mineral trioxide aggregate ( mta ) is a useful material for vital pulp therapy , such as pulp capping and pulpotomy . in these clinical situations ,
mta is used in a coronal position ; therefore , the color of mta is considered key to the final esthetic result . in the 1990s , torabinejad et al .
introduced gray mta ( gmta ) , which has a similar chemical constitution to that of portland cement except for the addition of bismuth oxide ( bi2o3 ) ( 1718 wt% ) to improve radioopacity . despite its favorable biological features , gmta causes tooth discoloration as well as discoloration of the adjacent gingiva .
because of this aesthetic problem , white mta ( wmta ) was developed through the exclusion of iron compounds rather than by the addition of other elements ( proroot ( pr ) ; dentsply , tulsa , ok , usa ) .
recently , belobrov and parashos reported a case of tooth discoloration after use of wmta as a pulp - capping material . in response to these issues ,
a pozzolan - based mta - derived material ( endocem ( ec ) ; maruchi , wonju , korea ) was introduced into the endodontic market .
furthermore , the biocompatibility and osteogenicity / odontogenicity of ec are similar to those of pr . however , there is still the possibility of tooth discoloration when using this material .
recent studies of color stability of wmta have suggested that the formation of metallic bismuth under light irradiation can lead to color change .
thus , the use of an alternative radiopacifier to replace bismuth in wmta is needed .
recently , the manufacturer that produces ec released another fast - setting pozzolan cement that contains zirconium oxide ( zro2 ) as a radiopacifier ( endocemzr [ ecz ] ; maruchi ) to overcome the gray color of original ec . it has been reported that zro2 has acceptable radiopacity and does not participate in the hydration reaction of portland cement .
recently , the addition of 30% zro2 to pc exhibited physical and antimicrobial properties comparable to those of pc
. however , there is little information about the effect of ecz on tooth discoloration . in various clinical situations , such as pulp capping and perforation repair , mta or mta - derived materials
furthermore , even blood in the root canal adjacent to the wmta has been shown to influence discoloration .
therefore , the aim of this study was to evaluate tooth discoloration induced by contact with various calcium silicate - based pulp - capping materials in the presence or absence of blood in vitro .
exclusion criteria were the presence of caries , visible structural defects , coronal staining , and narrow crown width ( labial surface area > 10  10 mm ) .
the samples were formed with reference to a new model introduced by a previous study , as shown in figure 1 .
briefly , the teeth were disinfected in 1% chloramine - t solution ( sigma - aldrich , st .
louis , mo , usa ) and stored in normal saline at room temperature for 1-month .
the roots were resected using a diamond - coated disc , and extrinsic stains and calculus on the labial surface were removed with an ultrasonic scaler .
a cuboid enamel - dentin block ( 10 mm  10 mm  3.5 mm ) was prepared from the middle third of each crown using a low - speed microtome ( isomet , buehler , il , usa ) .
the labial enamel surface was finished and polished with 220 , 600 , 1200 , and 2000-grit abrasive papers ( cc261 ; deerfos , seoul , korea ) .
then , a box - form cavity ( 6 mm  6 mm  1.5 mm ) was prepared with a # 330 carbide bur in the middle of each specimen , leaving 2 mm of the labial tooth structure ( 1 mm each of enamel and dentin ) .
sodium hypochlorite ( 1.5% naocl ) irrigation solution was placed in the cavity for 30 min , and 17% ethylene diamine tetraacetic acid ( edta ; prevestdentpro , jammu , india ) was applied for 2 min to remove the smear layer and to expose the dentinal tubules .
a standardized cuboid enamel - dentin block ( 10 mm  10 mm  3.5 mm ) , which was prepared by removing the middle third of a bovine incisor human blood was used to simulate the clinical situation of vital pulp therapy . in this study ,
the blood collection tubes ( vacuette ; greiner bio - one , kremsmnster , austria ) were sterile and spray - coated with the anticoagulant k2edta to prevent clotting .
the three materials included in this study were pr , ec , and ecz [ table 1 ] . the specimens were randomly divided into six experimental groups and two control groups according to the material used and the presence or absence of contamination with blood [ table 2 ] .
each material was mixed according to the manufacturer 's instructions and was placed into the cavity . in the blood - contaminated groups , 5 l of blood was placed on top of the material .
a negative control ( nc ) was filled with a cotton pellet moistened with normal saline , and a positive control was filled with a cotton pellet moistened with blood .
then , the cavities were sealed with a self - adhesive resin material ( u - cem universal ; vericom , chuncheon , korea ) .
an led curing light ( elipar s10 ; 3 m espe , munich , germany ) was used to irradiate the specimens for 20 s. all specimens were stored at room temperature and 100% relative humidity .
composition of tested materials teeth and materials in experimental and control groups as shown in figure 2 , a standardized acrylic resin mold was developed to allow measurement of the same position in each sample and to increase reproducibility .
the tooth color was recorded at baseline ( d0 ; after tooth preparation and placement of materials ) and at 1-week ( d7 ) , 2 weeks ( d14 ) , 4 weeks ( d28 ) , and 8 weeks ( d56 ) using a spectrophotometer ( nf999 ; nippon denshoku , tokyo , japan ) .
the color differences ( e ) between the baseline and the d7 , d14 , d28 , d56 measurements were calculated using the following formula : e = ( [ l * 2  l * 1]+ [ a * 2  a * 1 ] + [ b * 2  b * 1 ] ] .
the value of the luminosity ( l * ) represents the lightness values , a * represents the values of red - green , and b * represents the values of yellow - blue .
standardized acrylic resin mold used to repeatedly measure the same position in each sample in each group , a representative sample was selected and sectioned in the center by using a low - speed microtome ( isomet ) .
the sectioned surface was examined under a stereomicroscope ( leica mz16fa ; leica , wezler , germany ) .
the data were evaluated using spss software ( pasw statistics 20 ; spss inc . ,
statistical analysis was performed using repeated measures analysis of variance to examine the e values as the dependent variables and time as a factor .
tukey 's post hoc test was used to assess significant differences between the tested materials .
exclusion criteria were the presence of caries , visible structural defects , coronal staining , and narrow crown width ( labial surface area > 10  10 mm ) .
the samples were formed with reference to a new model introduced by a previous study , as shown in figure 1 .
briefly , the teeth were disinfected in 1% chloramine - t solution ( sigma - aldrich , st .
louis , mo , usa ) and stored in normal saline at room temperature for 1-month .
the roots were resected using a diamond - coated disc , and extrinsic stains and calculus on the labial surface were removed with an ultrasonic scaler .
a cuboid enamel - dentin block ( 10 mm  10 mm  3.5 mm ) was prepared from the middle third of each crown using a low - speed microtome ( isomet , buehler , il , usa ) .
the labial enamel surface was finished and polished with 220 , 600 , 1200 , and 2000-grit abrasive papers ( cc261 ; deerfos , seoul , korea ) .
then , a box - form cavity ( 6 mm  6 mm  1.5 mm ) was prepared with a # 330 carbide bur in the middle of each specimen , leaving 2 mm of the labial tooth structure ( 1 mm each of enamel and dentin ) .
sodium hypochlorite ( 1.5% naocl ) irrigation solution was placed in the cavity for 30 min , and 17% ethylene diamine tetraacetic acid ( edta ; prevestdentpro , jammu , india ) was applied for 2 min to remove the smear layer and to expose the dentinal tubules .
a standardized cuboid enamel - dentin block ( 10 mm  10 mm  3.5 mm ) , which was prepared by removing the middle third of a bovine incisor
human blood was used to simulate the clinical situation of vital pulp therapy . in this study ,
the blood collection tubes ( vacuette ; greiner bio - one , kremsmnster , austria ) were sterile and spray - coated with the anticoagulant k2edta to prevent clotting .
the three materials included in this study were pr , ec , and ecz [ table 1 ] . the specimens were randomly divided into six experimental groups and two control groups according to the material used and the presence or absence of contamination with blood [ table 2 ] .
each material was mixed according to the manufacturer 's instructions and was placed into the cavity . in the blood - contaminated groups , 5 l of blood was placed on top of the material .
a negative control ( nc ) was filled with a cotton pellet moistened with normal saline , and a positive control was filled with a cotton pellet moistened with blood .
then , the cavities were sealed with a self - adhesive resin material ( u - cem universal ; vericom , chuncheon , korea ) . an led curing light ( elipar s10 ; 3 m espe , munich , germany ) was used to irradiate the specimens for 20 s. all specimens were stored at room temperature and 100% relative humidity . composition of tested materials teeth and materials in experimental and control groups
as shown in figure 2 , a standardized acrylic resin mold was developed to allow measurement of the same position in each sample and to increase reproducibility .
the tooth color was recorded at baseline ( d0 ; after tooth preparation and placement of materials ) and at 1-week ( d7 ) , 2 weeks ( d14 ) , 4 weeks ( d28 ) , and 8 weeks ( d56 ) using a spectrophotometer ( nf999 ; nippon denshoku , tokyo , japan ) .
the color differences ( e ) between the baseline and the d7 , d14 , d28 , d56 measurements were calculated using the following formula : e = ( [ l * 2  l * 1]+ [ a * 2  a * 1 ] + [ b * 2  b * 1 ] ] .
the value of the luminosity ( l * ) represents the lightness values , a * represents the values of red - green , and b * represents the values of yellow - blue .
in each group , a representative sample was selected and sectioned in the center by using a low - speed microtome ( isomet ) .
the sectioned surface was examined under a stereomicroscope ( leica mz16fa ; leica , wezler , germany ) .
the data were evaluated using spss software ( pasw statistics 20 ; spss inc . ,
chicago , il , usa ) . statistical analysis was performed using repeated measures analysis of variance to examine the e values as the dependent variables and time as a factor .
tukey 's post hoc test was used to assess significant differences between the tested materials .
the mean , standard deviation , and significance of the color differences are shown in figure 3 . when the e value is higher than 3.3 , it is possible to distinguish the color difference of the tooth .
after 1 week , the e value was higher than 3.3 in all experimental groups .
the pr group and ec group showed significantly higher mean values than the nc group after 2 weeks and 1 week , respectively ( p < 0.05 ) . on the contrary , in the ecz group , the color of the tested specimens was not significantly different from that of the nc , with the exception of data at d7 ( p > 0.05 ) .
e values for the different groups at the different time points ( group 14 ) .
the same letters indicate statistically similar groups ( p = 0.05 ) there were larger color differences ( e value ) when in contact with blood irrespective of time or type of material [ figure 4 ] . in comparison with the positive control , the color changes of all experimental groups were not significantly different at most time points ( p > 0.05 ) .
after 2 weeks , the pr / b group showed a higher e value than the ecz / b group ; however , a significant difference was only observed at d56 ( p < 0.05 ) .
furthermore , the ec / b group ( g7 ) showed a larger color difference than the ecz / b group , but the difference was only significant at d28 ( p < 0.05 ) .
e values for the different blood contaminated groups at the different time points ( group 58 ) .
the mean , standard deviation , and significance of the color differences are shown in figure 3 . when the e value is higher than 3.3 , it is possible to distinguish the color difference of the tooth .
after 1 week , the e value was higher than 3.3 in all experimental groups .
the pr group and ec group showed significantly higher mean values than the nc group after 2 weeks and 1 week , respectively ( p < 0.05 ) . on the contrary , in the ecz group , the color of the tested specimens was not significantly different from that of the nc , with the exception of data at d7 ( p > 0.05 ) .
e values for the different groups at the different time points ( group 14 ) .
there were larger color differences ( e value ) when in contact with blood irrespective of time or type of material [ figure 4 ] . in comparison with the positive control ,
the color changes of all experimental groups were not significantly different at most time points ( p > 0.05 ) .
after 2 weeks , the pr / b group showed a higher e value than the ecz / b group ; however , a significant difference was only observed at d56 ( p < 0.05 ) .
furthermore , the ec / b group ( g7 ) showed a larger color difference than the ecz / b group , but the difference was only significant at d28 ( p < 0.05 ) .
e values for the different blood contaminated groups at the different time points ( group 58 ) .
several case reports have revealed discoloration produced by mta when the material was used as a pulp - capping or pulpotomy agent .
however , few studies have evaluated the discoloration of various mta - derived materials , especially zro2-containing materials . in the present study ,
the e value was higher than 3.3 in all experimental groups after 1-week [ figure 3 ] .
we observed all teeth to have experienced color alteration after contact with mta materials , which is consistent with findings of previous studies .
the present study showed that the pr and ec groups experienced significantly more discoloration than the nc group after 2 weeks .
however , there were no significant color changes between the ecz group and the nc .
a possible explanation for these results is that the bi2o3 included in pr and ec causes discoloration .
one possible mechanism of this change is that the bi2o3 interacts with collagen in dentin and is converted to a black precipitate in the material - dentin interface .
a recent investigation showed that collagen , which is present in organic dentin matrix , reacts with bi2o3 , resulting in grayish discoloration . another hypothesis to explain the discoloration is the formation of metallic bismuth under specific conditions . irradiated bi2o3 under oxygen - free conditions is excited and changed to dark grayish metallic bismuth .
a recent study also reported that contact of wmta and other bismuth - containing materials with naocl produces a change to a darker color because the oxide is converted to bismuth metal in contact with sodium hypochlorite and oxygen is lost .
our stereomicroscopic examination of the pr group revealed grayish discoloration in the interfacial dentin and the material itself [ figure 5a ] .
however , the color of the ec changed the color of the specimen to dark gray [ figure 5b ] .
a recent in vitro study supported our results that significant discoloration occurs in the cervical area in the presence of pr , mta angelus , and ec .
one possibility is that the organic matrix in the dentin surface reacts with bi2o3 , a possible major factor contributing to discoloration , and the remaining naocl solution in the cavities of the specimens might exacerbate the change in color through reaction with the bi2o3 included in the pr and ec . on the other hand , in the present study , the color of the ecz group remained relatively stable according to spectrophotometric [ figure 3 ] and stereomicroscopic analyses [ figure 5c ] .
these results might be explained by the inclusion of zro2 as a radiopacifier instead of bi2o3 , as well as the light color of the material itself . in other words , aside from the original dark color of the material , calcium silicate - based materials containing bi2o3 undergo discoloration under conditions that simultaneously include contact with an organic dentin matrix , an oxygen - free environment , and irradiation with a curing light .
therefore , when selecting pulp - capping materials for areas of aesthetic concern , ecz , which includes zro2 rather than bi2o3 , might be a better option .
stereomicroscopic appearance of a representative sample from each group after 8 weeks ; ( a ) pr , ( b ) ec , ( c ) ecz , ( d ) negative control , ( e ) proroot with blood , ( f ) ec with blood , ( g ) ecz with blood , and ( h ) positive control .
( i ) samples show the initial color of each material ; pr : proroot , ec : endocem , ecz : endocemzr the sequential color change ( e ) of teeth in contact with materials and blood is presented in figure 4 .
the specimens showed an increased e value after contamination with blood at each time point .
however , a significant difference was observed in the pr / b and ec / b groups only after 8 weeks .
on stereomicroscopic examination , the pr / b specimen [ figure 5e ] showed greater discoloration of the material itself , demonstrating a dark orange to gray color change .
this result is in agreement with the findings of a previous study that reported greater discoloration of the wmta / blood group compared to the wmta / saline group .
therefore , we conclude that the presence of blood within the cavity exacerbates tooth discoloration induced by mta - based pulp capping materials . one possible mechanism may be the interaction between erythrocytes in the blood and the unset mta - based pulp - capping materials .
incompletely set materials may allow absorption of blood and hemolysis of erythrocytes from the vital pulp tissue .
however , ec is known as a fast setting material , eliminating the possibility of this explanation here .
another hypothesis is that the material includes bi2o3 as a radiopacifier , which might result in progressive mass darkening due to interaction with the organic matrix in blood .
however , after 8 weeks , the discoloration value of the ecz group was not significant regardless of the presence or absence of blood .
we speculated that , because ecz is a fast setting material , it does not allow for absorption of blood , and it does not include bi2o3 , which might explain the absence of progressive discoloration of the specimens .
consequently , the pr / b and ec / b groups showed greater discoloration compared with the ecz group , and contamination with blood adversely affected the color stability of the tooth .
however , the exact mechanism remains unknown , indicating the need for additional research . in this study ,
bovine incisors were used to evaluate tooth discoloration because they have many advantages compared with human teeth .
second , the number of tubules per mm and the tubule diameter of bovine incisors are not significantly different than those of the coronal dentin of human teeth . furthermore , bovine incisors have wide and flat surfaces ; therefore , a standardized sample can be easily obtained .
although bovine incisors have been widely used as specimens for in vitro studies of discoloration , there is a need for additional research using human incisors .
within the limitations of the present study , we concluded that the largest color alteration was observed in the presence of pr and ec compared to ecz .
furthermore , the presence of blood within the cavity adjacent to the materials exacerbated tooth discoloration induced by mta - based pulp - capping materials . however , the type and compounds of the mta - based pulp - capping material play the most significant role in the discoloration .
therefore , ecz induces less discoloration and has the potential as an alternative pulp - dressing material for use in aesthetically sensitive areas .
however , more research is required to evaluate the color stability of mta - based materials in clinical settings .
 objectives : the aim of this study was to evaluate tooth discoloration induced by contact with various calcium silicate - based pulp capping materials in the presence or absence of blood in vitro.materials and methods : eighty bovine samples were divided into six experimental groups and two control groups according to the type of material used ( proroot [ pr ] , endocem [ ec ] , or endocemzr [ ecz ] ) and the presence or absence of contamination with blood . 
 a spectrophotometer was used to calculate the color difference ( e ) between the baseline measurement ( after placement of materials ) and measurements taken 1 , 2 , 4 , and 8 weeks . 
 the results were analyzed with repeated measures analysis of variance , tukey 's post - hoc tests and independent t - tests ( p = 0.05).results : the pr group and ec group showed significantly higher mean values of e than the negative control group after 2 weeks ( p < 0.05 ) , whereas ecz did not . there were larger e values when there was contact with blood , especially in pr and ec group ( p < 0.05).conclusions : ecz which contains zirconium oxide as a radiopacifier showed less discoloration irrespective of blood contamination compared to pr and ec . 
INTRODUCTION
MATERIALS AND METHODS
Tooth preparation
Blood collection
Sample preparation for experimental and control groups
Evaluation of tooth discoloration
Stereomicroscopic observation of representative samples
Statistical analysis
RESULTS
Color differences (
Color differences (
DISCUSSION
CONCLUSION