common failure of the root canal obturation process is because of the presence of gaps and porosities at the sealer / dentin interface . several studies have shown that filled root canals can allow the re - colonization of micro - organisms . the presence of micro - organisms and their products in the treated root canal can led to failure of the root canal treatment and the necessity of retreatment . the analysis of the dentin / sealer interface allows the determination of which materials and filling techniques could obturate the root canals with less gaps and voids . several microscopy techniques are currently used to evaluate the sealer / dentin interface , including stereomicroscopy , scanning electron microscopy ( sem ) , transmission electron microscopy , and confocal laser scanning microscopy ( clsm ) . in comparison to conventional sem , clsm has the advantage of providing detailed information about the presence and distribution of sealers or dental adhesives inside dentinal tubules in the total circumference of the root canal walls at relative low magnification as 100x through the use of fluorescent rhodamine marked sealers . placement and activation of a sealer into the root canal system should be done in a manner which is predictable and completely covers the dentin walls . accepted means of sealer placement include the use of endodontic files or reamers , lentulo spirals , gutta - percha cones , ultrasonic files , and counter - clockwise motion of rotary systems . very few studies have been done to evaluate the efficacy of different methods of sealer placement and activation effectively . mta fillapex is a sealer that is composed of mta and has excellent flow , radiopacity , easy handling , working time , and low solubility . because of its suitable physicochemical properties and excellent biocompatibility and bioactivity , hence , this study was planned to evaluate percentage and the depth of dentinal tubule sealer penetration of mta fillapex when agitated with three different techniques and to compare it with ah plus using clsm as the evaluative tool . sixty teeth with single canal and free of cracks , caries , resorption , calcification , previous endodontic treatment , and less than 10- root curvature were selected and stored in distilled water until use . for standardization of the root length ( 10 mm ) , the crowns were resected with a diamond disc in a slow speed straight handpiece under constant water cooling . the working length was determined by inserting # 15 k - file ( dentsply maillefer ) into the canal until it was just seen at the apical foramen and then 0.5 mm was subtracted from this length . the roots were instrumented by using the protaper universal root canal files ( dentsply maillefer ) in a sequential manner from s1 till f5 . canals were irrigated between files with 2 ml of 3% naocl ( sree rayalaseema alkalies and allies chemicals limited , india ) . to eliminate the smear layer 2 ml of 17% ethylenediaminetetraacetic acid ( edta ) ( ph 7.7 ) for 3 min was used , followed by a final rinse of 2 ml distilled water . the roots were randomly divided into three groups on the basis of sealer activation technique utilized : g1 : ultrasonic ( woodpecker dte - d5 ultrasonic scaler , china ) ; g2 : lentulo spiral ( dentsply , maillefer ) ; g3 : counter - clockwise motion ( x smart , densply , maillefer , ballaigues , switzerland ) . each group was further divided into two subgroups on the basis of type of sealer . subgroup a utilized ah plus sealer while subgroup m utilized mta fillapex sealer . ah plus sealer and mta fillapex sealer were mixed according to the manufacturer instructions and to allow analysis under the clsm , each sealer was labelled with rhodamine b ( mayor diagnostics , mumbai , india ) to an approximate concentration of 0.1%(by weight ) . group 1 ( g1 ) : the ultrasonic unit ( woodpecker dte - d5 ultrasonic scaler , china ) was used in endo mode with a satelec ultrasonic endodontic tip k15 sonofile ( dentsply tulsa ) for activation . the file was inserted 2 mm short of the working length inside the canal and was ultrasonically activated for 20 seconds . gutta - percha was compacted 1 mm below the canal orifice and the teeth were sealed with cavit ( 3 m , espe ) . in g1a ah plus sealer and in g1 m , mta fillapex sealer was used . group 2 ( g2 ) : a size 30 lentulo spiral ( dentsply , maillefer ) was selected that would not bind in the prepared canal and that would reach the prepared working length . the lentulo spiral was rotated at speed of 300 rpm keeping the instrument 2 mm from apex for 20 seconds . thereafter , the obturation of canal was conducted as described for g1 . in g2a ah plus sealer and in g2 m , mta fillapex sealer was used . group 3 ( g3 ) : f1 protaper file attached in handpiece of endodontic micromotor ( x - smart , dentsply tulsa ) was kept 2 mm short of apex and rotated at 300 rpm in reverse mode for 20 seconds . thereafter , the obturation of canal was conducted as described for g1 . in g3a ah plus sealer and in g3 m , mta fillapex sealer was used . the specimens were kept in an incubator at 37c and 100% humidity for 2 days . each root was sectioned at 90- to the long axis by using diamond disc removing two , 1 mm sections at 3 mm and 6 mm from the apex . coronal surface of each section was polished with sand paper ( politriz , arotec , cotia , sp , brazil ) . the dentin segments were examined on a confocal microscope ( olympus fluoview fv 1000 ) . the respective absorption and emission wave lengths for the rhodomine b were 540 nm and 590 nm . dentin samples were analyzed using the 10x lens . to calculate the percentage of sealer penetration around the root canal , first each image was imported into the iob software and the circumference of root canal measured using its ruler tool . next , areas along the canal walls in which the sealer penetrated into dentinal tubules were outlined and measured using the same method [ figure 1 ] . subsequently , the percentage of root canal sealer penetration in that section was established . to determine the maximum depth of penetration , the point of deepest penetration was measured from the canal wall to the maximum depth of penetration [ figure 2 ] . the effects of sealer agitation techniques and type of sealer on depth and percentage of sealer penetration were analyzed , by performing nonparametric kruskal - wallis for overall analysis , and a series of mann - whitney u tests for pairwise comparison using statistical package of social sciences ( spss ) statistics 20 software . confocal laser scanning microscopic photograph showing calculation of percentage of penetration of sealer in the dentinal tubules ( original magnification 10 ) confocal laser scanning microscopic photograph showing calculation of depth of penetration of sealer in the dentinal tubules ( original magnification 10 ) sixty teeth with single canal and free of cracks , caries , resorption , calcification , previous endodontic treatment , and less than 10- root curvature were selected and stored in distilled water until use . for standardization of the root length ( 10 mm ) , the crowns were resected with a diamond disc in a slow speed straight handpiece under constant water cooling . the working length was determined by inserting # 15 k - file ( dentsply maillefer ) into the canal until it was just seen at the apical foramen and then 0.5 mm was subtracted from this length . the roots were instrumented by using the protaper universal root canal files ( dentsply maillefer ) in a sequential manner from s1 till f5 . canals were irrigated between files with 2 ml of 3% naocl ( sree rayalaseema alkalies and allies chemicals limited , india ) . to eliminate the smear layer 2 ml of 17% ethylenediaminetetraacetic acid ( edta ) ( ph 7.7 ) for 3 min was used , followed by a final rinse of 2 ml distilled water . the roots were randomly divided into three groups on the basis of sealer activation technique utilized : g1 : ultrasonic ( woodpecker dte - d5 ultrasonic scaler , china ) ; g2 : lentulo spiral ( dentsply , maillefer ) ; g3 : counter - clockwise motion ( x smart , densply , maillefer , ballaigues , switzerland ) . each group was further divided into two subgroups on the basis of type of sealer . ah plus sealer and mta fillapex sealer were mixed according to the manufacturer instructions and to allow analysis under the clsm , each sealer was labelled with rhodamine b ( mayor diagnostics , mumbai , india ) to an approximate concentration of 0.1%(by weight ) . group 1 ( g1 ) : the ultrasonic unit ( woodpecker dte - d5 ultrasonic scaler , china ) was used in endo mode with a satelec ultrasonic endodontic tip k15 sonofile ( dentsply tulsa ) for activation . the file was inserted 2 mm short of the working length inside the canal and was ultrasonically activated for 20 seconds . canal was obturated with protaper f5 gutta - percha cone . gutta - percha was compacted 1 mm below the canal orifice and the teeth were sealed with cavit ( 3 m , espe ) . in g1a ah plus sealer and in g1 m , mta fillapex sealer was used . group 2 ( g2 ) : a size 30 lentulo spiral ( dentsply , maillefer ) was selected that would not bind in the prepared canal and that would reach the prepared working length . the lentulo spiral was rotated at speed of 300 rpm keeping the instrument 2 mm from apex for 20 seconds . thereafter , the obturation of canal was conducted as described for g1 . in g2a ah plus sealer and in g2 m , mta fillapex sealer was used . group 3 ( g3 ) : f1 protaper file attached in handpiece of endodontic micromotor ( x - smart , dentsply tulsa ) was kept 2 mm short of apex and rotated at 300 rpm in reverse mode for 20 seconds . thereafter , the obturation of canal was conducted as described for g1 . in g3a ah plus sealer and in g3 m , mta fillapex sealer was used . the specimens were kept in an incubator at 37c and 100% humidity for 2 days . each root was sectioned at 90- to the long axis by using diamond disc removing two , 1 mm sections at 3 mm and 6 mm from the apex . coronal surface of each section was polished with sand paper ( politriz , arotec , cotia , sp , brazil ) . the dentin segments were examined on a confocal microscope ( olympus fluoview fv 1000 ) . the respective absorption and emission wave lengths for the rhodomine b were 540 nm and 590 nm . dentin samples were analyzed using the 10x lens . to calculate the percentage of sealer penetration around the root canal , first each image was imported into the iob software and the circumference of root canal measured using its ruler tool . next , areas along the canal walls in which the sealer penetrated into dentinal tubules were outlined and measured using the same method [ figure 1 ] . subsequently , the percentage of root canal sealer penetration in that section was established . to determine the maximum depth of penetration , the point of deepest penetration was measured from the canal wall to the maximum depth of penetration [ figure 2 ] . the effects of sealer agitation techniques and type of sealer on depth and percentage of sealer penetration were analyzed , by performing nonparametric kruskal - wallis for overall analysis , and a series of mann - whitney u tests for pairwise comparison using statistical package of social sciences ( spss ) statistics 20 software . confocal laser scanning microscopic photograph showing calculation of percentage of penetration of sealer in the dentinal tubules ( original magnification 10 ) confocal laser scanning microscopic photograph showing calculation of depth of penetration of sealer in the dentinal tubules ( original magnification 10 ) the percentage and depth of sealer penetration of different groups and intergroup comparison are summarized in tables 1 and 2 , respectively . shows mean and standard deviation of depth and percentage of sealer penetration of various groups at 3 and 6 mm levels intergroup comparison of depth and percentage of sealer penetration at 6 and 3 mm levels mean percentage and depth of sealer penetration of mta fillapex and ah plus sealer was greatest when ultrasonics was used for agitation and statistically significant ( p < 0.001 ) than lentulo spiral and counter - clockwise agitation techniques . irrespective of method used for agitation , the percentage and depth of sealer penetration for mta fillapex was significantly greater ( p < 0.001 ) than ah plus . the percentage and depth of sealer penetration for mta fillapex and ah plus were greater and highly significant ( p < 0.001 ) at 6 mm than 3 mm . removal of smear layer and use of sealer to attain and impervious seal between the core material and root canal walls is considered an essential step of root canal treatment . sealers which can penetrate into the dentinal tubules exert bactericidal effect by having a closer contact with the residual bacteria within the tubules . in addition , sealer plugs inside the dentinal tubules provide a mechanical interlocking , thereby improving the retention of the filling material and reducing the microleakage along the root canal walls . thus , both percentage and depth of sealer penetration might influence the outcome or success rate of endodontic therapy . factors influencing sealer depth penetration in dentinal tubules are presence / absence of dentinal permeability ( the number and the diameter of tubules ) , root canal dimension , presence of water , and physical and chemical properties of the sealer . the compound effect of physical properties viz flow , surface tension , solubility , viscosity , chemical composition , and working and setting time may influence penetration . flow which is defined as the ability of a sealer to penetrate in irregularities , lateral canals , or dentinal tubules of the root canal system is one of the main physical factors to influence the tubular penetration . as most endodontic sealers are pseudoplastic , their flow increases with increase of shear rate . on comparison with conventional sealers patel dv et al . , and gharib sr et al . , found better depth of penetration of resin - based sealer in dentinal tubules . mta fillapex has excellent biocompatibility and bioactivity and displayed significantly higher ph up to 7 days period than ah plus . studies based on the physical properties reported significantly greater flow of mta fillapex than ah plus . therefore , this comparative study was conducted to evaluate and compare the quality of sealing of ah plus and mta fillapex . clinically , rapid insertion or activation of a pseudoplastic sealer into the canal would decrease viscosity and increase the flow of sealer . as very few studies have been conducted on the effect of sealer activation / placement and activation on sealing ability of root canal sealers therefore in the present study three activation techniques ( ultrasonics , lentulo spiral , and rotary counter - clockwise motion ) were chosen and the sealer distribution was analyzed . amount of sealer , extent of activating instrument , and time for activation were standardized to minimize the errors . all the analyzed activation techniques failed to show a consistent adaptation of both the sealers to the total circumference of the root canal walls . however , mta fillapex showed significantly better ( p < 0.001 ) percentage and depth of sealer penetration than ah plus with all the techniques of sealer activation . kuc , ia et al . , also reported greater mta fillapex sealer penetration than ah plus . zhou h et al . , and silva ej et al . , found mta fillapex significantly more flowable than ah plus and attributed difference in composition and smaller particle size of the sealer for this property . application of ultrasonics for activation led to significantly more ( p < 0.001 ) percentage and depth of both the sealers and is accordance to guimaraes bm et al . , the explanation for this is that the oscillating files in ultrasonics transmits the acoustic microstreaming energy and cause a greater depth of dentinal sealer penetration and coverage of root canal walls in the same manner as it promotes the penetration of irrigants in an area of anatomic complexities and the dentinal tubules . although activation of both the sealers with lentulo spiral resulted in more percentage and depth of penetration as compare to counter - clockwise rotation however , the difference was statistically insignificant ( p > 0.05 ) . this is one of the pioneer studies evaluating effect of methods of activation on mta fillapex so it was difficult to compare the results obtained with previous studies . lentulo spiral rotates and the spring portion pushes the sealer centrifugally while in counter - clockwise rotation with a file , the sealer tends to pool toward the tip of file but is not forced toward the walls . irrespective of technique of activation and type of sealer , the depth , and percentage of penetration of sealer was significantly better at the 6 mm level than 3 mm level . these findings are similar to other studies and could be because the number and diameter of dentinal tubules decreases on descending apically in the root canal and superior removal of smear layer . in addition , the apical dentin is irregular in direction and density ; even some areas are devoid of dentinal tubules . under the parameters of present study , it can be concluded that mta fillapex sealer exhibited better percentage and depth of penetration in the radicular dentinal tubules than ah plus . ultrasonic activation of pseudoplastic sealers can significantly increase its percentage and depth of penetration and level of root canal can affect both , percentage , and depth of penetration of sealers in the dentinal tubules .
aim : to compare the effect of three root canal sealer activation techniques on percentage and depth of sealer penetration of mta fillapex and ah plus sealers.materials and methods : sixty teeth prepared till f5 protaper size were divided into three equal groups on the basis of sealer activation technique ( g1 : ultrasonics , g2 : lentulo spiral , and g3 : counter - clockwise rotary motion ) . 
 each group was further divided into two equal subgroups on the basis of type of sealer used : ah plus ( denstply , konstanz , germany ) or mta fillapex ( angelus , londrina , pr , brazil ) and obturated with gutta - percha . 
 horizontal sections at 3 and 6 mm from the apex were obtained and the percentage and depth of penetration of sealers into dentinal tubules were measured using confocal laser scanning microscopy ( clsm ) . 
 statistical analysis was performed utilizing kruskal - wallis and mann - whitney u tests with a significance level of 5%.results : g1 showed significantly ( p < 0.001 ) high percentage and depth of sealer penetration than g2 and g3 while the difference was insignificant ( p > 0.05 ) between g2 and g3.conclusion:percentage and depth of sealer penetration are influenced by the type of sealer used sealer activation technique and by the root canal level . 
 ultrasonic method of sealer activation and mta fillapex showed the best results .