during endodontic treatment it may desirable or necessary to use an intracanal 
 medication . in pulpectomy therapy intracanal medication
is used between appointments 
 with the objective of reducing pain and inflammatory processes in pulp and periapical 
 tissues .
the choice of this agent depends on its biologic characteristics : be 
 non - irritant , control of the intensity and duration of inflammatory processes and 
 infection , and have potential healing induction . several chemicals and therapeutic agents have been suggested as 
 an intracanal medication : in this case , a combined corticosteroid - antibiotic could be 
 selected 
  in spite of being originally designed as an otology medication , a commercial 
 corticosteroid - antibiotic preparation , otosporin is currently used in 
 dentistry as an intracanal medication due to its components : polymyxin b sulphate , 
 neomycin sulphate , hydrocortisone and an aqueous base .
this 
 preparation reduces vasodilation , decreases liquid exudation , and also presents a direct 
 vasoconstrictive action on small blood vessels .
nevertheless , though this combination has many properties that 
 are desirable as a pulpectomy intracanal medication , if it is used for prolonged periods 
 it can reduce cellular metabolic activity and even impede or delay tissue 
 repair . in relation to tissue repair
, propolis has been known to be a natural antibiotic and has 
 been the subject of medical and dental research .
propolis is a complex mixture of 
 resinous and balsamic substances collected from plants by apis 
 mellifera bees , which transport them to their hives , modifying them with the 
 addition of their own secretions , pollen and wax .
propolis contains flavonoids which are 
 considered as the main primary biologically active component , responsible for a large 
 proportion of its known therapeutic properties . in dentistry
, propolis 
 has been used for the treatment of aphthous ulcers , candida albicans , 
 acute necrotizing ulcerative gingivitis ( unug ) , gingivitis and periodontitis , and it has been proven to have antimicrobial activity against 
 streptococcus mutans and polymicrobial cultures collected from necrotic root 
 canals . among the properties of propolis are : analgesic action , antiviral , 
 antifungal and antiprotozoal actions , immunostimulation , antioxidant 
 effects , antiinflammatory 
 action .
all these properties led to the question whether it 
 would be possible to use propolis as an intracanal medication in pulpectomy where there 
 is a need for both analgesic and anti - inflammatory effects .
the objective of this study was therefore to evaluate the periapical tissue response to 
 an experimental propolis paste when used as intracanal medication in dog 's teeth after 
 pulpectomy .
a total of 72 incisors ' root canals of 6 dogs aged 2 to 4 years were selected for the 
 experiment .
the protocol used was approved by the local ethics committee ( cep ulbra : 
 2003 - 015a ; cep upf : 276/2004 ) .
the animals were kept under general anesthetic supplemented by infiltration of local 
 anesthetic into the procedure area throughout the operation .
soft tissues and teeth were 
 sterilized using alcohol - iodine solution ( vico - farma , lages , sc , brazil ) .
the root canals of the 66 teeth were accessed on the buccal surface of the tooth using a 
 diamond bur # 1012 ( metalrgica fava indstria e comrcio ltda .
, franco da rocha , sp , 
 brazil ) at high speed with water - air cooling . after carrying out radiography on each 
 tooth individually ,
the working length was established at 1 mm short of the radiographic 
 root apex . following root canal cleaning ,
the root canal was instrumented using flexofile 
 k files ( dentsply , maillefer instruments s.a . , ballaigues , switzerland ) , finishing with 
 an instrument # 40 .
canals were irrigated with 2 ml of solution at each instrument 
 change , alternating between 1% sodium hypochlorite ( vico - farma ) and 17% trisodium 
 ethylenediaminetetraacetic acid ( edta ) ( vico - farma ) with ph 7.3 .
once chemomechanical 
 preparation was complete , the apex was breached passing 2 mm beyond working length a 
 file # 20 .
canals were then dried with absorbent paper points # 40 ( tanari indstria e 
 comrcio ltda .
the root canals were then packed with the medications to be analyzed : in each of the 6 
 dogs , five root canals were packed with a solution of 10 mg / ml hydrocortisone/3,400 u / ml 
 neomycin sulfate/10,000 u / ml polymyxin b sulfate , a corticosteroid - antibiotic 
 preparation ( zest farmacutica ltda . , so paulo , sp , brazil ) and another 5 were packed 
 with experimental propolis paste .
one root canal of each dog was packed with glycerin , 
 which was considered as vehicle group ( negative control ) , and another tooth did not 
 receive any treatment , considered as the non - treated group ( positive control ) .
corticosteroid - antibiotic preparation was stored in a clean and sterilized anesthetic 
 tube and the solution was introduced into the canal with a carpule syringe and a 27 g 
 needle set 1 mm short of the working length .
the experimental propolis paste was previously manipulated following the composition : 1% 
 propolis extract , titanium dioxide , glycerin .
the paste was stored in a sterilized tube 
 and at the moment of the root canal filling it was introduced using a # 35 lentulo spiral 
 ( pr dent , so paulo , sp , brazil ) , calibrated in two millimeters from the root canal 
 working length and running at low speed in a clockwise direction . in the cases of both 
 types of medication ,
the cavity was then cleaned , filled with sterile cotton and sealed with a 2 mm layer of 
 glass ionomer cement ( vidrion r - ss white artigos dentrios ltda .
, rio de 
 janeiro , rj , brazil ) , prepared in accordance with its manufacturer 's instructions .
the animals spent up to 28 days with intracanal medication in place , during which time 
 they were monitored by a veterinarian .
after 7 , 14 or 28 days with the intracanal 
 medication in place , the dogs were euthanized and the samples were collected .
tissues were preserved by intravenous perfusion of 4% buffered paraformaldehyde . the 
 maxilla and mandible were removed and postfixed in 10% neutral formaldehyde .
the samples 
 were demineralized in edta 4.13% at ph 7.2 , following the process protocol for paraffin , 
 five micrometers histological sections and hematoxylin and eosin staining .
the sections 
 were analyzed using an optical microscope ( olympus , tokyo , tokyo , japan ) at 100 , 200 
 and 400 magnification by a single examiner , who was an experienced histologist and 
 blind to the tested materials .
the subject of analysis was the periapical region and the 
 specimen with the greatest degree of inflammation was chosen from each sample .
the following qualitative observations were recorded : the presence of polymorphonuclear 
 neutrophils , polymorphonuclear eosinophils , lymphocytes and plasma cells , macrophages 
 and/or giant cells , fibrous condensation and abscesses .
each element was attributed a 
 numerical value according to the protocol proposed by figueiredo , et al .
approximately 30 days after the 
 original analysis of the sections , 10% of them were reanalyzed to test for intraexaminer 
 agreement between events observed and scores awarded .
the results were tested using factorial anova , followed by the mann whitney and tukey 's 
 tests , all to a significance level of 5% ( spss 12.0 ) .
intraexaminer agreement was 
 assessed using kendall 's w , with a 5% significance level ( spss 12.0 ) .
the negative control slices showed a normal histological periapical tissue , without a 
 significant inflammatory cellular event . on the other hand , positive control presented 
 neutrophils , lymphocytes and macrophages with higher scores than negative control 
 showing the inflammatory processes that occur after pulpectomy chemical - mechanical 
 preparation .
regarding to the cellular events listed for analysis , fibrous condensation was not 
 included , once its origin was not clear in the analysis : whether due to a natural 
 response of the periodontal ligament to the pulpectomy technique or tissue reaction to 
 the test materials .
no eosinophils were observed on any of the sections from either of 
 the groups , being scored 1 for the analysis .
kendall 's w showed that the intra - examiner 
 agreement was 98.91% , represented by w=0.119 .
figure 1 illustrates variations in the number of neutrophils when analyzed with 
 reference to the groups and experimental periods .
the non - treated group had the lowest 
 mean scores and had no variation throughout the time . analyzing this event with relation 
 to corticosteroid - antibiotic preparation
, it is observed that there was a continuous 
 increase in the mean scores as the experimental period elapsed , with statistically 
 significant differences ( p=0.007 ) detected by the tukey 's test between 7 and 28 and 
 between 14 and 28 days , which was quite similar to the vehicle group .
analyzing the mean 
 scores for these cells for the propolis paste , there was no significant difference 
 between the periods analyzed ( p=0.47 ) .
it was also possible to apply the mann - whitney 
 nonparametric test in order to compare the substances at each experimental time in 
 isolation for each of the different inflammatory events .
it was not found any 
 statistically significant differences between the tested substances for 7 days ; 
 statistically significant differences were detected for neutrophils between propolis 
 paste and corticosteroid - antibiotic preparation , and between propolis paste and vehicle 
 group at 14 and 28 days , with p=0.05 and 0.02 , consecutively , with higher scores for 
 corticosteroid - antibiotic preparation and vehicle group ; no difference between 
 corticosteroid - antibiotic preparation and vehicle group ; no difference between propolis 
 paste and non - treated group .
( a ) significant difference from propolis paste in 14 days ; ( b ) significant 
 difference from propolis paste in 28 days ; ( * ) significant difference in the 
 substance over time with relation to lymphocytes and plasma cells , illustrated in figure 2 , the non - treated 
 group had the lowest mean scores and had no variation throughout the time .
although the 
 test substances exhibited similar values in 7 days , by the end of the experimental 
 period a large difference was observed between the experimental groups with higher 
 medium score for corticosteroid - antibiotic preparation and vehicle group .
when each of 
 the test substances were analyzed in isolation with relation to the variable time , 
 corticosteroid - antibiotic preparation exhibited significant differences ( p=0.012 ) , 
 detected by the tukey test , between 7 and 14 and between 7 and 28 days . in this 
 analysis ,
the propolis paste once more exhibited no significant differences between 
 times ( p=0.41 ) .
the mann - whitney nonparametric test showed no statiscally significant 
 differences between the tested substances for 7 days .
significant difference between 
 corticosteroid - antibiotic preparation and propolis paste , and between propolis paste and 
 vehicle group at 14 and 28 days , with p=0.01 and 0.02 , consecutively , with higher scores 
 for corticosteroid - antibiotic preparation and vehicle group ; no difference between 
 corticosteroid - antibiotic preparation and vehicle group ; no difference between propolis 
 paste and non - treated group .
( a ) significant difference from propolis paste in 14 days ; ( b ) significant 
 difference from propolis paste in 28 days ; ( * ) significant difference in the 
 substance over time the non - treated group had the lowest mean scores and had no variation throughout the 
 time for macrophages and giant cells .
analysis of the mean scores between the tendencies 
 of cellular expression for vehicle group , propolis paste and corticosteroid - antibiotic 
 preparation showed that these events were constantly present in the tissues analyzed , 
 with no significant differences between them .
there were no significant differences when 
 each of the experimental groups was analyzed alone with relation to the variable time 
 ( p>0.05 ) ( figure 3 ) .
progression of macrophages medium scores of the groups over the experimental 
 time figure 4 illustrates the mean scores for abscesses for the tested groups . in 7 days 

the non - treated group and the propolis 
 paste group did not exhibit any significant difference over time ( p>0.05 ) . however , 
 as time passed , there was a drastic increase in the corticosteroid - antibiotic 
 preparation and vehicle group with statistically significant differences ( p<0.05 ) , 
 detected by the tukey test , between 7 and 14 and between 7 and 28 days . for this event , 

corticosteroid - antibiotic preparation and vehicle group had the higher scores when 
 compared with propolis paste and non - treated group at 28 days , with a significant 
 difference ( p<0.05 ) as detected by mann - whitney nonparametric test ; there was no 
 statiscally significant differences between the substances for 7 and 14 days .
( b ) 
 significant difference from propolis paste in 28 days ; ( * ) significant difference 
 in the substance through the time figure 5 shows representative images of samples tested after 28 days in contact with 
 periapical tissues .
figure 5a is a representative image of the non - treated group : normal 
 periapical tissue , with no inflammatory cells . in the corticosteroid - antibiotic 

preparation ( figure 5e - f ) , it is possible to observe the presence of intense 
 inflammation after 28 days , whereas there is merely mild periapical inflammation after 
 exposure to the propolis paste ( figure 5b - d ) .
a- non - treated sample  image of the periapical area of the root with a normal 
 appearance ; b- propolis after 28 days  mild periapical inflammatory reaction ; c- 
 propolis after 28 days 
macrophages present in periapical region ( arrowed ) ; d- 
 otosporin 28 days  intense inflammatory reaction with abscess ( arrowed ) ; e- 
 otosporin 28 days  greater magnification than ( d ) .
during pulpectomy the pulp removal and chemical - mechanical preparation will lead the 
 periapical tissues into an acute inflammatory process , which explains 
 the result of the vehicle group in this experiment . as it is known by the literature , 
 the main cellular events to be taken into consideration when attempting to observe an 
 initial inflammatory response in tissues are neutrophils and the presence of abscesses . 

when neutrophils are present it signify that an acute process is occurring , during which 
 these cells are constantly recruited to contain inflammation .
these are the first cells 
 to migrate from blood vessels and to reach the site of aggression .
nevertheless , if the 
 aggression is intense and neutrophils are unable to contain it rapidly , abscesses may 
 form from a buildup of dead neutrophils , tissue liquids and abnormal material , creating 
 pus .
corticosteroid based medicaments acts on the synthesis of lipocortin and vasocortin , 
 inhibiting the formation of edema and a2 phospholipasis enzymes , once inhibiting this 
 enzyme , membrane phospholipids can not be converted into arachidonic acid .
corticosteroid 
 preparation also acts on histamine , heparin , and bradicinin , which are important 
 chemical mediators in the initial phases of acute inflammation . 

propolis - based medicaments contains flavonoids which are considered as the main primary 
 biologically active component , responsible for a large proportion of its known 
 therapeutic properties .
the flavonoids interfere in the 
 arachidonic acid metabolism and so interfere in the cyclooxygenase and lypooxygenase 
 synthesis .
some of the substances derivate in this cascade are the prostaglandins , 
 thromboxane and leukotriene which are important to keep the integrity of the 
 inflammatory reaction .
therefore , it is expected that the use 
 of corticosteroid preparations and propolis - based medicaments cause changes in the 
 inflammatory response in the tissue as it was observed in this study and discussed in 
 the following sentences . when the results were analyzed , corticosteroid - antibiotic preparation had high scores 
 for the presence of neutrophils and abscesses , primarily from 14 days onwards .
the 
 intense neutrophil infiltrate , which was continuously present and had not receded by 28 
 days , demonstrates the severity of the inflammatory response to 
 corticosteroid - antibiotic preparation .
it was already discussed in the literature that 
 if this medication is left in contact with remaining apical tissues for longer periods 
 the tissue response in this region can become completely compromised . on the other hand
, it could be possible 
 that after 28 days , no more corticosteroid - antibiotic should be in the root canal and 
 this fact could explain why corticosteroid - antibiotic preparation had the same 
 inflammatory reaction in 28 days as the vehicle group ( glycerin ) , which is considered an 
 inert substance .
in contrast , the scores for neutrophils and abscesses in the propolis paste group 
 illustrate a tissue reaction that is more favorable to the repair process , since their 
 means were low and remained constant as the experimental period elapsed , quite similar 
 to the non - treated group .
( 2004 ) , which have demonstrated that 
 administering propolis - based substances leads to tissues exhibiting normal 
 reorganization without increased vascular or cellular events , maintaining low levels of 
 inflammation .
it is important to emphasize that , in this study , after 7 days of experiment , all tested 
 substance had the same behavior : low levels of inflammatory cells .
the differences 
 between the two substances were primarily observed after the 14 and 28-day experimental 
 periods , with the scores for neutrophils and abscesses being similar in 7 days .
this 
 result supports the indication for using corticosteroid - based medications for periods no 
 longer than 7 days . with relation to the presence of lymphocytes and plasma cells , these events characterize 
 the presence of a chronic process .
the mean values for the corticosteroid - antibiotic 
 preparation used at 14 and 28 days appear to be more worrying since they indicate that 
 there was intense lymphoplasmacytic infiltrate , which did not recede as the experimental 
 period elapsed . regarding the absence of eosinophils , their presence
is intimately related with allergic 
 processes and thus , despite the literature describing cases of allergy to 
 propolis , at least in 
 this study , this effect was not observed . analyzing cellular events as a whole , it was observed that in the 
 corticosteroid - antibiotic preparation group , they increased as the experimental period 
 elapsed , indicating a deterioration of the inflammatory process .
this was not the case 
 with the propolis paste , since no significant difference between the events analyzed 
 occurred during the experimental period .
( 2004 ) , where it was demonstrated that 
 propolis is more effective at reducing acute inflammatory processes than 
 corticosteroid - antibiotic preparation .
these findings confirm the anti - inflammatory 
 activity mentioned in earlier published studies . based on the obtained results
, this study shows that propolis paste is a viable 
 alternative for intracanal medication in cases of pulp or periapical inflammatory 
 processes .
length of time in contact with these tissues is a critical factor and , if an 
 endodontic treatment can not be completed in a single session , using 
 corticosteroid - antibiotic preparation for a period of more than 7 days may worsen the 
 inflammatory process . in this experiment ,
propolis paste in contact with periapical 
 tissues after pulpectomy did not appear to be a problem .
furthermore , the propolis paste 
 was more effective at controlling the inflammatory response over the 28-day experimental 
 period .
here it was demonstrated that the experimental propolis paste had the lowest value of 
 inflammatory cells events , with mean scores similar to the negative control group .
this 
 indicates that the propolis paste could be considered as an intracanal medication 
 possibility .
however , it is important to emphasize that this is part of a primary study 
 with the experimental propolis paste .
basic science and clinical experimental should be 
 performed to better know its properties and possible use in endodontic treatments .
within the context of the methodology proposed and results observed , it is possible to 
 conclude that propolis paste caused a periapical mild inflammatory response with no 
 variation throughout the experimental period when compared with 
 corticosteroid - antibiotic preparation ; corticosteroid - antibiotic preparation caused an 
 increasing degree of inflammatory response that was directly related to the length of 
 time , being most severe at 28 days .
 intracanal medication in pulpectomy therapy is used between appointments with the 
 objective of reducing pain and inflammatory processes in pulp and periapical tissues . 
 
 propolis has been known as a natural antibiotic and has been subject of medical and 
 dental research due to its therapeutic properties such as antibiotic , analgesic and 
 anti - inflammatory effects . 
 objectivethe aim was to carry out an in vivo evaluation of the periapical 
 tissue response to propolis paste when used as an intracanal medication in the 
 teeth of dogs after pulpectomy . 
 material and methods72 dog 's incisors were selected for the experiment . 
 after biomechanical 
 preparation the root canal was filled with a corticosteroid - antibiotic 
 preparation , experimental propolis paste , non - medicament ( negative control ) or 
 non - pulpectomy at all ( positive control ) . 
 the medications were left inside the 
 root canal for 7 , 14 or 28 days . at the end of the experimental period 
 
 histological sections were prepared and all laboratories processes for harris 
 hematoxylin and eosin staining was proceeded followed by the analysis using an 
 optical microscope . 
 sections were classified according to a score representing the 
 inflammatory events observed : the presence of polymorphonuclear neutrophils , 
 polymorphonuclear eosinophils , lymphocytes and plasma cells , macrophages and/or 
 giant cells , fibrous condensation and abscesses . 
 resultsthere were statistically significant differences between the tissue reactions 
 caused by the two substances being tested , after different experimental periods , 
 with the periapical tissue that was in contact with propolis paste exhibiting 
 fewer inflammatory reactions in comparison to corticosteroid - antibiotic 
 preparation . 
 conclusionsthe low tissue responses from propolis paste suggest that this material could be 
 considered as an option for root canal medication after pulpectomy . 
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
CONCLUSION