INTRODUCTION

The presence of stains and irregularities on the enamel surface are factors that may compromise dental aesthetics1), when the stains are located in the outer layers of enamel, those outer layers may be removed using enamel microabrasion. Enamel microabrasion provides satisfactory aesthetics, even though an insignificant amount of enamel is lost1-6).

Croll7) and Sundfeld et al.2,3) observed that the enamel surface of microabraided teeth exhibited considerable surface smoothness and regularity after time; while also presenting a greater resistance to both demineralization7,8) and colonization by Streptococcus mutans2,7,8). However, the microabraided teeth may acquire a darker color, due to the micro-reduction of the enamel surface caused by the acid-abrasive action1,2). Therefore, the remaining enamel becomes thinner, which exposes a greater amount of the dentin color. This certainly justifies the application of a bleaching technique using peroxide-based products, after performing enamel microabrasion1,3,9). Studies have observed that bleached teeth have a reduced resistance to demineralization10), increased roughness, reduced hardness, and considerable histomorphological changes of the dental elements11-13).

In vitro studies are widely used and have fundamental importance for the planning of clinical studies, although there is a limit to the comparability of results from different studies. In situ studies represent an intermediary stage between laboratory and clinical experiments11,14) because there is an attempt to reproduce the process to be studied under the influence of biological factors, such as the protective effect of saliva14) thus

better mimicking the oral environment.There are few studies assessing the combination

of enamel microabrasion with tooth bleaching, especially in determining if there is some structural risk to the enamel, especially immediately after enamel microabrasion. The aim of this present study was to assess whether the combination of enamel microabrasion and dental bleaching would cause any structural damage to the enamel, using a comparative between in vitro and in situ study models.

Null hypothesis: The combination of enamel microabrasion and tooth bleaching does not influence the hardness and surface roughness of microabraided teeth.

MATERIALS AND METHODS

Experimental designThe factors under study were: procedures at 5 levels —control, microabrasion (Opalustre, Ultradent Products, Utah, USA), dental bleaching (Opalescence Boost PF 38%, Ultradent Products) and a combination of both (immediate and later); as well as the analysis time of the specimens, in situ or in vitro (Table 1): 0, 1, 3 and 7 days. The response variables were microhardness and surface roughness.

Preparation of samplesOne hundred sound bovine incisors were cleaned and disinfected using 0.1% thymol. The teeth were sectioned at the enamel/cementum junction using a precision saw (Isomet 4000; Buehler, IL, USA). Enamel/dentin discs that were 5.7 mm in diameter were obtained from the mesial/cervical third of each crown using a cut

Surface effects after a combination of dental bleaching and enamel microabrasion: An in vitro and in situ studyLaura Molinar FRANCO, Lucas Silveira MACHADO, Fabio Martins SALOMÃO, Paulo Henrique DOS SANTOS, André Luiz Fraga BRISO and Renato Herman SUNDFELD

São Paulo State Univertsity, Department of Restorative Dentistry, Araçatuba Dental School, UNESP, 1193, José Bonifácio, 16015-050 Araçatuba, SP, BrazilCorresponding author, Laura Molinar FRANCO; E-mail: lauramolinarfranco@hotmail.com

This study evaluated the effects of combining enamel microabrasion and dental bleaching on the physical properties of enamel, using in vitro and in situ conditions and evaluating surface roughness, enamel microhardness and scanning electron microscopy images. One hundred sound bovine teeth were sectioned and cut into discs and randomly divided into 10 study groups (n=10). The results were submitted to Analysis of Variance (ANOVA) for repeated measures, followed by the Tukey test, with significance at 5%. Enamel surface roughness was significantly influenced by microabrasion, regardless of being combined with dental bleaching, for both HS (Human Saliva) or AS (Artificial Saliva) condition. Enamel microhardness was significantly decreased in the groups in which enamel microabrasion was performed, regardless its combination with dental bleaching; although storage in HS reestablished the initial enamel microhardness. It was concluded that dental bleaching does not cause major damage to microabraided enamel, and that only human saliva recovered the initial enamel microhardness.

Keywords: Dental enamel, Microhardness test, Electron microscopy

Received Dec 3, 2014: Accepted Feb 26, 2015doi:10.4012/dmj.2014-334 JOI JST.JSTAGE/dmj/2014-334

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Table 1 Sample distribution according to the surface treatment, combination and storage time in situ and in vitro

TimesGroups

T0 T1 T2 T3 T4

ControlHS/ ControlAS Baseline no treatment 1 day in HS/AS 3 days in HS/AS 7 days in HS/AS

Group IHS/ Group IAS Baseline Dental Bleaching 1 day in HS/AS 3 days in HS/AS 7 days in HS/AS

Group IIHS/ Group IIAS Baseline Microabrasion 1 day in HS/AS 3 days in HS/AS 7 days in HS/AS

Group IIIHS/ Group IIIAS BaselineMicroabrasion+Dental

Bleaching1 day in HS/AS 3 days in HS/AS 7 days in HS/AS

Group IVHS/ Group IVAS Baseline Microabrasion 1 day in HS/AS 3 days in HS/AS7 days in HS/

AS+Dental Bleaching

glass diamond bur (Dinser Ferramentas Diamantadas Ltda., Sacomã, SP, Brazil) and a drill bench (FGC-16 model, Ferrari, São Paulo, SP, Brazil), under constant irrigation. The samples were covered, except for the enamel surface, in a polystyrene resin to facilitate handling while using silicon carbide discs of increasing granulation (#600, #800 and #1200-Special Silicon Carbide, Buehler, IL, USA) on a Politriz Aropol E (Arotec Indústria e Comércio Ltda., Cotia, São Paulo, SP, Brazil). Subsequently, the samples were polished with felt (Extec, Enfield, USA) and diamond pastes (6, 3, and 1 µm) (Extec Corp. Enfield, CT, USA) for 3 min. Between each diamond paste, the samples were washed for 6 min in an ultrasonic cleaner using distilled water, to remove any residue from the enamel surface.

Ethical aspects and volunteer selection (In Situ study)The in situ study was submitted to the Research Ethics Committee of the Araçatuba School of Dentistry —UNESP. Ten volunteers, ranging in age between 18 and 25 years, enrolled in the Undergraduate course of the Araçatuba Dental School, participated in this study. The volunteers were selected through anamnesis and clinical exam, following the inclusion and exclusion criteria (Table 2). The selected volunteers received detailed oral and written explanations of the study, and gave their written consent to participate. Each volunteer received a kit with a Colgate Extra Clean toothbrush, Colgate dental floss, Colgate Total 12 toothpaste (Colgate, Palmolive Ind. e Com. Ltda, São Bernado do Campo, SP, Brazil) and a plastic case in which they could store the device.

Impressions of the upper arch of each patient were made using alginate (Hydrogum 5, Zhermack SpA, Badia Polesine, RO, Italy) and were poured up in dental stone (Durone IV, Dentsply Indústria e Comércio Ltda) to manufacture the intraoral palatal devices (IPDs). The palatal devices were made of self-cured acrylic resin containing 5 niches to hold the samples. After the procedures for each group, selected samples were attached to the intraoral device using sticky wax (Kota Industrial e Comércio Ltda, Sao Paulo, Brazil), and were kept in the oral cavities of the volunteers for 7 days.

Storage in artificial saliva (In Vitro study)After the procedures for each group, selected samples were stored in AS (Artificial Saliva) (Apothicário Farmácia de Manipulação, Araçatuba, SP, Brazil) (Table 3) for 7 days. To perform this procedure, cotton balls soaked in artificial saliva were placed in close contact with the enamel surface and stored at 37°C; the cotton balls were changed twice daily.

Enamel microabrasion techniqueThe microabrasion product, Opalustre (Ultradent Products), was applied on the enamel surface using standardized amounts as measured with a metering spoon. Ten applications, 10 s each, were performed using an abrasive rubber cup (Ultradent Products) mounted on a slow-speed handpiece with a 10:1 gear reduction. The samples were rinsed with water/air spray after each application. The samples were polished with fluoridated prophylaxis paste (Herjos F, Vigodent AS. Ind e Com., Rio de Janeiro, Brazil) for a period of 2 min at intervals of 30 s, followed by rinsing and drying, and were placed in an ultrasonic cleaner for 6 min.

Dental bleaching techniqueThe bleaching gel was applied 3 times on the enamel surface, remaining in contact with the tooth surface for 15 min each, according to the manufacturer’s instructions. The product was removed using cotton rolls and the teeth were washed and dried. The samples were placed in an ultrasonic cleaner with distilled water for 6 min.

Roughness test The samples were submitted for roughness evaluation using a portable profilometer, SJ-401 (Mitutoyo, Kanagawa, Japan), which had been calibrated before the readings. The standard roughness (Ra) was measured using a static load of 5 N and a speed of 0.05 mm/s. Ra represents the arithmetic mean between the recorded peaks and valleys, while using a 0.25 mm cut-off to filter out any surface ripple. At each analysis time and on each surface, three measurements were made at different positions, and the arithmetic mean was calculated. Measurements were performed at baseline

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Table 2 Inclusion and exclusion criteria for selection of volunteers for the in situ study

Inclusion Exclusion

Normal salivary flow Regular use of alcohol

Absence of caries Smokers

Absence of periodontal disease Orthodontic appliance

Proper oral hygiene Fixed and removable prosthesis

Absence of digestive disorders Systemic diseases

Available to attend the department Pregnant or breastfeeding volunteers

Table 3 Composition of artificial saliva

Component Concentration Component Concentration

Phosphate potassium, dibasic 4.35 g/L Magnesium chloride 0.14 g/L

Phosphate potassium, monobasic 3.2 g/L Calcium chloride 0.16 g/L

70% Sorbitol 42.7 g/L Sodium benzoate 5.0 g/L

Sodium fluoride 0.043 g/L Carboxymethycellulose 5.0 g/L

Potassium fluoride 0.62 g/L Destilled water 1,000 mL

Sodium chloride 5.85 g/L pH 7.0

(T0), immediately after the procedures (T1), and after 1 (T2), 3 (T3) and 7 (T4) days of in situ and in vitro storage.

Microhardness analysisSamples were submitted to microhardness evaluation using a microhardness tester (HMV 2000, Shimadzu, Tokyo, Japan) and a static load of 25 g for 10 s. At each analysis, 5 indentations were performed in the central region of the sample surface with a distance of 100 μm between each indentation. Measurements were performed at baseline (T0), immediately after the procedures (T1), and after 1 (T2), 3 (T3) and 7 (T4) days of in situ and in vitro storage.

Analysis in scanning electron microscopyThe five most representative samples of each group were selected and prepared for scanning electron microscope analysis (JSM 5600LV, JEOL, Tokyo, Japan). After gradual dehydration with alcohol, the specimens were plated with a layer of gold–palladium (Quorum, Q150T E, Quorum Technologies, West Sussex, UK). The most representative images of each group were stored for illustration and were not submitted to any statistical analysis.

RESULTS

The data were submitted to analysis using a statistical program “SigmaPlot 12.0”. Analysis of Variance (ANOVA) was used for repeated measures, considering

‘technique’ and ‘time’ as factors, followed by Tukey test, at a significance level of 5%.

RoughnessAt baseline, there were no statistically significant differences between the means of the study groups, for both the in vitro and in situ conditions. Both conditions presented statistically significant differences immediately after microabrasion (GIIHS and GIIAS) and after association with dental bleaching (GIIIHS, GIVHS and GIIIAS, GIVAS), with means when compared to baseline, for periods T1, T2, T3 and T4. The groups CAS and CHS, as well as those groups that only received dental bleaching (GIHS and GIAS), had statistically similar roughness means at all times analyzed (Tables 4 and 5).

MicrohardnessThere were no statistically significant differences between the means of the study groups at baseline (T0) for the in vitro and in situ conditions. When evaluating the effects of HS, there were no statistically differences between the CHS and GIHS groups (dental bleaching) at each analysis time. A significant decrease in microhardness occurred for all study groups at T1, and it remained the same for all analysis times when stored in AS. Lower values of microhardness were observed for the in situ condition at T1 and T2, for the groups in which only microabrasion was performed (GIIHS) or when microabrasion was combined with dental bleaching (GIIIHS and GIVHS), which were both statistically similar.

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Table 5 Roughness means Ra (μm) (standard deviation) for the in vitro study

T0 (baseline) T1 (immediate) T2 (1 day) T3 (3 days) T4 (7 days)

CAS 0.03 (0,01) A,a 0.03 (0,02) A,a 0.02 (0,01) A,a 0.03 (0,02) A,a 0.02 (0,01) A,a

GIAS 0.03 (0,01) A,a 0.02 (0,00) A,a 0.03 (0.01) A,a 0.02 (0,00) A,a 0.02 (0,00) A,a

GIIAS 0.03 (0,00) A,a 0.15 (0,05) C,b 0.16 (0,07) C,b 0.15 (0,05) C,b 0.15 (0,05) C,b

GIIIAS 0.02 (0,00) A,a 0.11 (0,03) B,b 0.12 (0,03) B,b 0.12 (0,04) B,b 0.12 (0,04) B,b

GIVAS 0.03 (0,01) A,a 0.14 (0,04) BC,b 0.14 (0,04) C,b 0.14 (0,03) B,b 0.14 (0,03) BC,b

(*) Means followed by different letters (uppercase in the vertical and lowercase in the horizontal) differ according to Analysis of Variance and Tukey’s test (p<0.05).

Table 7 Microhardness means (KHN) (standard deviation) for the in vitro study

T0 (baseline) T1 (immediate) T2 (1 day) T3 (3 days) T4 (4 days)

CAS 372 (27,02) A,a 358 (28,14) A,ab 348 (20,40) A,bc 332 (20,39) A,c 343 (35,45) A,bc

GIAS 366 (25,84) A,a 349 (29,76) A,b 332 (35,71) AB,bc 325 (25,75) AB,c 339 (26,49) A,bc

GIIAS 361 (18,75) A,a 296 (36,2) B,b 301 (43,51) C,b 293 (42,78) B,b 299 (50,84) B,b

GIIIAS 363 (20,62) A,a 311 (32,62) B,b 316 (34,94) BC,b 303 (32,74) B,b 307 (44,43) B,b

GIVAS 363 (26,46) A,a 298 (41,28) B,b 300 (28,31) C,b 289 (24,51) B,b 301 (25,09) B,b

(*) Means followed by different letters (uppercase in the vertical and lowercase in the horizontal) differ according to Analysis of Variance and Tukey’s test (p<0.05).

Table 6 Microhardness means (KHN) (standard deviation) for the in situ study

T0 (baseline) T1 (immediate) T2 (1 day) T3 (3 days) T4 (7 days)

CHS 344 (7,97) A,a 348 (19,52) A,a 341 (11,17) A,a 344 (21,85) A,a 348 (28,61) A,a

GIHS 349 (15,73) A,a 341 (46,53) A,a 341 (35,85) AB,a 333 (43,12) A,a 339 (32,61) A,a

GIIHS 351 (11,03) A,b 293 (46,47) B,a 301 (36,36) B,ab 328 (47,82) A,ab 353 (71,15) A,b

GIIIHS 354 (14,49) A,ab 319 (43,16) A,ab 304 (41,57) B,a 359 (40,93) A,b 365 (21,38) A,b

GIVHS 349 (16,74) A,ab 306 (59,02) AB,b 300 (68,08) B,b 339 (38,87) A,ab 358 (42,20) A,a

(*) Means followed by different letters (uppercase in the vertical and lowercase in the horizontal) differ according to Analysis of Variance and Tukey’s test (p<0.05).

Table 4 Roughness means Ra (μm) (standard deviation) for the in situ study

T0 (baseline) T1 (immediate) T2 (1 day) T3 (3 days) T4 (7 days)

CHS 0.05 (0,03) A,a 0.02 (0,01) A,a 0.02 (0,00) A,a 0.03 (0,00) A,a 0.02 (0,00) A,a

GIHS 0.02 (0,00) A,a 0.02 (0,00) A,a 0.02 (0,00) A,a 0.02 (0,00) A,a 0.02 (0,00) A,a

GIIHS 0.02 (0,01) A,a 0.13 (0,03) B,b 0.14 (0,04) B,b 0.15 (0,04) B,b 0.14 (0,04) B,b

GIIIHS 0.02 (0,01) A,a 0.14 (0,03) B,b 0.13 (0,03) B,b 0.13 (0,03) B,b 0.13 (0,03) B,b

GIVHS 0.03 (0,01) A,a 0.14 (0,05) B,b 0.14 (0,05) B,b 0.13 (0,04) B,b 0.13 (0,03) B,b

(*) Means followed by different letters (uppercase in the vertical and lowercase in the horizontal) differ according to Analysis of Variance and Tukey’s test (p<0.05).

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Fig. 1 SEM images of groups CHS (A) and CAS (B).

Fig. 2 SEM images of groups GIHS (A) and GIAS (B); A1 and B1 after dental bleaching (T1); A2 and B2 after 7 days in situ/in vitro (T4).

However, there was a gradual increase in microhardness values up to 7 days after performing the procedures (T4) for groups GIIHS, GIIIHS and GIVHS, with an eventual return to the values observed at baseline (T0). Regardless of the experimental condition (in situ or in vitro), dental bleaching did not intensify the alterations observed when microabrasion was performed, either with the immediate or later combination (Tables 6 and 7).

Scanning electron microscopySEM images showed a smooth surface for groups CHS and CAS, without alterations or signs of decalcification

(Fig. 1). For the groups which only received dental bleaching (GIHS and GIAS), the enamel surfaces did not suffer significant alterations (Fig. 2). For the groups in which only microabrasion was performed (GIIHS and GIIAS), the microabrasion effects were noted as decalcification and scratches on the enamel surface (Fig. 3), characteristics that were not pronounced when the immediate combination with dental bleaching was performed (GIIISHS and GIIIAS) (Fig. 4). After 7 days in situ, changes observed right after microabrasion (GIIHS), or it’s combination with dental bleaching (GIIIHS), were reduced; no significant alteration were observed when

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Fig. 3 SEM images of groups GIIHS (A) and GIIAS (B); A1 and B1 logo after enamel microabrasion (T1); A2 and B2 after 7 days in situ/in vitro (T4).

Fig. 4 SEM images of GIIIHS (A) and GIIIAS (B); A1 and B1 after combination (T1); A2 and B2 after 7 days in situ/in vitro (T4).

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Fig. 5 SEM images of groups GIVHS (A) and GIVAS (B); A1 and B1 after enamel microabrasion (T1); A2 and B2 after 7 days in situ/in vitro and dental bleaching (T4).

dental bleaching was applied on in situ microabraided samples (GIVHS) (Fig. 5). After 7 days of AS immersion, surface alterations were still noticed as signs of decalcification and structural loss (Fig. 5).

DISCUSSION

The null hypothesis was accepted because the combination of enamel microabrasion and dental bleaching did not influence microhardness and surface roughness of microabraided teeth, either with an immediate or late combination.

All study groups demonstrated a decrease in enamel microhardness after enamel microabrasion and its combination with dental bleaching, with or without the in situ and in vitro conditions. This significant reduction may be justified by the presence of 6.6% hydrochloric acid in the microabrasive compound. This is because the stain removal depends on the erosive action of the acid present in the microabrasive product2,3,5,15), justifying the reduction in mineralization of the enamel surface after microabrasion. Those current observations are in agreement with other studies13,16).

When combined with dental bleaching, the reduction in microhardness of the enamel was not significantly different when compared with values observed after microabrasion only (GIIHS/GIIAS) for all conditions. Thus, it was noted that dental bleaching did not significantly affect the surface of the microabraided enamel.

However, after 3 days in HS (in situ), a significant increase in enamel microhardness was observed in the groups that had a reduction in microhardness; suggesting that the in situ condition was fundamental for the remineralization of the specimens, probably by enamel surface remineralization13). Remineralization of the in vitro specimens was not sufficient to restore the tooth enamel, a result in accordance to that reported by Fragoso et al.15).

The interaction of HS with the components of the microabrasive product seems to be significant with respect to its remineralizing action on microabraided enamel. Compression of silica particles in the enamel may result in a tricalcium silicate bioactive compound (Ca3SiO5), which induces the formation of a new apatite layer on etched enamel17), which adds to the enamel hardness. Likewise, the mineral content and bicarbonate buffer of saliva may facilitate the deposition of calcium and phosphate on the enamel surface, thus increasing its microhardness18,19). Thus, the presence of external fluid seems to be significantly important and crucial when assessing the real effects of enamel microabrasion. This potentially explains the excellent clinical results observed over time with enamel microabrasion1-3,6,7,20).

Attin et al.21) stated that in vitro conditions are not useful when evaluating the real effects of dental bleaching. Studies that approximate intra-oral conditions, such as in situ studies, have shown that reductions in enamel microhardness after bleaching

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are insignificant11), and do not affect the wear and microhardness of enamel22). These considerations were confirmed by the results of this present study, because a positive effect was noted for HS after dental bleaching when evaluating decalcification, surface alteration and reduction of enamel microhardness.

When evaluating enamel surface roughness, significant changes were noted when microabrasion was performed, which may be explained by the acid-abrasive action provided by the components of the microabrasive product, such as hydrochloric acid and silica carbide particles. However, it is believed that the surface alterations observed after enamel microabrasion are clinically acceptable1-3,5-7,23).

The results obtained from the quantitative analyzes in this present study are confirmed by the qualitative analysis using scanning electron microscopy, confirming the values found for hardness and surface roughness. Signs of demineralization and surface irregularities, which are characteristic of the erosive/abrasive action of the microabrasive15), were noted in the images of the groups that received microabrasion. No surface morphology changes were observed when only the bleaching agent was applied, indicating that the SEM images were in agreement with the results of the surface roughness and microhardness tests that were used in the present study.

Despite the limitations, it was clear that dental bleaching on microabraided teeth did not accentuate the morphological changes normally found on a surface that had already been treated with microabrasion. Furthermore, the results of the current study also emphasize the importance of the effect HS has on the remineralization of microabraided enamel. Future studies should evaluate factors, such as sensitivity and penetration of peroxide, to assess whether these factors could be influenced by immediate or late combination of microabrasion and dental bleaching.

CONCLUSION

The immediate or late combination of dental bleaching with enamel microabrasion did not negatively influence the surface roughness or hardness of enamel.

ACKNOWLEDGMENTS

The authors thank “Fundação de Amparo à Pesquisa do Estado de São Paulo —FAPESP” (São Paulo State Research Support Foundation) for the financial support to develop the project, process no. 2012/06271-3.

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