aspects provided more detailed 
 information , showing different failure mode and tag formation according to the type of 
 coke drink .
further investigation is required to evaluate the impact of the type of coke 
 drink on the adhesion to enamel and also to dentin over time , using higher erosive 
 challenges and different bonding systems .
 objectivethis study aimed to assess the impact of in vitro erosion 
 provoked by different cola - based drinks ( coke types ) , associated or not with 
 toothbrushing , to bonding to enamel.material and methodsfifty - six bovine enamel specimens were prepared and randomly assigned into seven 
 groups ( n=8 ) : c- control ( neither eroded nor abraded ) , ero - rc : 3x/1-minute 
 immersion in regular coke ( rc ) , ero - lc : 3x/1-minute immersion in light coke ( lc ) , 
 ero - zc : 3x/1-minute immersion in zero coke ( zc ) and three other eroded groups , 
 subsequently abraded for 1-minute toothbrushing ( eroab - rc , eroab - lc and eroab - zc , 
 respectively ) . 
 after challenges , they were stored overnight in artificial saliva 
 for a total of 24 hours and restored with adper single bond 2/filtek z350 . 
 buildup 
 coronal surfaces were cut in 1 mm2 -specimens and subjected to a 
 microtensile test . 
 data were statistically analyzed by two - way anova / bonferroni 
 tests ( =0.05 ) . 
 failure modes were assessed by optical microscopy ( x40 ) . 
 the 
 interface of the restorations were observed using confocal laser scanning 
 microscopy ( clsm).resultsall tested cola - based drinks significantly reduced the bond strength , which was 
 also observed in the analyses of interfaces . 
 toothbrushing did not have any impact 
 on the bond strength . 
 clsm showed that except for zero coke , all eroded specimens 
 resulted in irregular hybrid layer formation.conclusionsall cola - based drinks reduced the bond strength . 
 different patterns of hybrid 
 layers were obtained revealing their impact , except for zc . 
INTRODUCTION
MATERIAL AND METHODS
Experimental design
Preparation of the specimens
Erosive protocols
Restorative procedures
Microtensile bond strength
Statistical analysis
Stereomicroscopy analysis
Confocal Laser Scanning Microscopy (CLSM)
RESULTS
DISCUSSION
CONCLUSIONS