 this structure seals the dentin , prevents post - operative sensitivity and secondary caries , and may act as an elastic buffer that compensates the tensions generated by the polymerization shrinkage of the restorative composite . in order to achieve such conditions ,
the adhesives systems can interact with the dentin by etch - and - rinse or self - etch approaches .
the etch - and - rinse approach is considered a time - consuming and technique - sensitive bonding strategy since separate etching and rinsing steps are required .
multi - bottle etch - and - rinse adhesives involve three steps : acid etching ( mostly 30 - 40% phosphoric acid ) followed by priming and the application of a fluid resin . the most popular two - step or 
1-bottle  systems combine the primer and adhesive resin into one application . despite the conventional three - step etch - and - rinse
adhesives still perform most favorably and are most reliable in the long term , the two - step systems usually achieve acceptable adhesion to dentin
. however , the higher degree of hydrophilicity of the two - step etch - and - rinse adhesives makes them behave as permeable membranes after polymerization .
moreover , a decreasing concentration gradient of resin monomer diffusion within the acid - etched dentin naturally occurs as a consequence of the etch - and - rinse approach , creating a porous zone especially at the bottom of the hybrid layer with increased nanoleakage expression .
this condition may cause post - operative sensitivity and accelerate the resin elution from hydrolytically unstable polymeric hydrogels within the hybrid layers . as a consequence ,
the collagen fibrils remain unprotected and vulnerable to degradation by endogenous metalloproteinases , undermining the long - term durability of the restorations . in the case of
self - etch adhesives , the non - rinsing acidic monomers are able to simultaneously etch and prime the dental tissues . therefore , they attend the demand for simpler , more - user - friendly and less - technique - sensitive adhesives .
however , nanoporosities and slight areas with exposed collagen fibrils without resin encapsulation can still be observed even with the reduced exposition of the collagen matrix network by the self - etch approach .
such imperfections are impregnated by the silver nitrate in nanoleakage investigations , revealing areas of increased permeability within the polymerized resin matrix . according to tay
et al . , this condition is promoted by the remaining water used as an ionizing medium for the self - etch adhesives , creating regions of incomplete polymerization and/or hydrogel formation .
the self - etch systems can hybridize the dental hard tissues through one or two operatory steps . in part depending on ph , the self - etch adhesives may be classified according to the interaction depth at dentin into ultra - mild ( ph > 2.5   nano - interaction  ) , mild ( ph  2  interaction depth about 1 m ) , intermediate strong ( ph 12 , interaction depth between 1 and 2 m ) , and strong ( ph  1  interaction of several micrometers depth ) .
the latter are able to produce an interfacial ultra - morphology resembling that typically produced by etch - and - rinse adhesives .
nevertheless , several in vitro studies have shown that despite the reasonable bonding potential of the strong self - etch adhesives , reduced bond strength and increased interfacial nanoleakage are recorded in comparison to the multi - step adhesives , especially regarding the strong simplified adhesives rich in hema .
in fact , these less - favorable in vitro results were confirmed by the inferior clinical performance of the strong one - step self - etch adhesives .
the adhesive - dentin bond strength can be affected by a number of factors such as the dentin region used for bonding , the composition of the adhesive system and the application mode , polymerization characteristics ( especially regarding energy dose and conversion degree ) , and variables related with the method used to evaluate the bond strength . with regards to the hybridization quality , revealed by the nanoleakage investigation
, it is associated in most studies with the degradation phenomena and bonding durability , but little is known about its influence on the immediate bond strength to dentin .
therefore , the purpose of this study was to investigate the structural integrity of the hybrid layers and peripheral areas of two single - step self - etch adhesives , two two - step self - etching primer systems , and one one - step etch - and - rinse adhesive using silver methenamine or ammoniacal silver nitrate dyes for scanning electron microscopy ( sem ) nanoleakage investigation , as well as evaluate the resin - dentin bond strength .
the hypotheses tested were that ( i ) the interaction form of the adhesives to dentin affects hybridization quality and bond strength , and that ( ii ) the hybridization quality can not be related with the immediate bond strength .
forty caries - free , human third molars were collected according to the local institutional review board ( # 073/2007 ) with the informed consent of the donors .
the teeth were disinfected in 1% thymol , stored in distilled water , and used within one month following extraction .
the roots of the teeth were severed along the cement - enamel junction using a diamond - impregnated disk ( extec , enfield , ct , usa ) under water lubrication in a specific cutter machine ( isomet 1000 , buehler , lake bluff , il , usa ) .
after that , the occlusal enamel of the teeth was ground using a wet # 180-grit silicon carbide ( sic ) paper in order to expose a flat dentin surface .
five adhesive systems were used : the single - step self - etch adhesives  adper prompt ( adp ) and xeno iii ( xe ) , the two - step self - etching primer systems  clearfil se bond ( se ) and adhe se ( adse ) , and the single - step etch - and - rinse adhesive  adper single bond ( sb ) .
the brands , components , ph values , and application protocols of the adhesives evaluated are listed in table 1 .
materials , brands , components , ph values , and application protocols of the investigated adhesives ten teeth were used .
each tooth received two longitudinal sections , perpendicular to each other , using a diamond disk ( kg sorensen , barueri , sp , brazil ) mounted in a slow - speed handpiece .
these sections produced four tooth - quarters that were randomly distributed in five groups with 8 tooth - quarters each according the adhesive evaluated .
prior to bonding procedures , the exposed dentin surfaces were wet - polished with # 600-grit sic paper under running water for 60 s to create a standard smear layer .
the adhesives were applied on the prepared dentin surfaces and light - cured using a quartz - tungsten - halogen curing unit ( xl 3000 - 3 m espe , st .
paul , mn , usa ) with a power density of 600 mw / cm .
after that , three 2-mm - thick increments of a resin composite ( filtek z250 , 3 m espe ) were built up on the bonded dentin surface of each tooth - quarter and individually light - cured for 20 s , constituting a crown of 6.0 mm in height .
the restored tooth - quarters were stored in distilled water at 37 c for 24 h. after this period of storage , they were longitudinally sectioned in both the  x  and  y  directions across the bonded interface with a diamond - impregnated disk ( extec , enfield ) under water cooling at 300 rpm in a specific cutter machine ( isomet 1000 , buehler ) to obtain 3 - 4 sticks of approximately 0.8 mm in cross - sectional area for each restored tooth - quarter ( 25 sticks per group ) .
the sticks were individually fixed to a custom - made testing jig ( geraldeli 's device ) with a cyanoacrylate glue ( model repair ii blue , dentsply - sakin , japan ) and subjected to tensile load using a universal testing machine ( instron 4411 , instron corporation , canton , ma , usa ) at a crosshead speed of 0.5 mm / min until failure .
after the microtensile bond testing , the dentin and composite sides of the failed specimens were sputter coated ( 40 ma for 120 s ) with gold / palladium ( scd 050 ; balzers , schaan , leichtenstein ) and then examined through sem ( jsm 5600lv ; jeol , tokyo , japan ) with 85 magnification , operating in secondary electron mode and accelerating voltage of 15 kv .
failure modes were classified into one of the following categories : type i : cohesive failure in the hybrid layer ; type ii : cohesive failure in the resin composite ; type iii : cohesive failure in the dentin ; and type iv : cohesive failure in the adhesive layer and resin composite ; type v : cohesive failure in the adhesive resin and hybrid layer ; or type vi : cohesive failure in the adhesive resin and dentin . in cases of uncertainty ,
examination under higher magnifications ( 650 - 5000 ) was performed in order to confirm the nature of the failure .
statistical differences between the mean bond strength of the five bonding systems tested were submitted to one - way anova and tukey 's hsd post - hoc test , at a pre - set alpha of 0.05 .
two discs of 1 mm ( 0.5 ) in thickness were prepared from each tooth by means of two parallel sections of the remaining coronary portion .
the discs were then laminated into disc - pairs using a thin layer ( 1.0 mm in thickness ) of the flowable composite filtek flow ( 3 m espe ) , similar to the sandwich technique first described by inokoshi et al .
the dentin - resin - dentin sandwiches were light - cured ( xl 3000 - 3 m espe ) for 160 s in four different directions .
after that , each sandwich was sectioned perpendicularly to the resin - dentin bonding interface , producing 12 half - sandwiches by group that were immediately immersed in 2.5% glutaraldehyde in 0.1 m sodium phosphate buffer at 7.2 ph , for 24 h at 37c .
the half - sandwiches were further subdivided into three groups according to the specific specimens preparation for the following types of sem ( jsm 5600lv ; jeol ) analysis : resin - dentin ultramorphological analysis ( control ) , hybridization quality investigation using silver methenamine , or ammoniacal silver nitrate dyes .
for the ultramorphological analysis of the resin - dentin bonding interfaces , common procedures were employed to specimen preparation including demineralization in 50% phosphoric acid for 5 s , deproteinization by immersion in 10% naocl for 15 min , dehydration in ascending ethanol series ( 20% , 30% , 40% , 50% , 70% , 90% , and 100% for at least 20 min per step ) , and immersion in hexamethyldisilazane ( electron microscope sciences , fort washington , pn , usa ) for 10 min .
after chemical dehydration , the processed bonding interfaces were mounted in aluminum stubs , sputter coated ( 40 ma for 120 s ) with gold / palladium ( scd 050 ; balzers ) and observed under a sem ( jsm 5600lv ; jeol ) , operating in secondary electron mode , working distance 20 mm and accelerating voltage of 20 kv .
for both silver methenamine and ammoniacal silver nitrate dyes uptake , the half - sandwiches were coated with two layers of nail varnish applied to within 1 mm of the bonded interfaces .
ammoniacal silver nitrate was prepared according to the protocol first described by tay et al .
the specimens were placed in the ammoniacal silver nitrate in darkness for 24 h , rinsed thoroughly in distilled water , and immersed in photo developing solution for eight hours under a fluorescent light to reduce silver ions into metallic silver grains within voids along the bonded interface .
the specimens were immersed in the freshly prepared solution and left for 90 min at 60c in a pre - heated oven . after this period , the specimens were rinsed in distilled water for 3 min .
specimens were then transferred to a 0.2% gold chlorine bath for 30 s , rinsed in distilled water for 1 min , placed in a 3% sodium thiosulfate bath for 3 min , and finally rinsed profusely in tap water .
after that , the specimens were wet - polished with # 600-grit sic paper to remove the nail varnish .
they were then placed inside an acrylic ring attached to double - sided adhesive tape and embedded in epoxy resin ( buehler ltd , lake bluff , il , usa ) .
after the epoxy resin set , the specimens were lightly finished with # 1000-grit and # 2000-grit sic papers under water and polished with 6- , 3- , 1- , and 0.25-m - grit diamond pastes ( buehler ltd , usa ) .
the specimens were ultrasonically cleaned with distilled water and air - dried at room temperature for 24 h. the resin - dentin interfaces were mounted in aluminum stubs , submitted to carbon evaporation ( scd 050 ; balzers ) , and analyzed using a sem ( jsm 5600lv ; jeol ) , operating in back - scattering electron mode , working distance 20 mm and accelerating voltage of 20 kv .
each tooth received two longitudinal sections , perpendicular to each other , using a diamond disk ( kg sorensen , barueri , sp , brazil ) mounted in a slow - speed handpiece .
these sections produced four tooth - quarters that were randomly distributed in five groups with 8 tooth - quarters each according the adhesive evaluated .
prior to bonding procedures , the exposed dentin surfaces were wet - polished with # 600-grit sic paper under running water for 60 s to create a standard smear layer .
the adhesives were applied on the prepared dentin surfaces and light - cured using a quartz - tungsten - halogen curing unit ( xl 3000 - 3 m espe , st .
paul , mn , usa ) with a power density of 600 mw / cm .
after that , three 2-mm - thick increments of a resin composite ( filtek z250 , 3 m espe ) were built up on the bonded dentin surface of each tooth - quarter and individually light - cured for 20 s , constituting a crown of 6.0 mm in height .
the restored tooth - quarters were stored in distilled water at 37 c for 24 h. after this period of storage , they were longitudinally sectioned in both the  x  and  y  directions across the bonded interface with a diamond - impregnated disk ( extec , enfield ) under water cooling at 300 rpm in a specific cutter machine ( isomet 1000 , buehler ) to obtain 3 - 4 sticks of approximately 0.8 mm in cross - sectional area for each restored tooth - quarter ( 25 sticks per group ) .
the sticks were individually fixed to a custom - made testing jig ( geraldeli 's device ) with a cyanoacrylate glue ( model repair ii blue , dentsply - sakin , japan ) and subjected to tensile load using a universal testing machine ( instron 4411 , instron corporation , canton , ma , usa ) at a crosshead speed of 0.5 mm / min until failure .
after the microtensile bond testing , the dentin and composite sides of the failed specimens were sputter coated ( 40 ma for 120 s ) with gold / palladium ( scd 050 ; balzers , schaan , leichtenstein ) and then examined through sem ( jsm 5600lv ; jeol , tokyo , japan ) with 85 magnification , operating in secondary electron mode and accelerating voltage of 15 kv .
failure modes were classified into one of the following categories : type i : cohesive failure in the hybrid layer ; type ii : cohesive failure in the resin composite ; type iii : cohesive failure in the dentin ; and type iv : cohesive failure in the adhesive layer and resin composite ; type v : cohesive failure in the adhesive resin and hybrid layer ; or type vi : cohesive failure in the adhesive resin and dentin . in cases of uncertainty ,
examination under higher magnifications ( 650 - 5000 ) was performed in order to confirm the nature of the failure .
statistical differences between the mean bond strength of the five bonding systems tested were submitted to one - way anova and tukey 's hsd post - hoc test , at a pre - set alpha of 0.05 .
two discs of 1 mm ( 0.5 ) in thickness were prepared from each tooth by means of two parallel sections of the remaining coronary portion .
the discs were then laminated into disc - pairs using a thin layer ( 1.0 mm in thickness ) of the flowable composite filtek flow ( 3 m espe ) , similar to the sandwich technique first described by inokoshi et al .
the dentin - resin - dentin sandwiches were light - cured ( xl 3000 - 3 m espe ) for 160 s in four different directions .
after that , each sandwich was sectioned perpendicularly to the resin - dentin bonding interface , producing 12 half - sandwiches by group that were immediately immersed in 2.5% glutaraldehyde in 0.1 m sodium phosphate buffer at 7.2 ph , for 24 h at 37c .
the half - sandwiches were further subdivided into three groups according to the specific specimens preparation for the following types of sem ( jsm 5600lv ; jeol ) analysis : resin - dentin ultramorphological analysis ( control ) , hybridization quality investigation using silver methenamine , or ammoniacal silver nitrate dyes .
for the ultramorphological analysis of the resin - dentin bonding interfaces , common procedures were employed to specimen preparation including demineralization in 50% phosphoric acid for 5 s , deproteinization by immersion in 10% naocl for 15 min , dehydration in ascending ethanol series ( 20% , 30% , 40% , 50% , 70% , 90% , and 100% for at least 20 min per step ) , and immersion in hexamethyldisilazane ( electron microscope sciences , fort washington , pn , usa ) for 10 min .
after chemical dehydration , the processed bonding interfaces were mounted in aluminum stubs , sputter coated ( 40 ma for 120 s ) with gold / palladium ( scd 050 ; balzers ) and observed under a sem ( jsm 5600lv ; jeol ) , operating in secondary electron mode , working distance 20 mm and accelerating voltage of 20 kv .
for both silver methenamine and ammoniacal silver nitrate dyes uptake , the half - sandwiches were coated with two layers of nail varnish applied to within 1 mm of the bonded interfaces .
ammoniacal silver nitrate was prepared according to the protocol first described by tay et al .
the specimens were placed in the ammoniacal silver nitrate in darkness for 24 h , rinsed thoroughly in distilled water , and immersed in photo developing solution for eight hours under a fluorescent light to reduce silver ions into metallic silver grains within voids along the bonded interface .
the specimens were immersed in the freshly prepared solution and left for 90 min at 60c in a pre - heated oven .
specimens were then transferred to a 0.2% gold chlorine bath for 30 s , rinsed in distilled water for 1 min , placed in a 3% sodium thiosulfate bath for 3 min , and finally rinsed profusely in tap water .
after that , the specimens were wet - polished with # 600-grit sic paper to remove the nail varnish .
they were then placed inside an acrylic ring attached to double - sided adhesive tape and embedded in epoxy resin ( buehler ltd , lake bluff , il , usa ) .
after the epoxy resin set , the specimens were lightly finished with # 1000-grit and # 2000-grit sic papers under water and polished with 6- , 3- , 1- , and 0.25-m - grit diamond pastes ( buehler ltd , usa ) .
the specimens were ultrasonically cleaned with distilled water and air - dried at room temperature for 24 h. the resin - dentin interfaces were mounted in aluminum stubs , submitted to carbon evaporation ( scd 050 ; balzers ) , and analyzed using a sem ( jsm 5600lv ; jeol ) , operating in back - scattering electron mode , working distance 20 mm and accelerating voltage of 20 kv .
the microtensile bond strength ( -tbs ) results of the adhesives evaluated are presented in table 2 .
clearfil se bond ( se ) reached significantly higher bond strength than that of obtained by the others adhesives ( p < 0.05 ) .
no significance was found when the bond strength of adhe se ( adse ) was compared to that of produced by xeno iii ( xe ) ( p > 0.05 ) ; as well , the results of adper single bond ( sb ) and adper prompt ( adp ) did not differ significantly ( p > 0.05 ) .
the bond strength of adse and xe were significantly higher than those produced by sb and adp ( p < .05 ) . bond strength ( meansstandard deviations ) of the adhesives evaluated the distribution of the failure pattern ( % ) as analyzed by sem can be observed in figure 1 .
representative sem images of the predominant failure mode for all of the adhesives evaluated are in figure 2 .
the type v failure mode was predominant for all of the adhesives except se , which showed higher incidence of the type iv failure mode .
the one - step self - etch adhesives xe and adp did not exhibit the type ii failure mode .
the type iii failure mode was observed only for the two - step self - etching primers se and adse .
as well , the type vi failure mode was seen only for sb and se .
the type iv failure mode was not found for the etch - and - rinse sb .
distribution of the failure pattern ( % ) of the adhesives evaluated representative sem images of the dentin side of fractured specimens bonded with the adhesives evaluated .
( a - a2 ) specimens bonded with adper single bond ( sb ) ; ( b - b2 ) specimens bonded with clearfil se bond ( se ) ; ( c - c2 ) specimens bonded with adhese se ( adse ) ; ( d - d2 ) specimens bonded with adper prompt ( adp ) ; ( e - e2 ) specimens bonded with xeno iii ( xe ) .
( a - e ) low - power magnification of whole area of the fractured specimens .
( b ) bottom of the hybrid layer ; ( t ) top of the hybrid layer ; ( ad ) adhesive resin ; ( hl ) hybrid layer ; ( rc ) resin composite ; ( white arrows ) dentinal tubules filled by resin tags ; ( asterisk ) intertubular dentin covered by the adhesive ; ( pointer ) exposed collagen fibrils ; ( between black arrows ) blistering microstructures representative sem micrographs of the bonding interface of the adhesives studied are depicted in figures 35 .
the hybrid layer of sb was about 56 m in thickness with funnel - shaped resin tags distributed along the interface [ figure 3a ] .
when ammoniacal silver nitrate was used , specimens bonded with sb showed the most intense pattern of silver nitrate uptake , in which the whole extension of the hybrid layer seemed to be impregnated [ figure 3c ] . in addition , tree - like silver deposits called  water trees  protruding from the adhesive interface toward the adhesive layer could also be observed .
when silver methenamine was used , the nanoleakage expression was predominately observed at the bottom of the hybrid later [ figure 3b ] .
( a ) secondary electron image of unstained specimen ( control ) . an authentic hybrid layer with 5 - 6-m - thick was observed .
funnel - shaped resin tags ( tg ) with lateral branches ( white arrow ) were noted .
the silver nitrate uptake was observed at the bottom of the hybrid layer ( between white arrows ) .
( c ) back - scattering image of ammoniacal - silver - nitrate - stained specimen .
( rc ) resin composite ; ( ad ) adhesive layer ; ( hl ) hybrid layer ; ( d ) dentine representative sem images of the resin - dentin interfaces bonded with clearfil se bond ( se ) ( a - c ) and adhese se ( adse ) ( d - f ) .
secondary electron images of unstained specimens ( control ) bonded with se ( a ) and adse ( d ) , showing the interaction zone ( arrow ) with thin hybrid layers ( hl ) and cylindrical resin tags ( tg ) .
back - scattering images of stained specimens ( se - b and c ; adse - e and f ) showing the hybridization quality .
when silver methenamine was used , a spotted nanoleakage pattern was observed for se ( b ) , which was characterized by silver clusters outlining the bottom of the hybrid layer ( between arrows ) .
adse ( e ) exhibited a diffuse nanoleakage pattern at the bottom of the hybrid layer ( between arrows ) .
an intermittent nanoleakage pattern was produced for se ( c ) ( between arrows ) , while it was more continuous for adse ( f ) ( between arrows ) .
both se and adse exhibited some extensions of silver deposits toward the adhesive layer ( pointer ) .
( rc ) resin composite ; ( ad ) adhesive layer ; ( d ) dentine representative sem images of the resin - dentin interfaces bonded with adper prompt ( adp ) ( a - c ) and xeno iii ( xe ) ( d - f ) .
secondary electron images of unstained specimens ( control ) showing a hybrid layer about 3 - 4 m thick for adp ( a ) with funnel - shaped resin tags ( tg ) ; xe ( d ) exhibited a thin hybrid layer with cylindrical tgs . when silver methenamine was used , both adp ( b ) and xe ( e ) exhibited spotted nanoleakage patterns at the bottom of the hybrid layer .
ammoniacal silver nitrate yielded intense silver observed in the bonding interfaces for both adp ( c ) and xe ( f ) .
clusters of silver deposits were observed at the interaction area between the adhesive and resin composite for xe (  ) .
( rc ) resin composite ; ( d ) dentine the self - etch systems se , adse , and xe exhibited a shallow interaction with the dentin that was characterized by thin hybrid layers and cylindrical resin tags [ figures 4a , 4d , and 5d ] .
a thicker hybrid layer about 34 m thick with funnel - shaped resin tags was observed for adp [ figure 5a ] , which was comparable to that produced by sb .
silver penetration for both two - step self - etch systems se and adse , using ammoniacal silver nitrate , was practically restricted to the hybridization zone [ figure 4c and f ] .
it was concentrated mainly in the region between the non - demineralized dentin and the hybrid layer , showing also some extensions for the adhesive layer .
an intermittent silver deposition along the bonding interface was observed for se [ figure 4c ] , while a more continuous nanoleakage pattern occurred for adse [ figure 4f ] .
the amount of silver deposits for both se and adse was evidently smaller when compared to the deposits observed for sb , adp , and xe [ figures 3c , 5c , and 5f ] .
the analysis of the hybrid layer formed by se , after the action of the silver methenamine , exhibited silver clusters outlining the bottom of the hybrid layer to form a spotted nanoleakage pattern [ figure 4b ] . in the case of adse ,
the silver deposits are closer to each other to form a nanoleakage pattern with a diffuse aspect [ figure 4e ] .
the ammoniacal silver nitrate produced intense silver deposition in the bonding interface for both one - step self - etch adhesives xe and adp [ figures 5c and f ] .
the nanoleakage uptake was predominantly at the hybridization zone for xe , but clusters of silver deposits were also observed at the interaction zone between the adhesive and resin composite .
the specimens bonded with adp showed a complex silver nitrate uptake at the hybrid layer with water trees in a similar way to that observed for the etch - and - rinse sb . when submitted to silver methenamine , both adp and xe exhibited spotted nanoleakage patterns [ figure 5b and e ] similar to that produced by se at the bottom of the hybrid layer [ figure 4b ] .
the microtensile bond strength ( -tbs ) results of the adhesives evaluated are presented in table 2 .
clearfil se bond ( se ) reached significantly higher bond strength than that of obtained by the others adhesives ( p < 0.05 ) .
no significance was found when the bond strength of adhe se ( adse ) was compared to that of produced by xeno iii ( xe ) ( p > 0.05 ) ; as well , the results of adper single bond ( sb ) and adper prompt ( adp ) did not differ significantly ( p > 0.05 ) .
the bond strength of adse and xe were significantly higher than those produced by sb and adp ( p < .05 ) . bond strength ( meansstandard deviations ) of the adhesives evaluated the distribution of the failure pattern ( % ) as analyzed by sem can be observed in figure 1 .
representative sem images of the predominant failure mode for all of the adhesives evaluated are in figure 2 .
the type v failure mode was predominant for all of the adhesives except se , which showed higher incidence of the type iv failure mode .
the one - step self - etch adhesives xe and adp did not exhibit the type ii failure mode .
the type iii failure mode was observed only for the two - step self - etching primers se and adse .
as well , the type vi failure mode was seen only for sb and se .
the type iv failure mode was not found for the etch - and - rinse sb .
distribution of the failure pattern ( % ) of the adhesives evaluated representative sem images of the dentin side of fractured specimens bonded with the adhesives evaluated .
( a - a2 ) specimens bonded with adper single bond ( sb ) ; ( b - b2 ) specimens bonded with clearfil se bond ( se ) ; ( c - c2 ) specimens bonded with adhese se ( adse ) ; ( d - d2 ) specimens bonded with adper prompt ( adp ) ; ( e - e2 ) specimens bonded with xeno iii ( xe ) .
( a - e ) low - power magnification of whole area of the fractured specimens .
( b ) bottom of the hybrid layer ; ( t ) top of the hybrid layer ; ( ad ) adhesive resin ; ( hl ) hybrid layer ; ( rc ) resin composite ; ( white arrows ) dentinal tubules filled by resin tags ; ( asterisk ) intertubular dentin covered by the adhesive ; ( pointer ) exposed collagen fibrils ; ( between black arrows ) blistering microstructures
representative sem micrographs of the bonding interface of the adhesives studied are depicted in figures 35 .
the hybrid layer of sb was about 56 m in thickness with funnel - shaped resin tags distributed along the interface [ figure 3a ] .
when ammoniacal silver nitrate was used , specimens bonded with sb showed the most intense pattern of silver nitrate uptake , in which the whole extension of the hybrid layer seemed to be impregnated [ figure 3c ] . in addition , tree - like silver deposits called  water trees  protruding from the adhesive interface toward the adhesive layer could also be observed .
when silver methenamine was used , the nanoleakage expression was predominately observed at the bottom of the hybrid later [ figure 3b ] .
( a ) secondary electron image of unstained specimen ( control ) . an authentic hybrid layer with 5 - 6-m - thick was observed .
funnel - shaped resin tags ( tg ) with lateral branches ( white arrow ) were noted .
the silver nitrate uptake was observed at the bottom of the hybrid layer ( between white arrows ) .
( c ) back - scattering image of ammoniacal - silver - nitrate - stained specimen .
( rc ) resin composite ; ( ad ) adhesive layer ; ( hl ) hybrid layer ; ( d ) dentine representative sem images of the resin - dentin interfaces bonded with clearfil se bond ( se ) ( a - c ) and adhese se ( adse ) ( d - f ) .
secondary electron images of unstained specimens ( control ) bonded with se ( a ) and adse ( d ) , showing the interaction zone ( arrow ) with thin hybrid layers ( hl ) and cylindrical resin tags ( tg ) .
back - scattering images of stained specimens ( se - b and c ; adse - e and f ) showing the hybridization quality .
when silver methenamine was used , a spotted nanoleakage pattern was observed for se ( b ) , which was characterized by silver clusters outlining the bottom of the hybrid layer ( between arrows ) .
adse ( e ) exhibited a diffuse nanoleakage pattern at the bottom of the hybrid layer ( between arrows ) .
an intermittent nanoleakage pattern was produced for se ( c ) ( between arrows ) , while it was more continuous for adse ( f ) ( between arrows ) . both se and adse exhibited some extensions of silver deposits toward the adhesive layer ( pointer ) .
( rc ) resin composite ; ( ad ) adhesive layer ; ( d ) dentine representative sem images of the resin - dentin interfaces bonded with adper prompt ( adp ) ( a - c ) and xeno iii ( xe ) ( d - f ) .
secondary electron images of unstained specimens ( control ) showing a hybrid layer about 3 - 4 m thick for adp ( a ) with funnel - shaped resin tags ( tg ) ; xe ( d ) exhibited a thin hybrid layer with cylindrical tgs .
when silver methenamine was used , both adp ( b ) and xe ( e ) exhibited spotted nanoleakage patterns at the bottom of the hybrid layer .
ammoniacal silver nitrate yielded intense silver observed in the bonding interfaces for both adp ( c ) and xe ( f ) .
clusters of silver deposits were observed at the interaction area between the adhesive and resin composite for xe (  ) .
( rc ) resin composite ; ( d ) dentine the self - etch systems se , adse , and xe exhibited a shallow interaction with the dentin that was characterized by thin hybrid layers and cylindrical resin tags [ figures 4a , 4d , and 5d ] . a thicker hybrid layer about 34 m thick with funnel - shaped resin tags was observed for adp [ figure 5a ] , which was comparable to that produced by sb .
silver penetration for both two - step self - etch systems se and adse , using ammoniacal silver nitrate , was practically restricted to the hybridization zone [ figure 4c and f ] .
it was concentrated mainly in the region between the non - demineralized dentin and the hybrid layer , showing also some extensions for the adhesive layer .
an intermittent silver deposition along the bonding interface was observed for se [ figure 4c ] , while a more continuous nanoleakage pattern occurred for adse [ figure 4f ] .
the amount of silver deposits for both se and adse was evidently smaller when compared to the deposits observed for sb , adp , and xe [ figures 3c , 5c , and 5f ] .
the analysis of the hybrid layer formed by se , after the action of the silver methenamine , exhibited silver clusters outlining the bottom of the hybrid layer to form a spotted nanoleakage pattern [ figure 4b ] . in the case of adse ,
the silver deposits are closer to each other to form a nanoleakage pattern with a diffuse aspect [ figure 4e ] .
the ammoniacal silver nitrate produced intense silver deposition in the bonding interface for both one - step self - etch adhesives xe and adp [ figures 5c and f ] .
the nanoleakage uptake was predominantly at the hybridization zone for xe , but clusters of silver deposits were also observed at the interaction zone between the adhesive and resin composite .
the specimens bonded with adp showed a complex silver nitrate uptake at the hybrid layer with water trees in a similar way to that observed for the etch - and - rinse sb . when submitted to silver methenamine ,
both adp and xe exhibited spotted nanoleakage patterns [ figure 5b and e ] similar to that produced by se at the bottom of the hybrid layer [ figure 4b ] .
the hybridization quality is key to achieving reliable bond strength and hermetic seal of the dentin surface .
the results of the present study showed that each adhesive system resulted in different bond strength and degree of nanoleakage , which corroborated the results of previous studies .
it was also demonstrated that the interaction form of the adhesives systems to dentin importantly affected the hybridization quality and bond strength ; hypothesis ( i ) was accepted .
sano et al . first described the hybridization quality investigation to explore the sealing ability of the dentin adhesives using a silver nitrate tracer .
however , the remnant dentin apatites and amorphous calcium phosphates , which are re - precipitated in the bonded interfaces of self - etching adhesives , may be dissolved when immersed in such a mildly acidic solution of silver nitrate ( ph 4.2 ) , producing artifactual microporosities , and then false - positive results .
therefore , a basic version of this solution , the ammoniacal silver nitrate ( ph 9.5 ) , was used to eliminate unwanted acidity of the conventional silver nitrate tracer solution . besides ammoniacal silver nitrate , the silver methenamine dye ( ph 8.1 ) was used in the present study to complement the investigation of the hybridization quality of the adhesives to dentin .
this silver dye was claimed to offer reduced risks of super - estimating the nanoleakage expression in a similar way to ammoniacal silver nitrate .
the silver methenamine was basically designed by gromori for histologic visualization of carbohydrate derivatives in animal tissues .
many years later , the silver methenamine was applied to dental hard tissues for visualization of hypo - mineralized areas in human teeth .
these studies concluded that the silver methenamine can stain collagen of hypo- and unmineralized areas of the dentin and should be employed to demonstrate abnormal patterns of mineralization .
de goes and montes ( 2004 ) confirmed this information and demonstrated that the silver methenamine is a useful method for nanoleakage investigation , marking the exposed collagen fibrils within the hybrid layer .
the results of the immediate bond strength were related with the hybridization quality of the adhesives evaluated . according to this analysis ,
the less - favorable hybridization patterns , considering the intensity , distribution , and morphological aspect of the silver uptake were associated with lower bond strength to dentin ; hypothesis ( ii ) was rejected .
one may argue that no quantitative assessment of the nanoleakage is a limitation of the study .
however , it is important to consider that the results of the present study demonstrated that the hybridization quality have relevant influence in the immediate bond strength to dentin , while the amount of silver uptake was supposed to be more properly associated with the long - term bonding durability . for a better understanding of the results of the present study , it is of paramount importance to take into account the composition of the adhesives .
one common characteristic among them is the presence of water as an organic solvent [ table 1 ] , which plays an important role in both etch - and - rinse and self - etch approaches .
regarding the etch - and - rinse adhesives , water is responsible for carrying resinous monomers into dentin after the etching step , and contributes to support the collagen fibrils expanded according to the wet - bonding technique .
however , the solvent can not be completely removed after the application of the adhesive , especially from water - based adhesives ( for instance , sb  water / ethanol solvent ) that show minor solvent evaporation in comparison with ethanol - based or acetone - based ones . as a consequence ,
the solvent remains in the bonding interface and may disturbs the polymerization reaction of the resin systems , which is extremely deleterious for the adhesion .
this condition could have importantly influenced the intense silver uptake observed for sb [ figure 3b and c ] , and also the ultimate bond strength to dentin , which was significantly lower than that produced by the self - etch systems se , adse , and xe .
another aspect that may importantly have affected the hybridization quality was the adhesion approach of the adhesive systems to the dentin .
the etch - and - rinse approach used the phosphoric acid to etch the dentin prior the resin monomers infiltration , creating a demineralization zone of 5 to 8 m in thickness .
however , this deep and complex etching pattern was not completely filled by the resin monomers of the adhesive , especially at the bottom of the hybrid layer , where the collagen fibrils remain exposed . using methenamine silver solution ,
the presence of silver impregnated predominantly at the bottom of the hybrid layer for the specimens bonded with sb was evident [ figure 3b ] ; this characterize the discrepancy between the depths of demineralization and resin infiltration . when bonding interfaces of sb [ figure 3b ] and adp [ figure 5b ] are compared , the discrepant zone for sb is evidently thicker . in spite of the similarity in terms of aggressiveness between adp and sb , which results in similar hybrid layers regarding the morphologic aspect and thickness [ figures 3a and 5a ] , the discrepant zone of adp
such a condition can be considered a goal of this adhesion strategy once it contributes to reduce the nanoleakage at the bottom of the hybrid layer , which represents a more susceptible area for degradation by the action of the endogenous metalloproteinases . despite the minimization of the discrepant zone due to the self - etch approach
, adp showed an intense silver uptake after ammoniacal silver nitrate staining [ figure 5c ] , and the bond strength was not significantly different from that obtained by sb .
in general , the most simple - to - use one - step self - etch adhesives are composed of intricate mixes of hydrophilic and hydrophobic monomers , requiring relatively high amounts of water to make possible this complex chemical combination . due to the deleterious effects of water upon adhesion
compromise materials , which have been documented to possess several shortcomings . as a consequence ,
the immediate bond strength of adp was reduced , and the degree of the nanoleakage observed is at the same level of the etch - and - rinse sb .
the analysis of the failure mode distribution [ figure 1 ] shows predominance of type v failure mode ( cohesive in the adhesive resin and hybrid layer ) for both sb [ figures 2a , 2a , and 2a  ] and adp [ figures 2d , 2d , and 2d  ] , suggesting that these regions are less resistant against mechanical efforts .
regarding the etch - and - rinse sb , the authors speculated that there was a tendency for failure to initiate at the severely demineralized dentin not infiltrated by the resin monomers ( discrepant zone ) , propagating then within the adhesive resin . on the other hand ,
the predominance of type v failure mode for adp may be explained based on the presence of water blisters and poly - hema hydrogels into both hybrid and adhesive layers .
after the evaporation of the water , in which the monomers are mutually soluble , the chemical equilibrium of the adhesive is broken , resulting in above mentioned microstructures that capture the water , and are responsible to create a complex pattern of silver deposition [ figure 5b and c ] . a similar situation that results in intense silver uptake was also observed with the one - step self - etch xe [ figure 5e and f ] . especially for xe , the blistering microstructures could be identified in the sem analysis of the failure pattern [ figure 2e2 ] .
the sorption of water by these microstructures causes the internal plasticization of the resinous matrix , reducing the intensity of the chemical bond between the polymer chains .
thus , the absorbed moisture acts as a plasticizer agent , lowering the glass transition temperature of cured resin , and then its physical properties and bond strength of the one - step self - etches adhesives to dentin .
the self - etch adhesives are in general composed of acidic resin monomers , hydrophilic monomers ( hema ) , and functional dimethacrylates .
the presence of water is also crucial for this category of bonding systems . in this case
, water ionizes the acidic monomers , providing the ability to etch and infiltrate the tooth substrates simultaneously .
the presence of low molecular weight and hydrophilic resin monomers such as 2-hydroxyethyl methacrylate ( hema ) is necessary , acting as a kind of solvent that enhances the penetration capability of dentinal substrates and can be polymerized with the others resin monomers .
so , this category of resin monomers works as an auxiliary of the acidic monomers not readily soluble in water , attending in the demineralization and polymerization processes .
other bi- or multifunctional monomers are included in the formulation of the adhesives to increase the resistance of the adhesive via cross - links between polymer chains . despite the similar basic composition , the self - etch adhesives exhibit particularities in their chemical components and water content .
consequently , the adhesives show particular interaction and bond strength with the dentin , as observed in the present study . unlike the one - step self - etch adhesives ( ad and xe ) , the two - step self - etch systems have lower acidities and degrees of hydrophilicity , which minimize the possibility of creating water blisters .
such characteristics determined the particular interaction of se with dentin [ figure 4a  c ] , and are thought to significantly contribute to the higher magnitude of bond strength when compared with those produced by the other adhesives evaluated [ table 2 ] .
the superior hybridization quality of se was considered decisive for the low incidence of cohesive fractures in the hybrid layer ( type i ) , suggesting that the hybridization zone is mechanically resistant against tensile tensions .
another important aspect to be considered is the chemical bonding between the monomer 10-mdp , present in the composition of se [ table 1 ] , and the hydroxyapatite - crystals that remain around the collagen fibrils due to the ultra - mild demineralization .
the resultant two - fold micro - mechanical and chemical bonding mechanisms of se help explain the better results regarding the hybridization quality and bond strength . besides the more - favorable degree of hydrophilicity and ph of the two - step self - etch adhesives se and adse in comparison with the simplified ones , the improved hybridization quality se and adse was also importantly influenced by the application of a hydrophobic adhesive layer .
adhesive systems that have the hydrophobic monomers stored in a separate bottle tend to produce a more - resistant barrier for water permeability , and thus a more - effective sealing of the dentin . in spite of the similar characteristics of the two self - etch systems
adse provided significantly lower bond strength values than those obtained for se and predominance of cohesive failure in the adhesive resin and in the hybrid layer ( type v ) [ figure 2c , c1 and c2 ] .
the authors of the present study speculated that a larger amount of water is required for ionizing the acidic monomers of the more - aggressive adse , giving the adhesive a higher degree of hydrophilicity . as a result ,
adse exhibited a more - intense and continuous silver uptake after ammoniacal silver staining [ figure 4f ] than that observed for se [ figure 4c ] , which is thought to have negatively influenced the bond strength and failure pattern distribution .
the same magnitude of bond strength between the two - step adse and the one - step xe self - etch adhesives may be attributed to its similar hybridization quality [ figures 4e , 4f , 5e , and 5f ] and also upon the aggressiveness , which in part depends upon the ph of the adhesives .
adse and xe are considered mild and intermediate strong adhesives , respectively , for which the interaction depth with the dentin is about 12 m . comparing the one - step self - etch adhesives adp and xe , the significant difference in the bond strength observed between them was influenced not only by the difference in aggressiveness and hybridization quality to dentin , but it may also have be affected by the presence of inorganic fillers in the composition of xe [ table 1 ] .
the inorganic phase contributes to form a thicker adhesive layer over the hybrid layer that is better polymerized due to the proportionally lower oxygen inhibition .
the failure pattern distributions are consistent with this interpretation , since xe showed lower incidence of cohesive failure involving the adhesive resin when compared to that observed in adp .
the results of the present study demonstrated that the simplified and more - hydrophilic systems sb , adp , and xe underperform when compared to the two - step self - etch adhesives se and adse , which show less silver uptake and higher bond strength .
therefore , the hybridization was key to providing good dentin bonding , corroborating the results of a previous study . especially regarding the one - step self - etch adhesives , represented in the present study of adp and xe , the severely compromised bonding
to dentin apparently pushed them today toward the more - promising mild self - etch adhesives . in an effort to improve the performance of the one - step self - etch adhesives , a new generation of  single - component 
self - etch adhesives with higher ph was recently introduced . in general , the increase in ph leads to a reduction in the degree of hydrophilicity , since less water would be required for the ionization of acidic monomers .
therefore , further studies are necessary to investigate the performance of this recent concept for the one - step self - etch adhesives .
within the limits of the present study , the following can be concluded : the adhesive systems evaluated showed particular hybridization quality and bond strength , which depended upon not only the actual category of the adhesives , but also on the composition of the adhesives and the interaction to dentin.the hybridization quality is related with the immediate -tbs to dentin , and is essential to improve the bonding effectiveness .
the adhesive systems evaluated showed particular hybridization quality and bond strength , which depended upon not only the actual category of the adhesives , but also on the composition of the adhesives and the interaction to dentin .
the hybridization quality is related with the immediate -tbs to dentin , and is essential to improve the bonding effectiveness .
 objective : to evaluate the hybridization quality and bond strength of adhesives to dentin.materials and methods : ten human molars were ground to expose the dentin and then sectioned in four tooth - quarters . they were randomly divided into 5 groups according to the adhesive used : two single - step self - etch adhesives  
 adper prompt ( adp ) and xeno iii ( xe ) , two two - step self - etching primer systems  clearfil se bond ( se ) and adhe se ( adse ) , and one one - step etch - and - rinse system  adper single bond ( sb ) . 
 resin composite ( filtek z250 ) crown buildups were made on the bonded surfaces and incrementally light - cured for 20 s. the restored tooth - quarters were stored in water at 37c for 24 h and then sectioned into beams ( 0.8 mm2 in cross - section ) . 
 maximal microtensile bond strength ( -tbs ) was recorded ( 0.5 mm / min in crosshead speed ) . 
 the results were submitted to one - way anova and tukey 's test (  = 0.05 ) . 
 thirty additional teeth were used to investigate the hybridization quality by sem using silver methenamine or ammoniacal silver nitrate dyes.results:se reached significantly higher -tbs ( p < 0.05 ) ; no significance was found between adse and xe ( p > 0.05 ) , and between sb and adp ( p > 0.05 ) ; adse and xe were significantly higher than sb and adp ( p < 0.05 ) . 
 the bonding interface of sb showed the most intense silver uptake . 
 se and adse showed more favorable hybridization quality than that observed for adp and xe.conclusions:the bond strength and hybridization quality were affected by the interaction form of the adhesives with dentin . 
 the hybridization quality was essential to improve the immediate -tbs to dentin . 
INTRODUCTION
MATERIALS AND METHODS
Microtensile bond testing
Failure mode classification
Scanning electron microscopy for hybridization quality evaluation
RESULTS
Bond strength results and failure analysis
Hybridization quality
DISCUSSION
CONCLUSIONS