to achieve adequate bonding for a resin - dentin bond with etch - and - rinse adhesives two clinical techniques , that is , dry bonding and wet bonding , have been suggested .
water - wet bonding ( wwb ) technique may contribute to the technique sensitivity of these adhesives , and most of the contemporary dentin adhesives are also hydrophilic to adhere to wet dentin substrates .
thus , bonding deteriorates overtime , due to a variety of physical and chemical factors including hydrolysis and enzymatic degradation by host - derived matrix metalloproteinases ( mmps ) .
hence , wwb technique with simplified hydrophilic adhesives would not be able to achieve durable adhesion to dentin .
( ewb ) has been postulated in which ethanol as a polar solvent is used to dehydrate and support the acid etched demineralized dentin collagen matrix for the application of etch - and - rinse adhesives .
it is used to create a less hydrophilic more durable hybrid layer and also prevent phase separation of the hydrophobic adhesive monomer .
dentin bonding using the ewb technique produces the highest microtensile bond strengths when compared to dentin bonding with water under moist or dry conditions .
the use of collagen cross - linkers ( ccls ) has been proposed to enhance the properties and longevity of the dentin - resin bonds .
proanthocyanidins ( pa ) are natural biocompatible ccls , mmp inhibitors , anti - bacterial and anti - oxidants that interact with proteins to stabilize and increase the type - i collagen cross - linkage , by promoting hydrogen bond formation between the protein amide carbonyl and the phenolic hydroxyl .
grape seed extracts are a rich source of pa , yielding a 10% higher concentration of pa .
proanthocyanidins have shown to improve the resin - dentin bond strength of etch - and - rinse adhesives to both sound dentin and caries - affected dentin .
assuming that the infiltration of resins would be facilitated by ewb and the presence of ccls within the hybrid layer would build the ideal interface resistant to hydrolysis and enzymatic degradation , this study was designed with the aim to evaluate whether the application of two simplified etch - and - rinse adhesives to biomodified dentin using ewb with and without ccl pretreatment improves their sealing ability .
the null hypothesis tested was that there is no difference between the microleakage of simplified etch - and - rinse adhesives bonded to pulp - chamber dentin with wwb or ewb with and without pa application .
the teeth were cleaned of any debris and stored in 0.1% thymol in distilled water at room temperature and used within 6 months of the extraction . using a carborundum disc and water coolant , the roof of the pulp - chamber was decoronated perpendicular to the longitudinal axis
, 1.5 mm coronal to the cement - enamel junction and roots were sectioned 2 mm apical to the bifurcation .
the canal orifices were widened with gates glidden drill no . 2 - 3 ( gates drills , mani inc .
, tochigi , japan ) and the pulp tissue was extirpated carefully with the help of excavator and broaches .
the root ends were sealed with amalgam ( dpi alloy , dental products of india , mumbai , india ) and later the pulp - chambers were irrigated with normal saline .
proanthocyanidins powder was derived after removing the coating from the grape seed extract capsules ( 95% purity vitis vinifera , zenith nutrition ) . to obtain 15% pa solutions ,
15.79 g powder was dissolved in 84.21 ml of 100% ethanol and distilled water separately at normal temperature and pressure .
the ph of the slightly acidic solutions was adjusted to 7.2 using 0.5 mol / l naoh ( hdb chemical engineer co. ltd . ,
after double filtration through a filter paper , the supernatant fluid was collected in a closed beaker .
samples were equally divided into eight groups ( n = 22 ) according to the four bonding techniques to be performed using adper single bond 2 ( asb ) ( 3 m espe , st .
paul , usa ) in group 1 ( a - d ) and xp bond ( xpb ) ( dentsply , detrey gmbh , germany ) in group 2 ( a - d ) [ tables 1 and 2 ] .
pulp - chamber in all the groups was acid etched with 37% phosphoric acid for 15 s , followed by rinsing and blot drying .
materials / adhesive systems , composition and manufacturer 's instructions experimental groups and their bonding protocols after bonding procedures , composite restorations were placed in the pulp - chamber of all the samples using a dual cure composite , core x flow ( dentsply caulk , milford , usa ) .
two increments of 2 mm thickness were placed using a dual - barreled syringe and each increment was light cured at 500 mw / cm by spectrum 800 ( dentsply , caulk , milford , usa ) for 20 s according to the manufacturer 's instructions [ table 1 ] .
the restored specimens were finally placed in distilled water at 37c for 24 h and subjected to thermo - cycling at a temperature range of 5c and 55c for 500 cycles , with 30 s dwell time at each bath and 10 s for specimen transfer .
twenty samples from each group were subjected to dye penetration test , and two samples per group were evaluated for interfacial morphology under scanning electron microscope ( sem ) .
the samples were coated with 2 layers of nail polish leaving 1 mm window around the composite restored margins and immersed in 2% methylene blue dye for 2 days .
they were washed under running water , and air dried at room temperature for 24 h. all samples were prepared by one operator and later sectioned longitudinally along the bucco - lingual direction corresponding to the center of the restorations .
dye penetration at the tooth - restoration interface was assessed by stereomicroscope at 10 by two independent precalibrated examiners who were unaware of the treatment groups . in case of any disagreement
the maximum degree of dye penetration was recorded for each specimen and dye penetration was scored on a nonparametric scale from zero to three for microleakage analysis .
dye leakage score criteria for scoring : 
 0 no leakage.1 leakage extending into pulp - chamber.2 leakage involving pulp floor.3 leakage involving root canal . 
  1 leakage extending into pulp - chamber .
acid - base treatment ( 6n hcl for 30 s , followed by 4% naocl for 10 min ) was done , and the samples were dehydrated in ascending ethanol concentration ( 50% , 75% , and 95% for 20 min each and 100% for 1 h ) , then transferred to a critical point dryer for 30 min .
gold sputter coating was carried out under reduced pressure in an inert argon gas atmosphere in an agar sputter coater p7340 ( agar scientific ltd . ,
all the sixteen gold - coated samples were examined under sem ( leo 430 , england ) operated at 15 kv to observe the micromorphology of the resin - dentin interface .
dye leakage scores obtained were subjected to statistical analysis using kruskal - wallis and mann - whitney u - tests statistical package for social sciences version 15.0 ( spss inc . ,
the samples were coated with 2 layers of nail polish leaving 1 mm window around the composite restored margins and immersed in 2% methylene blue dye for 2 days .
they were washed under running water , and air dried at room temperature for 24 h. all samples were prepared by one operator and later sectioned longitudinally along the bucco - lingual direction corresponding to the center of the restorations .
dye penetration at the tooth - restoration interface was assessed by stereomicroscope at 10 by two independent precalibrated examiners who were unaware of the treatment groups . in case of any disagreement
the maximum degree of dye penetration was recorded for each specimen and dye penetration was scored on a nonparametric scale from zero to three for microleakage analysis .
dye leakage score criteria for scoring : 
 0 no leakage.1 leakage extending into pulp - chamber.2 leakage involving pulp floor.3 leakage involving root canal . 
  1 leakage extending into pulp - chamber . 2 leakage involving pulp floor . 3 leakage involving root canal .
acid - base treatment ( 6n hcl for 30 s , followed by 4% naocl for 10 min ) was done , and the samples were dehydrated in ascending ethanol concentration ( 50% , 75% , and 95% for 20 min each and 100% for 1 h ) , then transferred to a critical point dryer for 30 min .
gold sputter coating was carried out under reduced pressure in an inert argon gas atmosphere in an agar sputter coater p7340 ( agar scientific ltd . ,
all the sixteen gold - coated samples were examined under sem ( leo 430 , england ) operated at 15 kv to observe the micromorphology of the resin - dentin interface .
dye leakage scores obtained were subjected to statistical analysis using kruskal - wallis and mann - whitney u - tests statistical package for social sciences version 15.0 ( spss inc . ,
intergroup comparisons using mann - whitney u test and corresponding p values are presented in table 4 .
the maximum leakage scores were found in wwb groups of both the adhesives , that is , groups 1a and 2a ( wwb ) and 1c and 2c
( wwb + ccl ) with no significant difference between groups 1a and 1c ( p = 0.510 ) and 2a and 2c ( p = 0.821 ) .
least dye leakage scores were observed in ewb groups ( 1b , 1d and 2b , 2d ) of both the adhesives regardless of ccl application , with no significant difference between groups 1b and 1d ( p = 0.918 ) and 2b and 2d ( p = 0.399 ) .
ewb groups of both the adhesives depicted significantly improved sealing ability as compared to wwb groups irrespective of ccl pretreatment . while ccl pretreatment had no effect on the sealing ability of either of the adhesives applied following wwb or ewb technique .
observed in the study groups ( n = 20 ) intergroup comparisons using mann - whitney u - test the representative sem photomicrographs of each group are shown in figures 1a - d and 2a - d . sem observation of resin - dentin interfaces demonstrated generalized gap along the entire interface showing poor adaptation after bonding with asb / xpb using wwb technique in both groups 1a and 2a ( wwb ) and groups 1c and 2c ( wwb + ccl ) [ figures 1a , 1c , 2a , and 2c ] .
ewb groups 1b and 2b ( ewb ) and groups 1d and 2d ( ewb + ccl ) revealed perfect adaptation depicting the absence of interfacial gaps after bonding with asb / xpb [ figures 1b , 1d , 2b , and 2d ] .
( a ) scanning electron microscopic ( sem ) view depicting generalized gap at resin - dentin interface after bonding with adper single bond 2 ( asb ) using water - wet bonding ( wwb ) technique ( group 1a - asb  wwb ) .
( b ) sem view depicting perfect interfacial seal after 100% ethanol pretreatment and bonded with asb 2 ( group 1b - asb  ethanol - wet bonding [ ewb ] ) .
( c ) sem microscopic view depicting poor adaptation at resin - dentin interface with asb 2 bonded to 15% proanthocyanidins pretreated pulp chamber dentin using wwb technique ( group 1c - asb  wwb + collagen cross - linker [ ccl ] ) .
( d ) sem view depicting absence of gap and good interfacial adaptation after 100% ethanol and 15% proanthocyanidins pretreatment of pulp chamber dentin bonded using asb 2 ( group 1d - asb  ewb + ccl ) ( a ) scanning electron microscopic ( sem ) view depicting generalized gap at resin - dentin interface after bonding with xp bond ( xpb ) using water - wet bonding ( wwb ) technique ( group 2a - xpb  wwb ) .
( b ) sem view depicting perfect interfacial seal after 100% ethanol pretreatment and bonded with xpb ( group 2b - xpb  ethanol - wet bonding [ ewb ] ) .
( c ) sem view depicting poor adaptation at resin - dentin interface with xpb bonded to 15% proanthocyanidins pretreated pulp - chamber dentin using wwb technique ( group 2c - xpb  wwb + collagen cross - linker [ ccl ] ) .
( d ) sem view depicting absence of gap and good interfacial adaptation after 100% ethanol and 15% proanthocyanidins pretreatment of pulp - chamber dentin bonded using xpb ( group 2d - xpb  ewb + ccl )
intergroup comparisons using mann - whitney u test and corresponding p values are presented in table 4 .
the maximum leakage scores were found in wwb groups of both the adhesives , that is , groups 1a and 2a ( wwb ) and 1c and 2c
( wwb + ccl ) with no significant difference between groups 1a and 1c ( p = 0.510 ) and 2a and 2c ( p = 0.821 ) .
least dye leakage scores were observed in ewb groups ( 1b , 1d and 2b , 2d ) of both the adhesives regardless of ccl application , with no significant difference between groups 1b and 1d ( p = 0.918 ) and 2b and 2d ( p = 0.399 ) .
ewb groups of both the adhesives depicted significantly improved sealing ability as compared to wwb groups irrespective of ccl pretreatment . while ccl pretreatment had no effect on the sealing ability of either of the adhesives applied following wwb or ewb technique .
microleakage scores observed in the study groups ( n = 20 ) intergroup comparisons using mann - whitney u - test
the representative sem photomicrographs of each group are shown in figures 1a - d and 2a - d .
sem observation of resin - dentin interfaces demonstrated generalized gap along the entire interface showing poor adaptation after bonding with asb / xpb using wwb technique in both groups 1a and 2a ( wwb ) and groups 1c and 2c ( wwb + ccl ) [ figures 1a , 1c , 2a , and 2c ] .
ewb groups 1b and 2b ( ewb ) and groups 1d and 2d ( ewb + ccl ) revealed perfect adaptation depicting the absence of interfacial gaps after bonding with asb / xpb [ figures 1b , 1d , 2b , and 2d ] .
( a ) scanning electron microscopic ( sem ) view depicting generalized gap at resin - dentin interface after bonding with adper single bond 2 ( asb ) using water - wet bonding ( wwb ) technique ( group 1a - asb  wwb ) .
( b ) sem view depicting perfect interfacial seal after 100% ethanol pretreatment and bonded with asb 2 ( group 1b - asb  ethanol - wet bonding [ ewb ] ) .
( c ) sem microscopic view depicting poor adaptation at resin - dentin interface with asb 2 bonded to 15% proanthocyanidins pretreated pulp chamber dentin using wwb technique ( group 1c - asb  wwb + collagen cross - linker [ ccl ] ) .
( d ) sem view depicting absence of gap and good interfacial adaptation after 100% ethanol and 15% proanthocyanidins pretreatment of pulp chamber dentin bonded using asb 2 ( group 1d - asb  ewb + ccl ) ( a ) scanning electron microscopic ( sem ) view depicting generalized gap at resin - dentin interface after bonding with xp bond ( xpb ) using water - wet bonding ( wwb ) technique ( group 2a - xpb  wwb ) .
( b ) sem view depicting perfect interfacial seal after 100% ethanol pretreatment and bonded with xpb ( group 2b - xpb  ethanol - wet bonding [ ewb ] ) .
( c ) sem view depicting poor adaptation at resin - dentin interface with xpb bonded to 15% proanthocyanidins pretreated pulp - chamber dentin using wwb technique ( group 2c - xpb  wwb + collagen cross - linker [ ccl ] ) .
( d ) sem view depicting absence of gap and good interfacial adaptation after 100% ethanol and 15% proanthocyanidins pretreatment of pulp - chamber dentin bonded using xpb ( group 2d - xpb  ewb + ccl )
despite apical leakage being considered an important factor in the endodontic failure , more attention is now being focused on procedures performed to achieve an effective coronal seal soon after the completion of root canal therapy using adhesive restoration .
the results of the present study depicted that the use of both simplified etch - and - rinse adhesives ; asb and xpb , on 100% ethanol - saturated pulp - chamber dentin achieved significantly lower microleakage values than wwb with and without pa application .
in the current study , evaluation of coronal seal of composite restorations in pulp - chamber revealed extensive leakage in both the adhesives when used with conventional wwb technique .
as compared to coronal dentin , bonding to pulp - chamber dentin is more difficult due to its complex hierarchical structure .
thus , the bond strengths in deep dentin are reported to be lower than superficial dentin due to less amount of inter - tubular dentin available for resin bonding . to optimize the bonding effectiveness of adhesives in pulp - chamber , dentin biomodification with ewb technique
originally designed to be used with experimental hydrophobic adhesives it can be performed in either of the two versions : in the progressive ethanol replacement , the ethanol saturation is achieved using a series of ascending ethanol concentrations , taking approximately 3 - 4 min , which defies the principles of user - friendliness and technique simplification . to overcome this obstacle , clinically relevant simplified dehydration protocol with single - step 1 min application of 100% ethanol has been developed . however , it is extremely technique - sensitive and does not completely replace residual water and , therefore , may not be successfully used with hydrophobic adhesives . on the other hand , it may be assumed that contemporary hydrophilic adhesives will be more tolerant to the presence of residual water as compared to hydrophobic adhesives after ewb in a simplified dehydration protocol .
therefore , the alternative simplified version of simplified ewb technique was used in the current study where contemporary hydrophilic etch - and - rinse adhesives were applied to ethanol - saturated demineralized dentin . a significant reduction in dye leakage
this may be attributed to the fact that ethanol , is a desirable solvent for most hydrophobic resin monomers and it can slowly replace residual water in the dentin matrix by chemical dehydration , thus preventing collagen hydrolysis , plasticization of resin and enzymatic degradation of resin - dentin interface . as mmps are hydrolases , they will also not become active in ethanol - saturated acid - etched dentin .
ethanol - saturated demineralized dentin results in better resin - collagen interactions / encapsulation as ethanol has a vapor pressure of 52.50 mmhg at 23.8c , whereas water has a value of 21.05 mmhg .
this means that ethanol evaporates much more quickly than water ; thereby removing / evaporating more water - ethanol from around the collagen fibrils for the adhesive monomers for adhesion .
ethanol used to wet the collagen matrix may create some interpeptide hydrogen bonds which will stiffen the matrix to minimize its collapse and produces shrinkage of the collagen fibrils thereby enlarging the interfibrillar spaces , which allow better infiltration of the monomers into the demineralized collagen matrix .
also , it is speculated that ethanol might collapse the highly hydrated glycosaminoglycans in interfibrillar spaces .
moreover , ewb preserves the resin - dentin bond integrity without the adjunctive use of mmp inhibitors .
all these factors result in improved bonding efficacy of contemporary etch - and - rinse adhesives with ewb depending on the composition of adhesives .
similar to our study , li et al . also demonstrated improved bonding efficacy of contemporary hydrophilic etch - and - rinse adhesives with simplified ewb technique ( 100% ethanol for 1 min ) .
they attributed this to the good wettability of ethanol - saturated dentin and more stable hybrid layer , as the hoy 's solubility parameter of hydrophilic adhesives better matches with that of partially dehydrated dentin matrix ( using simplified protocol ) , which also contains some residual water . on the other hand ,
complete water replacement by ethanol ( using ascending concentrations ) would result in hoy 's solubility parameter value , which is better matched with hydrophobic adhesives .
collagen cross - linker like pa may play an important role in the establishment of tissue engineering / biomimetics approaches to improve the intrinsic properties of the dentin bonding substrate .
the use of pa is a clinically feasible and promising approach for efficiently cross - linking and stabilizing collagen along with improving the durability of current dentin adhesives .
however in our study , application of pa did not improve the sealing ability of simplified etch - and - rinse adhesives .
the reason might be the shorter duration of application ( 1 min ) or the high c - factor in pulp - chamber as opposed to flat tooth surface mostly used in bond strength studies .
contrary to our study , in some studies pa was applied for 1 h and 5 - 10 min respectively that might account for the discrepancy from our results .
however , such application protocols are time - consuming and clinically unrealistic to justify the use of pa , which is not so in this study . in summary ,
ewb technique allows for higher infiltration of hydrophobic monomers and better collagen encapsulation , which would lead to more stable bonds .
further studies should aim to delineate guidelines for the time , concentration and source of pa and development of simplified ewb technique for use with different adhesives under simulated pulpal pressure conditions .
within the limitation of this in vitro study , it can be concluded that the sealing ability of both asb and xpb simplified etch - and - rinse adhesives significantly improved after bonding to ethanol - saturated pulp - chamber dentin using 100% ethanol application for 1 min ewb .
while dentin biomodification using ccl pretreatment with the application of 15% pa in clinically relevant time period had no significant effect on the sealing ability of the adhesives .
 aim : to evaluate whether the application of two simplified etch - and - rinse adhesives to biomodified dentin using ethanol - wet bonding ( ewb ) and collagen cross - linker ( ccl ) pretreatment improves their sealing ability.materials and methods : in 176 extracted human molars , the pulp - chambers were deroofed , and teeth were sectioned horizontally . 
 samples were randomly divided into eight groups according to four bonding techniques using two simplified etch - and - rinse adhesives ; adper single bond 2 ( asb ) and xp bond ( xpb ) . 
 the bonding protocols included : ( a ) water - wet bonding ( wwb ) ; ( b ) ewb ; ( c ) wwb and ccl application ; ( d ) ewb and ccl application . 
 after composite resin restorations , dye leakage evaluation and scanning electron microscope analysis were done . 
 leakage scores were statistically analyzed using kruskal - wallis and mann - whitney u tests at a significance level of p < 0.05.result:for both asb and xpb adhesives , least dye leakage was observed in ewb groups ( b and d ) ( p = 0.918 and p = 0.399 respectively ) which showed no significant difference , while maximum leakage scores were seen in wwb groups ( a and c ) . 
 regardless of ccl application and adhesives used , ewb technique depicted ( p = 0.003 and p = 0.004 ) significantly greater sealing ability than wwb.conclusion:bonding of asb and xpb using ewb significantly improved their sealing ability . 
 biomodification using ccl pretreatment had no significant effect on the sealing ability of adhesives bonded with either wwb or ewb . 
INTRODUCTION
MATERIALS AND METHODS
Sealing ability evaluation
Scanning electron microscopic evaluation
Statistical analysis
RESULTS
Sealing ability
Scanning electron microscopic
DISCUSSION
CONCLUSION