the major aim of root canal therapy is to prevent and treat periradicular inflammation 
 by eliminating microorganisms from the root canal system .
the methods commonly used for 
 this purpose include root canal preparation using different instruments and irrigants , 
 adequate filling , and coronal restoration .
chemical irrigants are essential for successful debridement of root canals during 
 cleaning and shaping procedures . 

they are used during chemo - mechanical procedures not only as antimicrobial agents , but 
 also to lubricate the dentinal walls , flush out debris and dissolve organic and 
 inorganic components of the smear layer , thus cleaning the dentin surface .
different irrigants have been proposed and used , including : 5.25% 
 sodium hypochlorite , 2% chlorhexidine , 17% edta , 10% citric acid and 37% phosphoric acid 
 solution .
chlorhexidine has been used as irrigant during root canal therapy because of its 
 antibacterial effects , substantivity , and relative absence of cytotoxicity , even though 
 this solution is unable to dissolve the tissue .
additionally , chlorhexidine has been 
 suggested as a final irrigant . 
 regarding its use as final irrigant , a final flush with 2% chlorhexidine favors the 
 wettability of ah plus and real seal se sealers on the dentin surface .
furthermore , it 
 was verified that the bond strength of activ gp , a glass ionomer based system , was 
 improved by using 2% chlorhexidine in the final irrigation after 17% edta .
the development and maintenance of the sealing of the root canal system is the key to 
 the success of root - canal treatment .
the resin - based adhesive material has the potential 
 to reduce the microleakage of the root canal because of its adhesive properties and 
 penetration into dentinal walls . 

moreover , the irrigation protocols may have an influence on the adhesiveness of 
 resin - based sealers to root dentin .
a variety of experimental models are used to detect and measure any leakage along 
 endodontic fillings , such as dye penetration , clearing of the teeth , radioisotope tests , 
 bacterial penetration , electrochemical tests , fluid filtration , and glucose penetration 
 model .
the aim of the present study was to evaluate the effect of different irrigant protocols 
 on coronal bacterial microleakage of gutta - percha / ah plus and resilon / real seal 
 self - etch systems .
one hundred ninety single - rooted pre - molars with straight roots , mature root apices 
 and similar anatomical characteristics were used in this study .
all instruments used 
 in the root canal preparation were sterilized previously to the procedure .
the teeth 
 were positioned on a metallic apparatus that allowed for all procedures to be carried 
 out without manual contact with the roots .
a size 10 k - file ( dentsply maillefer , petrpolis , rio de 
 janeiro , brazil ) was used to verify the patency of the canals and to determine the 
 total length of the root canal , i.e. the work length .
next , the foramina were standardized by using 
 a size 20 k - file and root canals were shaped by using mtwo niti rotary system ( vdw , 
 mnich , bavaria , germany ) .
the sequence employed was the following : 10/.04 , 15/.05 , 
 20/.06 , 25/.06 , 30/.05 , 35/.04 , 40/.04 , and 25/.07 .
the teeth were divided into 
 groups of ten according to the irrigation regimen ( figure 1 ) and root canal filling .
protocols for irrigation before the insertion of each file , 2% chlorhexidine ( chx ) gel ( drogal , piracicaba , 
 so paulo , brazil ) or 5.25% sodium hypochlorite ( naocl ) ( drogal , piracicaba , so 
 paulo , brazil ) were used as chemical - auxiliary substance .
once the preparation was 
 finished , 10 ml of distilled water ( dw ) was used to remove the chemical - auxiliary 
 substance .
next , 17% edta or 37% phosphoric acid solution ( drogal , piracicaba , so 
 paulo , brazil ) was used for 3 minutes to remove the smear layer , with changes every 1 
 minute ( 1 ml per minute ) .
finally , 2% chlorhexidine solution ( drogal , piracicaba , so paulo , brazil ) 
 was used for final flush . during the chemo - mechanical preparation ,
all teeth had 
 their apices sealed with utility wax ( technew , rio de janeiro , rio de janeiro , 
 brazil ) to prevent flow through them .
the root canals were dried with sterilized medium - sized paper points ( endopoints , 
 paraba do sul , rio de janeiro , brazil ) .
groups 1 to 9 had the canals filled with 
 gutta - percha cones ( odous , belo horizonte , minas gerais , brazil ) associated with ah 
 plus sealer ( dentsply , petrpolis , rio de janeiro , brazil ) , whereas groups 10 to 18 
 had the canals filled with resilon associated with real seal se ( sybronendo , orange , 
 california , usa ) .
the cones packages were opened and they were used immediately , and 
 the sealers were manipulated in sterile plates according to the manufacturer 's 
 recommendations . a system - b endodontic heat source unit ( sybronendo , orange , california , usa ) was used 
 to down - pack and obtura system ( j morita , so paulo , so paulo , brazil ) to backfill . 

subsequently , teeth were 
 radiographed mesiodistally and buccolingually to assess the quality of the 
 filling .
all roots were kept on gauzes at 37c and 100% humidity for 2 weeks before leakage 
 measurement in order to allow the materials to set properly .
next , the external root surface of all specimens was sealed with two layers of red 
 nail varnish ( revlon , new york , new york , usa ) , except the last 1 mm of the apex .
glass 
 vials with rubber stoppers were adjusted for use . by using a shear ,
a hole was made 
 at the center of each rubber stopper ( figure 
 2b ) , through which each tooth was inserted under pressure up to the 
 cementoenamel junction , so that its crown was outside the vial and its root inside 
 ( figure 2c ) .
cyanoacrylate glue ( cg ) was 
 applied at the interface between tooth and stopper for sealing .
cylinders prepared from 10 ml 
 plastic syringes were adapted to the outer surface of the stoppers to create a 
 chamber around the crown of the tooth ( figure 
 2d ) .
again , cg was used at the interface between syringes and stoppers , 
 followed by a parafilm layer ( american national cantm , menasha , wisconsin , usa ) to 
 help in the sealing .
the syringe / stopper / tooth sets were submitted to sterilization 
 by gamma - rays ( embrarad , so paulo , so paulo , brazil ) .
, a hole 
 was made at the center of the rubber stopper ( b ) , through which the tooth was 
 inserted ( c ) .
a cylinder prepared from a 10 ml plastic syringe was adapted to 
 the outer surface of the stopper to create a chamber around the crown of the 
 tooth ( d ) .
the glass flask filled with bhi broth was connected to the 
 syringe / stopper / tooth set ( e ) .
turbidity in the bhi broth was evaluated ( f ) the sterilized glass flasks were then filled with sterile brain heart infusion broth 
 ( bhi ; oxoid , so paulo , so paulo , brazil ) so that a 2-mm length of root apex was 
 immersed in the broth .
cg and parafilm were used to seal the interface between 
 stopper and flask ( figure 2e ) . in all samples , 
 in order to ensure the efficiency of the seal , 2 ml of 1% sterile methylene blue dye 
 was placed into the tube until the coronal portion of the sample was 
 reached .
after the third day , the 
 methylene blue was removed with sterile distilled water by using a pipette . when a 
 green medium was observed the specimen was discarded .
the green color was due to the 
 blue dye association with the yellow medium .
for preparation of microbial medium , enterococcus faecalis ( atcc 29212 ) was grown on 
 bhi agar plates ( brain heart infusion agar ; oxoid , so paulo , so paulo , brazil ) and 
 supplemented with 5% sheep blood for 24 hours at 37c in co2 .
then , the 
 enterococcus faecalis was inoculated into tubes containing 5 ml 
 sterile bhi suspension , which were adjusted spectrophotometrically at 800 nm ( od800 ) 
 to a turbidity of 1.5x108 
 colony - forming units ( cfu)/ml . with the aid of a pipette
, 5 ml of the 
 suspensions were placed into the syringe apparatuses ( in the upper region , removing 
 the gauze stop ) , which were left at 37c for 90 days in co2 and checked 
 daily for turbidity in the bhi broth .
every 2 days , 3 ml of the suspension ( bhi+microorganisms ) were removed from the 
 chamber and replaced by 3 ml of bhi to avoid saturation and to confirm the growth of 
 enterococcus faecalis .
positive 
 cultures were confirmed by using gram staining ( gram - positive ) , colony morphology on 
 blood agar plates ( cocci ) and biochemical identification kits ( rapid i d
32 strep , 
 biomrieux sa , marcyl'etoile , charbonnieres - les - bains , france ) .
ten samples were used as positive ( n=5 ) and negative ( n=5 ) controls .
the positive 
 controls consisted of instrumented teeth without obturation , while negative controls 
 consisted of sound teeth , both with no contamination .
the results were analyzed with kaplan - meier survival test , kruskal - wallis and 
 mann - whitney tests ( p<0.05 ) .
one hundred ninety single - rooted pre - molars with straight roots , mature root apices 
 and similar anatomical characteristics were used in this study .
all instruments used 
 in the root canal preparation were sterilized previously to the procedure .
the teeth 
 were positioned on a metallic apparatus that allowed for all procedures to be carried 
 out without manual contact with the roots .
a size 10 k - file ( dentsply maillefer , petrpolis , rio de 
 janeiro , brazil ) was used to verify the patency of the canals and to determine the 
 total length of the root canal , i.e. the work length .
next , the foramina were standardized by using 
 a size 20 k - file and root canals were shaped by using mtwo niti rotary system ( vdw , 
 mnich , bavaria , germany ) .
the sequence employed was the following : 10/.04 , 15/.05 , 
 20/.06 , 25/.06 , 30/.05 , 35/.04 , 40/.04 , and 25/.07 .
the teeth were divided into 
 groups of ten according to the irrigation regimen ( figure 1 ) and root canal filling .
protocols for irrigation before the insertion of each file , 2% chlorhexidine ( chx ) gel ( drogal , piracicaba , 
 so paulo , brazil ) or 5.25% sodium hypochlorite ( naocl ) ( drogal , piracicaba , so 
 paulo , brazil ) were used as chemical - auxiliary substance .
once the preparation was 
 finished , 10 ml of distilled water ( dw ) was used to remove the chemical - auxiliary 
 substance .
next , 17% edta or 37% phosphoric acid solution ( drogal , piracicaba , so 
 paulo , brazil ) was used for 3 minutes to remove the smear layer , with changes every 1 
 minute ( 1 ml per minute ) .
finally , 2% chlorhexidine solution ( drogal , piracicaba , so paulo , brazil ) 
 was used for final flush . during the chemo - mechanical preparation ,
all teeth had 
 their apices sealed with utility wax ( technew , rio de janeiro , rio de janeiro , 
 brazil ) to prevent flow through them .
the root canals were dried with sterilized medium - sized paper points ( endopoints , 
 paraba do sul , rio de janeiro , brazil ) .
groups 1 to 9 had the canals filled with 
 gutta - percha cones ( odous , belo horizonte , minas gerais , brazil ) associated with ah 
 plus sealer ( dentsply , petrpolis , rio de janeiro , brazil ) , whereas groups 10 to 18 
 had the canals filled with resilon associated with real seal se ( sybronendo , orange , 
 california , usa ) .
the cones packages were opened and they were used immediately , and 
 the sealers were manipulated in sterile plates according to the manufacturer 's 
 recommendations . a system - b endodontic heat source unit ( sybronendo , orange , california , usa ) was used 
 to down - pack and obtura system ( j morita , so paulo , so paulo , brazil ) to backfill . 

subsequently , teeth were 
 radiographed mesiodistally and buccolingually to assess the quality of the 
 filling .
all roots were kept on gauzes at 37c and 100% humidity for 2 weeks before leakage 
 measurement in order to allow the materials to set properly .
next , the external root surface of all specimens was sealed with two layers of red 
 nail varnish ( revlon , new york , new york , usa ) , except the last 1 mm of the apex .
glass 
 vials with rubber stoppers were adjusted for use . by using a shear ,
a hole was made 
 at the center of each rubber stopper ( figure 
 2b ) , through which each tooth was inserted under pressure up to the 
 cementoenamel junction , so that its crown was outside the vial and its root inside 
 ( figure 2c ) .
cyanoacrylate glue ( cg ) was 
 applied at the interface between tooth and stopper for sealing .
cylinders prepared from 10 ml 
 plastic syringes were adapted to the outer surface of the stoppers to create a 
 chamber around the crown of the tooth ( figure 
 2d ) .
again , cg was used at the interface between syringes and stoppers , 
 followed by a parafilm layer ( american national cantm , menasha , wisconsin , usa ) to 
 help in the sealing .
the syringe / stopper / tooth sets were submitted to sterilization 
 by gamma - rays ( embrarad , so paulo , so paulo , brazil ) .
the apparatus used to evaluate coronal leakage ( a ) . by using a shear ,
a hole 
 was made at the center of the rubber stopper ( b ) , through which the tooth was 
 inserted ( c ) .
a cylinder prepared from a 10 ml plastic syringe was adapted to 
 the outer surface of the stopper to create a chamber around the crown of the 
 tooth ( d ) .
the glass flask filled with bhi broth was connected to the 
 syringe / stopper / tooth set ( e ) .
turbidity in the bhi broth was evaluated ( f ) the sterilized glass flasks were then filled with sterile brain heart infusion broth 
 ( bhi ; oxoid , so paulo , so paulo , brazil ) so that a 2-mm length of root apex was 
 immersed in the broth .
cg and parafilm were used to seal the interface between 
 stopper and flask ( figure 2e ) . in all samples , 
 in order to ensure the efficiency of the seal , 2 ml of 1% sterile methylene blue dye 
 was placed into the tube until the coronal portion of the sample was 
 reached .
after the third day , the 
 methylene blue was removed with sterile distilled water by using a pipette . when a 
 green medium was observed the specimen was discarded .
the green color was due to the 
 blue dye association with the yellow medium .
for preparation of microbial medium , enterococcus faecalis ( atcc 29212 ) was grown on 
 bhi agar plates ( brain heart infusion agar ; oxoid , so paulo , so paulo , brazil ) and 
 supplemented with 5% sheep blood for 24 hours at 37c in co2 .
then , the 
 enterococcus faecalis was inoculated into tubes containing 5 ml 
 sterile bhi suspension , which were adjusted spectrophotometrically at 800 nm ( od800 ) 
 to a turbidity of 1.5x108 
 colony - forming units ( cfu)/ml . with the aid of a pipette
, 5 ml of the 
 suspensions were placed into the syringe apparatuses ( in the upper region , removing 
 the gauze stop ) , which were left at 37c for 90 days in co2 and checked 
 daily for turbidity in the bhi broth .
every 2 days , 3 ml of the suspension ( bhi+microorganisms ) were removed from the 
 chamber and replaced by 3 ml of bhi to avoid saturation and to confirm the growth of 
 enterococcus faecalis .
positive 
 cultures were confirmed by using gram staining ( gram - positive ) , colony morphology on 
 blood agar plates ( cocci ) and biochemical identification kits ( rapid i d 32 strep , 
 biomrieux sa , marcyl'etoile , charbonnieres - les - bains , france ) .
ten samples were used as positive ( n=5 ) and negative ( n=5 ) controls .
the positive 
 controls consisted of instrumented teeth without obturation , while negative controls 
 consisted of sound teeth , both with no contamination . the results were analyzed with kaplan - meier survival test , kruskal - wallis and 
 mann - whitney tests ( p<0.05 ) .
no differences were found following the use of chlorhexidine or naocl associated with 
 edta or phosphoric acid for smear layer removal .
however , it was clearly observed that 
 groups receiving a final flush with chlorhexidine showed a lower number of contaminated 
 samples .
graph showing the number of contaminated samples according to the irrigation 
 protocol * different letters indicate statistically significant values ( p<0.05 ) regarding the root canal filling system ( gutta percha / ah plus and resilon / real seal se ) , 
 there was no statistically significant differences in relation to the coronal 
 microleakage .
statistic analysis of the contamination days revealed 
 difference in the groups receiving the final flush with chlorhexidine .
chlorhexidine 
 groups started to contaminate only in the 6week , while in the others the 
 microbial growth was verified in the 1or 2week .
the control 
 apparatuses showed broth turbidity within 1 day of incubation in all samples , whereas no 
 microbial growth was found in the negative control throughout the experiment .
number of contaminated samples per week dw- distilled water ; chx- chlorhexidine ; gpah- gutta - percha / ah plus ; rrs- 
 resilon / real seal se
leakage of the root canal has been defined as the passage of bacteria , fluids , and 
 chemical substances between the dentinal wall and the root canal filling material , and 
 results from the presence of space at the interface of the filling material and the root 
 canal wall .
this space can result from deficient adaptation of the filling material to 
 the root dentin , solubility of the sealer , or sealer expansion or shrinkage .
there are 2 
 possibilities of leakage : at the interface between the main filling material and sealer , 
 or between the sealer and root canal wall . in the present study ,
this methodology is 
 reproducible and has clinical relevance , presenting reliable data and simulating 
 clinical conditions .
this 
 methodology allows the observation of the exact day of the sample contamination , showed 
 by the broth turbidity .
the 37% phosphoric acid was evaluated in comparison with edta because this solution is 
 effective for smear layer removal , showing better results than edta in the apical third 
 during 3 minutes . additionally , the 
 protocols associating naocl with phosphoric acid showed higher bond strength values when 
 compared with naocl associated with edta .
although phosphoric acid had showed better performance in removing 
 the smear layer from the apical third , it did not influence the results of coronal leakage .
the 2% chlorhexidine , a cationic bisbiguanide , was used here for the final flush after 
 smear layer removal .
this substance has a broad - spectrum mmp - inhibitory effect that 
 improves the integrity of the hybrid layer6 
 and the resin - dentin bond stability .
additionally , the use of chx increases the wettability of 
 endodontic sealers on dentin , which 
 can be explained by the presence of surface surfactant in chx , increasing the surface 
 energy and promoting higher wetting ability to dentin .
our results showed that a final 
 flush with chlorhexidine significantly reduced the coronal microleakage , when compared 
 to the other experimental groups .
it might be explained by the fact that chlorhexidine 
 is adsorbed onto dentin and prevent microbial colonization , increasing the time required for recontamination of 
 filled rootup to 12 weeks due to 
 its substantivity . regarding the effect of a final flush with chlorhexidine on adhesion
, previous studies 
 showed that this solution did not affect the bond strength of resin - based 
 sealersand improved the adhesion of hydrophilic bonded 
 materials such as activ gp and epiphany .
naocl was not used as a final irrigant because a previous study showed that this 
 solution decreased the bond strength between epoxy resin and dentin and increased the 
 leakage . regarding the root canal filling system ( gutta percha / ah plus and resilon/ real seal 
 se )
, there was no statistically significant differences in relation to the coronal 
 microleakage , agreeing with previous findings
. however , some studies reported that resilon / epiphany sealer was 
 more efficient than gutta - percha / ah plus , whereas others found 
 the opposite .
in conclusion , a final flush with 2% chlorhexidine after smear layer removal reduces 
 coronal microleakage of teeth filled with gutta - percha / ah plus or resilon / real seal 
 self - etch . the bacterial leakage methodology used made possible the verification of this 
 behavior .
 the development and maintenance of the sealing of the root canal system is the key to 
 the success of root canal treatment . 
 the resin - based adhesive material has the 
 potential to reduce the microleakage of the root canal because of its adhesive 
 properties and penetration into dentinal walls . 
 moreover , the irrigation protocols 
 may have an influence on the adhesiveness of resin - based sealers to root dentin.objectivethe objective of the present study was to evaluate the effect of different 
 irrigant protocols on coronal bacterial microleakage of gutta - percha / ah plus and 
 resilon / real seal self - etch systems.material and methodsone hundred ninety pre - molars were used . 
 the teeth were divided into 18 
 experimental groups according to the irrigation protocols and filling materials 
 used . 
 the protocols used were : distilled water ; sodium hypochlorite ( naocl)+edta ; 
 naocl+h3po4 ; naocl+edta+chlorhexidine ( chx ) ; 
 naocl+h3po4+chx ; chx+edta ; chx+ 
 h3po4 ; chx+edta+chx and 
 chx+h3po4+chx . 
 gutta - percha / ah plus or resilon / real seal se 
 were used as root - filling materials . 
 the coronal microleakage was evaluated for 90 
 days against enterococcus faecalis . 
 data were statistically 
 analyzed using kaplan - meier survival test , kruskal - wallis and mann - whitney 
 tests.resultsno significant difference was verified in the groups using chlorhexidine or sodium 
 hypochlorite during the chemo - mechanical preparation followed by edta or 
 phosphoric acid for smear layer removal . 
 the same results were found for filling 
 materials . 
 however , the statistical analyses revealed that a final flush with 2% 
 chlorhexidine reduced significantly the coronal microleakage.conclusiona final flush with 2% chlorhexidine after smear layer removal reduces coronal 
 microleakage of teeth filled with gutta - percha / ah plus or resilon / real seal 
 se . 
INTRODUCTION
MATERIAL AND METHODS
Sample preparation
Analysis of coronal bacterial microleakage
RESULTS
DISCUSSION
CONCLUSION