the use of hydrogen peroxide was first reported by harlan in 1884 , which is currently the most effective bleaching material .
bleaching is known to stimulate patients toward acceptance of additional esthetic dental procedures . with the increasing number of patients seeking esthetic correction
several studies in the literature have shown that the bond strength of enamel decreased after bleaching with concentrated 35% hydrogen peroxide .
a period of up to 3 weeks is required for resin  enamel bond strengths to return to values obtained for unbleached enamel .
this decrease in bond strength can also be reversed by applying a biocompatible and neutral antioxidant such as sodium ascorbate before resin  composite application .
ascorbic acid and its sodium salt are potent antioxidants capable of quenching reactive free radicals in biological systems .
because vitamin c and its salts are non - toxic and widely used in the food industry as antioxidants , it is unlikely that their use on enamel and dentin will create any adverse biological effect or clinical hazards . in the past decade , numerous studies evaluated the effects of peroxide - containing bleaching agents on tooth hard tissues .
however , there are no reports in the literature regarding the effects of 10% sodium ascorbate on the enamel surface .
the molecular constituents of dental hard tissues have been the subject of many studies using a variety of methods , including infrared spectroscopy , electronic microprobe , raman spectroscopy and energy - dispersive x - ray analysis ( edax ) .
x - ray microanalysis using edax is routinely used in the study of matrix elements like phosphate and calcium .
thus , the aim of this in vitro study was to evaluate quantitatively the calcium and phosphorous loss from the enamel surface following bleaching with 35% hydrogen peroxide and reversal with 10% sodium ascorbate using edax .
a total of eight non - carious , freshly extracted human permanent maxillary central incisors without any visible defects were used in this study . after extraction , the teeth were cleaned of any residual tissue tags , pumiced and washed under running tap water .
they were stored in distilled water at + 4c until needed for the study , a period not exceeding 1 week .
the labial surfaces of the teeth were polished with fine grit silicon carbide paper on a water - irrigated metallurgical polishing wheel .
the specimens were stored in artificial saliva except during the bleaching , bonding and testing procedures .
the samples were stored in artificial saliva as described in british standard 7115 , part 2 , bsi london , 1988 .
the artificial saliva with an electrolyte composition similar to human saliva was prepared by mixing sodium chloride  0.5 g , sodium bicarbonate  4.2 g , sodium nitrate 
all the teeth were then mounted in an elastomeric impression material ( aquasil ; dentsply ) for the purpose of applying the bleaching agent and antioxidant gel .
the specimens were bleached with 35% hydrogen peroxide ( opalescence extra ) , exposed to a fast halogen - curing light ( 3000 mw / cm ) for 20 s and left standing for 15 min .
a fresh layer was reapplied , light activated , left standing for another 15 min and further washed away .
sodium ascorbate ( sodium salt of ascorbic acid ) was used for the antioxidant preparation . the antioxidant gel
( 2.5% [ wt / wt ] ) containing sodium ascorbate ( 10% ) was prepared by dispersing the carbopol 976p resin polymer in purified water containing sodium ascorbate under gentle mixing .
the mixture was stirred until thickening occurred and then neutralized by dropwise addition of thriethanolamine until a transparent gel appeared .
sodium ascorbate gel was placed on the enamel surfaces of the embedded teeth for 120 min .
after the antioxidant treatment , the enamel surface was thoroughly rinsed with distilled water for 30 s. the mineral content of each specimen after reversal was measured using edax .
three peak plots per specimen were taken : 
 the first was to evaluate the normal enamel surface prior to any procedure.the second was to evaluate the surface change of the same specimen after bleaching with 35% hydrogen peroxide.the third was to evaluate the surface change of the same specimen after reversal with 10% sodium ascorbate . 
 
the second was to evaluate the surface change of the same specimen after bleaching with 35% hydrogen peroxide .
the third was to evaluate the surface change of the same specimen after reversal with 10% sodium ascorbate .
the calcium and phosphorous content in weight percent of sound , bleached and reversed enamel was tabulated and statistically analyzed .
the calcium and phosphorous content was then converted into ca / p ratio in each group and a range of ca / p ratio was calculated from the obtained data .
results of the present study were subjected to statistical analysis to interpret the significant differences between various ca / p ratios of normal , bleached and reversed specimens .
the specimens were bleached with 35% hydrogen peroxide ( opalescence extra ) , exposed to a fast halogen - curing light ( 3000 mw / cm ) for 20 s and left standing for 15 min .
a fresh layer was reapplied , light activated , left standing for another 15 min and further washed away .
sodium ascorbate ( sodium salt of ascorbic acid ) was used for the antioxidant preparation . the antioxidant gel
( 2.5% [ wt / wt ] ) containing sodium ascorbate ( 10% ) was prepared by dispersing the carbopol 976p resin polymer in purified water containing sodium ascorbate under gentle mixing .
the mixture was stirred until thickening occurred and then neutralized by dropwise addition of thriethanolamine until a transparent gel appeared .
sodium ascorbate gel was placed on the enamel surfaces of the embedded teeth for 120 min . after the antioxidant treatment
, the enamel surface was thoroughly rinsed with distilled water for 30 s. the mineral content of each specimen after reversal was measured using edax .
three peak plots per specimen were taken : 
 the first was to evaluate the normal enamel surface prior to any procedure.the second was to evaluate the surface change of the same specimen after bleaching with 35% hydrogen peroxide.the third was to evaluate the surface change of the same specimen after reversal with 10% sodium ascorbate . 
 
the second was to evaluate the surface change of the same specimen after bleaching with 35% hydrogen peroxide .
the third was to evaluate the surface change of the same specimen after reversal with 10% sodium ascorbate .
the calcium and phosphorous content in weight percent of sound , bleached and reversed enamel was tabulated and statistically analyzed .
the calcium and phosphorous content was then converted into ca / p ratio in each group and a range of ca / p ratio was calculated from the obtained data .
three peak plots per specimen were taken : 
 the first was to evaluate the normal enamel surface prior to any procedure.the second was to evaluate the surface change of the same specimen after bleaching with 35% hydrogen peroxide.the third was to evaluate the surface change of the same specimen after reversal with 10% sodium ascorbate . 
 
the second was to evaluate the surface change of the same specimen after bleaching with 35% hydrogen peroxide .
the third was to evaluate the surface change of the same specimen after reversal with 10% sodium ascorbate .
the calcium and phosphorous content in weight percent of sound , bleached and reversed enamel was tabulated and statistically analyzed .
the calcium and phosphorous content was then converted into ca / p ratio in each group and a range of ca / p ratio was calculated from the obtained data .
results of the present study were subjected to statistical analysis to interpret the significant differences between various ca / p ratios of normal , bleached and reversed specimens .
the elements detected in one representative sample of sound , bleached and reversed enamel are shown in figure 1 , which shows a typical edax plot obtained from the enamel .
table 1 gives the calcium and phosphorous wt% in all eight specimens of sound , bleached and reversed enamel and their calcium : phosphorous ratio .
table 2 gives the mean calcium wt% , phosphorous wt% and mean ca / p ratio of sound , bleached and reversed enamel .
table 3 gives the comparisons of ca / p ratio in sound , bleached and reversed enamel .
energy - dispersive x - ray analysis plot obtained from the enamel spectral calcium and phosphorous concentrations with their corresponding ca / p ratios mean calcium and phosphorous content and the ca / p ratios for sound , bleached and reversed enamel comparison of the ca / p ratios for sound , bleached and reversed enamel the ca / p ratio of sound enamel was 2.13  0.17 . in the bleached enamel ,
this ratio kept on increasing to reach a value of 2.15  0.08 on reversal with antioxidant gel .
the results showed that all the samples subjected to bleaching using 35% hydrogen peroxide showed a statistically significant decrease in the ca / p ratio as compared with samples in which no bleaching procedure was performed ( p - value < 0.01 ) .
the striking finding was that there was a significant increase in the ca / p ratio on application of sodium ascorbate antioxidant gel when compared with the bleached enamel ( p - value < 0.01 ) .
bleaching agents release free radicals as nascent oxygen and hydroxyl or peri - hydroxyl ions when they are applied to dental structures .
these molecules react with electron - rich regions of pigments inside the dental structure , breaking down large pigmented molecules into smaller , less - pigmented ones .
one of the possible adverse effects is that the enamel structure may be weakened by oxidation of the organic and inorganic elements .
hydrogen peroxide and the released free radicals could react with both the organic and the inorganic structures of the enamel .
the reaction between peroxide and the organic materials on the surface or in the subsurface enamel can result in morphological alterations .
ernst et al . evaluated four bleaching agents using scanning electron microscopy ( sem ) .
tong et al . found that 30% hydrogen peroxide treatment with a bleaching light for 30 min caused no measurable loss in the enamel , while another study reported a decrease in the ca / p ratio on the enamel surface with the use of 30% hydrogen peroxide . on the other hand , this dissolved peroxide remnant is also deleterious to the bonding of resinous materials .
this may pose a problem in certain clinical conditions where bleaching may not be the only treatment of choice and requires additional bonding procedures .
in such conditions , recommendations of waiting periods for the application of composite materials on to the bleached enamel surface ranges from 1 day to 6 weeks . however , in order to remove the dissolved peroxide remnants on the bleached enamel , it has been demonstrated that the application of a catalase or 10% sodium ascorbate on the bleached enamel and dentin immediately after the bleaching treatment makes the above - mentioned waiting periods unnecessary . it is proposed that sodium ascorbate may remove the oxidative effect of the bleaching agent .
it is also possible that , by restoring the altered redox potential of the oxidized bonding substrate , sodium ascorbate allows free radical polymerization of the adhesive to proceed without premature termination and hence reverses the compromised bonding .
the bombarding electrons collide with the specimen atoms own electrons , knocking off some of them in the process . a position vacated by an ejected inner shell electron
to do so however the transferring electron must give up some energy by emitting an x - ray .
the edx spectrum is just a plot of how frequently an x - ray is received for each energy level .
an edx spectrum normally displays peaks corresponding to the energy levels for which most of the x - rays had been received .
each of these peaks is unique to the atom and , therefore , corresponds to a single element .
the higher a peak in a spectrum , the more concentrated the element is in the specimen .
calcium and phosphorous are present in the hydroxyapatite crystal , which is the main building block of dental hard tissues .
changes in the calcium / phosphate ratio indicate alterations in the inorganic components of hydroxyapatite .
previous studies have shown that bleaching causes calcium and phosphate ion loss in the dental hard tissues , change in the calcium / phosphate ratio and surface alterations depending on their concentration . in the present study
, the ca / p ratio was altered on the application of 35% hydrogen peroxide .
there was a decrease in the ca / p ratio on the enamel surface following an application of 35% hydrogen peroxide , which was consistent with earlier studies .
this suggested that the resistance of enamel surfaces decreased in these groups when there was an acid attack .
reported a loss of strength and higher solubility of enamel , dentin and cementum after bleaching .
the change in the original ratio between the organic and the inorganic components of the tissues increased the solubility .
the present study showed that there was an increase in the ca / p ratio following the application of sodium ascorbate antioxidant gel , reaching a value equivalent to that of normal enamel .
this increase in the ca / p ratio may be either due to an increase in the phosphorous content or a decrease in the calcium content .
it is well known that the antioxidant gel is not a source of phosphorous ions .
thus , this increase in the ca / p ratio can be attributed to the further loss of calcium ions and , therefore , it is proposed from the present study that antioxidant application restores the normal ca / p ratio of the tooth but causes a decrease in the actual calcium content of the tooth .
the findings of this in vitro study may not be representative of the in vivo condition in which the oral cavity is continuously bathed with saliva that contains various minerals , lipids , carbohydrates , proteins and other substances .
according to the results and within the limitations of this in vitro study , it can be concluded that 35% hydrogen peroxide causes a significant decrease in the ca / p ratio .
this decrease in the ca / p ratio can be restored by the application of 10% sodium ascorbate antioxidant gel .
further studies are required to evaluate the actual calcium loss , the reactions of the antioxidant gel with tooth structure and the alterations of enamel surface caused by antioxidant application .
 objectives : the study assessed quantitatively the calcium and phosphorous loss from the enamel surface following bleaching with 35% hydrogen peroxide and reversal with 10% sodium ascorbate using energy - dispersive x - ray analysis ( edax).materials and methods : eight non - carious , freshly extracted human permanent maxillary central incisors without any visible defects were used . 
 each specimen was bleached with 35% hydrogen peroxide activated by light and reversed with sodium ascorbate antioxidant gel . 
 the calcium and phosphorous content in weight percent of sound , bleached and reversed enamel was acquired using edax . 
 the ca / p ratio was calculated from the obtained data . 
 one - way anova followed by post hoc tukey test was used for comparing the ca / p ratio of sound , bleached and reversed enamel.results:all the samples subjected to bleaching using 35% hydrogen peroxide showed a statistically significant decrease in the ca / p ratio as compared with samples in which no bleaching procedure was performed ( p - value < 0.01 ) . 
 the striking finding was that there was a significant increase in the ca / p ratio on application of sodium ascorbate antioxidant gel when compared with the bleached enamel ( p - value < 0.01).conclusion : the authors concluded that 35% hydrogen peroxide causes a significant decrease in the ca / p ratio . 
 this decrease in the ca / p ratio can be restored by the application of 10% sodium ascorbate antioxidant gel . 
Introduction
Materials and Methods
Bleaching procedure
Preparation of antioxidant gel
Antioxidant application
Quantitative evaluation
EDAX analysis
Statistical analysis
Results
Discussion
Conclusion