digital intraoral radiographic systems have several advantages compared with 
 conventional film - based radiography .
these advantages , in endodontics , include the 
 potential of a lower radiation exposure to the patient and faster image 
 display .
moreover , it was 
 introduced the opportunity of evaluating the progression of periapical bone loss by 
 digital tools as the pixel values .
radiographs do not always reflect the extent of the destructive process in the 
 periapical tissues and generally under - represent the size of a lesion .
studies have suggested that pathologic 
 involvement of the cortical plate , or at least junctional trabeculae , is a prerequisite 
 for radiographic detection of periapical pathoses . nevertheless , periapical lesions confined to cancellous bone 
 could be detected radiographically using pixel values using one of the tools of the 
 digital image , the histogram .
only a 
 few studies employing animal models 
 or human biopsies have compared 
 the diagnostic and quantifying accuracy of radiographs to the gold standard represented 
 by the histology .
the aim of this study was to induce periapical disease in rats to 
 assess the pixel values in different experimental periods to evaluate the bone loss 
 progression .
this study was approved by the animal research ethics committee of the bauru school 
 of dentistry , university of so paulo .
the experimental groups were composed of 27 
 wistar rats ( rattus novergicus ) , divided into three groups with nine 
 animals each , according to the sacrifice date , group 1 : 2 days , group 2 : 15 days , 
 group 3 : 30 days .
, a cavity was made on 
 the occlusal face of the mandibular first molar on the right side with a spherical 
 burr size 1/4 , at high speed without cooling .
the material for study was obtained after euthanizing the animal with an anesthetic 
 overdose .
the right and left hemi - mandibles of each rat were separated and 
 radiographed with two phosphor plates ( psps/ digora - soredex - tuusula , finland ) .
the 
 exposition was performed according to the parameters : 70 kvp , 7 ma , focus - film 
 distance of 30 cm , exposure time of 0.09 s. a standard aluminum stepwedge was set in 
 each image , and an acrylic plate measuring 0.3x0.4x0.2 cm was positioned over the 
 hemi - mandibles to simulate soft tissue .
the stepwedge was used as a standard image 
 technique , which is essential for control of day - to - day variations in the sensitivity 
 of the detector .
the radiographic evaluation was performed using the public domain software image j 
 ( version 1.33  , national institute of health , washington , dc ) a region of interest 
 ( roi/252 pixels ) was opened in the periapical region of the every tooth , closest to 
 the mesial root , without touching it . only the periapical bone was enclosed in the 
 roi . using the histogram tool ,
the 
 normalized pixel values of the images ( npi ) were then obtained by using the following 
 equation : npi = pi / cr . where 
 pi is the mean pixel intensity of the roi , and cr is the pixel intensity of the roi 
 of the standard aluminum stepwedge .
the pixel value of zero corresponded to black and 
 the pixel value of 255 to white .
hemi - mandibles were fixed in formalin 10% , decalcified in edta and processed 
 histologically .
five semi - serial sections of each hemi - mandible with 5 m thickness 
 were cut and stained with hematoxylin and eosin .
all histological sections were 
 identified with a random numerical sequence and periapical tissues were 
 histomorphometrically analyzed for both test and control sites . in the test site
it 
 was noted whether the tooth had pulpitis , necrosis or presented any sign of 
 inflammation , and whether or not there was presence of periapical bone resorption . 
 the percentages of bone tissue in the apical region of the root , soft tissue 
 ( connective tissue and inflammatory infiltrate , when present ) , bone marrow and 
 inferior alveolar nerve were obtained in a digital image analysis system , composed of 
 a zeiss axioskop ii microscope , ccd - iris rgb - sony camera ( sony dxc-151a rgb video 
 camera - sony corporation , tokyo , japan ) and kontron ks300 software 
 ( kontron electronic gmbm , munich , germany ) . for this purpose ,
an image was captured 
 at 10x objective from each histologic slice , which contained the apical portion of 
 the mesial root perpendicular to the alveolar nerve , positioned centrally .
the area ( in pixels ) was 
 measured to quantify the area occupied by bone tissue in the apical region of the 
 root , soft tissue ( connective tissue and inflammatory infiltrate , when present ) , bone 
 marrow and inferior alveolar nerve .
this study was approved by the animal research ethics committee of the bauru school 
 of dentistry , university of so paulo .
the experimental groups were composed of 27 
 wistar rats ( rattus novergicus ) , divided into three groups with nine 
 animals each , according to the sacrifice date , group 1 : 2 days , group 2 : 15 days , 
 group 3 : 30 days .
, a cavity was made on 
 the occlusal face of the mandibular first molar on the right side with a spherical 
 burr size 1/4 , at high speed without cooling .
the material for study was obtained after euthanizing the animal with an anesthetic 
 overdose .
the right and left hemi - mandibles of each rat were separated and 
 radiographed with two phosphor plates ( psps/ digora - soredex - tuusula , finland ) .
the 
 exposition was performed according to the parameters : 70 kvp , 7 ma , focus - film 
 distance of 30 cm , exposure time of 0.09 s. a standard aluminum stepwedge was set in 
 each image , and an acrylic plate measuring 0.3x0.4x0.2 cm was positioned over the 
 hemi - mandibles to simulate soft tissue .
the stepwedge was used as a standard image 
 technique , which is essential for control of day - to - day variations in the sensitivity 
 of the detector .
the radiographic evaluation was performed using the public domain software image j 
 ( version 1.33  , national institute of health , washington , dc ) a region of interest 
 ( roi/252 pixels ) was opened in the periapical region of the every tooth , closest to 
 the mesial root , without touching it . only the periapical bone was enclosed in the 
 roi . using the histogram tool ,
the 
 normalized pixel values of the images ( npi ) were then obtained by using the following 
 equation : npi = pi / cr . where 
 pi is the mean pixel intensity of the roi , and cr is the pixel intensity of the roi 
 of the standard aluminum stepwedge .
the pixel value of zero corresponded to black and 
 the pixel value of 255 to white .
hemi - mandibles were fixed in formalin 10% , decalcified in edta and processed 
 histologically .
five semi - serial sections of each hemi - mandible with 5 m thickness 
 were cut and stained with hematoxylin and eosin .
all histological sections were 
 identified with a random numerical sequence and periapical tissues were 
 histomorphometrically analyzed for both test and control sites . in the test site
it 
 was noted whether the tooth had pulpitis , necrosis or presented any sign of 
 inflammation , and whether or not there was presence of periapical bone resorption . 
 the percentages of bone tissue in the apical region of the root , soft tissue 
 ( connective tissue and inflammatory infiltrate , when present ) , bone marrow and 
 inferior alveolar nerve were obtained in a digital image analysis system , composed of 
 a zeiss axioskop ii microscope , ccd - iris rgb - sony camera ( sony dxc-151a rgb video 
 camera - sony corporation , tokyo , japan ) and kontron ks300 software 
 ( kontron electronic gmbm , munich , germany ) . for this purpose ,
an image was captured 
 at 10x objective from each histologic slice , which contained the apical portion of 
 the mesial root perpendicular to the alveolar nerve , positioned centrally .
the area ( in pixels ) was 
 measured to quantify the area occupied by bone tissue in the apical region of the 
 root , soft tissue ( connective tissue and inflammatory infiltrate , when present ) , bone 
 marrow and inferior alveolar nerve .
the radiographic analysis showed that the pixel values in the three experimental 
 periods increased on the test side in comparison with the control side , indicating 
 bone loss in a similar manner both psps plates used .
the student 's t test showed a 
 statistically significant difference between control and test sites for the three 
 groups tested ( p<0.05 ) ( table 1 ) .
the 
 influence of the time on periapical disease was studied using one - way anova and 
 tukey 's tests .
anova showed no statistically significant variation for any of the 
 time intervals in plate 1 ( p>0.05 ) , as well as for the control side of plate 2 
 ( p>0.05 ) .
this variation was only shown to be significant in plate 2 ( p<0.05 ) 
 between the test groups 2 and 3 ( figures 1a , 
 d , g , 
 j )
. means and standard deviation of the pixel values on the test and control sides 
 for the digora system phosphorus plates 1 and 2 , in the rats of groups i , ii 
 and iii mean pixel value from 9 animal samples .
control group : a ) radiographic image of hemi - mandible ; b - c ) photomicrograph of 
 the left first molar showing healthy pulp ( black arrow ) and periapical tissues 
 with cementum ( ce ) , fibers of periodontal ligament ( li ) and alveolar bone ( ab ) . 

group 1 : d ) radiographic image of hemi - mandible similar at the control group ; 
 e - f ) photomicrograph of the right first molar exhibiting inflammatory cells 
 ( black arrow ) in the periapical region .
group 2 : g ) radiographic image of 
 hemi - mandible showing radiolucent area in the periapical region ; h - i ) 
 photomicrograph of the right first molar showing extensive bone ( b ) resorption 
 by osteoclasts ( red arrow ) and connective tissue ( ct ) .
group 3 : j ) radiographic 
 image of hemi - mandible exhibiting extensive radiolucent area in the periapical 
 region ; k - l ) photomicrography of the right first molar showing abscess ( red 
 arrow ) , increase of connective tissue area ( ct ) and decrease of bone tissue ( b ) 
 in relation to the other groups the histological analysis of the left first molars of the rats , which served as 
 control , showed no alterations in the pulp or periapical tissues , and evidenced that 
 100% of these teeth presented healthy pulp and periapical tissues ( figures 1b , c ) .
the teeth submitted to coronal opening in group 1 , showed in 100% of 
 the cases , necrosis in the coronal region and pulp inflammation with the presence of 
 inflammatory cells and increased vascularization in the medial and apical thirds .
it 
 was possible to observe inflammatory cells and small bone and cementum resorption in 
 the periapical region ( figures 1e , f ) .
group 2 showed necrosis in the coronal and 
 middle radicular pulp and extensive inflammatory process in the apical third . in the 
 periapical region exhibited increase of bone resorption and replaced by conjunctive 
 tissue rich in inflammatory cells ( figures 1h , 
 i ) .
the group 3 showed total pulp necrosis , 
 extensive area of abscess and bone , cementum and dentine resorption and few bone 
 trabeculae surrounded by conjunctive tissue in the apical portion ( figures 1k , l ) .
the total 
 numbers of pixels obtained for each of the measured regions in the control and tests 
 sides , in the various experimental periods are presented in table 2 .
two - way anova and tukey 's tests showed statistically 
 significant difference between the test and control sides and among the experimental 
 periods as well . mean and standard deviation of the total number of pixels of each structure 
 present in the periapical region of the mesial root of the mandibular first 
 molar in the control and test groups in the experimental periods mean pixel value from 9 animal samples .
the radiographic analysis showed that the pixel values in the three experimental 
 periods increased on the test side in comparison with the control side , indicating 
 bone loss in a similar manner both psps plates used .
the student 's t test showed a 
 statistically significant difference between control and test sites for the three 
 groups tested ( p<0.05 ) ( table 1 ) .
the 
 influence of the time on periapical disease was studied using one - way anova and 
 tukey 's tests .
anova showed no statistically significant variation for any of the 
 time intervals in plate 1 ( p>0.05 ) , as well as for the control side of plate 2 
 ( p>0.05 ) .
this variation was only shown to be significant in plate 2 ( p<0.05 ) 
 between the test groups 2 and 3 ( figures 1a , 
 d , g , 
 j )
. means and standard deviation of the pixel values on the test and control sides 
 for the digora system phosphorus plates 1 and 2 , in the rats of groups i , ii 
 and iii mean pixel value from 9 animal samples .
control group : a ) radiographic image of hemi - mandible ; b - c ) photomicrograph of 
 the left first molar showing healthy pulp ( black arrow ) and periapical tissues 
 with cementum ( ce ) , fibers of periodontal ligament ( li ) and alveolar bone ( ab ) . 

group 1 : d ) radiographic image of hemi - mandible similar at the control group ; 
 e - f ) photomicrograph of the right first molar exhibiting inflammatory cells 
 ( black arrow ) in the periapical region .
group 2 : g ) radiographic image of 
 hemi - mandible showing radiolucent area in the periapical region ; h - i ) 
 photomicrograph of the right first molar showing extensive bone ( b ) resorption 
 by osteoclasts ( red arrow ) and connective tissue ( ct ) .
group 3 : j ) radiographic 
 image of hemi - mandible exhibiting extensive radiolucent area in the periapical 
 region ; k - l ) photomicrography of the right first molar showing abscess ( red 
 arrow ) , increase of connective tissue area ( ct ) and decrease of bone tissue ( b ) 
 in relation to the other groups
the histological analysis of the left first molars of the rats , which served as 
 control , showed no alterations in the pulp or periapical tissues , and evidenced that 
 100% of these teeth presented healthy pulp and periapical tissues ( figures 1b , c ) .
the teeth submitted to coronal opening in group 1 , showed in 100% of 
 the cases , necrosis in the coronal region and pulp inflammation with the presence of 
 inflammatory cells and increased vascularization in the medial and apical thirds .
it 
 was possible to observe inflammatory cells and small bone and cementum resorption in 
 the periapical region ( figures 1e , f ) .
group 2 showed necrosis in the coronal and 
 middle radicular pulp and extensive inflammatory process in the apical third . in the 
 periapical region
exhibited increase of bone resorption and replaced by conjunctive 
 tissue rich in inflammatory cells ( figures 1h , 
 i ) .
the group 3 showed total pulp necrosis , 
 extensive area of abscess and bone , cementum and dentine resorption and few bone 
 trabeculae surrounded by conjunctive tissue in the apical portion ( figures 1k , l ) .
the total 
 numbers of pixels obtained for each of the measured regions in the control and tests 
 sides , in the various experimental periods are presented in table 2 .
two - way anova and tukey 's tests showed statistically 
 significant difference between the test and control sides and among the experimental 
 periods as well .
mean and standard deviation of the total number of pixels of each structure 
 present in the periapical region of the mesial root of the mandibular first 
 molar in the control and test groups in the experimental periods mean pixel value from 9 animal samples .
in the present study , the periapical inflammation was induced by coronal opening of the 
 mandibular right first molar and confirmed that only coronal opening and pulp contact 
 with the oral cavity were sufficient for inducing periapical disease .
various other 
 studies have described this technique for induction of pulpal and periapical 
 inflammatory reactions in rat teeth . the inflammatory process was observed 
 in the pulp region in the experimental period of 2 days .
this is in agreement with the 
 findings of a previous study that showed that pulp inflammation is already observed 6 h 
 after the operative procedure . in 
 the present study
, pulp necrosis was observed in the experimental periods of 15 and 30 
 days .
this is also in agreement with a previous study that showed the presence of 
 complete pulp necrosis between the 8 and 20 days .
( 1995 ) affirmed that 
 bone resorption is present from the 3 day after the operative procedure . 

bone resorption in the present study , although very subtle , could already be observed in 
 the group 1 .
resorptive bone activity was actively present on the 15 day 
 ( group 2 ) .
some authors affirmed that this activity begins to diminish from then on , 
 with a decrease in the number of osteoclasts .
although the lesion appeared to be stabilized , with diminished 
 bone resorption , new bone formation did not occur due to the presence of aggressive 
 factors . in the 30-day 
 experimental period , a large area of resorption
the amount of periapical bone was minimal , and it was 
 replaced by inflammatory tissue .
statistical analysis showed a significant difference between test and control sides in 
 the 3 experimental periods , except for the root region , in which this difference was not 
 significant between the test and control sides , in the 2-day period , and between the 
 experimental periods of 15 and 30 days .
this was probably because resorption in 
 periapical disease is greater in the bone region than in the root region , which was not 
 very evident .
the present study confirmed radiographically the presence and intensity of bone 
 resorption observed by the histological study .
( 2003 ) removed medullar bone from cadaver mandibles , 
 radiographed the areas to measure the pixel value respectively .
these studies showed 
 that it is possible to detect bone loss in the mandibular region by analyzing the pixel 
 value , even if it is not yet visible radiographically .
conversely , studies that compared 
 images obtained by conventional radiographs ( without pixel value analysis ) and bone 
 resorption seen microscopically , showed that this relationship is not established with 
 precision 
  de rossi , et al .
( 2007 ) concluded 
 that although image digitization could not improve the detection of the early stages of 
 periapical lesions , it provides valuable quantitative assessment of extensive periapical 
 lesions . in order to diminish the noise level in the images captured
directly , the phosphorus 
 plates were covered by a protective envelope , as they are sensitive to light .
the images were always downloaded 
 immediately after the radiograph was taken , as the time between taking the radiograph 
 and downloading the plates also influences the pixel values . in the direct photostimulable phosphorus plate systems , such as digora , 

there is a correlation between the radiation dose and the quantity of capture plate 
 luminescence .
the capture 
 plates present less noise and a better quality image than the other direct systems , in 
 addition to being more sensitive to small variations of the exposure source in 
 comparison with conventional films , and should be used when small differences in 
 contrast are important .
the noise and coefficient of 
 variability of pixel value calculations showed low noise and small variation in the 
 images in this study . in spite of resistance by many professionals , the high cost of the technology and the 
 ethical implications because of the risk of manipulating the images , the use of digital 
 imaging has increased and is constantly being perfected .
the methodology applied was efficient for causing pulpitis and pulpal necrosis in the 
 studied rats .
teeth with pulpitis microscopically presented periapical bone resorption , 
 but the teeth with necrosis presented greater resorption . the control teeth presented 
 healthy pulp , without signs of periapical bone resorption .
the means of the pixel values 
 in the areas of periapical disease induced in rats indicated greater bone resorption 
 than the means of these values on the control side for the three experimental periods 
 ( p<0.05 ) .
even small periapical bone resorption was already sufficient for 
 determining changes in the pixel values of that area in the direct digital method , when 
 compared to their respective controls .
 objectivethis study evaluated experimentally - induced periapical bone loss sites using 
 digital radiographic and histopathologic parameters . material and methodstwenty - seven wistar rats 
 were submitted to coronal opening of their mandibular 
 right first molars . 
 they were radiographed at 2 , 15 and 30 days after the 
 operative procedure by two digital radiographic storage phosphor plates 
 ( digora ) . 
 the images were analyzed by creating a region of interest 
 at the periapical region of each tooth ( imagej ) and registering the corresponding 
 pixel values . 
 after the sacrifice , the specimens were submitted to microscopic 
 analysis in order to confirm the pulpal and periapical status of the tooth . 
 resultsthere was significant statistically difference between the control and test sides 
 in all the experimental periods regarding the pixel values ( two - way anova ; 
 p<0.05 ) . 
 conclusionsthe microscopic analysis proved that a periapical disease development occurred 
 during the experimental periods with an evolution from pulpal necrosis to 
 periapical bone resorption . 
INTRODUCTION
MATERIAL AND METHODS
General and radiographic procedures
Radiographic analysis
Histomorphometrical analysis
RESULTS
Radiographic analysis
Histopathological analysis
DISCUSSION
CONCLUSIONS