EVALUATION OF THE BLEACHED HUMAN ENAMEL BY SCANNING ELECTRON MICROSCOPY

ince bleaching has become a popular procedure, the effect of peroxides on dental hard tissues is of great interest in research. Purpose: The aim of this in vitro study was to perform a qualitative analysis of the human enamel after the application of in-office bleaching agents, using Scanning Electron Microscopy (SEM). Materials and Methods: Twenty intact human third molars extracted for orthodontic reasons were randomly divided into four groups (n=5) treated as follows: G1storage in artificial saliva (control group); G2four 30-minute applications of 35% carbamide peroxide (total exposure: 2h); G3four 2-hour exposures to 35% carbamide peroxide (total exposure: 8h); G4two applications of 35% hydrogen peroxide, which was light-activated with halogen lamp at 700mW/cm2 during 7min and remained in contact with enamel for 20min (total exposure: 40min). All bleaching treatments adopted in this study followed the application protocols advised by manufacturers. Evaluation of groups submitted to 35% carbamide peroxide was carried out after two time intervals (30 minutes and 2 hours per session), following the extreme situations recommended by the manufacturer. Specimens were prepared for SEM analysis performing gold sputter coating under vacuum and were examined using 15kV at 500x and 2000x magnification. Results: Morphological alterations on the enamel surface were similarly detected after bleaching with either 35% carbamide peroxide or 35% hydrogen peroxide. Surface porosities were characteristic of an erosive process that took place on human enamel. Depression areas, including the formation of craters, and exposure of enamel rods could also be detected. Conclusion: Bleaching effects on enamel morphology were randomly distributed throughout enamel surface and various degrees of enamel damage could be noticed. Clinical significance: In-office bleaching materials may adversely affect enamel morphology and therefore should be used with caution. Uniterms: Hydrogen peroxide; Carbamide peroxide; Tooth bleaching.


INTRODUCTION
Esthetics has strongly influenced patient's perceptions and appealed to the development and expansion of the cosmetic industry.Inevitably, the request of whiter teeth has become a major concern in dental practice since discoloration of a single tooth or a group of teeth negatively interferes with the harmony of smile.Although tooth color is only one of the aspects involved in facial harmony, it represents the most important isolated factor because it is immediately noticed 1 .
Esthetic dentistry has introduced various corrective measures to treat discolored teeth, such as laminate veneers, direct resin restorations, crowns or fixed prostheses.Dental bleaching represents a conservative treatment option or may become an auxiliary therapy when restorative procedures are required to eliminate color abnormalities 2 .
Bleaching agents are provided for in-office or at-home therapies.In-office bleaching requires less patient's cooperation, but longer sessions are necessary in the dental office, which increases the treatment costs.Otherwise, athome bleaching is less expensive, but patient's collaboration is essential to obtain successful treatment results 3 .
Hydrogen peroxide is the main active ingredient of bleaching agents and the whitening effect it produces is due to its low molecular weight and its ability to penetrate through enamel and dentin 1,4 .The basic process involves oxidation and reduction reactions that convert organic pigments into carbon dioxide and water 1,5 .Carbamide peroxide decomposes into urea and hydrogen peroxide 5 .Therefore, products containing the same concentration but different active ingredients are expected to behave differently on dental hard tissues.Such behavior might become even more evident when considering regular or light-activated gels or distinct exposure periods.
Although dental hard tissues are highly mineralized, their organic content can play an important role in the bleaching process.It is speculated that the reaction between peroxide and organic materials on the surface or in the subsurface of enamel can result in morphological alterations 6 .
Despite the advantages offered by the bleaching treatment, the effect of bleaching agents on dental hard tissues is rather controversial.Some studies have demonstrated no significant effects of bleaching agents on human enamel 4,7 .Contrary results have shown morphological alterations on bleached enamel, thus suggesting an erosive process 8,9,10,11 .
Since bleaching has been widely advocated by dental professionals, the effect of bleaching agents on dental hard tissues is of great interest in research.Therefore, the purpose of this in vitro study was to investigate the influence of inoffice bleaching agents on human enamel morphology, comparing 35% carbamide peroxide or 35% light-activated hydrogen peroxide.

MATERIALS AND METHODS
This study was developed in accordance with the Code of Ethics in Research (nº063/2001 PH/CEP).Twenty intact human third molars extracted for orthodontic reasons were selected for this study.Immediately after extraction, periodontal tissue was removed and teeth were stored in saline solution at -20°C for a maximum period of 28 days 12 .Prior to group division, teeth were examined at a stereomicroscope Stemi 2000 (Carl Zeiss, Jena, Germany) at 20x magnification to select teeth without cracks or structural defects that could compromise the results of the study.
All teeth were submitted to mesio-distal crosscut sections to separate buccal and lingual fragments.Horizontal sections were performed at the cementoenamel junction, separating coronary and root pieces.Buccal sections were selected for the SEM analysis and randomly divided into four groups of five specimens each, treated as follows: • Group 1: no bleaching treatment was performed.All buccal sections were kept in artificial saliva at 37ºC until SEM analysis was performed.
• Group 2: buccal sections were placed in contact with 35% carbamide peroxide (Opalescence Quick, batch #479B, Ultradent, South Jordan, Utah, United States), facing a Petri dish containing the gel.Product was heated in warm water prior to use, following the manufacturer's recommendation.Specimens were kept in contact with the bleaching agent for 30 minutes.Next, specimens were rinsed with distilled water and stored in artificial saliva at 37°C.This procedure was repeated weekly in four applications (total exposure: 2h).
• Group 3: bleaching protocol followed steps determined for group 3, but adopting a longer exposure period.Twohour applications of 35% carbamide peroxide (Opalescence Quick, batch #479B, Ultradent, South Jordan, Utah, United States) were performed weekly for four weeks (total exposure: 8h).Variation in the exposure period was determined since the manufacturer suggested a minimum of 30-minute and maximum of 2-hour exposure.Therefore, minimum time exposure was investigated in group 2 while maximum exposure period was evaluated in group 3.
• Group 4: two 20-minute applications of 35% hydrogen peroxide (Opalescence X-tra, batch #532B, Ultradent, South Jordan, Utah, United States) were performed in accordance to manufacturer's recommendation.Gel was applied on enamel specimens and light-activation was performed initially for 5 minutes.The light source used was Curing Light XL3000 (3M ESPE) at 700mW/cm 2 .Four extra 30second light-activations were carried out every 3.5min during the 15-minute period that the gel remained in contact with enamel.Therefore, total light activation was performed for 7 minutes during gel contact of 20 minutes.Teeth were rinsed with distilled water to remove the gel and bleaching protocol was repeated (total exposure: 40 minutes).

Scanning Electron Microscopy (SEM) investigation
Twenty-four hours after bleaching, specimens were dehydrated in ascending grades of ethanol.Twenty-minute immersion in each solution was performed at concentrations of 50%, 60%, 70%, 80%, and 90% ethanol.Sixty-minute immersion in 100% ethanol was carried out.Specimens dried at room temperature during 12 hours.Crowns were fixed in aluminum stubs and then sputter-coated with goldpalladium (Desk II, Denton Vacuum, Moorestown, NJ, United States) during 2 minutes.
Enamel surface was examined at 500x and 2000x magnification with scanning electron microscope (JMS 5310, JEOL, Tokyo, Japan), using 15kV.A scanning of the entire buccal surface was performed and most critical areas were selected for scanning electron microscope photomicrographs.

RESULTS
Figure 1 depicts unbleached enamel.Although the surface is not completely smooth, uniformity of the aprismatic surface layer can be observed.Pores and superficial irregularities were also present.
Effects of bleaching were randomly distributed on enamel surface after the application of in-office products (Figures 2, 3, 4, and 5).In fact, different types of defects and distinct severity of such events could be observed throughout the enamel surface.Modification of enamel morphology was detected either after 35% carbamide peroxide (Figures 2 and  3) or 35% hydrogen peroxide applications (Figures 4 and 5).In general, it seems that bleaching evidenced original irregularities present on enamel surface.Morphologic surface alterations became much more pronounced after bleaching, including partial removal of the aprismatic layer, increased depth of enamel grooves, and exposure of the enamel prisms (Figures 2B, 4A, 4B and 5B).An increased number of porosities on enamel surface could be noticed (Figures 2A and 5A), when compared to the control group (Figure 1).Intermittent depressions of various depths were present (Figures 3A, 3B, 4A and 5B); craters (Figure 2B), and shallow erosions (Figures 2A and 5A) could also be observed.
Similar observations were detected on enamel morphology of teeth treated with either conventional carbamide peroxide gel or light-activated hydrogen peroxide.It must be emphasized, however, that the irregularities found on enamel surface were more frequently distributed in the experimental groups, but were also detected on the unbleached enamel.

DISCUSSION
Effects of bleaching agents on enamel surface have been discussed recently.When considering in-office bleaching agents, controversial results have been reported.Thirtyfive percent hydrogen peroxide applications did not produce morphological alterations on enamel in research conducted by Ernst, el al. 7 .Likewise, Gultz, et al. 13 , who adopted the same methods of this study and tested the same bleaching agents (Opalescence Quick and Opalescence X-tra), did not report significant alterations on the enamel surface.However, exposure periods determined in their study were shorter (total exposures of 2 hours for 35% carbamide peroxide and 8 to 10 minutes for 35% light-activated hydrogen peroxide) than those stipulated for this investigation (total exposures of 2 or 8 hours for 35% carbamide peroxide and 40 minutes for 35% light-activated hydrogen peroxide).
In this study, defects could be randomly detected on the enamel surface with distinct severity after bleaching.Similar behavior has been reported in previous studies, even after 10% carbamide peroxide bleaching, which releases lower amounts of hydrogen peroxide when compared to in-office bleaching agents.Surface erosion 9 , depressions 14 , porosity, and increased depth of enamel grooves 6 were some of the alterations mentioned.
More severe alterations could be expected on enamel surface after in-office bleaching.Increased porosity 14,15 and precipitate deposition 16 characterized enamel erosion 9,17,18 .
Depressions 6,19 with crater formation, removal of the aprismatic layer and exposure of the enamel prisms 15,17 could also be detected.Hegedüs, et al. 6 detected a pronounced increase in the depth of enamel grooves after 30% hydrogen peroxide bleaching.McGuckin, et al. 19 reported morphological alterations on enamel surface, describing enamel patterns similar to type II acid etching, after 30% hydrogen peroxide applications.
Although 35% hydrogen peroxide presents higher concentration of free hydrogen peroxide when compared to 35% carbamide peroxide gel, no visual difference was detected between groups treated with such products.Similar results were probably observed due to the shorter exposure periods and fewer sessions determined for the light-activated gel.Authors believe that enamel alterations are probably due to an initial process of demineralization.McCracken and Haywood 20 and Rotstein, et al. 21have proven loss of calcium in teeth exposed to peroxide.Rotstein, et al. 22 observed lower resistance and higher solubility of dental hard tissues after bleaching procedures, possibly due to modification of the organic and inorganic ratio of the tissues.Oltu and Gürgan 23 observed changes in the inorganic composition of the bleached enamel after 35% carbamide peroxide exposure.Indeed, most bleaching agents are acidic, which is not favorable to enamel, dentin, and cement.The pH of bleaching agents investigated in this study was 6.4 and 3.8 for 35% carbamide peroxide (Opalescence Quick) and 35% hydrogen peroxide (Opalescence X-tra), respectively.
Although artificial saliva was used in this study to simulate oral conditions, its remineralization potential is not equivalent to natural saliva in vivo.Fewer alterations are expected in vivo, due to the buffer capacity of saliva.Rodrigues, et al. 24 reminded that saliva and fluoride ions present in oral care substances are essential to equilibrate the demineralization and remineralization processes.According to Lopes, et al. 25 , pH values as low as 6.4 may remove minerals from teeth in in vitro situations; thus, artificial saliva might not be able to guarantee remineralization.
In vivo or in vitro bleaching, various concentrations and exposure periods to bleaching agents, use of different substances prior to treatment, sample size, and evaluation criteria are distinct among different research studies.Thus, a comparison between studies becomes rather difficult.However, most papers are in agreement with findings reported in this study, which detected significant changes on the enamel surface after bleaching with concentrated hydrogen or carbamide peroxide.Therefore, caution is required when performing in-office bleaching.
Significant changes in the enamel morphology and microstructure produced by bleaching agents are suggestive of alterations in physical and mechanical properties of dental hard tissues 10,25,26,27 .Indeed, further studies are necessary to completely understand the effects of bleaching agents on dental tissues.

CONCLUSION
SEM qualitative investigation demonstrated that inoffice bleaching agents affected human enamel morphology, producing porosities, depressions, craters, increased depth of enamel grooves, and partial removal of enamel prisms.These defects were randomly distributed and affected the enamel surface at various degrees.

Clinical implications
In-office bleaching materials may adversely affect enamel morphology and therefore should be used with caution.

FIGURE 2 -
FIGURE 2-Photomicrographs of bleached enamel after 35% carbamide peroxide applications at 500x (A and B) and 2000x magnifications (C and D): removal of the aprismatic layer, craters, pores, and erosion 207

FIGURE 3 -
FIGURE 3-Photomicrographs of bleached enamel after 35% carbamide peroxide applications at 500x (A and B) and 2000x magnifications (C and D): depressions and increased depth of enamel irregularities

FIGURE 4 -
FIGURE 4-Photomicrographs of bleached enamel after 35% hydrogen peroxide applications at 500x (A and B) and 2000x magnifications (C and D): depressions, removal of the aprismatic layer, and increased depth of enamel irregularities

FIGURE 5 -
FIGURE 5-Photomicrographs of bleached enamel after 35% hydrogen peroxide applications at 500x (A and B) and 2000x magnifications (C and D): partial removal of the aprismatic layer, pores, depressions and erosions