Green LED Associated to 20%Hydrogen Peroxide for Dental Bleaching: Nanomorfologic Study of Enamel by Scanning Electron Microscopy

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1 Green LED Associated to 20%Hydrogen Peroxide for Dental Bleaching: Nanomorfologic Study of Enamel by Scanning Electron Microscopy Susana C. P. S. Oliveira a,b, Gustavo M. P. Santos c, Juliana S. C. Monteiro a,b, Fernando J. P. Sampaio a,b, Maria F. M. Gesteira a,b, Fátima A. A. Zanin a,b,d, Marcos A. V. Santos c, Antônio L. B. Pinheiro* a,b,d,e. a Center of Biophotonic, School of Dentistry, Federal University of Bahia, Salvador, BA, Brazil, ; b National Institute of Optics and Photonics, São Carlos, SP, Brazil, ; c Laboratory Parasitic of Biomorphology, Gonçalo Muniz Institute, Oswaldo Cruz Foundation, Ministry of Health, Salvador, BA, Brazil ; d Brugnera & Zanin Institute, São Paulo, SP, Brazil ; e Camilo Castelo Branco University, São José dos Campos, SP, Brazil, ABSTRACT Dental bleaching is a much requested procedure in clinical dental practice and widely related to dental esthetics. The literature is contradictory regarding the effects of bleaching agents on the morphology and demineralization of enamel after bleaching. The aim of this study was to analyze in vitro by scanning electron microscopy (SEM) the effect of hydrogen peroxide at 20% at neutral ph, cured by the green LED, to evaluate the action of these substances on dental enamel. We selected 15 pre-molars, lingual surfaces were sectioned and previously marked with a central groove to take the experimental and control groups on the same specimen. The groups were divided as follows. The mesial hemi-faces were the experimental group and distal ones as controls. For morphological analysis were performed 75 electron micrographs SEM with an increase of X 43, X 220 and X 1000 and its images were evaluated by tree observers. Was also performed quantitative analysis of the determination of the surface atomic composition of the samples through microanalysis with the aid of scanning electron microscopy. The use of hydrogen peroxide at a concentration of 20% at photoactivated green LED showed no significant changes in mineral composition of the samples or the dental morphological structure of the same when compared to their controls, according to the study protocol. Keywords: Dental Bleaching, SEM, Hydrogen Peroxide, Light 1. INTRODUCTION Tooth bleaching photoassisted is an innovative technique that wants to employ a treatment that meets the expectations of the patient and not promotes damage in its dental structure 1. The expectation of being able to provide better results, offering safety and comfort, are constant concerns in research with bleaching dental 2,3. Similarly to the technique, the bleaching materials have evolved. The adverse effects of these materials on the tooth and periodontium still have being investigated. The bleaching techniques employ oxygen, obtained from the decomposition of hydrogen peroxide, which can occur by enzymatic catalysis, thermal, light, laser and LED. We now market a wide range of equipment-based light emitted from diodes (LEDs), which modified the techniques of tooth whitening, facilitating these procedures in dentistry clinics 4. *albp@ufba.br; phone ; fax ; Mechanisms for Low-Light Therapy VIII, edited by Michael R. Hamblin, Praveen R. Arany, James D. Carroll, Proc. of SPIE Vol. 8569, 85690M 2013 SPIE CCC code: /13/$18 doi: / Proc. of SPIE Vol M-1

2 Nowadays the desire for aesthetic smile causes immediate whitening is one of the most popular treatments in the dental office, thanks to the existence of simple techniques and quick, where in less than an hour the patient can get "teeth more white" 5. The advantage of whitening in a single session is shortening the time of product contact with the tooth, which takes place in about an hour. If there is sensitivity, it will be mild and easily controlled. The enhancement of oxide-reduction reaction decreases the application time 6. An important factor in the study of bleaching gels is their ph and the effects of mineral loss that may occur in the surface enamel 7. These analyzes usually done by scanning electron microscopy, allow us to infer that there are pronounced differences in morphology of enamel between different samples of teeth, emphasizing thus the need for the use of a single tooth for comparative morphological studies. The analysis of the hemi-faces of the same tooth, with the same color, which has passed the same challenges biochemical and functional facilitates analysis of the results obtidos 7. The aim of this study was to analyze the surface of enamel after use of hydrogen peroxide to 20% by scanning electron microscopy (SEM). For both, were treated with hydrogen peroxide premolars, employed the concentration of 20% compared to the controls and the possible morphological changes caused by photoactivated bleaching substances by the green LED. 2.1 Sample preparation 2. METHODOLOGY In this study, it was used 15 human teeth, premolars removed surgically, which had as selection criteria the integrity of the crown. The teeth were prepared until the beginning of experimento 8. We performed a section of dental crowns units through vertical cut towards occluded cervical, in the area corresponding to the central sulcus of the tooth crown separating the buccal and palatine. To carry out this study, the teeth still had their faces interrupted by a cleft palate splitting into hemi-faces: palatal-mesial subjected to the action of hydrogen peroxide (20%), their distal hemi-faces were used as negative control. Exp Group I - Hydrogen peroxide (20%) photoactivated with LED green 15 hemi-palatal-mesial faces of the dental crown. Control Group I - 15-sided hemi-palate distal dental crown. 2.2 Photoactivated bleaching protocol The teeth were fixed in "stubs" (metal brackets used to hold the specimens in SEM) and had their faces distal (control) and slit covered with Topdam (gingival barrier, FGM, Joinville, SC). The substance used was whitening Whiteform Perox Red (Formula Action & Pharmaceutical Laboratory Ltd - São Paulo-SP), the concentration (20%), was manipulated specifically for this study, retains the red color and is presented in syringes with disposable tips. The gel was applied in uniform layer thickness measured with probe was (sswhite - DUFLEX, Rio de Janeiro, RJ) of approximately 1mm in G.Exp.I. Then, we performed activation gel with LED (Light Green D - Kondortech, São Carlos, SP, Brazil), the device was positioned at a distance of 5mm from the tooth surface, measured with a millimeter ruler (Maqueira, Maringa, PR, Brazil). The protocol elected 9, the parameters of LED in Table1. Parameters Power Density Wavelength Table 1: Summary of the parameters used on the study. LED D-Light Green 180 mw / cm 2 520±30nm Proc. of SPIE Vol M-2

3 2.3 Scanning Electron Microscopy (SEM) Upon completing the photoactivated bleaching procedure, their tooth faces were dried at 50 C for 24 hours. Sample analysis was performed using the Scanning Electron Microscope (JEOL JSM 6390 LV), belonging to the Electron Microscopy Unit of the Gonçalo Moniz Research Center - FIOCRUZ - Salvador, Bahia. The specimens thus prepared were fixed in small metal brackets "stubs" through a carbon double-sided tape and then analyzed. There was the standardization of the region for analysis, being given the middle third of the tooth crown, being an intermediate region where there is less likelihood of physiological wear of tooth enamel. Electron micrographs were acquired dental crowns using the program "the JEOL SEM Control Program" and obtained an increase of X 43, X 220 and X 1000 in all study groups, in order to allow detailed qualitative evaluation of the surface enamel. The characteristics of the morphology of the tooth enamel assessed in SEM was according to the qualitative assessment performed by three examiners, where the evaluation criterion and comparative examination of palatal groups and their controls. We also carried out a quantitative determination of the atomic composition of the surface of the samples through microanalysis with the aid of a scanning electron microscope. 2.4 Statistical analysis For the statistical analysis it was used the computer software 'GraphPad Prism', the test was part listed the Kruskal- Wallis test, it was considered as statistically significant results with P < RESULTS The morphological evaluation of the surface enamel subjected to the action of the bleaching agent hydrogen peroxide (20%) had no change worthy of observations, even in samples with different morphologies as can be seen in Figure 1. Being, 1B and 1D faces treated 1A and 1C and their respective controls (Fig.1). 15kV X pm Fiocruz 15kV X m Fiocruz Figure 1. A) GCI (20%): Electron (a) aprismatic layer, (b) prisms, (c) interprismatic layer, (d) coverslip. B) GExp.I (20%): Electron (a) aprismatic layer, (b) grooves. C) GCI (20%): Electron (a) prisms, (b) interprismatic layer, (c) perikymatas. D) GExp.I (20%): Electron (a) prisms, (b) interprismatic layer, (c) perikymatas. The quantitative assessment of the distribution of calcium and phosphorus in the tooth surface can be observed in Fig. 2, which also showed no statistically significant differences. The figures for calcium and phosphorus are graphically represented in Fig.3. C D 15kV X pm 0000 Fiocruz 15kV X pm 0000 Fiocruz A a g Proc. of SPIE Vol M-3

4 o m oo Figure 1: The distribution of calcium and phosphate to the tooth surface was shown to be uniform and no difference between control and treated (20%). O co a) có,),; o/ _, o/ -1Oo ó- Ca concentration N N U1 O Ut I P concentration Fig.3: Graphs of relative concentration of calcium and phosphorus in the enamel (the ratio between the treated group and its control). By analyzing graphs can be seen that the median concentrations of calcium and phosphorus are lower than the median of the controls, however despite these reductions were not statistically significant. 4. DISCUSSION The aim of the study was to evaluate the enamel nanomorfology, and the whole experiment was planned for the same photobleached tooth was its own control. This standardization is necessary because of the differences and deficiencies in existing enamel specimens, it provides a more accurate assessment of the research. The standardization is outlined in some studies, which all tests were performed in just one tooth, when measuring different light sources in relation to the variation temperatura The pigments of red color having a peak absorption wavelength of 512nm 12-13, which corresponds to green light, so that light would increase the efficiency in activating the red gel and therefore chosen for this study. Proc. of SPIE Vol M-4

5 The protocol of choice becomes more dependent on consistent information that may be provided by scientific literature, from the analysis of images of tooth surfaces obtained by scanning electron microscopy, as has often been a record of morphological changes arising by peroxide bleaching hidrogênio 14. This study showed that there are morphological changes, but not due to bleaching. While acknowledging the conservative nature of bleaching, studies suggest the occurrence of hypersensitivity, which arises from the infiltration capacity of these chemicals in dental tissues 15. The results of this study show the presence of cracks in the surface of dental enamel does not visualized clinically, may explain the occurrence of hypersensitivity in some patients, since these cracks were not caused by photobleaching. The enamel surface shows irregularities, "physiological slots" that can be alarged 16. These observations are consistent with the present study, which was observed irregularities of tooth surfaces and the presence of grooves. The changes that are observed in the morphology of the tooth enamel whitening are associated with the areas of depression, with cratering, impairment of microhardness, roughness and surface wear this structure, and exposure over the surfaces of the prisms affected 17. The present study found those features on the surfaces of the experimental group and its control which had similar characteristics. There is an understanding that these changes depend on the previous history of this dental unit. Morphological changes in the enamel surface after completion of bleaching, are directly related to the contact time of bleaching substances with the surface of the tooth and its concentração1. The study protocol was aimed at a shorter contact of the substance with the tooth surface, as was done in one session, this time reduction is a condition that most researchers consider as the most relevant for understanding that expanding the application time is the main factor to aggression teeth. The single-session protocol may have contributed to the successful outcome of this study. The concentration of hydrogen peroxide constitutes an important causal variable changes the appearance of enamel 18. This factor was not relevant in the present study, it was the use of concentration (20%) and compared with their controls, which was the tooth itself, there was no morphological changes. The ph of the hydrogen peroxide is an important causal variable configures the appearance of lesions enamel 18. The photo bleaching gels have assisted the ph buffered, differing from the home whitening gels, which have acidic ph, around two 1. This factor may have contributed to the results of this study, since the bleaching agent used was neutral ph, preventing demineralization of tooth enamel. Since the protocol is performed correctly bleaching, independent of product concentration, its mode of activation or no production of harmful changes considered clinical standpoint, since the results obtained show good clinical significance which was reduced staining and low sensibility 19. The result of this study agrees with the understanding that the protocol can be the difference of the results found in the literature. 5. CONCLUSION Analysis by scanning electron microscopy allowed us to infer that there are marked morphological differences between the enamel samples. Evidencing the need to use the same tooth for comparative morphological studies. Photoassisted Bleaching with green LED and treatment with 20% concentration of hydrogen peroxide, did not promote morphological changes in mineral or surface enamel when compared to their respective controls. REFERENCES [1] Pinheiro, A.L.B., Brugnera, Jr. A., Zanin, F., [Aplicação do laser na Odontologia], São Paulo, (2010). [2] Saba-Chujfi, E., Cicareli, A. J., Zanin, F. [Odontologia: resultados e integração], São Paulo, (2008). [3] Haywood, V.B., Heymann, H.O. Nightguard Vital bleaching, Quint. Int. 20, (1989). Proc. of SPIE Vol M-5

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