vital tooth bleaching has widely been performed , and several materials and techniques have been presented for this purpose16 .
vital tooth bleaching is considered a safe , effective , minimally invasive , non - destructive and well - accepted procedure for the treatment of discolored teeth27,29 .
a number of methods and approaches have been described for the bleaching of vital teeth17 .
however , basically , there are three techniques : in - office or power bleaching , mass market bleaching products , and dentist - supervised home bleaching15 . since its introduction by haywood and heymann14 ( 1989 ) , several products have been employed for home bleaching .
these products are mainly available in gels or strips containing several concentrations of carbamide peroxide or hydrogen peroxide29 .
carbamide peroxide gel at a 10% concentration is one of the most commonly employed home bleaching agents due to its safety and effectiveness12 .
although different concentrations of carbamide and hydrogen peroxides have shown similar results10,18,29 , increased concentrations and application times may cause enamel surface alterations , such as loss of mineral content and microhardness2,22 .
thus , considering the divergences on the effects of bleaching agents on properties of hard tooth tissues , the purposes of this study were to evaluate the effectiveness of different home bleaching agents and their effects on surface and subsurface microhardness of bovine enamel .
the hypotheses tested in this study were that the type of bleaching agent influences the color alteration in stained teeth , but does not influence the surface and subsurface microhardness of bovine enamel .
forty - five freshly extracted sound bovine incisors were selected and stored under refrigeration in a saturated thymol solution until preparation for testing .
teeth had their roots removed 2 mm apically to the cementoenamel junction in a sectioning machine with a water - cooled diamond saw ( minitom , struers a / s , copenhagen , d2610 , denmark ) .
the buccal surfaces of the crowns were cut longitudinally and transversely , using double - faced diamond discs mounted at a low - speed handpiece in order to obtain fragments with dimensions of 11 mm x 11 mm . to obtain plane surfaces , required for microhardness tests ,
the external enamel surfaces of the fragments were flattened in a water - cooled polishing machine ( politriz ; struers a / s ) using 600- , 1000- and 1200-grit silicon carbide abrasive papers sequentially , followed by a final polishing with a 0.3-m - gamma alumina suspension on felt wheel .
, so carlos , sp , brazil ) in distilled and deionized water for 10 min to remove polishing debris .
artificial staining of the specimens was performed following a modification5 of the method proposed by freccia and peters8 .
blood samples collected from adult male wistar rats ( weighing 200 to 250 g ) were heparinised to avoid coagulation , and were centrifuged at 10,000 rpm for 10 min .
the blood serum was discarded and 40 ml of distilled and deionized water were added to 60 ml of the precipitated blood .
this mixture was centrifuged at 10,000 rpm for 20 min , resulting in a rich - hemoglobin hemolyzate blood solution .
specimens were immersed in this solution for 4 days , performing a centrifugation cycle ( 10,000 rpm , 20 min ) at every 24 h. after 4 days , the specimens were removed , washed in distilled water , dried with absorbent paper and kept at 37c in 100% relative humidity for 15 days . after staining , the specimens were randomly assigned to 3 groups ( n=15 ) according to the bleaching treatment : 10% carbamide peroxide gel ( cp10 ) , 16% carbamide peroxide gel ( cp16 ) and 6.5%-hydrogen - peroxide - based strip ( hp6.5 ) .
the bleaching treatment was performed over 21 days , according to the manufacturers ' instructions .
for each specimen of the groups treated with the bleaching gels ( cp10 and cp16 ) , a tray was fabricated using a low - density polyethylene sheet and a vacuum - thermoforming machine .
every day , a thin layer of each gel was applied on the enamel surface and the tray was placed on each specimen , which was stored for 8 h at 37c in a recipient containing artificial saliva ( ph 7.0 - figure 1 ) .
after 8 h , the gel was removed from enamel surface with running distilled water for 15 s. enamel surfaces of the group treated with the bleaching strip ( hp6.5 ) were covered with the strip for 30 min twice a day .
after each application , the strips were removed from enamel surfaces , which were rinsed with running distilled water for 15 s. in all groups , when the specimens were not in contact with the bleaching agents , they were kept at 37c immersed in artificial saliva , which was changed daily .
the color of the specimens was measured after the artificial staining ( baseline ) and 7 , 14 and 21 days after the beginning of the bleaching treatment . before each color measurement
color was measured over a white background employing a color spectrophotometer ( color guide 45/0 , pcb 6807 ; byk - gardner , gmbh gerestsried , germany ) , which records the color variables l * , a * , b * according to the ciel*a*b * ( commission internationale de l'eclairage l * , a * , b * ) color system18 , where l * stands for luminosity dimension or whiteness , ranging from 0 ( pure black ) to 100 ( reference white ) , a * for green - red contrast ( -a*= green and + a*= red ) and b * for blue - yellow contrast ( -b*= blue and + b*= yellow ) . color change ( e ) was calculated from l * , a * and b * values employing the following formula : e= [ ( l * ) 
  + ( a * ) 
  + ( ab * ) 
 ] .
positive l * means the specimens became whiter , while negative l * means specimens became darker .
knoop microhardness measurements of the enamel surface were performed before the staining procedure ( initial values ) and following the bleaching treatment ( after 21 days ) using a microhardness tester ( hmv-2000 ; shimadzu corporation , kyoto , japan ) .
the specimens were individually fixed in the device in such a way that the test surface was perpendicular to the micro - indenter tip .
three indentations ( 100 g load , 30 s ) equally spaced over a circle and not closer than 1 mm to the adjacent indentations or the margin of the specimen were taken and the average was calculated .
the post - bleaching measurements were repeated for all the specimens at locations near to the previous series of indentations .
specimens were individually included in acrylic resin blocks to facilitate that they were perpendicularly bisected by halves .
one crosssectioned face of each specimen was ground and polished following the protocol initially described .
measurements of subsurface microhardness were accomplished at distances of 50 m , 100 m , 150 m and 200 m from the external enamel surface . at each distance , three linearly 100-m - spaced indentations were performed and the mean was calculated .
data from color change and microhardness testing were analyzed for homogeneity and normality and were subjected to anova and tukey 's test at a 5% significance level .
forty - five freshly extracted sound bovine incisors were selected and stored under refrigeration in a saturated thymol solution until preparation for testing .
teeth had their roots removed 2 mm apically to the cementoenamel junction in a sectioning machine with a water - cooled diamond saw ( minitom , struers a / s , copenhagen , d2610 , denmark ) .
the buccal surfaces of the crowns were cut longitudinally and transversely , using double - faced diamond discs mounted at a low - speed handpiece in order to obtain fragments with dimensions of 11 mm x 11 mm . to obtain plane surfaces , required for microhardness tests ,
the external enamel surfaces of the fragments were flattened in a water - cooled polishing machine ( politriz ; struers a / s ) using 600- , 1000- and 1200-grit silicon carbide abrasive papers sequentially , followed by a final polishing with a 0.3-m - gamma alumina suspension on felt wheel .
, so carlos , sp , brazil ) in distilled and deionized water for 10 min to remove polishing debris .
artificial staining of the specimens was performed following a modification5 of the method proposed by freccia and peters8 .
blood samples collected from adult male wistar rats ( weighing 200 to 250 g ) were heparinised to avoid coagulation , and were centrifuged at 10,000 rpm for 10 min .
the blood serum was discarded and 40 ml of distilled and deionized water were added to 60 ml of the precipitated blood .
this mixture was centrifuged at 10,000 rpm for 20 min , resulting in a rich - hemoglobin hemolyzate blood solution .
specimens were immersed in this solution for 4 days , performing a centrifugation cycle ( 10,000 rpm , 20 min ) at every 24 h. after 4 days , the specimens were removed , washed in distilled water , dried with absorbent paper and kept at 37c in 100% relative humidity for 15 days .
after staining , the specimens were randomly assigned to 3 groups ( n=15 ) according to the bleaching treatment : 10% carbamide peroxide gel ( cp10 ) , 16% carbamide peroxide gel ( cp16 ) and 6.5%-hydrogen - peroxide - based strip ( hp6.5 ) .
the bleaching treatment was performed over 21 days , according to the manufacturers ' instructions .
for each specimen of the groups treated with the bleaching gels ( cp10 and cp16 ) , a tray was fabricated using a low - density polyethylene sheet and a vacuum - thermoforming machine .
every day , a thin layer of each gel was applied on the enamel surface and the tray was placed on each specimen , which was stored for 8 h at 37c in a recipient containing artificial saliva ( ph 7.0 - figure 1 ) .
after 8 h , the gel was removed from enamel surface with running distilled water for 15 s. enamel surfaces of the group treated with the bleaching strip ( hp6.5 ) were covered with the strip for 30 min twice a day .
after each application , the strips were removed from enamel surfaces , which were rinsed with running distilled water for 15 s. in all groups , when the specimens were not in contact with the bleaching agents , they were kept at 37c immersed in artificial saliva , which was changed daily .
the color of the specimens was measured after the artificial staining ( baseline ) and 7 , 14 and 21 days after the beginning of the bleaching treatment . before each color measurement
color was measured over a white background employing a color spectrophotometer ( color guide 45/0 , pcb 6807 ; byk - gardner , gmbh gerestsried , germany ) , which records the color variables l * , a * , b * according to the ciel*a*b * ( commission internationale de l'eclairage l * , a * , b * ) color system18 , where l * stands for luminosity dimension or whiteness , ranging from 0 ( pure black ) to 100 ( reference white ) , a * for green - red contrast ( -a*= green and + a*= red ) and b * for blue - yellow contrast ( -b*= blue and + b*= yellow ) . color change ( e ) was calculated from l * , a * and b * values employing the following formula : e= [ ( l * ) 
  + ( a * ) 
  + ( ab * ) 
 ] .
positive l * means the specimens became whiter , while negative l * means specimens became darker .
knoop microhardness measurements of the enamel surface were performed before the staining procedure ( initial values ) and following the bleaching treatment ( after 21 days ) using a microhardness tester ( hmv-2000 ; shimadzu corporation , kyoto , japan ) .
the specimens were individually fixed in the device in such a way that the test surface was perpendicular to the micro - indenter tip .
three indentations ( 100 g load , 30 s ) equally spaced over a circle and not closer than 1 mm to the adjacent indentations or the margin of the specimen were taken and the average was calculated .
the post - bleaching measurements were repeated for all the specimens at locations near to the previous series of indentations .
specimens were individually included in acrylic resin blocks to facilitate that they were perpendicularly bisected by halves .
one crosssectioned face of each specimen was ground and polished following the protocol initially described .
measurements of subsurface microhardness were accomplished at distances of 50 m , 100 m , 150 m and 200 m from the external enamel surface . at each distance , three linearly 100-m - spaced indentations were performed and the mean was calculated .
data from color change and microhardness testing were analyzed for homogeneity and normality and were subjected to anova and tukey 's test at a 5% significance level .

 table 1 displays the mean values ( and standard deviations ) of color change ( e * , l * , a * , and b * values ) at each time point .
the e * values produced by the bleaching agent cp16 were similar to those of cp10 , and the e * values produced by both cp16 and cp10 were significantly superior to those of hp6.5 .
the e * values obtained at each time point revealed that the color changes at 21 days were superior to those observed at 7 days , but similar were similar to those occurred at 14 days .
the bleaching agents x time interaction showed that , for cp16 , there was no statistically significant difference between 14 and 21 days . for both cp10 and hp6.5 , the e * values were statistically significant only at 21 days .
the means and standard deviations of surface microhardness and subsurface microhardness are shown in table 2 and table 3 , respectively .
the analysis of data did not reveal significant differences among the studied factors neither in the surface nor in the subsurface microhardness .

 table 1 displays the mean values ( and standard deviations ) of color change ( e * , l * , a * , and b * values ) at each time point .
the e * values produced by the bleaching agent cp16 were similar to those of cp10 , and the e * values produced by both cp16 and cp10 were significantly superior to those of hp6.5 .
the e * values obtained at each time point revealed that the color changes at 21 days were superior to those observed at 7 days , but similar were similar to those occurred at 14 days .
the bleaching agents x time interaction showed that , for cp16 , there was no statistically significant difference between 14 and 21 days . for both cp10 and hp6.5 , the e * values were statistically significant only at 21 days .
the means and standard deviations of surface microhardness and subsurface microhardness are shown in table 2 and table 3 , respectively .
the analysis of data did not reveal significant differences among the studied factors neither in the surface nor in the subsurface microhardness .
when the objective of an in vitro study is to evaluate the effectiveness of bleaching agents , it is very difficult to observe differences in the effect of bleaching techniques on samples without staining7 .
thus , the present study used the method proposed by freccia and peters8(1982 ) for artificial staining of extracted teeth .
in addition to being reliable , safe and easily reproducible , this method simulates one of the main causes of intrinsic tooth discoloration that is the oxidation of hemoglobin inside dentinal tubules3 .
shade guides , photography , colorimeters or computer digitization can be employed to assess tooth color changes20,25,28 .
tooth color determination employing the commonly cited shade - based guides have limitations as they are subjected to examiner and environmental factors that can potentially influence classification of color9,19 and hamper the visualization of color variation existent among the thirds of a tooth crown26 .
the ciel*a*b * three - dimensional color space system is the most frequently quoted index employed in dental bleaching research and can be generated from colorimeters , spectrophotometers or digital image analysis7,11,28 
  in the present study , the 16% carbamide peroxide bleaching agent was more effective than the 10% carbamide peroxide gel and the strip containing 6.5% hydrogen peroxide .
the reason why 16% carbamide peroxide gave the highest change in l * values is probably its extended contact time ( 8 h / day ) with enamel compared to the considerably shorter contact time of the 6.5% hydrogen peroxide gel ( 30 min twice a day ) .
it is known that the outcome of a bleaching procedure depends mainly on the concentration of the bleaching agent , the ability ofthe agent to reach the chromophore molecules , and the duration and frequency that the agent is in contact with chromophore molecules6 .
moreover , the difference among the bleaching systems could be explained by the different kinetics of hydrogen peroxide and carbamide peroxide .
hydrogenperoxide acts as a strong oxidizing agent through the formation of free radicals23 , reactive oxygen molecules , and hydrogen peroxide anions13 .
these reactive molecules attack the long - chained , dark - colored chromophore molecules and split them into smaller , less colored and more diffusible molecules .
carbamide peroxide also yields urea that theoreticallycan be further decomposed to carbon dioxide and ammonia .
the influence of hydrogen peroxide and carbamide peroxide on enamel and dentin properties , such as surface morphology and chemistry , surface and subsurface ultrastructure and microhardness , has been extensively investigated in the literature . according to a recent review16 ,
the most frequently employed technique for evaluating the effects of peroxide and bleaching products on enamel and dentin has been surface microhardness .
surface microhardness testing is a simple method for determining the mechanical properties of enamel and dentin surfaces and is related to a loss or gain of mineral of the dental structure16 . on account of the acidic properties of the bleaching agents , changes in the mineral content of dental hard tissues may occur , leading a decrease in the microhardness values after the bleaching treatment22 .
however , in the present study , neither the enamel surface microhardness nor the enamel subsurface microhardness was affected by the different bleaching agents employed .
as the specimens were maintained in artificial saliva during and after the bleaching treatment , it may be assumed that this solution promoted a continuous mineral intake due to the presence of calcium and phosphate ions in its composition , justifying the absence of microhardness changes .
this also demonstrates that peroxide bleaching products have no significant effects on microhardness , and contrasting results , in general , reflect some methodological limitations that do not represent the clinical situation or employ highly acidic agents16 .
furthermore , decrease in microhardness can be recovered in a post - bleaching period , following remineralization from saliva storage21,22 .
it may be concluded that the 16% carbamide peroxide gel was the most effective in bleaching stained bovine enamel without causing changes in the microhardness of this substrate .
 objectives : this study evaluated the effectiveness of different home bleaching agents on color alteration and their influence on surface and subsurface microhardness of discolored bovine enamel.material and methods : forty - five fragments of bovine incisors were randomly allocated into 3 groups ( n=15 ) according to the bleaching agent : 10% carbamide peroxide gel ( cp10 ) , 16% carbamide peroxide gel ( cp16 ) and 6.5%-hydrogen - peroxide - based strip ( hp6.5 ) . before bleaching treatment , initial values of knoop surface microhardness and color ( ciel*a*b * ) were obtained and the fragments were artificially stained in hemolyzed rat blood . 
 then , bleaching treatments were performed over a 21-day period . 
 color changes ( e ) were assessed at 7 , 14 and 21 days , and final surface microhardness reading was done after 21 days . 
 thereafter , the fragments were bisected to obtain subsurface microhardness . 
 data were subjected to anova and tukey 's tests ( =5%).results : color changes produced by cp16 were similar to those of cp10 , and the color changes produced by these materials were significantly superior to those produced by hp6.5 . 
 color changes at 21 days were superior to 7 days and similar to 14 days . 
 the time did not influence color changes for cp16 , which showed similarity between the 14- and 21-day results . 
 no statistically significant differences were found among the home bleaching agents for surface and subsurface microhardness.conclusions:microhardness of bovine enamel was not affected by the bleaching agents . 
 the 16% carbamide peroxide gel was the most effective for bleaching the stained substrate . 
INTRODUCTION
MATERIAL AND METHODS
Enamel Specimen Preparation
Staining Procedure
Bleaching Treatment
Color Measurement
Microhardness Testing
Statistical Analysis
RESULTS
Color Change
Microhardness Test
DISCUSSION
CONCLUSION