one disadvantage of these resins is polymerization shrinkage , which can lead to leakage of bacteria and fluid through the tooth adhesive and bracket - adhesive interfaces [ 
 1 
 , 
 2 
 ] .
microleakage beneath the orthodontic brackets may be followed by enamel discoloration and decalcification as well as decreased bond strength .
enamel decalcification ( white spot lesion ) is one of the main concerns of patients receiving fixed orthodontic therapy [ 
 3 
 ] .
this lesion can develop within four weeks in patients with poor oral hygiene [ 
 4 
 ] . on the other hand ,
one of the common problems during bracket bonding is poor isolation [ 
 5 
 , 
 6 
 ] , which can result in surface contamination by saliva , blood , gingival crevicular fluid , or water [ 
 7 
 ] .
saliva contact with the etched tooth results in plugging of porosities produced by acid etching ; therefore the micromechanical retention and bond strength of adhesive may decrease [ 
 8 
 , 
 9 
 ] .
to overcome the clinical problems related to poor isolation , some manufacturers introduced hydrophilic bonding materials , which can be used on moistened surfaces .
transbond moisture intensive primer ( tmip ; 3 m unitek ) + transbond plus color change adhesive ( 3 m unitek ) and assure primer ( reliance orthodontic products ) + assure adhesive paste ( reliance orthodontic products ) are two examples , which have been reported to have higher bond strength on saliva or water contaminated surfaces compared with conventional or hydrophobic systems [ 
 10 
 , 
 11 
 ] .
many studies have evaluated the effect of saliva contamination on bond strength of hydrophilic adhesive systems ; however , we could not find any study assessing the effect of saliva contamination during bonding process on microleakage beneath bonded brackets .
hence , we aimed to study the effect of saliva contamination on microleakage score of brackets bonded with two hydrophilic adhesive systems .
then , scaling and rubber cup prophylaxis with pumice paste were carried out and the teeth were rinsed with water and air - dried .
the teeth were acid etched by 37% phosphoric acid ( etching gel , 3 m unitek , monrovia , ca , usa ) for 30 seconds , water rinsed for 10 seconds , and then dried for 20 seconds with oil - free compressed air until a chalky surface appeared . in this study , standard 18-slot edgewise brackets ( equilibrium dentaurum inc .
after surface preparation , bonding process was carried out for each group based on one of the following protocols : g1 ( control ) : assure primer ( reliance orthodontic product , itasca , il , usa ) was applied to freshly etched enamel surface with a microbrush .
a small amount of assure adhesive ( reliance orthodontic product , itasca , il , usa ) was squeezed into the mesh on the back of each bracket and was then pressed on the tooth surface .
the excess adhesive material was removed and it was light - cured by led light curing unit ( smart life iq2 , dentsply - milford , usa ) at 400 nm wavelength for 40 seconds ( 10 seconds at each direction).g2 ( contaminated after etching ) : prior to the primer application , the teeth were contaminated with saliva . unstimulated human saliva was collected from one of the authors who had not consumed any food or liquids for 60 minutes prior to the collection [ 
 10 
 , 
 11 
 ] .the rest of the procedure was the same as in g1.g3 ( contaminated after priming ) : assure primer was applied to freshly etched enamel surface and gently air - dried for five seconds .
then , fresh saliva was applied with a microbrush on the labial surface until they were totally contaminated .
the rest of the procedure was the same as in g1 and g2.g4 ( control ) : the teeth in this group were treated as in g1 except that the moisture intensive primer transbond ( tmip , 3 m unitek , monrovia , ca , usa ) and transbond plus color change adhesive were used.g5 ( contaminated after etching ) : the teeth in this group were treated as in g2 except that transbond plus was used.g6 ( contaminated after priming ) : the teeth in this group were treated as in g3 except that the tmip and transbond plus were used .
after the bonding procedure , the samples were stored in distilled water for four weeks .
all samples were thermocycled for 500 cycles between 5c and 55c with 30 seconds of dwell time and 10 seconds of transfer time .
g1 ( control ) : assure primer ( reliance orthodontic product , itasca , il , usa ) was applied to freshly etched enamel surface with a microbrush .
a small amount of assure adhesive ( reliance orthodontic product , itasca , il , usa ) was squeezed into the mesh on the back of each bracket and was then pressed on the tooth surface .
the excess adhesive material was removed and it was light - cured by led light curing unit ( smart life iq2 , dentsply - milford , usa ) at 400 nm wavelength for 40 seconds ( 10 seconds at each direction ) .
g2 ( contaminated after etching ) : prior to the primer application , the teeth were contaminated with saliva .
unstimulated human saliva was collected from one of the authors who had not consumed any food or liquids for 60 minutes prior to the collection [ 
 10 
 , 
 11 
 ] .
g3 ( contaminated after priming ) : assure primer was applied to freshly etched enamel surface and gently air - dried for five seconds .
then , fresh saliva was applied with a microbrush on the labial surface until they were totally contaminated .
g4 ( control ) : the teeth in this group were treated as in g1 except that the moisture intensive primer transbond ( tmip , 3 m unitek , monrovia , ca , usa ) and transbond plus color change adhesive were used .
g5 ( contaminated after etching ) : the teeth in this group were treated as in g2 except that transbond plus was used .
g6 ( contaminated after priming ) : the teeth in this group were treated as in g3 except that the tmip and transbond plus were used .
after the bonding procedure , the samples were stored in distilled water for four weeks .
all samples were thermocycled for 500 cycles between 5c and 55c with 30 seconds of dwell time and 10 seconds of transfer time .
following thermocycling , root apices of the teeth were sealed using sticky wax ; the entire teeth surfaces were coated with nail polish except for 1 mm rim of tooth structure around the bracket ( fig .
teeth sealed with wax bonding procedure of study groups 
 the teeth were stored in 2% methylene blue solution for 24 hours at room temperature .
they were then cleaned by a toothbrush under running water , dried by air ; and mounted in blocks containing auto - polymerizing acrylic resin .
four parallel longitudinal sections were made using low - speed diamond saw ( auccutom-50 , struers , denmark ) in buccolingual direction . 
 

dye penetration at the bracket / adhesive and enamel / adhesive interfaces was evaluated by a blinded tester under a stereomicroscope ( olympus szx7 , olympus optical , tokyo , japan ) at 16 magnification ( 
 figs . 
 2 
 and 
 3 
 ) . 
 
no micro leakage on none of the surfaces microleakage of enamel - adhesive surface according to arhun et al , [ 
 12 
 ] dye penetration at both incisal and gingival margins of the brackets at the bracket - adhesive and the adhesive - enamel interfaces was scored using a digital caliper ( shoka golf , tokyo , japan ) .
scoring was done according to the following criteria : 0 : no dye penetration at the bracket - adhesive or the adhesive - enamel interface ; 1 : dye penetration restricted to 1 mm at the bracket - adhesive or the adhesive - enamel interface ; 2 : dye penetration into the inner half ( 2 mm ) of the bracket - adhesive or adhesive - enamel interface ; 3 : dye penetration into 3 mm of the bracket - adhesive or adhesive - enamel interface . to determine the measurement error
inter and intra - examiner kappa scores of both observers were over 0.75 . in case of disagreement
the frequency of each score at the two sides ( occlusal and gingival ) and surfaces ( enamel - adhesive and enamel - bracket ) of each group was demonstrated in the frequency table .
descriptive table ( table 2 ) shows the microleakage of the study groups at occlusal and gingival margins in both interfaces of adhesive .
the microleakage score in all samples was higher at gingival margins of both adhesive interfaces compared to the occlusal margins ; although it was not statistically significant .
descriptive statistics and comparison of the microleakage scores between occlusal and gingival sides at the enamel - adhesive and adhesive - bracket interfaces n = number ; ns = non significant group 1=assure primer+assure adhesive ; group 2= saliva+assure primer+assure adhesive ; group 3=assure primer+saliva+assure past ; group 4= tmip+transbond plus .
group 5=saliva+tmip+transbond plus ; group 6= tmip+ saliva+transbond plus multiple comparison of the microleakage scores of all the six study groups is shown in table 3 . when comparing the microleakage score at the enamel / adhesive interface , samples contaminated with saliva showed greater microleakage than control groups and the highest microleakage score
was observed in g2 and g3 , which were bonded with assure and contaminated with saliva .
multiple comparisons of the microleakage scores among the groups for occlusal and gingival sides at the enamel - adhesive and adhesive - bracket interface group 1= assure primer+ assure adhesive ; group 2= saliva+ assure primer+ assure adhesive ; group 3= assure primer+ saliva+ assure past ; group 4= tmip+ transbond plus .
group 5= saliva+ tmip+ transbond plus ; group 6= tmip+ saliva+ transbond plus the results showed that the microleakage score at the adhesive - bracket interface was not significantly different between the groups .
comparison of the microleakage scores among the six study groups is shown in 
 table 3 
 . in all contaminated groups ,
microleakage score at the enamel - adhesive interface ( in both gingival and occlusal margins ) was significantly higher than that at the adhesive - bracket interface ( p < 0.01 ) .
in restorative dentistry , passage of fluids and molecules through the tooth - restoration interface is called microleakage , which can not be diagnosed clinically and may cause secondary caries and post - operative tooth hypersensitivity [ 
 13 
 ] . in orthodontics ,
microleakage is defined as the passage of liquids and particles through the tooth - adhesive and adhesive - bracket interfaces , which may cause enamel decalcification , corrosion , discoloration and decreased bond strength [ 
 3 
 ] .
white spot lesions were found in a quarter of patients treated with fixed orthodontic appliances [ 
 14 
 ] . water [ 
 15 
 , 
 16 
 ] and saliva [ 
 15 
 , 
 17 
 ] can significantly reduce the bond strength between bracket and enamel and are reported to be the most common reasons for bond failure . in an attempt to overcome this problem , hydrophilic bonding systems such as transbond plus color change and assure were introduced .
these products have been proven to be moisture resistant and can acceptably bond to saliva - contaminated enamel surfaces [ 
 10 
 , 
 11 
 ] . in the current study ,
the microleakage score of brackets bonded with the above - mentioned two hydrophilic bonding systems was measured in presence or absence of saliva contamination using dye penetration method [ 
 3 
 , 
 12 
 , 
 18 
 , 
 19 
 ] . to determine the measurement error , two observers assessed the samples .
the results showed that microleakage score at the gingival margin of all samples was higher than that at the occlusal margin , although it was not significant .
this finding is consistent with previous studies [ 
 12 
 , 
 18 
 , 
 20 
 ] .
[ 
 12 
 ] reported that the premolar teeth morphology is the main reason of this finding .
thicker adhesive on the gingival side , due to prominent buccal contour can cause higher microleakage score at this area . also , ramoglu et al .
[ 
 21 
 ] believed that thinner adhesive on the occlusal side was the reason for lower microleakage score at this side . in dry conditions ,
no significant differences were observe between microleakage scores of the two adhesive systems in the two sides and the two interfaces , which is similar to the results of previous studies .
arhun et al , [ 
 12 
 ] alkis et al , [ 
 18 
 ] and yagci et al .
[ 
 22 
 ] concluded that the amount of microleakage was not affected by the type of adhesive . in this study , all groups except for g1 and g4 ( control groups ) showed significantly higher microleakage at the enamel - adhesive interface at both occlusal and gingival sides compared with bracket - adhesive interfaces .
being closer to saliva at the enamel  adhesive interface rather than the bracket - adhesive interface might be a possible reason . in the current study , brackets bonded with both bonding systems , when contaminated immediately after etching , showed higher microleakage compared with those contaminated after priming although the difference was not statistically significant .
saliva contamination after etching causes the water and glycoproteins in the saliva to interfere with proper adhesion .
when surfaces are contaminated with saliva after application of primer and adhesive , saliva can affect the degree of conversion and bond strength ; because hydroxyethyl methacrylate ( hema ) molecules with their hydrophilic nature may retain water within the adhesive layer ; thus , they can no longer participate in chain growth during polymerization [ 
 23 
  
 26 
 ] .
the results showed that microleakage scores at the enamel - adhesive interface at both gingival and occlusal sides were lower for transbond plus / tmip adhesive system in wet conditions ( p<0.05 ) .
the reason for better performance of transbond plus adhesive in wet conditions may be its chemical properties .
tmip primer like other moisture resistant primers is produced by dissolving hydrophilic primers in ethanol or acetone . a reversible hydrolytic bond , beside micromechanical retention , is created by breaking and reforming of carboxylate salt complexes between ionized carboxyl groups of primer and calcium of enamel .
the polyethylene glycol dimethacrylate ( pegdma ) is one of the transbond plus color chang adhesive components , which might be another factor improving its resistance in wet environment .
the lower bisphenol a diglycidyl ether dimethacrylate ( bis - gma ) content ( > 2% ) in this adhesive , compared with traditional adhesives e.g. transbond xt ( 1020% ) , resulted in higher proportional content of pegdma that might enhance the infiltration of bis - gma adhesive into moistened enamel [ 
 27 
 ] .
thermocycling was performed to consider the unmatched coefficients of thermal expansion of adhesive material and tooth substance [ 
 3 
 , 
 12 
 ] .
thermocycling was not performed in some recent studies because it was assumed to be ineffective on microleakage [ 
 20 
 , 
 21 
 ] .
however , vicente et al . [ 
 28 
 ] reported that bovine incisors bonded with transbond xt adhesive and thermocycled showed significantly higher microleakage score at the enamel - adhesive interface . this might be an explanation for the higher microleakage scores found in this study compared to previous studies [ 
 20 
 , 
 21 
 ] . several studies
reported that there was a correlation between the microleakage score at the bracket - adhesive interface and bond strength [ 
 19 
 , 
 20 
 ] .
short - term evaluation of microleakage was a limitation of this study while in clinical conditions , brackets remain in the mouth for a long time ( about two years ) .
the small sample size was another limitation of the current study , which might affect the generalizability of the findings ; therefore , we recommend further clinical studies with larger sample sizes and longer evaluation periods to investigate the effect of saliva contamination during the bonding process on microleakage .
enamel - adhesive interface showed a higher microleakage score following saliva contamination compared to bracket - adhesive interface . in saliva contaminated groups ,
lower microleakage score was observed at the enamel - adhesive interface of transbond plus / timp compared to assure .
 objectives : this study aimed to evaluate the effects of saliva contamination on the metallic bracket microleakage bonded with two moisture - tolerant bonding systems.materials and methods : ninety freshly extracted premolar teeth were randomly divided into six groups of 15 with the following treatments : g1 ( control ) : after acid etching , assure primer and assure adhesive were applied to non - contaminated enamel surfaces . 
 g2 ( contaminated after etching ) : the etched enamel surface was exposed to saliva , then assure primer and assure adhesive were applied . 
 g3 ( contaminated after priming ) : saliva contamination was done after application of assure primer . 
 the exact same procedures were applied to groups g4 to g6 except that timp primer and transbond plus adhesive system were used.to measure the microleakage score , the teeth were stained with 2% methylene blue for 24 hours , sectioned and examined under a stereomicroscope at 16 magnification . 
 data analysis was performed using fisher s exact test.results:in dry conditions , assure and tmip were not significantly different in terms of microleakage scores . 
 all contaminated groups exhibited higher microleakage score at the enamel / adhesive interface compared to the bracket / adhesive interface ( p < 0.01 ) . 
 in wet conditions , assure groups showed higher microleakage at the enamel - adhesive interface compared to the tmip groups ( p<0.05 ) . at the bracket - adhesive interface , 
 the microleakage scores were not significantly different in saliva contaminated groups compared to the controls.conclusion:saliva contamination caused greater microleakage at the enamel - adhesive interface compared to the adhesive - bracket interface . 
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION