Danilo Barral de Araújo, Luciana Rodrigues Silva, Elisângela de Jesus Campos, Roberto Paulo Correia de Araújo

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1 Original Research In vitro study on tooth enamel lesions related to whitening dentifrice Danilo Barral de Araújo, Luciana Rodrigues Silva, Elisângela de Jesus Campos, Roberto Paulo Correia de Araújo Institute of Health Sciences, Federal University of Bahia, Salvador, Bahia, Brazil Received : Review completed : Accepted : ABSTRACT Background: The tooth whitening substances for extrinsic use that are available in Brazil contain hydrogen peroxide or carbamide peroxide. Several studies have attributed the appearance of lesions in the enamel morphology, including hypersensitivity, to these substances. Such lesions justify fluoride therapy and application of infrared lasers, among other procedures. However, there is no consensus among researchers regarding the relevance of the severity of lesions detected on the tooth surface. Objectives: The present study was carried out with an aim of evaluating in vitro the effects of the hydrogen peroxide, carbamide peroxide and sodium bicarbonate contained in dentifrice formulations, on human tooth enamel. Materials and Methods: After darkening process in laboratory, human premolars were brushed using dentifrice containing the two whitening substances (Rembrandt carbamide peroxide and Mentadent hydrogen peroxide) and the abrasive product (Colgate sodium bicarbonate). The degree of specimen staining before and after this procedure was determined using spectrophotometry. Scanning electron microscopy (SEM) was used to obtain images, which were analyzed to show the nature of the lesions that appeared on the enamel surface. Results: The effectiveness of the whitening caused by hydrogen peroxide and carbamide peroxide and the abrasion caused by bicarbonate were confirmed, given that the treated test pieces returned to their original coloration. Based on SEM, evaluation of the enamel surfaces subjected to the test products showed that different types of morphologic lesions of varying severity appeared. Conclusions: Whitening dentifrice containing hydrogen peroxide and carbamide peroxide produced lesions on the enamel surface such that the greatest sequelae were associated with exposure to hydrogen peroxide. Key words: Enamel lesions, hydrogen peroxide, sodium bicarbonate, tooth whitening, whitening dentifrice Tooth whitening is a dental procedure that fulfills an esthetic function. This technologic resource for resolving tooth color changes can be classified as extrinsic or intrinsic. Staining of internal origin may arise pre-or post-eruptively. Such staining also results from trauma to the teeth that causes internal hemorrhage, independent of whether pulp vitality Address for correspondence: Dr. Danilo Barral de Araújo danilobarral81@hotmail.com Quick Response Code: Access this article online Website: PMID: *** DOI: / is maintained or of the natural aging process. [1,2] Extrinsic whitening is a procedure considered to be effective for reestablishing the esthetics of darkened teeth and the appearance of the smile, given that it makes it possible to remove stains, consequently changing the color of the tooth without causing great harm to the tooth enamel. [3] Both the darkening process and the whitening mechanism are only possible because of the relative permeability of the tooth structures. Thus, the greater the penetration of the whitening substance into the tooth, the greater is the quantity of pigmentation that can be removed, and, therefore, the better are the esthetic results obtained. Whitening substances are characterized by the biochemical reaction that triggers the rupture of pigmented molecules that have impregnated the tooth structures, thereby 770

2 lightening them. They consequently produce a significant reduction in the intensity of staining, thereby whitening the tooth under treatment. [4] The capacity of whitening agents to promote the decomposition of pigment-generating organic materials has been attributed by several authors to their oxidizing power, through the release of free radicals, including oxygen. Because these free radicals have unpaired electrons in the outermost electron shell of their atoms, they have a strong tendency toward interaction with other electrons that are under the same conditions, in order to stabilize their incomplete orbits. [4-6] Some important reports have recorded the appearance of adverse effects on tooth structure (loss of substance) and on the neighboring tissues (irritation and/or scaling). The appearance of hypersensitivity during or after whitening has been mentioned as the most frequently seen effect. This finding raises the hypothesis that once whitening agents have infiltrated into the tooth tissue, they are capable of producing morphologic changes to the structure or molecular composition of the tooth. [7] Nevertheless, this point is still very controversial, considering the lack of consensus among researchers. Carbamide peroxide is an effective whitening agent and is very safe to use, with regard to the risk of tooth structure demineralization. [8] This safety is due to the products that result from the decomposition of this whitening agent: urea, ammonia, carbonic acid and hydrogen peroxide. The low molecular weight of urea (60 g/mol) allows it to flow freely through the enamel and dentin, thereby raising the ph of this medium. [9,10] Although whitening carried out using 10% carbamide peroxide may cause reactions in the pulp tissue, such reactions are considered mild, given that this substance does not seem to change the integrity of this tissue and considering that the symptoms disappear after 2 weeks. [11,12] Addition of whitening agents to dentifrice formulations has been justified through studies which have found that the probable mineral loss is more due to the mechanical act of brushing (which abrades the tooth surface and gives rise to erosion) than to the action of whitening agents themselves. [13,14] The main factor associated with degradation of the tooth surface seems to be the duration of exposure of the enamel to the whitening agent and not the concentration of this agent. [13,15,16] Various changes to the morphology of the enamel structure have been associated with whitening procedures. [17] However, this deleterious effect on the enamel seems to be reduced on surfaces treated with carbamide peroxide as compared to those treated with hydrogen peroxide. 771 Although several studies have recorded the occurrence of sequelae in the enamel morphology, resulting from the action of whitening products, others of equal relevance have stressed that the lesions produced do not have clinical significance. [18] There is a lack of consensus in the scientific literature reporting the occurrence of lesions that affect the integrity of the morphology of the enamel surfaces of human teeth, which result from the application of dentifrices containing hydrogen peroxide and carbamide peroxide. [19] This raises the questions of whether the lesions that affect the enamel morphology might be of different types and whether they might have different degrees of severity. Considering the null hypothesis that bleaching agents do not alter the tooth surface, the present study aimed to elucidate the nature and severity of possible sequelae from applications of whitening agents that are present in the formulations of two toothpastes, in comparison with the action of a dentifrice with an abrasive agent, through electron micrograph analysis. MATERIALS AND METHODS Twenty-five vestibular faces of upper and lower human premolars were obtained from the tooth bank of the UNIME Schools, Bahia, Brazil. These were divided into five study groups in accordance with the pre-established biosafety norms and criteria. There were three test groups defined according to the chemical agent present in the formulation of each dentifrice applied when brushing: GT 1 (Mentadent hydrogen peroxide, Chesebrough Pond s USA Co., Oral Division, Greenwich, CT, USA); GT 2 (Rembrandt carbamide peroxide, Den-Mat Corporation, Santa Maria, CA, USA) and GT 3 (Colgate sodium bicarbonate, Colgate-Palmolive Company, S. Paulo, Brazil). There was one negative control group (GC 1 ) in which the enamel was brushed using only dentifrice without whitening agent (Colgate Total 12, Colgate-Palmolive Company) and one repetitive control group (GDC 2 ), in which the enamel underwent a laboratory process of darkening and was brushed using dentifrice without whitening agent. Because this was a double-blind study, the five study groups were set up randomly in blocks with five test pieces each. The selected teeth were conditioned in saline solution (0.9% sodium chloride) that was renewed every 24 hours and were refrigerated after cleaning and the removal of possible soft tissue residue. This was followed by separating the crowns from the root portions by means of a horizontal cut in the area corresponding to the enamel cement boundary. This cut was made from the vestibular side to the lingual side. The crown pieces were sealed using adhesive glue, and the lingual or palatine face was then embedded in ο-phthalic resin. [19] In this procedure, a piece of wax was placed in the central area of a ½-inch PVC cylinder with the aim of fixing

3 and isolating the vestibular face of each specimen used in the experiments. Each cylinder was then filled with ο-phthalic resin, which made it possible to embed the other faces of each test piece in the resin. After autopolymerization, the wax was removed, thereby exposing the vestibular face of each specimen. [19] The test pieces that formed the three test groups and the repetitive control group were subjected to a darkening process in the laboratory by means of immersion in a mixture made up of equal parts of coffee, black tea, cola-based drink, red wine and tobacco. [20] After the darkening process, the specimens were conserved in a remineralizing solution for 96 hours at a temperature of 37 C. Daily maintenance for the specimens consisted of rigorous control over the temperature and ph of the incubation medium. Technical brushings were applied three times a day, [21] standardized as follows: application of an Oral B toothbrush (Oral B Laboratories, S Paulo, Brazil) with dentifrice without whitening agent for 10 seconds, coupled to a dynamometer with the aim of controlling the brushing force for it to be equivalent to 0.2 N. [19] Over a 21-day period, dentifrice was applied to the specimens by means of three technical brushings a day, [21] adapted to the experimental conditions of this study. A portion of 0.4 g of each toothpaste [22] was added to the Oral B toothbrush coupled to the dynamometer. The dentifrice was applied to each test piece for 10 seconds. The daily maintenance of the specimens consisted of control over the temperature and ph of the medium and preservation in remineralizing solution. At the end of the 21-day whitening period, the test pieces were washed with deionized distilled water, dried in a glass chamber at 37 C for 24 hours and packed, group-by-group, in numbered colored receptacles, with the aim of ensuring that the analysis under the electron microscope would be conducted in a double-blind manner. Using the IC-50 Ion Coater device (Shimadzu Biotech., Tokyo, Japan), the vestibular faces of the test pieces were coated with a thin layer of gold, approximately 9 nm in thickness, so as to make the surface conductive and capable of secondary electron emission. The sample analysis was done by means of the Superscan SS-550 scanning electron microscope (SEM; Shimadzu Superscan SS-550/SSX-550 Scanning Electron Microscope, Tokyo, Japan). The analysis concentrated on the mid-zone of the specimens. For this, magnifications ranging from 500 to at 15 kv were used, aiming to enable detailed qualitative evaluation of the study surfaces, with a view to detect possible morphologic abnormalities. and the occurrence of grooves, along with artifacts without major relevance. The electron micrographs of the specimens that made up group GC 2 [Figure 2] presented regularity in certain areas of the enamel surface because of reductions in the aprismatic layer. Predominant irregularity, which is a particular characteristic of the aprismatic layer, made it easier to see the prismatic and interprismatic areas that formed the structure within the enamel (the prismatic layer). This showed that the prisms remained intact, as could be seen from the preservation of the interprismatic layer. Thus, there was no significant loss of mineral components. The images of the test pieces that were brushed using the dentifrice-containing hydrogen peroxide (group GT 1,) showed severe abnormalities on the surface of the tooth enamel [Figure 3], indicating significant aggressive action against the integrity of the prisms. These structures demonstrated a substantial reduction in the quantity of mineralized material and also presented marked disfiguration of the organizational patterns. Images suggestive of corrosion of the areas exposed to the whitening product were observed, resulting in harm to the integrity of the enamel morphology. These sequelae accentuated the surface irregularities that existed in this structure. Increased numbers of depressions, irregularities and isolated points of surface stripping were also observed, resulting from removing the surface limits of the area examined and the access to the more mineralized layer. This suggested deepening of the disorganization of the inorganic structure. There were also some artifacts without major significance. The electron micrographs of the specimens in group GT 2, which was treated with the dentifrice containing carbamide peroxide [Figure 4], showed changes to the enamel surface, of lesser severity. In comparison with the sequelae produced on the group GT 1 specimens, the sequelae affecting the morphology of the test pieces of this group were more RESULTS Evaluation of the images from the specimens in group GC 1 [Figure 1] revealed the striking presence of an aprismatic layer. Some irregularity on the enamel surface was observed, accompanied by occasional randomly scattered porous areas Figure 1: Electron micrographs of the enamel surface treated with dentifrice without whitening agent. Magnitude:

4 Figure 2: Electron micrographs of the enamel surface darkened in the laboratory and treated with dentifrice without whitening agent. Magnitude: 540 Figure 3: Electron micrographs of the enamel surface darkened in the laboratory and treated with dentifrice containing hydrogen peroxide. Magnitude: 500 Figure 4: Electron micrographs of the enamel surface darkened in the laboratory and treated with dentifrice containing carbamide peroxide. Magnitude: 1000 moderate. It was also noted that removal of the aprismatic layer occurred, together with initial compromising of the prisms, since these structures showed loss of definition. Occasional porous areas were also observed. The images of the test pieces brushed with dentifrice containing the abrasive sodium bicarbonate [Figure 5] showed that the enamel surfaces of these specimens presented the greatest numbers of affected areas. The morphologic damage to the surfaces was notably uniform, and it was seen that there were lesions of greater depth. The aprismatic layer had been almost completely removed, with loss of identity of the prismatic regions, and prisms could be delimited in only a few areas. The likely prismatic delimitations could not be identified in the very severe lesions. Because of the wear on the enamel surfaces, the resultant images showed a porous appearance with notable regularity. All the areas examined presented the same degree of damage, with fewer of the depressions and 773 Figure 5: Electron micrographs of the enamel surface darkened in the laboratory and treated with dentifrice containing the abrasive agent sodium bicarbonate. Magnitude: 1000 irregularities that are normally seen on healthy enamel. Independent of the magnitude of each image, significant loss of mineral structure was revealed through the aggressive action on the prismatic layer, which extended into the prisms. The images presented an appearance of surface pulverization, with the formation of erosion. DISCUSSION The electron micrographs obtained not only prove the existence of severe aggressive action on the morphology of the enamel surface but also contribute to an explanation of the origin of hypersensitivity events that are attributed to the period following applications of whitening agents. [7,23] Even though it is recognized that whitening is a technical procedure without great risk, compared with other types of esthetic treatments, hypersensitivity can result from the infiltrative capacity of these chemical products into tooth tissue, producing morphologic, structural or molecular composition changes.

5 From the results of this study, a variety of causes can explain the lesions detected after application of whitening dentifrices. Among these is the oxidation reaction that occurs during tooth whitening, [2,24] although the increased ph of the medium due to carbamide peroxide can be considered to be a suitable condition. The theory of this explanation is grounded in studies that considered tooth whitening to be a procedure of chemical nature consisting of an oxidation reduction reaction. Partial oxidation causes breakage of the cyclic carbon chains of pigmented compounds, rendering them acyclic, unsaturated, double-bonded and capable of incorporating hydroxyl groups. This process gives rise to lighter-toned products. [4-6] Nevertheless, it is also accepted that the whitened structure could be stripped off due to the loss of inorganic material (demineralization), as one of the threats to enamel following the initial fall in ph. [16,17] The hydrogen peroxide and carbamide peroxide concentrations (incidentally, not explicitly stated by the dentifrice manufacturers) were probably an important causal variable for the appearance of the enamel lesions, independent of their intensity. This finding is compatible with the scientific literature dealing with this topic. [13,15,16,25,26] The fact that these lesions resulted from in vitro experiments does not minimize the importance of their severity. It is emphasized that all the tests were carried out under rigorously controlled temperature and ph, with specimens maintained in artificial saliva. Furthermore, care was taken with the brushing technique and in following the manufacturers recommendations for use. A fall in ph induces mineral loss from teeth. Such losses tend to be worsened through the mechanical friction produced by brushing, or through the changes in enamel morphology produced by whitening agents, particularly if they are added to dentifrices. Whitening agents presenting ph conditions between 5.7 and 6.2 may trigger demineralization of the tooth enamel. [2] In the control group GC 1 specimens [Figure 1a and b], we observed the presence of an aprismatic layer characterized by surface irregularity and an organized structure. Analysis of the electron micrographs revealed occurrences of occasional porous areas scattered randomly on the enamel surface, along with grooves that were considered to be lesions of mild nature, resulting from widening of the grooves of the enamel. Analysis of the electron micrographs of control group GC 2 [Figure 2] suggested that there was some reduction of the aprismatic layer, given that some areas of the enamel surface were seen to be more organized and less irregular. From a morphologic point of view, this characteristic was certainly irrelevant, considering that an aprismatic layer was seen in several other areas of the enamel surface. Although darkening of the specimens in the laboratory was an essential condition for determining the whitening efficacy of the study substances and consequently the possible lesions that could form because of the whitening substances, the prismatic and interprismatic areas that made up the prismatic layer of the test pieces in the repetitive group (GC 2 ) remained mostly unaffected. In the electron micrographs for the specimens in group GT 1 [Figure 3], to which the dentifrice-containing hydrogen peroxide was applied, severe aggressive action on the enamel surface was indicated by the loss of prismatic organization patterns. The changes to the enamel morphology that were seen to be associated with the whitening procedures were areas of depressions, with formation of craters, impaired microhardness, rugosity and surface wear, along with exposure of the prisms in the areas most affected. [17,27-30] The areas showing stripping of the whitened structure were indicative of losses of inorganic substance: decalcification, with disfiguring of the prismatic patterns due to hollowing out of the interprismatic and intraprismatic zones, thereby accentuating and deepening the tooth surface irregularities [Figure 3]. If hydrogen peroxide is indicated clinically for tooth whitening, it should be used at a concentration of 35%, applied solely under professional care. [4] Although the concentration of the whitening products incorporated in dentifrices needs to be lower, their continual use within a given period of time, even when recommended by the manufacturers, implies a cumulative effect that may give rise to greater chemical aggression on the enamel. The lesions observed in the GT 1 group certainly resulted from the efficacy of hydrogen peroxide for penetrating the enamel because of its low molecular weight (34 g/mol). This substance not only promotes protein denaturing (a property that facilitates ion movement in the treated area) but also favors whitening action. Real changes to the enamel surface texture occurred, and depressions appeared. These situations implied loss of definition of the striae of Retzius and increased porosity in the whitened structure, thereby giving rise to an appearance resembling the effect caused by partial conditioning with acid. The lesions caused by carbamide peroxide [Figure 4], which was present in the formulation of the dentifrice evaluated using group GT 2 specimens, probably resulted from denaturing of the vestiges of low-molecular weight protein present in the organic structure of the enamel. The possibility that urea could penetrate the enamel surface and affect the intraprismatic and interprismatic areas contributed not only toward increasing the permeability of the product but also toward modifying the surface at the microstructural level. [24,31] The test pieces of the GT 2 group presented lesions of lower severity than those seen on the specimens whitened with hydrogen peroxide [Figure 3], even though partial removal of the prismatic layer and the beginning of damage to the enamel prisms were observed. 774

6 It should be considered that the only product recommended by the Food and Drug Administration (FDA, 2007) [32] for home use is a gel based on 10% carbamide peroxide. This is important in relation to comparison between the electron micrographs of the GT 1 and GT 2 groups, which revealed that the lesions in the group treated with carbamide peroxide were of lower severity. The oxygen released from the reaction with carbamide peroxide gave rise to increased porosity of the tooth structure surface, thereby enabling greater penetration and propagation, and hence greater efficacy of the whitening agent. On the other hand, the oxygen free radicals resulting from the decomposition of this peroxide were able to react with the organic structures of the tooth tissue, thereby favoring mineral loss. [33] Nonetheless, the ph of this dentifrice was found to be around 7.5, and this situation resulted in a lower degree of aggression on the morphologic structure of the enamel. The test pieces of the group treated with dentifrice containing sodium bicarbonate (GT 3 ) showed severe morphologic damage and lesions of greater depth. Consequently, a greater number of areas were affected. The images shown in Figure 5 indicate that there was marked loss of the aprismatic layer, impairment of the identity of the enamel prism regions and a porous appearance with significant regularity. The aggressive action on the prismatic layer, reaching the prisms, denoted significant loss of the mineral structure, giving rise to an appearance of surface pulverization, with the formation of eroded areas. [34] Certainly, such lesions result from abrasive action of sodium bicarbonate. Brushing using abrasive agents implies considerable mineral loss. This is because such agents lead to an important increase in the rugosity of enamel surface. [34] These findings are in agreement with the results of relevant studies that recommend usage of products for oral hygiene featured by low abrasivity, especially when initial white spot lesions are present. [35] The sodium bicarbonate present in dentifrice, which is considered a potent abrasive agent, caused more damage when compared to the bleaching agents used. Taking into account that there is a possibility of arising lesions as a result of the exposure to bleaching agents, topical usage of fluoride after bleaching procedure has been recommended. In spite of the benefits of fluoride ion, patients should be evaluated in order to identify those who may be prone to caries, before going through the bleaching procedure. [36] This caution is necessary since there is no consensus about the remineralizing efficacy of fluoride combined to bleaching agents. 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