apical periodontitis is an infection of the area around the root of a tooth , usually caused by bacteria .
although bacterial infection can be substantially reduced by standard intracanal procedures such as intracanal medication and root canal treatment , it is very difficult to render the root canal free of bacteria .
this is because bacteria are located in inaccessible areas such as deep inside the dentinal tubules and lateral canals and it is difficult for any intracanal medication to reach these locations .
moreover , bacteria may survive and re - colonize in the areas around the root canal whenever there is opportunity and this may become a primary source of persistent infection .
bacteria are commonly found within dentinal tubules of clinically infected canals . among these bacteria ,
enterococcus faecalis is of interest because it is the most frequently detected species in root - filled teeth with persistent lesions .
some possible factors facilitating the long - term survival of e. faecalis in the root canal system are its ability to invade dentinal tubules , where it can survive for a prolonged period under adverse conditions such as starvation , high ph of calcium hydroxide medication and adhesion of e. faecalis to collagen .
angiotensin - converting enzyme ( ace ) and a serine protease ( spr ) are the collagen binding proteins produced by e. faecalis . with the help of these proteins ,
ace promotes the binding of e. faecalis to type i collagen and in vitro ace gene expression at 37c was enhanced in the presence of collagen . in this study ,
the interaction of e. faecalis with root cementum and its role in persistent infection was investigated .
this in vitro study was conducted in the department of conservative dentistry and endodontics . teeth sterilization ( gamma irradiation at 25 kgy ) was performed at microtol , bangalore .
data was obtained using an inverted confocal laser scanning microscope ( clsm ) ( zeiss lsm 510 meta .
gmbh , mannheim , germany ) at the indian institute of science , bangalore . a total of 60 human single - rooted teeth recently extracted for orthodontic reasons were collected for the study .
, caries - free teeth were examined under 20 microscope to rule out any cracks , caries , fractures or craze lines and radiographed to confirm the presence of a single canal .
teeth that had already undergone root canal treatment or teeth with more than one canal , immature root apices , root caries , restorations , fractures or craze lines , thin curved roots and calcified canals were excluded from the study .
the teeth were cleaned off soft - tissues , calculus and stains using sharp hand scalers and thoroughly washed under running tap water to remove any tissue remnants sticking to the tooth surface .
all the 60 specimens were randomly divided into three experimental groups as follows : group i ( n = 20 ) : ( control group ) intact teeth with no access cavity preparation and sealing of the root apex was done using varnish .
group ii ( n = 20 ) : access opening was done to gain access to the root canal .
group iii ( n = 20 ) : 1 mm of the root apex was exposed to lactic acid ( organic acid ) at ph below 5.5 to mimic apical demineralization .
apical root cementum was roughened using diamond point to mimic apical resorption that is seen in apical periodontitis cases , followed by access opening to gain access to the root canal .
for the samples in group i ( control group ) , no access preparation was done and apical 1 - 2 mm of the teeth was sealed with three coats of varnish followed by gamma irradiation of all the samples to eradicate any bacteria that was previously present . for the specimens in groups
the teeth were then subjected to gamma irradiation , followed by inoculation with the e. faecalis broth within the root canal with the help of a micropipette .
simultaneously , apical one - third of the teeth were submerged in the broth for all the teeth samples and incubated for 8 weeks to allow bacterial growth with alternate day refreshment .
streptomycin - resistant strains of e. faecalis ( atcc 29212 ) were cultured in tryptone soya bean agar broth prepared by mixing 1.8 g powder in 60 ml of distilled water .
the e. faecalis strain was inoculated in the broth and placed in an incubator to allow the bacteria to grow at 37c for 24 - 48 h. gram staining was done to confirm bacterial growth .
the e. faecalis broth was inoculated within the root canal of the teeth samples with a micropipette .
furthermore , apical one - third of the teeth were submerged in the broth to mimic primary infection .
after 8 weeks of culturing , the specimens of groups ii and iii were subjected to biomechanical preparation followed by obturation up to the working length ( root zx ii , j. morita , japan ) .
the teeth were instrumented using protaper ni - ti rotary instrument system in a contra - angle gear reduction handpiece ( x - smart dentsply ) and finally obturated with gutta - percha ( single - cone technique ) using ah plus sealer .
after coronal seal , apical one - third of all the samples were again immersed in the e. faecalis broth for 8 weeks with alternate day refreshment to show secondary infection . after the incubation period of 8 weeks , all the samples were washed using 1 ml phosphate buffered saline to remove non - adherent bacteria .
a vertical groove was made on buccolingual surface starting from occluso - apical of the all teeth samples using a tapered fissure diamond point . then with the help of a chisel ,
the teeth were stained with a fluorescent dye to observe under an inverted clsm ( zeiss lsm 510 meta .
the teeth were stained with 50 l fluorescein diacetate ( fda , sigma , st .
louis , mo ) and 50 l of propidium iodide ( pi , sigma ) .
, the dye crosses the cell membrane and gets metabolized by intracellular esterases and converted to fluorescein ( green ) so the viable cells appears green in color .
pi is a non - cell permeable , red fluorescent dye , which adheres to ruptured cell membranes so the dead bacteria appear red in color .
single - rooted , caries - free teeth were examined under 20 microscope to rule out any cracks , caries , fractures or craze lines and radiographed to confirm the presence of a single canal .
teeth that had already undergone root canal treatment or teeth with more than one canal , immature root apices , root caries , restorations , fractures or craze lines , thin curved roots and calcified canals were excluded from the study .
the teeth were cleaned off soft - tissues , calculus and stains using sharp hand scalers and thoroughly washed under running tap water to remove any tissue remnants sticking to the tooth surface .
all the 60 specimens were randomly divided into three experimental groups as follows : group i ( n = 20 ) : ( control group ) intact teeth with no access cavity preparation and sealing of the root apex was done using varnish .
( n = 20 ) : access opening was done to gain access to the root canal .
( n = 20 ) : 1 mm of the root apex was exposed to lactic acid ( organic acid ) at ph below 5.5 to mimic apical demineralization .
apical root cementum was roughened using diamond point to mimic apical resorption that is seen in apical periodontitis cases , followed by access opening to gain access to the root canal .
for the samples in group i ( control group ) , no access preparation was done and apical 1 - 2 mm of the teeth was sealed with three coats of varnish followed by gamma irradiation of all the samples to eradicate any bacteria that was previously present . for the specimens in groups ii and iii , access opening and canal debridement were done .
the teeth were then subjected to gamma irradiation , followed by inoculation with the e. faecalis broth within the root canal with the help of a micropipette .
simultaneously , apical one - third of the teeth were submerged in the broth for all the teeth samples and incubated for 8 weeks to allow bacterial growth with alternate day refreshment .
streptomycin - resistant strains of e. faecalis ( atcc 29212 ) were cultured in tryptone soya bean agar broth prepared by mixing 1.8 g powder in 60 ml of distilled water .
the e. faecalis strain was inoculated in the broth and placed in an incubator to allow the bacteria to grow at 37c for 24 - 48 h. gram staining was done to confirm bacterial growth .
the e. faecalis broth was inoculated within the root canal of the teeth samples with a micropipette .
furthermore , apical one - third of the teeth were submerged in the broth to mimic primary infection .
after 8 weeks of culturing , the specimens of groups ii and iii were subjected to biomechanical preparation followed by obturation up to the working length ( root zx ii , j. morita , japan ) .
the teeth were instrumented using protaper ni - ti rotary instrument system in a contra - angle gear reduction handpiece ( x - smart dentsply ) and finally obturated with gutta - percha ( single - cone technique ) using ah plus sealer .
after coronal seal , apical one - third of all the samples were again immersed in the e. faecalis broth for 8 weeks with alternate day refreshment to show secondary infection . after the incubation period of 8 weeks , all the samples were washed using 1 ml phosphate buffered saline to remove non - adherent bacteria .
a vertical groove was made on buccolingual surface starting from occluso - apical of the all teeth samples using a tapered fissure diamond point .
then with the help of a chisel , each tooth was split into two halves . after coding the teeth samples ,
the teeth were stained with a fluorescent dye to observe under an inverted clsm ( zeiss lsm 510 meta .
the teeth were stained with 50 l fluorescein diacetate ( fda , sigma , st .
louis , mo ) and 50 l of propidium iodide ( pi , sigma ) .
, the dye crosses the cell membrane and gets metabolized by intracellular esterases and converted to fluorescein ( green ) so the viable cells appears green in color .
pi is a non - cell permeable , red fluorescent dye , which adheres to ruptured cell membranes so the dead bacteria appear red in color .
for adhesion , we used scoring criteria of 0 ( for no adhesion ) and 1 ( for adhesion ) . for penetration measured in m , we used the depth - measuring tool from the clsm software .
accordingly , results were subjected to statistical analysis using median test , anova and student 's t - test .
figure 1 shows live bacteria ( green color ) and dead bacteria ( red color ) .
i ( control group ) no access cavity was prepared and at the same time apical one - third of the teeth were sealed with varnish .
results showed no adhesion [ table 1 ] , [ figure 1a ] and no penetration of e. faecalis into the root cementum in any of the samples [ table 2 ] , [ figure 2a ] . in group
ii few samples showed adhesion , mean of adhesion is 0.55 [ table 1 ] , [ figure 1b ] but no penetration was seen in any of the samples [ table 2 ] , [ figure 2b ] .
this means that if an intervention like root canal treatment was done at early stage of infection when no apical changes like demineralization or resorption had taken place , there would be lesser chances of e. faecalis penetration and persistent infection or re - infection . in group iii , apical one - third of the teeth
were exposed to acid and apical cementum was roughed to mimic apical periodontitis . in this group ,
all the samples showed adhesion [ table 1 ] , [ figure 1c ] and highest values of penetration was up to 160 m deep in root cementum [ table 2 ] , [ figure 2c ] .
this means that delay in treatment leads to changes in apical environment such as apical demineralization and apical resorption , which helps e. faecalis to penetrate deep into cementum and in favorable conditions chances of persistent infection or re - infection also increases .
a comparison of values shows high significant difference ( p < 0.01 ) between groups i and iii and groups ii and iii and significant difference ( p < 0.05 ) between groups
i and ii [ tables 1 and 2 ] . mean and sd of adhesion for three groups . comparisons between groups with median test ( a ) confocal image showing group i ( control group ) , absence of enterococcus faecalis .
red color shows dead e. faecalis and green shows live e. faecalis under confocal laser scanning microscope ( clsm ) .
red color shows dead e. faecalis and green shows live e. faecalis under clsm mean and sd of penetration for three groups .
f value calculated by one way anova and comparisons between groups with student 's t - test ( a ) depth measuring confocal image showing , group i ( control group ) absence of enterococcus faecalis .
( b ) group ii samples showing adhesion of e. faecalis up to 1 m deep under confocal laser scanning microscope ( clsm ) .
( c ) group iii samples showed presence of e. faecalis up to 160 m deep in root cementum under clsm
in this study , e. faecalis was chosen as the test organism because of its ability to penetrate the root dentin in vitro and it is found up to 90% in persistent infection .
e. faecalis has unique properties such as production of ace and spr that are collagen binding proteins , which help e. faecalis to adhere strongly to mainly type i and iv collagen present in the root dentin .
e. faecalis is a very virulent microorganism , which can survive in alkaline ph and during long starvation periods and then becomes viable in the presence of serum .
e. faecalis can survive long periods without any nutrient availability because it can derive nutrients from hyaluronan , which is converted by enzyme hyaluronidase and also derive energy sources from dentinal fluid even in a well - sealed root canal system . under stress ,
lipoteichoic acids protect e. faecalis against lethal conditions , cytolysin , as-48 and bacteriocin , which inhibits other bacterial growth .
cytolysin destroys cells such as erythrocytes , pnm cells , macrophages and kills gram - positive microorganisms . due to this effect
the adhesion of e. faecalis to type i and iv collagen is the basis of the present study because apical one - third of the root is made of cellular cementum primarily composed of type iv collagen .
adhesion is the first step in colonization and our study confirms adhesion and invasion by e. faecalis up to 160 m into the root cementum [ figure 2c ] .
however , a previous study showed e. faecalis penetration only up to 150 m deep into the root dentin .
deeper penetration of e. faecalis in our study is due to the change in apical environment such as demineralization and apical resorption . in group
i , there was no invasion or adhesion of e. faecalis as it is a control group . in group ii
, only few samples showed adhesion , mean of adhesion is 0.55 [ table 1 ] and no samples showed penetration . on the other hand in group iii , all samples showed adhesion [ table 1 ] and highest penetration into root cementum up to 160 m [ table 2 ] .
this shows that if early treatment is done after primary infection as in group ii , there are less chances of penetration of e. faecalis and re - infection or persistent infection .
whereas in group iii , there was delay in treatment at early stages of infection , leading to change in apical environment like demineralization of root cementum and root cementum resorption .
these apical changes helped e. faecalis to penetrate deep into the root cementum , thereby increasing the chances of persistent infection or re - infection .
in our study , we cultured for 8 weeks twice because 18 weeks showed primary infection followed by biomechanical preparation and obturation giving the impression of normal root canal treatment done to subside primary infection .
group ii showed only adhesion but no penetration [ figures 1b and 2b ] , whereas group iii showed deeper penetration .
this means irrespective of the primary or secondary infection , what matters most is the apical changes in environment like demineralization or apical resorption , which help e. faecalis to penetrate deep into root cementum and is the cause for re - infection or persistent infection , as confirmed by the samples in group iii .
we used gamma irradiation to sterilize the teeth because it does not alter collagen characteristics of the teeth .
e. faecalis produces collagen binding protein , with the help of which it adheres strongly to collagen
. other methods of sterilization of teeth samples are by autoclaving , using hot air oven , etc .
the disadvantage of autoclave is that it collapses the collagen strands and use of hot air oven makes teeth dehydrated and more brittle . in our study , data was collected using a clsm as it has advantages over other methods such as histological samples , which can not distinguish between viable and dead bacteria .
scanning electron microscope has the disadvantage of multiple steps for sample preparation making it time consuming and also it can not differentiate between dead and viable bacteria .
fluorescence probe has the disadvantage that it can not distinguish between viable and dead bacteria and also it can not show distribution of bacteria .
the clsm ( zeiss lsm 510 meta gmbh , mannheim , germany ) analysis has advantage over other methods to visualize bacteria .
our study confirms that clsm can give a clear picture about viability and spatial distribution of bacteria .
fda is a non - fluorescent cell - permeable dye , which in viable cells crosses the cell membrane , gets metabolized by intracellular esterases and is converted to fluorescein ( green ) .
pi is non - cell permeable fluorescent dye , which gets adhered to ruptured cell membranes and dead cells appear red in color .
we used an organic acid ( lactic acid ) for demineralization of root cementum because the end product of sucrose metabolism is lactic acid .
demineralization and apical resorption at root cementum plays a significant role in penetration of e. faecalis into root cementum .
the severity of infection is directly proportional to depth of penetration of e. faecalis into root cementum .
the progress of the disease plays a critical role in the adhesion and penetration of e. faecalis to root cementum .
 aim : the aim of this study is to address the cause of persistent infection of root cementum by enterococcus faecalis.materials and methods : a sample of 60 human single - rooted teeth were divided into three groups . 
 group i ( control group ) had no access opening and one - third of the apical root cementum was sealed using varnish . 
 group ii had no preparation of teeth samples . in group iii , apical root cementum was exposed to organic acid and roughened using diamond point to mimic apical resorption . 
 after access opening in groups ii and iii , all teeth samples were sterilized using gamma irradiation ( 25 kgy ) . 
 e. faecalis broth was placed in the root canal and apical one - third of the tooth was immersed in the broth for 8 weeks with alternate day refreshment followed by biomechanical preparation , obturation and coronal seal . 
 apical one - third of all teeth samples were again immersed in the broth for 8 weeks with alternate day refreshment to mimic secondary infection . 
 the samples were observed under a confocal microscope after splitting the teeth into two halves.results:e . 
 faecalis penetrated 160 m deep into the root cementum in group iii samples and only showed adhesion in group ii samples.conclusion:penetration and survival of e. faecalis deep inside the cementum in extreme conditions could be the reason for persistent infection . 
INTRODUCTION
MATERIALS AND METHODS
Inclusion criteria
Exclusion criteria
Procedure
Culturing procedure
RESULTS
DISCUSSION
CONCLUSION