the cleaning and disinfection role of the irrigating solutions in the complex protocol of the endodontic therapy is essential for treatment success .
as mccomb and smith10 ( 1975 ) demonstrated the presence of a dentin layer attached to the root canal walls , the irrigating solutions should act on both organic and inorganic compounds . among the irrigants used for removal of inorganic compounds are the chelating agents and inorganic acids represented by ethylenediaminetetraacetic acid ( edta ) and citric acid , respectively7,10,11,13 .
the biocompatibility of citric acid and edta in the pulp and periapical tissues induces an inflammatory response of variable intensity and depth depending on the methodology used .
cotton and siegel5 ( 1978 ) and baumgartner , et al.3 observed no severe or irreversible damage on the human dental pulp .
sterrett , et al.12 ( 1993 ) and malheiros , et al.8 ( 2005 ) stated that the effects observed on the human dentin may vary according to the concentration and application time .
mcinnes - ledoux , et al.9 ( 1985 ) reported that , in monkeys , 1% citric acid showed severe effects on the pulpal tissues .
leonardo , et al.6 ( 1984 ) observed a good tolerance of the periapical tissues to edta on dog 's teeth , while zina , et al.15 ( 1981 ) using trisodium edta in dog 's teeth did not observe any damage on the healing process of the pulp stump and periapical tissues .
chan , et al.4 ( 1999 ) observed that in human pulp cell cultures , the toxicity of 0.5% and 1% citric acid is proportional to the concentration and exposure time , whereas other studies observed in rats that citric acid was less irritating than edta and ethyleneglycol - bis(2-aminoethoxy)-tetraacetic acid ( egta)14 .
amaral , et al.1 ( 2007 ) observed that both edta and citric acid showed cytotoxic effects on macrophages , though citric acid produced less cytotoxic .
malheiros , et al.8 ( 2005 ) demonstrated in cultured fibroblasts and in rats ( rattus novergicus ) in vivo , respectively , that , if applied at high concentrations , citric acid leads to a delay in cell growth and has an irritating effect in vivo .
based on the endodontic biologic principles , the purpose of this study was to evaluate , histopathologically , the periapical tissue response of dog 's teeth submitted to root canal therapy with two demineralizing chemical substances : 1% citric acid and 17% trisodium edta salt .
three different solutions were used in this study : 17% trisodium edta salt ( , ph 8.0 ; chemical lab .
ucpel , pelotas , rs , brazil ) , 1% citric acid ( ph 2.0 ; curativa pharmacy , pelotas , rs , brazil ) and saline ( texon co. , rs , brazil ) as the control .
six adult dogs from the central laboratory of ufpel were selected as the biological model of the study .
the experimental sample comprised 56 roots of mandibular molars ( first and second ; n= 13 ) and premolars ( first , second and third ; n= 22 ) .
the animals were sedated with an intravenous injection of 2% virbaxyl ( virbac co. , sp , brazil ) and then anesthetized with thiopental ( cristlia chemical products , sp , brazil ) . after oral asepsis with 0.12% chlorhexidine
access to the pulp chamber was gained with # 1012 , 1015 and 1016 diamond burs ( kg sorensen , so paulo , sp , brazil ) and the convenience shape was given with an endo z bur ( dentsply - maillefer , ballaigues , switzerland ) .
pulpectomy was performed with a hedstren file and the working length was determined 1 mm short of the radiographic apex with k - files ( dentsply - maillefer ) .
root canals were instrumented with four k - files ( dentsply - maillefer ) of greater sizes than that of file used for root canal length determination . during biomechanical preparation , the number of irrigations and volume of the irrigating solution were standardized in 16 ml for premolars and 32 ml for molars with the aid of disposable syringes and 303 , 304 and 305 hypodermic needles ( becton - dickinson , so paulo , sp , brazil ) . in all phases of biomechanical preparation , either one of the test or the control substances were used as auxiliary irrigant .
when instrumentation was completed , the root canal was filled with the test solution for 5 minutes to allow an intimate contact . after drying ,
the canal entrance was sealed with disinfected gutta - percha followed by composite prisma aph ( dentsply ind .
ltda , petrpolis , rj , brazil ) below the occlusion line . after 24 and 48 hours of the treatment
, the animals were euthanized and the teeth removed and fixated in 10% formalin solution .
thereafter , the teeth were decalcified by immersion in 1:1 formic acid and sodium citrate solution followed by 5% nitrate acid .
the specimens were embedded in fused paraffin histosec ( merck kgaa , darmstadt , germany ) , cut with a microtome at the apical foramen in serial 6-m - thick sections , which were stained with hematoxylin and eosin and mounted on glass slides .
the sections that best represented the area of pulp - periodontium communication were selected and examined with a light microscopy at 100 magnification .
the histologic analysis considered the occurrence of an acute inflammatory response , so the presence of swelling , vasodilatation and inflammatory cells was evaluated and the degree of inflammation was determined for each case . for systematic examination of the specimens , a squared and numbered ( with 100 indexed crosses ( + ) of equal dimension ) eyepiece grid ( ne21 mm ; graticules ltd , kent , uk ) , was introduced into one of the eyepieces of the microscope . of the total of 100 crosses that composed the net , approximately 50 would cover the periodontal space , being the remainder occupied by the root , dental canal and alveolar bone , which were excluded from the analysis .
three fields of the periodontal ligament were evaluated : its most central area in relation to the apical foramen and two lateral margins . for each evaluated field
, we considered the number of points coinciding with the occurrence of the three primary events of inflammatory reaction : swelling , vasodilatation and presence of inflammatory cells .
the following criteria were used : mild inflammation - when each parameter was coincident with a maximum of 4 crosses ; moderate inflammation - when at least 2 parameters were coincident with at least 5 and at most 9 crosses ; severe inflammation - when at least 2 parameters were coincident with 10 or more crosses .
chi - square test could have been used to assess differences in the efficacy of the three substances . however , as the expected frequencies for a few cells were less than 5 , chi - square test was not deemed appropriate .
alternatively , fisher 's exact test was used to overcome the problem due to small cell frequencies because it requires a non - zero frequency in each cell .
hence , the category " severe " was eliminated because there was no observation for that level .
the resulting contingency table was used for fisher 's exact test is reproduced as table 2 and 4 .
the data were analyzed with a confidence interval of 95% ( p<0.05 ) using the spss software ( spss 14.0 , spss inc . ,
the two tailed p value generated by fisher 's exact test for the above contingency table was 0.482 ( for the 24-hour exposure time ) and 0.377 ( for the 48-hour exposure time ) . as both p values were greater than 0.05 ,
the null hypothesis of independence was accepted , which means that the three substances did not have different efficacy at the either of the study intervals .
the results of the histologic analysis of the inflammatory response observed in dogs ' teeth after contact with edta , citric acid and saline , after 24 and 48 hours , are presented in the tables 1 and 2 . in order to provide the necessary adjustment for the statistical analysis ,
the scores 1 , 2 and 3 were attributed to the mild ( figure 2 ) , moderate ( figure 3 ) and severe ( figure 4 ) inflammation degrees , respectively , which differ from the normal aspect ( figure 1 ) .
there was no significant difference in the inflammation degree among the tested substances within the 24 hour ( p=0.482 ) or 48 hour ( p=0.377 ) periods .
in the present study , the sample comprised the roots instead of molar and premolar teeth because , after pulpectomy , the surgical wounds behave independently even if submitted to the same treatment .
the 24- and 48- hour periods were established because , though presenting a relative antimicrobial activity , the tested substances , are not indicated for this use and after longer observation periods there would be the risk of bacterial contamination , especially in the control group .
the method proposed for inflammation degree evaluation consisted of using a net of eyepiece reticular scale adapted to the ocular of the microscope , which allowed the quantification of the events composing the inflammatory process , i.e. , swelling , vasodilatation and presence of inflammatory cells in the apical region .
therefore , the classification of the inflammatory response in mild , moderate and severe was determined by the counting .
the results obtained with edta in relation to the control group demonstrated a good tolerance by the periapical tissues .
similar findings have been reported by zina , et al.15 ( 1981 ) and leonardo , et al.6 ( 1984 ) , the latter evaluating the tissue response in dogs ' teeth .
the statistical analysis showed that even though there was a severe degree inflammation within 48 hours , there was no significant difference between both periods .
citric acid showed a similar behavior as that of edta and saline at both periods and , though not statistically significant , it showed a higher degree of inflammation at 48 hours compared to the shorter period .
this result differs from those of previous studies that used citric acid for cavity cleaning5,9 and evaluated the response to pulp tissues instead of to periapical tissues .
the findings of the present study and of other authors1,8 disagree from those reported by chan , et al.4 ( 1999 ) who observed an increase in the inflammation degree with a longer exposure time in cell cultures .
citric acid is an organic acid that takes part in the krebs cycle in the human cells , being therefore biologically acceptable .
the results of the control group showed that an inflammatory response is a natural consequence of pulpectomy .
comparison of the substances in both studied periods ( 24 and 48 hours ) did not show statistically significant differences .
this fact indicates that , although these two substances have a low superficial tension , even in multiple foramen conditions , other factors referring to their physicochemical properties and the site of application , control their diffusion .
regarding edta , its activity is limited by the presence of organic material2 , in addition to promoting the coagulation of proteins , which form of a mechanical barrier that avoids its diffusion towards the periapical tissues .
citric acid has the property of not promoting collagen selling , which occurs with most acids .
this justifies its use for histological preparations in the form of sodium citrate , in demineralization processes .
this aspect could allow a higher diffusion of this substance , but its acidic characteristics provide cauterizing properties , due to its low ph .
both edta and citric acid compared to the control group showed a potential for a mild inflammation , with no statistically significant difference to each other .
the clinical use of either edta or citric acid should be done with caution to prevent their extrusion to the periapical tissues .
citric acid showed similar conditions for clinical use as those of edta , as it has similar characteristics regarding the property as an inorganic solvent , removal of smear layer , improvements in naocl properties when associated to it and a satisfactory tissue tolerance . under the tested conditions ,
under the experimental conditions of this study , the obtained results showed that 17% edta and 1% citric acid caused inflammatory responses in dog 's periapical tissues with no significant differences from saline within the evaluated time periods ( 24 and 48 hours ) .
17% edta and 1% citric acid produced similar inflammatory reaction to each other at both the shorter and the longer time period . it may be concluded that the inflammatory response to the tested substances was of mild intensity .
 the purpose of this study was to analyze the inflammatory response of dog 's periapical tissues to 17% trisodium edta salt ( ph 8.0 ) and 1% citric acid ( ph 2.0 ) . 
 saline was used as a control . 
 six adult dogs were used as the biological model of the study . 
 the experimental units comprised 56 roots of mandibular molars ( first and second ) and premolars ( first , second and third ) . 
 after coronal opening , pulpectomy and root canal instrumentation were performed using the above - mentioned irrigating solutions . 
 after 24 and 48 hours , the animals were euthanized and the teeth and their supporting tissues were removed and histologically processed . 
 the sections were stained with hematoxylin and eosin and analyzed histopathologically with a light microscope at x100 magnification . 
 the histological analysis focused on the occurrence of acute inflammatory response . 
 the presence of swelling , vasodilatation and inflammatory cells were evaluated and the degree of inflammation was determined for each case . 
 data were analyzed by fisher 's exact test using the spss software with a confidence interval of 95% ( p<0.05 ) . 
 17% edta and 1% citric acid caused inflammatory responses in dog 's periapical tissues with no significant differences to each other or to saline ( control ) at either the 24-hour ( p=0.482 ) or 48-hour ( p=0.377 ) periods . 
 it may be concluded that the inflammatory response was of mild intensity for the tested substances . 
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS