INFLUÊNCIA DA TÉCNICA DO PREPARO CAVITÁRIO UTILIZANDO LASER DE Er:YAG E DOS TIPOS DE MATERIAIS RESTAURADORES NA PREVENÇÃO DE CÁRIE

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1 CEPPE Centro de Pós-Graduação e Pesquisa Curso de Mestrado em Odontologia área de concentração em Dentística MARIO ALBERTO MARCONDES PERITO INFLUÊNCIA DA TÉCNICA DO PREPARO CAVITÁRIO UTILIZANDO LASER DE Er:YAG E DOS TIPOS DE MATERIAIS RESTAURADORES NA PREVENÇÃO DE CÁRIE Guarulhos 2009

2 MARIO ALBERTO MARCONDES PERITO INFLUÊNCIA DA TÉCNICA DO PREPARO CAVITÁRIO UTILIZANDO LASER DE Er:YAG E DOS TIPOS DE MATERIAIS RESTAURADORES NA PREVENÇÃO DE CÁRIE Dissertação apresentada à Universidade Guarulhos para obtenção do título de Mestre em Odontologia. Área de Concentração em Dentística. Orientador Prof. Dr. José Augusto Rodrigues Co-orientadora Profa. Dra. Alessandra Cassoni Ferreira Guarulhos 2009

3 P446i Perito, Mario Alberto Marcondes Influência da técnica do preparo cavitário utilizando laser de ER: dos tipos de materiais restauradores na prevenção de cárie/ Alberto Marcondes Perito. Guarulhos, SP, f. ; 31 cm YAG e Mario Dissertação (Mestrado em Odontologia, área de concentração em Dentística) - Centro de Pós-Graduação e Pesquisa Universidade Guarulhos, Orientador: Prof. Dr. José Augusto Rodrigues Co-orientadora: Profa. Dra. Alessandra Cassoni Ferreira Bibliografia: f Laser. 2. Cárie dental. 3. Cimentos de ionômero de vidro. 4. Laser de Er: YAG I. Título. II. Universidade Guarulhos. CDD 22 st Ficha catalográfica elaborada pela Coordenação Biblioteca Fernando Gay da Fonseca

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5 Dedico este trabalho à minha esposa Patrícia e aos meus filhos Pedro e Giovana que me dão força e coragem para prosseguir.

6 AGRADECIMENTOS À Universidade Guarulhos, pela oportunidade dada na obtenção do título de Mestre. Ao Prof. Dr. José Augusto Rodrigues pelo estímulo, amizade e paciência, cuja dedicação o faz um exemplo de profissional. À Profa. Dra. Patrícia Moreira de Freitas do Laboratório Experimental de Laser em Odontologia (LELO) da Faculdade de Odontologia da Universidade de São Paulo por permitir a utilização dos equipamentos para o desenvolvimento deste trabalho. À Cirurgiã-Dentista Ana Carolina Tedesco Jorge pelo auxílio no desenvolvimento deste trabalho. A todos os professores do Curso de Mestrado em Odontologia da Universidade Guarulhos, especialmente ao Prof. Dr. André Figueiredo Reis e à Profa. Dra. Cláudia Ota- Tsuzuki pela compreensão e amizade. À Profa. Tânia Rocha Cabral Ribas pela amizade, confiança e incentivo. Aos funcionários do Curso de Odontologia da Universidade Guarulhos pela dedicação e apoio. Aos colegas de mestrado, Carlos Eduardo Pena, Luis Gustavo Barrotte Albino e Ronaldo Viotti pelo companheirismo e amizade.

7 RESUMO Este estudo in vitro avaliou a influência do preparo cavitário com laser de Er:YAG e materiais restauradores cariostáticos na prevenção de lesões de cáries secundárias. Em uma seqüência lógica, o assunto foi abordado por intermédio do desenvolvimento de quatro trabalhos. No primeiro foi realizada uma revisão bibliográfica sobre a utilização do laser na prevenção da cárie dental. No segundo e no terceiro trabalho, blocos de esmalte dental humano foram distribuídos em dois grupos para preparos cavitários (1,6 mm ), realizados com pontas diamantadas ou com laser de Er:YAG (LA - 6Hz, 300mJ), ambos refrigerados. Cada grupo foi dividido em 3 subgrupos e restaurados com ionômero de vidro (GI), ionômero de vidro modificado por resina (RM) ou resina composta (CR). Os blocos foram termociclados (5º - 55ºC ± 2ºC, 1000 ciclos) e submetidos a ciclagem de ph. No segundo trabalho foi realizada a análise visual da formação de lesões de cárie nas amostras, por três examinadores calibrados (Kappa> 0,73) de acordo com escala ordinal com escores de 0-3. Os resultados foram analisados pelo teste de Kruskal-Wallis e teste de Dunn (α=0,05). Não foi observado efeito cariostático nas cavidades preparadas com pontas diamantadas e restauradas com compósitos. Não foi observada nenhuma diferença no efeito cariostático nas cavidades restauradas com os mesmos materiais e preparadas com pontas diamantadas ou laser de Er:YAG. Entretanto, cavidades preparadas com laser mostraram menor formação de lesões cariosas que as cavidades preparadas com pontas diamantadas. No terceiro trabalho foi realizada análise de microdureza superficial (Knoop) das amostras a 100µm da margem das cavidades. A média de 4 indentações foi utilizada para ANOVA seguida pelo teste de Tukey. O desenvolvimento de lesões de cáries ao redor dos preparos por laser foi menor que nas cavidades preparadas por pontas diamantadas, contudo, nenhum efeito cariostático sinérgico foi observado entre o laser e o cimento de ionômero de vidro. No quarto trabalho foi avaliada a correlação de Spearman entre o diagnóstico de lesões artificiais de cárie secundária em esmalte in vitro por inspeção visual e por microdureza superficial (Knoop). Essa, foi estatisticamente significante e demonstrou uma fraca correlação negativa entre as variáveis de resposta. Com base nos trabalhos desenvolvidos, observou-se que o Laser de Er:YAG proporcionou efeito cariostático ao redor dos preparos cavitários sendo mais evidente nas análises realizadas pelo teste de microdureza. O GI apresentou maior efeito cariostático em relação à RM e não foi observado efeito cariostático na CR independente do tipo de preparo. Palavras-Chaves: Laser, cárie dental, compósitos resinosos, cimento de ionômero de vidro, flúor, fluoretos, esmalte dental, microdureza.

8 ABSTRACT The influence of the cavity preparation technique and the types of restorative materials containing fluorides in the prevention of the secondary caries lesions was evaluated in this in vitro study. In a logic sequence the subject was approach by four manuscripts. The first study made a bibliographic revision about the laser employment in the prevention of the secondary caries lesions. The second and the third manuscripts, human dental enamel blocks were distributed into 2 groups for cavity preparations (1.6 mm ), performed with diamond burs or Er:YAG laser (LA - 6Hz, 300mJ) both refrigerated. Each group was divided into 3 sub-groups that were restored using a glass-ionomer cement (GI), a resin-modified glass-ionomer (RM), or a composite resin (CR). The blocks were thermocycled (5º - 55ºC ± 2ºC, 1000 cicles) and submitted to a ph challenge. In the second work the slabs were analyzed by visual examination by 3 calibrated examiners (Kappa> 0.73) according to an ordinal scale ranked (0-3). The results were analyzed by the Kruskal-Wallis test and the Dunn test (α=0.05). Non cariostatic effect in the cavities performed with diamond burs and restored with composite resin was observed. No differences in the cariostatic effect of the cavities restored with the same material and prepared with diamond burs or Er:YAG laser was observed. However, cavities prepared with Er:YAG laser showed less caries lesions formation than cavity preparation with diamond burs. In the third study the blocks were analyzed by the microhardness test (Knoop) in a distance of 100µm from the cavity walls. The average of 4 indentations was used in the ANOVA followed by Tukey s test. The development of caries lesion around lased cavity preparation were lesser than the cavities prepared with diamond burs, however, no synergistic cariostatic effect was observed between Er:YAG laser and glass ionomer cement. In the fourth study the correlation of in vitro artificial secondary caries diagnosis on enamel between visual evaluation and superficial microhardness test (Knoop) was verified by Spearman s rho nonparametric correlation that showed a statistical significant weak negative agreement between the response variables. Based in the manuscripts presented it was observed that the Er:YAG laser provide cariostatic effect around the cavities preparation, which was more evidenced with the microhardness analysis. The GI presented more cariostatic effect than RM and no cariostatic effect was observed in CR despite the cavity preparation technique. Key words: Laser, dental caries, cariostatic agents, composite resin, glass-ionomer cement, fluoride, dental enamel, microhardness.

9 SUMÁRIO Página 1. INTRODUÇÃO PROPOSIÇÃO DESENVOLVIMENTO Capítulo 1 Uso do laser na prevenção da cárie dental Capítulo 2 Effect of the cavity preparation with Er:YAG laser and fluoride releasing materials in the prevention of caries lesions Capítulo 3 Cavity preparation and restorative materials influence on the prevention of secondary caries Capítulo 4 Correlation between visual and superficial microhardness evaluation of artificial secondary caries CONCLUSÕES REFERÊNCIAS ANEXOS... 73

10 7 1. INTRODUÇÃO Até o século passado a doença cárie era uma doença com alta incidência que ocorria em quase todos os indivíduos. Atualmente, com os conhecimentos sobre a etiologia, e desenvolvimento da doença, sabe-se que ela afeta indivíduos que possuem dentes, microrganismos patogênicos e consomem uma dieta rica em carboidratos, levando a freqüentes quedas de ph no meio bucal. Entretanto, seu desenvolvimento pode ser afetado por outros fatores moduladores, como a quantidade e a qualidade da saliva, a classe social, renda familiar, escolaridade, conhecimento e comportamento frente à doença (THYLSTRUP & FEJERSKOV, 1994; MOI et al., 2005) A presença de flúor na cavidade bucal também pode interferir nos fenômenos de desmineralização e potencializar a remineralização. Os fluoretos estão disponíveis para a maior parte da população na água de abastecimento, e na forma de dentifrícios, bochechos, aplicações tópicas em géis ou vernizes ou ainda pode ser liberado de materiais restauradores prevenindo as lesões secundárias (THYLSTRUP & FEJERSKOV, 1994; RODRIGUES et al., 2005; MOI et al., 2005). As lesões secundárias são lesões que se desenvolvem ao redor das restaurações, sendo ocasionadas pelo mesmo agente da lesão primária, o ácido gerado no biofilme bacteriano, promovendo um desequilíbrio entre a desmineralização e a remineralização, favorecendo a desmineralização. Entretanto, estas lesões podem se desenvolver em duas frentes: na superfície como a lesão primária e através da parede da cavidade quando há uma falha no selamento marginal da restauração (TANTBIROJN et al., 1997). Nesse contexto, o uso de materiais restauradores adesivos e que possuem a vantagem de liberação de flúor com propósitos preventivos vem recebendo muita ênfase e é amplamente discutido (TANTBIROJN et al., 1997; RODRIGUES et al., 2005; MOI et al., 2005). Essa técnica preventiva que emprega materiais que liberam flúor surgiu com os cimentos de silicato que proporcionavam às paredes das cavidades um alto grau de resistência à formação de lesões de cárie, causado pela alta liberação de fluoretos (HALS, 1975). Porém, estes cimentos eram muito solúveis e foram substituídos pelos cimentos de ionômero de vidro, que em relação aos cimentos de silicato, possuem menor solubilidade, mas mantém a ação anticariogênica pela liberação de flúor, considerada de grande importância na prevenção de cáries secundárias (HICKS et al., 1986; TANTBIROJN et al., 1997).

11 8 Apesar de melhoras nas propriedades estéticas, mecânicas e biocompatibilidade, os cimentos de ionômero de vidro ainda possuem algumas limitações podendo sofrer desequilíbrios hídricos que podem comprometer seu desempenho clínico (ARAÚJO et al., 2006). Materiais híbridos de ionômero de vidro e resina composta foram desenvolvidos no final da década de 80, apresentando como vantagens os resultados estéticos, a facilidade de aplicação e a presa imediata pela luz, com maior resistência ao desgaste e efeito cariostático semelhante aos ionômeros convencionais (DIJKMAN et al., 1993). Devido à necessidade estética, fluoretos também foram adicionados à fórmula de algumas resinas compostas e sistemas adesivos, mas o efeito cariostático destes materiais ainda é questionável pois para que o flúor tenha ação deve se tornar ionizado e, para tanto, deve se desprender da matriz resinosa a qual pode perder propriedades físicas. Poucos estudos demonstram a efetividade destes materiais (KERBER & DONLY, 1993; PARK & KIM, 1997; FERRACANE et al., 1998; LOBO et al., 2005; RODRIGUES et al., 2005). Paralelamente ao desenvolvimento dos materiais restauradores com ação cariostática, em 1965 estudos sugeriram a utilização do laser de alta potência, principalmente o laser de Er:YAG, como ferramenta na prevenção da cárie dental por promover uma maior ácido-resistência ao esmalte (YAMAMOTO & SATO, 1980). A grande parte dos estudos recentes está focada nos efeitos da irradiação laser sobre o esmalte desmineralizado isolada ou em associação aos fluoretos tópicos. Estes empregam ensaios de microradiografia, espectroscopia Raman, microscopia de luz polarizada, microscopia eletrônica de varredura, ensaios de microdureza e avaliação clínica. Tais estudos demonstram que os lasers que tem afinidade por hidroxiapatita e água como o de argônio, CO 2 ou os de Érbio podem reduzir a desmineralização do esmalte frente ao desafio cariogênico em 30 50% (CEBALLOS et al., 2001; HARAZAK et al., 2001; KLEIN et al., 2005; FREITAS et al., 2005; CECCHINI et al., 2005; KIM et al., 2006; LIU & HSU, 2007). O mecanismo pelo qual ocorre o ganho de ácido-resistência ainda não está totalmente claro, alguns autores atribuem ao efeito dos lasers de derretimento do esmalte dental sem a ocorrência do fenômeno de ablação. A ablação é um efeito do aquecimento e vaporização da água, resultando em altas pressões internas, com microexplosões resultando na remoção do conteúdo orgânico e inorgânico, alterando a superfície do esmalte (HIBST & KELLER, 1989). Este mesmo efeito é esperado para estes lasers nos preparos cavitários, por exemplo, o laser de Er:YAG causa uma efetiva ablação em tecido saudável, assim como em

12 9 lesões cariosas, sem causar danos térmicos aos tecidos adjacentes, e é indicado para a remoção de tecido dental no preparo de cavidades visto que possibilita o máximo de conservação de estrutura dental e não ocasiona danos a polpa (MISERENDINO & PICK, 1995; CORDEIRO et al., 2005). Ceballos et al. (2001), prepararam cavidades classe V e condicionaram com laser de Er:YAG ( mJ 2-2Hz) e restauraram com resina composta. Após um desafio cariogênico observaram através de microscopia de luz polarizada uma redução de 56% na profundidade de lesão. Concordando com Klein et al. (2005), que demonstraram que a irradiação da margem cavo-superficial de restaurações de resina composta com laser de CO 2 foi capaz de inibir a perda de minerais no esmalte humano e com Harazak et al. (2001); que observaram através da avaliação por fotografias que o laser de Nd:YAG (40J/cm 2-20Hz 5s) é efetivo na prevenção da formação de manchas brancas in vitro, em pré-molares humanos, imersos em ácido lático, bem como pode ser utilizado in vivo em associação com flúor na reversão de lesões iniciais de mancha branca ao redor de braquetes ortodônticos. No estudo in vitro, realizado por Freitas et al. (2005), observou-se que a irradiação do laser de ER,Cr:YSGG inibe o processo de desmineralização do esmalte e aumenta a sua ácido-resistência. Cecchini et al. (2005), avaliaram in vitro a eficácia do laser de Er:YAG no aumento da ácido-resistência do esmalte, por meio de espectrometria de força atômica verificando a quantidade de cálcio e fósforo do esmalte, e esta análise associada à microscopia eletrônica de varredura demonstrou que a aplicação do laser de Er:YAG com baixos níveis de energia oferece diminuição da solubilidade do esmalte sem causar alterações na estrutura superficial. Observa-se ainda por difração de Rx e espectrofotômetro de emissão de plasma atômico, que o esmalte bovino tratado com um pulso de laser de Er:YAG (33J/cm 2-2Hz) apresentaram uma maior quantidade de Ca, sendo uma perda de 10% de Ca e 13% de fosfato menor do que o esmalte bovino normal frente a um modelo de desafio cariogênico (KIM et al., 2006). Ainda através de espectroscopia Raman, Liu & Hsu (2007) demonstraram que dentes decíduos tornam-se mais resistentes à desafios cariogênicos após a aplicação do laser de Er:YAG (5.1 J/cm 2 2 Hz 5s). Por outro lado Apel et al. (2003), compararam a resistência ao desafio cariogênico de cavidades preparadas com lasers de Er:YAG e de Er,Cr:YSGG. Empregando microscopia de luz polarizada, não encontraram diferenças estatísticas entre os lasers, e o grupo que recebeu o preparo cavitário com pontas diamantadas apresentou profundidade de lesão estatisticamente menor que os grupos preparados com os lasers. Assim, concluíram que o preparo cavitário ou a aplicação de lasers não oferece resistência a cárie.

13 10 Assim, pode-se notar a existência de poucos estudos que avaliam o efeito de ácido-resistência sugerido ao laser durante o preparo cavitário e condicionamento da superfície, bem como a ausência da associação desta técnica com materiais que apresentam efeito cariostático, indicados para pacientes de alto risco de cárie. Dessa forma, não se sabe se a associação do preparo cavitário com laser e o uso de materiais restauradores pode ter um efeito sinérgico inibindo ainda mais o desenvolvimento de lesões cariosas secundárias.

14 11 2. PROPOSIÇÃO O propósito deste trabalho foi avaliar, in vitro, a influência da técnica do preparo cavitário convencional com alta rotação e pontas diamantadas e com laser de Er:YAG associadas a materiais restauradores cariostáticos na prevenção do desenvolvimento de cárie secundária.

15 12 3. DESENVOLVIMENTO Em uma seqüência lógica o tema deste trabalho foi estudado por intermédio do desenvolvimento de quatro estudos, aprovados no Comitê de Ética em Pesquisa da Universidade Guarulhos (Anexos A, B e C), apresentados a seguir como capítulos: Capítulo 1: Artigo de revisão de literatura: Uso do laser na prevenção da cárie dental, submetido à revista Dentística on line. Capítulo 2: Artigo em fase de redação: Effect of the cavity preparation with Er:YAG laser and fluoride releasing materials in the prevention of caries lesions, a ser submetido à revista Lasers in Medical Science. Capítulo 3: Artigo aceito na revista Photomedicine and Laser Surgery: Cavity preparation and restorative materials influence on the prevention of secondary caries. (Anexo D) Capítulo 4: Artigo aceito na revista Saúde da Universidade Guarulhos: Correlation between visual and superficial microhardness evaluation of artificial secondary caries. (Anexo E)

16 Capítulo 1 Artigo submetido à revista Dentística on line USO DO LASER NA PREVENÇÃO DA CÁRIE DENTAL USE OF LASER IN DENTAL CARIES PREVENTION Mario Alberto Marcondes Perito 1 Ana Carolina Tedesco Jorge 2 Alessandra Cassoni 3 José Augusto Rodrigues 4 ENDEREÇO PARA CORRESPONDÊNCIA: Prof. Dr. José Augusto Rodrigues Programa de Pós-Graduação em Odontologia Universidade Guarulhos - UnG Rua Dr. Nilo Peçanha, 81 Prédio U 6º Andar Centro Guarulhos - CEP Tel (+55 11) Fax (+55 11) jrodrigues@prof.ung.br ou guto_jar@yahoo.com 1 Prof. Assistente da Universidade Guarulhos (UnG) e Diretor do Curso de Odontologia da UnG 2 Cirurgiã-Dentista Graduada na UnG. 3 Mestre e Doutora em Odontologia (Dentística) pela Faculdade de Odontologia da USP- SP, Profa. Adjunta da UnG. 4 Doutor e Mestre em Dentística pela Faculdade de Odontologia de Piracicaba (UNICAMP); Professor Adjunto da UnG

17 14 Use of laser in dental caries prevention Uso do laser na prevenção da cárie dental Resumo Desde o desenvolvimento dos primeiros lasers, pesquisas estão sendo realizadas com a finalidade de aprimorar seu uso em diferentes áreas. Na Odontologia a luz laser pode ser utilizada em diferentes especialidades, incluindo a prevenção de lesões cariosas primárias e secundárias. Este trabalho tem como objetivo discutir o uso da luz laser na prevenção da cárie dental. Os lasers mais utilizados na prevenção da cárie dental são os de Argônio, Érbio e dióxido de carbono (CO 2 ). Cada um destes trabalha com padrões diferentes mas com a mesma finalidade, a modificação do tecido dental tornando-o mais ácido-resistente. Nota-se através da revisão de literatura que os resultados observados em laboratório são muito promissores e os lasers podem ser utilizados na prevenção da cárie dental. Palavras-chave: Lasers, uso terapêutico, cárie dentária, desmineralização dental

18 15 1- Introdução A cárie dental é uma doença infecciosa que acarreta o desenvolvimento de lesões nos tecidos dentais quando não controlada. As lesões cariosas são o resultado do metabolismo bacteriano, na presença de carboidratos provenientes da dieta, com a produção de ácidos orgânicos que causam a desmineralização do esmalte e da dentina 1. A prevenção da doença cárie é baseada no controle dos múltiplos fatores que podem determinar ou moderar seu desenvolvimento, ou seja, é baseada na avaliação do risco de cárie do paciente e instituição de medidas que possam diminuir este risco como aperfeiçoamento da técnica de higiene bucal e aumento do uso de fluoretos pelos pacientes 2. Nos casos em que os pacientes necessitam de tratamento restaurador o objetivo inicial deve ser a adequação do meio bucal e redução da atividade de cárie do paciente, para que em seguida, sejam realizadas as restaurações definitivas e não haja reincidência de lesões, ou seja, desenvolvimento de cárie secundária 2;3. O desenvolvimento de lesões cariosas secundárias ainda é um dos principais motivos para substituição de restaurações, e a possibilidade de evitar ou mesmo retardar este tipo de lesão pode reduzir a necessidade de substituição de restaurações 2;4. Para tanto, além da instrução sobre higiene bucal na fase de adequação do paciente, pode-se utilizar materiais cariostáticos restauradores, como os híbridos de ionômeros de vidro em pacientes de alto risco 5;6;7. O potencial cariostático dos materiais ionoméricos convencionais e dos híbridos vem sendo amplamente estudado desde a década de e o efeito cariostático dos materiais ionoméricos na prevenção de lesões de cárie secundária já é bem descrito na literatura e estes possuem grande aplicabilidade clínica 5;6;7;8. Paralelamente ao desenvolvimento destes materiais restauradores cariostáticos ocorreu a descoberta do laser e iniciaram-se os primeiros experimentos em Odontologia 9, nos quais foi notada a capacidade da luz laser de modificar os tecidos dentais duros tornando-os mais ácido-resistentes 10;11;12. Laser é o acrônimo de Light Amplification by Stimulated Emission of Radiation, que significa Ampliação de Luz por Meio da Emissão Estimulada de Radiação, ou seja, o laser nada mais é do que uma luz que quando emitida vai promover fenômenos físicos e interagir com os tecidos como qualquer outro tipo de luz, a diferença é que é uma luz com comprimento de onda específico emitida em um feixe monocromático, coerente e colimado

19 16 que pode ser facilmente focado para aplicação no tecido desejado obtendo interação ou efeito terapêutico 12. Entretanto, existem diferentes tipos de laser, que podem ser utilizados para o preparo cavitário ou mesmo para modificar o esmalte e dentina visando a prevenção do desenvolvimento de lesões cariosas e este trabalho tem como objetivo demonstrar os lasers indicados para prevenção da cárie dental. 2- Uso do laser na prevenção de lesões cariosas A luz laser quando incide sobre um material pode sofrer, em combinação ou não, quatro fenômenos físicos: reflexão, quando a luz é refletida em outra direção; transmissão, quando a luz atravessa diretamente o material e não causa nenhum efeito, difusão, quando a luz penetra no material mas se difunde no mesmo; e absorção, quando a luz é absorvida. Desses, a absorção é o fenômeno mais desejado sobre os tecidos dentais, pois é através deste que a energia luminosa do laser se transforma em calor e promove alterações que podem tornar os tecidos dentais mais ácido-resistentes 13. O primeiro laser desenvolvido foi o de rubi, e sua primeira tentativa de uso em Odontologia, como substituto das pontas diamantadas, foi pouco explorada no início, pois a quantidade de energia gerada era muito grande e somente 20% era absorvida e produzia uma grande quantidade de calor que se difundia por todo o tecido 14. A produção de calor em excesso pelos lasers é um efeito co-lateral não desejado, pois pode acarretar em danos nos tecidos pulpares e periodontais adjacentes. Assim, o laser ideal deve produzir a ação desejada gerando pouco calor, o qual deve se restringir ao local desejado 15. Com o avanço da pesquisa científica novos lasers que possuem maior absorção pela hidroxiapatita e pela água foram desenvolvidos e estes se destacaram para o uso em tecidos dentais duros e na prevenção de lesões de cárie dental 16;17. Esta prevenção é obtida pela modificação da estrutura do esmalte tornando-o mais ácido-resistente 16;18. A ácido-resistência é obtida com a absorção do laser pela hidroxiapatita e sua subseqüente conversão em calor. O calor gerado causa alterações microestruturais e químicas na hidroxiapatita, ocorre o derretimento da mesma e re-cristalização que gera modificações da estrutura da hidroxiapatita com o aumento da proporção de minerais e redução de carbonato e água que sofrem evaporação 14;16;18;19;20. Embora a presença de matéria orgânica seja pouca, sua eliminação

20 17 garante uma maior ácido-resistência e supõe-se que os micro-espaços formados são rapidamente mineralizados e re-cristalizados 10;21. Outro efeito observado após a aplicação do laser é a redução da permeabilidade dental, efeito que diminui a passagem dos ácidos gerados pelas bactérias através da estrutura dental dificultando a desmineralização e retardando a progressão de lesões cariosas 10. Assim, o uso do laser nas superfícies dentais, ao redor das restaurações, bem como a irradiação das paredes de preparos ou a total confecção dos mesmos com o laser pode ser considerada uma medida profilática para o desenvolvimento de lesões cariosas secundárias 4. Diversos tipos de laser estão sendo estudados para o uso profilático da cárie dental, entretanto, a inibição de lesões cariosas varia de acordo com o tipo de comprimento de onda, modo operacional e densidade de energia utilizada, o que torna difícil uma comparação entre eles 4. Os lasers utilizados para este fim são os de Argônio, CO 2, Nd:YAG, Er:YAG e Er,Cr:YSGG. O laser de CO 2, que possui meio ativo gasoso e como facilitadores os gases de He, N 2 e CO 2, com comprimento de onda entre 9,3 e 10,6 µm no espectro infravermelho, foi um dos primeiros aplicados na prevenção da cárie dental e se destacava por atuar com pequenas densidades de energia, com 13 a 50 J/cm 2 modificando o esmalte dental de uma forma similar ao laser de rubi (trabalhando de 200 a 700J/cm 2 ), diminuindo significativamente a produção de calor e de fissuras na superfície dental 22. Em uma revisão de literatura sobre laser de dióxido de carbono em prevenção de cáries, Rodrigues et al. 23 afirmam que irradiação do esmalte dental pelo laser de CO 2 altera os cristais de hidroxiapatita reduzindo a reatividade ácida dos minerais. Com o intuito de avaliar o efeito preventivo do laser de CO 2 in vivo, Brugnera Junior et al. 24, em 1997, trataram 112 primeiros molares permanentes de pré-adolescente com selante ou laser e observaram, após 4 anos, que a aplicação individual do laser não foi suficiente na prevenção de lesões cariosas, porém, pode apresentar um efeito preventivo mais vantajoso se associada à aplicação de selantes. Tsai et al. 25 avaliaram a resistência ácida de dentes humanos tratados com o laser de CO 2 e laser de Nd:YAG ao processo de desmineralização durante 24 e 72 hs e observaram que o grupo tratado com o laser de CO 2 apresentou menor concentração de cálcio dissolvida no tampão lactato do que o laser de Nd:YAG, e este não foi diferente do grupo controle em 24 hs. Mais focado na prevenção de lesões secundárias, Klein et al. 4, em 2005, irradiaram as paredes de preparos cavitários com o laser de CO 2 com comprimento de onda de 10,6 µm e

21 18 observaram a fusão e derretimento das mesmas em microscopia eletrônica de varredura. Quando os preparos restaurados foram submetidos ao desafio térmico e cariogênico observaram uma redução na perda mineral, sendo que a maior redução foi obtida quando utilizada densidade de energia de 16J/cm 2 comparada a de 8J/cm 2. Kantorowitz et al. 19, em um estudo in vitro também observaram que o aumento do número de pulsos do laser de CO 2 levou a um aumento da inibição de lesões cariosas, e que existe um ponto limite, após o qual, o aumento da densidade de energia não acarreta em uma maior ácido-resistência, sendo que o laser de CO 2 com comprimento de onda 10,6µm causou a fusão do esmalte dental e o de 9,6µm causou somente pequenos pontos de fusão, sendo o mais indicado. Fried et al. 17, em 2006, observaram que o laser de CO 2 com comprimento de onda de 9,3 µm utilizado com refrigeração reduz a dissolução do esmalte dental, e que o uso sem a refrigeração pode causar exposição excessiva ao calor e produzir cristais mais susceptíveis a dissolução, proporcionou um efeito inverso. Segundo Tepper et al. 26, em 2004, a associação da irradiação com o laser de CO 2 aos fluoretos pode promover um efeito sinérgico com maior incorporação de flúor no esmalte dental e um menor desenvolvimento de trincas visto que o flúor pode atuar refrigerando o esmalte durante a irradiação. Assim, observa-se que o laser de CO 2 possui efeito preventivo, sendo que o aumento da densidade de energia pode aumentar a ácido-resistência do esmalte. Entretanto, é extremamente necessário o uso de refrigeração para evitar a formação de trincas e poros, e a associação de flúor pode ser o veículo de refrigeração e potencializar o efeito de ácidoresistência 26;27;28. Outro laser muito utilizado em associação com a aplicação tópica de flúor é o de Argônio que apresenta como meio ativo o gás Argônio e possui comprimentos de onda na faixa do espectro eletromagnético visível 488nm (azul) e 514nm (verde) 29;30;31. Este é utilizado como co-adjuvante durante a aplicação tópica de flúor pois devido à baixa potência empregada, embora seja classificado como laser de alta potência, causa mínimos efeitos aos tecidos dentais duros e potencializa o efeito do flúor 21. Hicks et al. 32 notaram mudanças topográficas na superfície adjacente às restaurações de resinas compostas e cimento de ionômero de vidro modificado por resina ativados pelo laser de Argônio. Acredita-se que as alterações na estrutura mineral e componentes orgânicos produzem uma superfície menos susceptível à formação de cáries. Hicks et al. 33 investigaram o papel da radiação com laser de Argônio e sua combinação com aplicação tópica de flúor na redução da formação de lesões de cárie in vivo. Somente a aplicação prévia de laser de Argônio com baixa fluência (12J/cm 2 ) reduziu em 44% a profundidade das lesões. Quando

22 19 associada à aplicação tópica de flúor houve uma redução das lesões de cárie na ordem de 62%. Em 1995, Flaitz et al. 34 observaram uma redução de 26 a 32% das lesões no esmalte dental após a irradiação com o laser de Argônio e de mais de 50% quando o laser foi associado ao flúor. Da mesma forma, outros estudos tem demonstrado que o uso do laser de Argônio promove um pequeno grau de ácido-resistência, mas quando aplicado juntamente com o flúor pode-se aumentar significativamente a ácido-resistência do esmalte e dentina 29;30;35. O laser mais estudado e utilizado na prevenção da formação de lesões cariosas é o de Er:YAG, este apresenta como meio ativo sólido o cristal de ítrio-alumínio-granada dopado com érbio (2,94µm), e atua diretamente na estrutura do esmalte e dentina assim como o de CO 2, vaporizando a água e outros componentes orgânicos para aumentar a ácidoresistência 18;27. Hossain et al. 36, em 2003, demonstraram que após a irradiação com o laser de Er:YAG observa-se um aumento na proporção de cálcio e Fósforo no tecido dental, sem modificar a razão entre estes minerais e está de acordo com o estudo de Liu & Hsu 21, em 2007, que relatam que a quantidade de minerais após a irradiação com este laser é a mesma, o que ocorre é a diminuição do conteúdo orgânico, o que é associado ao aumento da ácido resistência. Este laser também pode ser utilizado para o preparo cavitário e seu uso associado à irrigação dos tecidos o torna mais eficiente e efetivo sem causar danos térmicos, apresentando como vantagem a modificação das paredes do preparo aumentando a cristalinidade e diminuindo a perda mineral, 37 ou seja, tornando-as mais ácido-resistentes, podendo resultar em uma redução de 56% em profundidade na formação de lesões cariosas secundárias em esmalte e 39% em superfície radicular 38. Liu et al. 39, em 2005, relatam que uma energia de 200mJ para o Er:YAG (sem spray de água) atingiram redução do tamanho das lesões de cárie em 32%. Efeitos similares aos do laser de Er:YAG na prevenção de lesões cariosas primárias ou secundárias tem sido observados com o laser de Er,Cr:YSGG (ítrio-scandiuum-gáliogranada dopado com érbio e comprimento de onda de 2,79µm), mesmo em doses subablativas utilizadas somente como medida preventiva 18. Yu et al. 40 afirmam após análise em microscopia atômica que o esmalte dental irradiado com Er,Cr:YSGG apresenta uma diminuição dos íons cálcio, porém, a proporção entre cálcio e fósforo permaneceu a mesma provavelmente devido a reorganização dos cristais de hidroxiapatita. Além destes usos, é sugerido o seu uso para modificar o esmalte e dentina promovendo uma melhor superfície para adesão, dispensando assim o condicionamento ácido,

23 20 visto que remove efetivamente toda a camada de esfregaço 41. Entretanto, os parâmetros testados ainda não permitem a formação de um padrão na superfície do esmalte que favorece a adesão e em dentina o efeito térmico parece penetrar em camadas sub-superficiais eliminando a água e desestruturando a dentina o que pode prejudicar a formação da camada híbrida 27;42. Assim, ainda existem dúvidas sobre os parâmetros mais adequados para utilização dos lasers de Érbio para obter uma boa adesão e evitar microinfiltração 27;36;42;43. Rolla et al. 44 obtiveram bons resultados com o uso do laser de Nd:YAG para o condicionamento e relatam ainda que este laser pode ser utilizado para prevenção da formação de lesões cariosas. A irradiação dos tecidos dentais com o laser de Nd:YAG, que apresenta como meio ativo sólido o cristal de ítrio-alumínio-granada dopado com neodímio e possui comprimento de onda no infravermelho (1064nm), promove ácido-resistência pela evaporação da água e conteúdo orgânico 20;44;45. Kwon et al. 46 afirma que o esmalte dental irradiado com Nd:YAG apresenta um aumento da proporção entre cálcio e fósforo após ablação devido a redistribuição dos minerais atuando de forma preventiva. Apesar de poucos estudos comparativos sobre os efeitos do laser de Nd:YAG na prevenção da cárie, ele parece ser tão eficiente quanto o laser de Er:YAG 47. Assim, apesar de existirem diversos tipos de lasers que podem ser utilizados na prevenção da cárie dental observa-se que todos promovem um aumento da ácido-resistência do esmalte e da dentina. Dentre eles, os de Érbio são os mais promissores, pois apresentam diversas indicações comprovadas quando comparados com outros lasers que possuem indicações mais específicas, tornando o uso dos demais lasers mais oneroso aos clínicos pois necessitariam adquirir diversos tipos de lasers. Porém, devido ao custo elevado para aquisição dos lasers e seus efeitos colaterais, ainda discutidos, como alterações no processo adesivo ainda são uma barreira para que os clínicos possam usufruir de seus benefícios, mas com o avanço tecnológico e da pesquisa científica em um breve intervalo de tempo os lasers poderão ter seu custo diminuído e os parâmetros de uso definidos para obter resultados ainda mais efetivos e possivelmente se tornarão uma realidade clínica 19.

24 21 3- Conclusão Observa-se na literatura que a irradiação laser pode tornar os tecidos dentais mais ácido-resistentes, o que pode evitar ou retardar o desenvolvimento de lesões cariosas primárias ou secundárias.

25 22 Abstract Since the development of the first lasers, research is being carried to improve its use in different areas. In dentistry the laser light can be used in different specialties, including the prevention of primary and secondary caries lesions. This literature review describes the laser light use in the prevention dental caries. The lasers more used in the prevention dental caries argon, erbium and CO 2. Each one of these works with different standards but with the same purpose, the modification of dental tissues and promoting it more acid resistance. Through the present literature review it was observed in laboratory researches that lasers are a very promising technology and they can be used in the prevention of dental caries development. Key-words: Lasers, therapeutic use, Dental Caries, Tooth Demineralization

26 23 Referências Bibliográficas 1. Clarkson BH, Rafter ME. Emerging methods used in the prevention and repair of carious tissues. J. Dent. Educ. 2001; 65: Fontana M, Zero DT. Assessing patients' caries risk. J. Am. Dent. Assoc. 2006; 137: Elderton RJ. Preventive (evidence-based) approach to quality general dental care. Med Princ Pract. 2003; 12 Suppl 1: Klein AL, Rodrigues LK, Eduardo CP, Nobre dos Santos M, Cury JA. Caries inhibition around composite restorations by pulsed carbon dioxide laser application. Eur. J. Oral Sci. 2005; 113: Serra MC & Rodrigues Júnior AL. Potencial Cariostático de Materiais Restauradores Contendo Flúor. Revista da Associação Paulista de Cirurgiões Dentistas 1998; 52: Rodrigues JA, Marchi GM, Serra MC, Hara AT. Visual evaluation of in vitro cariostatic effect of restorative materials associated with dentifrices. Braz Dent J. 2005;16: Hicks MJ, Flaitz CM. Resin-modified glass-ionomer restorations and in vitro secondary caries formation in coronal enamel. Quintessence Int. 2000; 31: Wilson AD, Kent BE. A new translucent cement for dentistry. The glass ionomer cement. Br. Dent. J. 1972; 132: Goldman L, Hornby P, Meyer R, Goldman B. Impact of the laser on dental caries. Nature. 1964; 203: Oho T, Morioka T. A possible mechanism of acquired acid resistance of human dental enamel by laser irradiation. Caries Res. 1990; 24: Blankenau RJ, Powell G, Ellis RW, Westerman GH. In vivo caries-like lesion prevention with argon laser: pilot study. J. Clin. Laser Med. Surg. 1999; 17: Sognnaes RF, Stern RH. Laser effect on resistance of human dental enamel to demineralization in vitro. J. S. Calif. Dent. Assoc. 1965; 33: Aoki A, Sasaki KM, Watanabe H, Ishikawa I. Lasers in nonsurgical periodontal therapy. Periodontol ; 36: Stern RH, Vahl JA, Sognnaes RF. Lased enamel: Ultrastructural observations of pulsed carbon dioxide laser effects J. Dent. Res. 1972; 51: Yamamoto H, Sato K. Prevention of dental caries by acousto-optically Q-switched Nd: YAG laser irradiation. J. Dent. Res. 1980; 59: 137.

27 Fried D, Featherstone JD, Le CQ, Fan K. Dissolution studies of bovine dental enamel surfaces modified by high-speed scanning ablation with a lambda = 9.3-microm TEA CO(2) laser. Lasers Surg. Med. 2006; 38: Fried D, Zuerlein M, Featherstone JDB, Seka W, McCormack SM. IR laser ablation of dental enamel: mechanistic dependence on the primary absorber. Appl. Surf. Sci. 1997: : Apel C, Birker L, Meister J, Weiss C, Gutknecht N. The caries-preventive potential of subablative Er:YAG and Er:YSGG laser radiation in an intraoral model: a pilot study. Photomed. Laser Surg. 2004;22: Kantorowitz Z, Featherstone JD, Fried D. Caries prevention by CO2 laser treatment: dependency on the number of pulses used. J. Am. Dent. Assoc. 1998; 129: Korytnicki D, Mayer MP, Daronch M, Singer Jda M, Grande RH. Effects of Nd:YAG laser on enamel microhardness and dental plaque composition: an in situ study. Photomedi Laser Surg. 2006; 24: Liu Y, Hsu CY. Laser-induced compositional changes on enamel: a FT-Raman study. J Dent. 2007; 35: Featherstone JD, Nelson DG. Laser effects on dental hard tissues. Adv Dent Res. 1987;1: Rodrigues LKA, Santos MN, Pereira D, Assaf AV, Pardi V. Carbon dioxide laser in dental caries prevention. J. Dent. 2004; 32: Brugnera Junior A, Rosso N, Duarte D, Pinto AC, Genovese W. The use of carbon dioxide laser in pit and fissure caries prevention: clinical evaluation. J Clin Laser Med Surg. 1997;15: Tsai C-L, Lin Y-T, Huang S-T, Chang H-W. In vitro acid resistance of CO 2 and Nd:YAG laser-treated human tooth enamel. Caries Res. 2002; 36: Tepper SA, Zehnder M, Pajarola GF, Schmidlin PR. Increased fluoride uptake and acid resistance by CO2 laser-irradiation through topically applied fluoride on human enamel in vitro. J. Dent. 2004; 32: Ceballo L, Toledano M, Osorio R, Tay FR, Marshall GW. Bonding to Er-YAG-lasertreated dentin. J. Dent. Res. 2002;81: Schmidlin PR, Dörig I, Lussi A, Roos M, Imfeld T. CO2 laser-irradiation through topically applied fluoride increases acid resistance of demineralised human enamel in vitro. Oral Health Prev. Dent. 2007; 5:

28 Westerman GH, Hicks MJ, Flaitz CM, Powell GL. In vitro caries formation in primary tooth enamel: role of argon laser irradiation and remineralizing solution treatment. J. Am. Dent. Assoc. 2006; 137: Westerman, GH, Hicks MJ, Flaitz CM, Ellis RW, Powell GL. Argon laser irradiation and fluoride treatment effects on caries-like enamel lesion formation in primary teeth: an in vitro study. Am. J. Dent. 2004;17: Sun G. The role of lasers in cosmetic dentistry. Dent. Clin. North Am. 2000; 44: Hicks J, Ellis R, Flaitz C, Werstermann G, Powell L. Restoration-enamel interface with argon laser and visible light polymerization of compomer and composite resin restorations: a polarized light and scanning electron microscopic in vitro study. J. Clin. Pediatr. Dent. 2003; 27: Hicks J, Winn D 2nd, Flaitz C, Powell L. In vivo caries formation in enamel following argon laser irradiation and combined fluoride and argon laser treatment: a clinical pilot study. Quintessence Int. 2004; 35: Flaitz CM, Hicks MJ, Westerman GH, Berg JH, Blankenau RJ, Powell GL. Argon laser irradiation and acidulated phosphate fluoride treatment in caries-like lesion formation in enamel: an in vitro study. Pediatr. Dent. 1995;17: Anderson JR, Ellis RW, Blankenau RJ, Beiraghi SM, Westerman GH. Caries resistance in enamel by laser irradiation and topical fluoride treatment. J. Clin. Laser Med. Sur. 2000;18: Hossain M, Nakamura Y, Murakami Y, Yamada Y, Matsumoto K. A comparative study on compositional changes and Knoop hardness measurement of the cavity floor prepared by Er:YAG laser irradiation and mechanical bur cavity. J. Clin. Laser Med. Surg. 2003; 21: Kim JH, Kwon OW, Kim HI, Kwon YH. Acid resistance of erbium-doped yttrium aluminum garnet laser-treated and phosphoric acid-etched enamels. Angle Orthod. 2006; 76: Ceballos L, Toledano M, Osorio R, Garcia-Godoy F, Flaitz C, Hicks J. ER-YAG laser pretreatment effect on in vitro secondary caries formation around composite restorations. Am. J. Dent. 2001;14: Liu JF, Liu Y, Stephen HC. Optimal Er:YAG laser energy for preventing enamel demineralization. J.Dent. 2006; 34: 62-6.

29 Yu D, Kimura Y, Kinoshita J, Matsumoto K. Morphological and atomic analytical studies on enamel and dentin irradiated by an Erbium,Chromium:YSGG laser. J. Clin. Laser Med. Surg. 2000; 18: Delme KI, Deman PJ, De Moor RJ. Microleakage of class V resin composite restorations after conventional and Er:YAG laser preparation. J. Oral Rehabil. 2005; 32: Chimello-Sousa DT, de Souza AE, Chinelatti MA, Pecora JD, Palma-Dibb RG, Milori Corona SA. Influence of Er:YAG laser irradiation distance on the bond strength of a restorative system to enamel. J. Dent. 2006; 34: Corona SA, Borsatto MC, Pecora JD, De SA Rocha RA, Ramos TS, Palma-Dibb RG. Assessing microleakage of different class V restorations after Er:YAG laser and bur preparation. J. Oral Rehabil. 2003; 30: Rolla JN, Mota EG, Oshima HM, Júnior LH, Spohr AM. Nd:YAG laser influence on microtensile bond strength of different adhesive systems for human dentin. Photomed. Laser Surg. 2006; 24: Naylor F, Aranha AC, Eduardo Cde P, Arana-Chavez VE, Sobral MA. Micromorphological analysis of dentinal structure after irradiation with Nd:YAG laser and immersion in acidic beverages. Photomed. Laser Surg. 2006; 24: Kwon YH, Kwon OW, Kim HI, Kim KH. Nd:YAG laser ablation and acid resistance of enamel. Dent. Mater. J. 2003; 22: Castellan CS, Luiz AC, Bezinelli LM, Lopes RM, Mendes FM, De P Eduardo C, De Freitas PM. In vitro evaluation of enamel demineralization after Er:YAG and Nd:YAG laser irradiation on primary teeth. Photomed. Laser Surg. 2007; 25:

30 Capítulo 2 Artigo em fase de redação a ser submentido à revista Lasers in Medical Science Effect of the cavity preparation with Er:YAG laser and fluoride releasing materials in the prevention of caries lesions Ana Carolina Tedesco Jorge 1, Mario Alberto Marcondes Perito 1, Patricia Moreira de Freitas 2, Alessandra Cassoni 3, Cristiane Mariote Amaral 3, José Augusto Rodrigues 3 1- DDS, Dental Research and Graduate Studies, Division Department of Restorative Dentistry, Guarulhos University, Guarulhos, SP, Brazil. 2- DDS, MS, PhD, Special Laboratory of Lasers in Dentistry, Department of Restorative Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil. 3- DDS, MS, ScD, Dental Research and Graduate Studies, Division Department of Restorative Dentistry, Guarulhos University, Guarulhos, SP, Brazil. Corresponding author: José Augusto Rodrigues R. Dr. Nilo Peçanha, 67 - Prédio U - 6º Andar - Centro - Guarulhos -SP, CEP: Brazil. / Phone: Fax: gutojar@yahoo.com jrodrigues@prof.ung.br ( to be published)

31 28 Effect of the cavity preparation with Er:YAG laser and fluoride releasing materials in the prevention of caries lesions Abstract The influence of the cavity preparation technique and the restorative materials containing fluoride in the prevention of the secondary caries were evaluated. Human teeth were sectioned into 72 blocks and distributed into 2 groups. Cavities measuring 1.6mm were performed with diamond burs or Er:YAG laser (6Hz, 300mJ, 47 J/cm 2 ). Each group was divided into 3 subgroups restored with a glass-ionomer cement, a resin-modified glass-ionomer, or a composite resin. The specimens were thermocycled and submitted to a ph cycling. Artificial caries were scored using an ordinal scale by visual inspection. Kruskal-Wallis and Dunn test (α=0.05) showed no differences in the cariostatic effect between the cavities restored with the same material and prepared with diamond burs or Er:YAG laser. Keywords: Erbium laser, dental caries, cariostatic agents, composite resins, glass ionomer cement, fluoride, dental enamel, secondary caries.

32 29 Introduction The metabolic bacteria processes in the biofilm are a physiological phenomenon that may lead to enamel mineral loss and subsequent cavity formation because of the imbalance in the dynamic equilibrium between tooth mineral and plaque fluid determining caries lesion development [1]. To avoid caries development, an individual preventive treatment based on the patients caries risk should be implemented [1]. Secondary caries is the lesion at the margin of an existing restoration similar to the primary caries but also may show lines of demineralized tissue on the cavity wall [2]. The presence of fluorides in the oral cavity may inhibit the demineralization process caused by bacteria acid production in the biofilm. Therefore the use of topical fluorides and restorative materials that release fluorides like glass ionomer based materials are useful tools to prevent secondary caries and also in enamel located at a considerable distance from the cavity margin [3-6]. However, some patients at high caries risk need additional care in preventive treatments to avoid primary or secondary caries development [1,5]. Some studies have shown the potential of laser irradiation on morphological and chemical changes in dental enamel by organic matrix decomposition and carbonate content reduction resulting in a less acidpermeable enamel with improved bacterial acid-resistance [6-8]. The most commonly used lasers for preventive procedures are CO 2 and Erbium lasers [6-8]. Although, they are classified as high intensity lasers, the energy densities required for caries preventive treatment are low and enamel ablation is avoided [7,9]. Ablation is a phenomenon that occurs when the laser energy is absorbed by water molecules and hydrous organic components of biological tissues, and the water vapor production induces an increase in internal pressure within the tooth tissue, resulting in microexplosions which cause dental tissue removal [10]. This way, ablative parameters are used to remove carious tissue and perform cavity preparations which shows as advantage, compared to conventional bur preparations, a significantly reduced need for local anesthesia, no vibratory or auditory irritation which is perceived by patients as more comfortable [11-12]. In spite of the energy densities used for cavity preparation are higher than densities used for caries prevention, heat is produced during ablation and transmitted through the cavity margins and this not ablated surface may be fused or melted with enamel recristallization resulting in a less permeable substrate to bacterial acid diffusion [13,14]. However it is not

33 30 known if the heat accumulation may be enough to thermally modify enamel chemical structure and improve its acid resistance as occurs by direct laser irradiation with subablative energy densities. In this way, if such increase in the acid resistance of enamel cavities margins are possible it may act synergistically with fluoride releasing restorative materials in the prevention of caries lesion development. Therefore, the present study aimed to investigate, in vitro by a visual evaluation, the effect of the cavity preparation with Er:YAG laser, on the inhibition of secondary caries around cavities filled with fluoride releasing restorative materials. Examiners evaluated, by visual examination, the presence and severity of caries lesions development around cavities prepared with burs or Er:YAG laser irradiation. Visual inspection is frequently used to quantify opacities, fluorosis and white spots lesions resulting from enamel demineralization in laboratorial and clinical studies [4,5,16-18]. Although this method may be considered as subjective compared to other methods such as microradiograph, polarized light microscopy or microhardness, visual inspection is simple, facilitates laboratory investigation and allows the inspection of the total net area resulting in a general result. In addition, it facilitates the conduction of studies faster and at lower costs and present correlation to other sophisticated methods [4,16]. Also, the examiners performed the diagnosis in a way similar to clinical diagnosis evaluating the absence or presence of white spot lesions, and quantified their activity and severity, considering that the opacity of the lesion increases as the mineral content decreases, by the use of a four-point ordinal scale [4,5] with the advantage of the magnification and room standardized conditions [16].

34 31 Material and Methods The Ethics Research Committee at the University Guarulhos approved the research protocol. The effects of the 3 restorative materials and 2 cavity preparation techniques with diamond burs or Er:YAG laser were evaluated by the use of human teeth. It resulted in 6 experimental groups (Table I). Table I. Restorative systems and cavity preparation Groups Cavity Preparation Restorative Systems G 1 G 2 G 3 G 4 Diamond burs (#2292, KG Sorensen, Barueri, SP, Brazil) Diamond burs Diamond burs Laser Er:YAG (Kavo Key II; Kavo, Biberach, Germany) Conventional glass ionomer cement (GI) (Ketac-Fil,3M/ ESPE, Seefeld, Germany) Resin-modified glass ionomer (RM) (Vitremer, 3M/ESPE, St. Paul, MN, USA) Composite resin (CR) (Z250, 3M/ESPE, St. Paul, MN, USA) Conventional glass ionomer cement G 5 Laser Er:YAG Resin-modified glass ionomer G 6 Laser Er:YAG Composite resin For blocks preparation, unerupted third molars were selected and stored in a 0.1% Thimol solution. The teeth were soft-tissue debrided and cleaned with water/pumice slurry and rubber cups in a low-speed handpiece (Kavo do Brasil, Joinville, SC, Brazil). The crowns were sectioned to obtain 72 dental enamel/dentin blocks (4x4x3mm 3 ) from the middle of the crows, using double-faced diamond discs #7020 (KG Sorensen, Barueri, SP, Brazil, ). Then, the blocks were stored in 100% humidity until cavity preparation. A total of 72 dental blocks (n=12/group) were restored in 12 steps. In each stage, 2 restoration of each restorative system in a cavity prepared with a diamond bur and in a cavity prepared with Er:YAG laser were made according to a randomized complete block design with 1 replication per block. The qualitative variable response development of artificial caries-like lesion was evaluated blindly and independently by 3 calibrated examiners using an ordinal scale based on visual examination. The blocks were distributed in two halves, one half had cylindrical class V cavities with approximately 1.6mm in diameter and 1.6mm depth prepared in high speedy with diamond burs #2292 using constant water spray coolant. The other half had the cavities prepared with Er:YAG laser working at 2,940 nm. The output power and pulse rate ranged from mj and 1 15 Hz, respectively. Working

35 32 with a distance of 12 mm from the lased surface, a handpiece (# 2056) with a 0.63 spot size, and energy of 300 mj with a repetition rate of 6 Hz, with an approximately energy density of 47 J/cm 2 was employed in a focused mode to prepare the cavities at continuous water spray (5 ml/min). The prepared blocks were randomly assigned to the 3 restorative materials subgroups (Table I). Restorations were done in 12 steps, in which one block per subgroup was filled. The sequence of restoration was determined at random and the materials were inserted according to the manufacturers instructions and photo-activated with an Optilux 501 device (Demetrom/Kerr, USA) with a mean of 700 mw/cm 2. In cavities filled with Ketac-Fil (3M/ESPE), the Ketac conditioner was applied for 10 s, rinsed and dried for 10 s. Ketac-Fil was prepared within s, inserted in the cavity with a centrix injector, protected with a lead strip for 5 min, coated with Vitremer Finish Gloss (3M/ESPE) and light-activated for 20 s to maintain the ionomer water stability. To Vitremer (3M/ESPE) restoration, the Primer was applied for 30 s, dried for 5 s and light-activated for 20 s. Vitremer was prepared within 45 s, inserted in the cavity with a centrix injector, photoactivated for 40 s, coated with Vitremer Finish Gloss and light-activated for 20 s. In cavities filled with composite resin, the 3M Scotch Bond etchant was applied for 15 s, rinsed for 10 s and air-dried. Two coats of Adper Single Bond 2 (3M/ESPE) were applied, air-dried for 5 s and light-activated for 10 s. The Z250 (3M/ESPE) composite resin was inserted and lightactivated for 20 s. All restored blocks were stored in 100% humidity for 24h and then polished using the Sof-lex (3M ESPE) disks for 15s with each disk under water-cooling at a low speed. The blocks were individually immersed in 1 ml of deionized distilled water to avoid ionic changes among them and thermocycled together for 1000 cycles in water between 5 ± 2ºC and 55 ± 2ºC with a dwell time of 2 min for each bath and a 15 s transfer time between baths [4]. A uniform area of exposed enamel surrounding the restorations was obtained by covering the remaining dental block with red wax. To simulate high caries risk conditions, the restored blocks were submitted to a demineralization/remineralization dynamic model, as proposed by Featherstone et al. [4,5,15]. This model simultaneously measures the net result of the inhibition of demineralization and the enhancement of remineralization. The demineralization stage uses an acid buffer containing 2 mmol/l Ca, 2 mmol/l PO 4, 0.075mol/L acetate at ph 4.3. The

36 33 remineralization solution contains calcium and phosphate at a know degree of saturation, to mimic the remineralizing properties of saliva, and 50 mmol/l KCl, 1.5 mmol/l Ca, 0.9 mmol/l PO 4, 20 mmol/l tri-hydroxymethylaminomathan buffered at ph 7.0 [5,15]. The blocks were immersed separately in 15 ml of demineralization solution for 6h, washed with deionized distilled water, immersed in 15 ml of remineralization solution for 18 h, washed and immersed in demineralization solution, thereby initiating a new cycle. The ph cycles were conducted during 14 days with 10 daily cycles. In the 6 th, 7 th, 13 th, and 14 th days of the cycle, the blocks were kept only in the remineralization solution [4,5,15]. After the 14 days the wax was removed, the blocks were air-dried for 15 s and standardized images were obtained from each slab with a Nikon D70 digital camera equipped with a macro #105 lens. Three calibrated examiners evaluated independently and blindly the images of all blocks projected in a dark room with approximately 100x magnification. The examiners evaluated these specimens scoring the presence and severity of caries-like lesions according to an ordinal scale ranked 0 to 3 based on visual examination, as described in Figure I [4]. Figure I Scores used to quantify artificial caries-like lesion development around restorative materials. A median was obtained from scores given by the 3 examiners for each block. Differences among the medians were analyzed by Kruskal-Wallis non-parametric test at a 95% confidence level and Dunn test. The calibration between examiners was verified by Kappa test.

37 34 Results The intra and inter-examiners kappa values are shown in Table II, and may be considered with good or excellent agreement. Table II- Kappa intra and inter-examiners values. Examiners The exploratory values to estimate of effect (medium) and variation (amplitude) and the results of Dunn test are shown in Table III. The greatest development of artificial caries lesions was in G3, which was prepared with DB and restored with CR, which showed statistical differences from G1, G2, G4, and G5. The G6 did not differ from G3 or from the other groups. The lowest incidence of artificial caries was observed in G4. Table III- Exploratory results of medium scores, median post, range from minimum to maximum scores (min-max), and Dunn test results per group. Glass-ionomer cement (GI), resin-modified glassionomer (RM), composite resin (CR), diamond bur (DB), Er:YAG laser (LA) Restorative material GI RM CR Cavity preparation DB LA DB LA DB LA Group G1 G4 G2 G5 G3 G6 Median Median post Min - Max Dunn test A A A A B AB

38 35 Discussion In the present study, the Er:YAG laser used for cavity preparation was not able to change enamel surface and guarantee a significantly higher acid-resistance than bur preparation against the acid challenge. The ph cycling model used to create the acid challenge and promote artificial caries like lesion is similar to the acid challenge found in a patient at high caries risk and shows a correlation with the onset and progression of caries lesions [15,19]. This method simulates the demineralization and remineralization phenomena occurring in oral environment and has often been recommended to investigate the effects of different substances in dental caries prevention aiming to correctly predict clinical outcomes [15,19]. There is an agreement that the fluoride released from restorative materials may inhibit secondary caries development [1-5,20-22]. Among the groups which cavities were prepared with burs, the group G1 restored with the glass ionomer cement showed the least artificial caries development. This result is in agreement with some previous studies that described the potential to prevent secondary caries by glass ionomer cements [4,5,22]. Also, some studies demonstrated that the resin-modified glass ionomer materials, which are hybrid materials, exhibit intermediate properties between their precursors glass ionomer cements and light-curing composite resin [4,5,23]. This result was observed in the present study, as G2 and G1 showed a similar anticariogenic effect, such effect was also observed among the lased preparations. Neither the composite resin nor the adhesive system used in the present study contains fluorides in their formulations, so it was observed that all blocks prepared with burs and restored with the composite resin showed artificial caries development, which scores ranged form 2 to 3. This result is in agreement with other studies that demonstrated that Z-250 did not present any cariostatic effect [4,5,16,20,25]. Chimello et al. reveal that after in situ caries development the Er:YAG laser did not differ from conventional cavity preparation with regard to enamel microhardness when restored with a composite resin [25]. Also a Polarized Light Microscopic analysis showed no differences irrespective of the Er:YAG laser parameters in comparison with the conventional bur cavity preparation [16]. However, after visual inspection of the specimens by image presentation in a dark room Chimello et al. observed that inhibition zone scores showed

39 36 significant difference among groups, which was ascribed to the control group which cavities were prepared with diamond burs and suggest a lower degree of demineralization at the restoration margin of the irradiated samples [16]. Although no statistical significant differences were found between the groups restored with composite resin (G3 and G6), all blocks in Group G3 presented caries development (scores 2-3) and the blocks prepared with Er:YAG laser (G6) ranged from 0 to 3. The presence of blocks without caries development in this group suggests some acid-resistance gained by enamel due to laser preparation that prevented the artificial caries development. This theory may be strongly reinforced by the absence of differences between the group prepared by Er:YAG laser and restored with composite resin (G6) and the group prepared with burs and restored with glass ionomer cement (G1). Also, from the comparison of scores range of groups G1 and G4 restored with glass ionomer cement, it can be observed that G1 present scores form 0 to 3 and G4 showed no advanced active caries like lesions (score 3) that also may suggest that some acidresistance may be promoted by laser preparation. Additionally, some studies showed that erbium lasers used with low energy densities may improve enamel acid-resistance [7,24], and a clinical trial showed that cavities prepared with Er,Cr:YSGG, after six months presented no secondary caries at the margins of the preparation sites [12]. In a previous study Perito et al. found less development of caries lesion around Er:YAG laser-prepared cavities than around the cavities prepared with diamond burs. However, no synergistic cariostatic effect was observed between the Er:YAG laser and glassionomer cement [26]. Despite of some evidence of acid-resistance gain was suggested, under the experimental conditions a synergic effect with glass ionomers materials or a simple improvement in the enamel acid-resistance after Er:YAG cavity preparation were not statistically confirmed. Conclusion In the present study, the Er:YAG laser used for cavity preparation did not show the ability to change enamel surface and guarantee significantly more acid-resistance than bur preparation against the acid challenge.

40 37 ACKNOWLEDGEMENTS We would like to thank the Special Laboratory of Lasers in Dentistry of the School of Dentistry of the University of São Paulo (LELO) for making their facilities available for us and for their friendly help during research. We also thank FAPESP (Grant n. 97/ ). DISCLOSURE STATEMENT The authors disclose any commercial or other associations that might pose a conflict of interest in connection with submitted material.

41 38 References 1. Elderton RJ (2003) Preventive (evidence-based) approach to quality general dental care. Med. Princ. Pract. 12, (Suppl 1) Mjör IA, Toffenetti F (2000) Secondary caries: a literature review with case reports. Quintessence Int 31: Tantbirojn D, Douglas WH, Versluis A (1997) Inhibitive effect of a resin-modified glass ionomer cement on remote enamel artificial caries. Caries Res 31(4): Rodrigues JA, Marchi GM, Serra MC, Hara AT (2005) Visual evaluation of in vitro cariostatic effect of restorative materials associated with dentifrices. Braz Dent J 16: DOI: /S Serra MC, Cury JA (1992) The in vitro effect of glass-ionomer cement restoration on enamel subjected to a demineralization and remineralization model. Quintessence Int 23: Fried D, Featherstone JD, Le CQ, Fan K (2006) Dissolution studies of bovine dental enamel surfaces modified by high-speed scanning ablation with a lambda = 9.3-microm TEA CO(2) laser. Lasers Surg Med 38(9): DOI: /lsm Kim JH, Kwon OW, Kim HI, Kwon YH (2006) Acid resistance of erbium-doped yttrium aluminum garnet laser-treated and phosphoric acid-etched enamels. Angle Orthod 76(6): Klein AL, Rodrigues LK, Eduardo CP, Nobre dos Santos M, Cury JA (2005) Caries inhibition around composite restorations by pulsed carbon dioxide laser application. Eur J Oral Sci 113(3): DOI: /j x 9. Kantorowitz Z, Featherstone JD, Fried D (1998) Caries prevention by CO 2 laser treatment: dependency on the number of pulses used. J Am Dent Assoc 129(5): Aoki A, Sasaki KM, Watanabe H, Ishikawa I Lasers in nonsurgical periodontal therapy. Periodontol , Keller U, Hibst R, Geurtsen W, Schilke R, Heidemann D, Klaiber B, Raab WH (1998) Erbium:YAG laser application in caries therapy. Evaluation of patient perception and acceptance. J Dent 26(8): Hadley J, Young DA, Eversole LR, Gornbein JA (2000) A laser-powered hydrokinetic system for caries removal and cavity preparation. J Am Dent Assoc 131(6):

42 Hossain M, Nakamura Y, Kimura Y, Yamada Y, Ito M, Matsumoto K 2000 Cariespreventive effect of Er:YAG laser irradiation with or without water mist. J Clin Laser Med Surg 18: Ying D, Chuah GK, Hsu CS. Effect of Er:YAG laser and organic matrix on porosity changes in human enamel. J Dent 2004;32: Featherstone JDB, O Really MM, Shariati M, Brugler S. Enhancement of remineralization in vitro and in vivo. In: Factors Relating to Demineralization and Remineralization of the Teeth. Leach SA (Editor). Oxford: IRL, p Chimello DT, Serra MC, Rodrigues AL Jr, Pécora JD, Corona SA (2008) Influence of cavity preparation with Er:YAG Laser on enamel adjacent to restorations submitted to cariogenic challenge in situ: a polarized light microscopic analysis. Lasers Surg Med 40(9): DOI: /lsm Gorelick L, Geiger AM, Gwinnet AJ (1982) Incidence of white spot formation after bonding and banding. Am J Orthod 81: Noel L, Rebellato J, Sheats RD (2003) The effect of argon laser irradiation on demineralization resistance of human enamel adjacent to orthodontic brackets: an in vitro study. Angle Orthod 73(3): Featherstone JD (1996) Modeling the caries-inhibitory effects of dental materials. Dent Mater 12(3): Pin ML, Abdo RC, Machado MA, da Silva SM, Pavarini A, Marta SN (2005) In vitro evaluation of the cariostatic action of esthetic restorative materials in bovine teeth under severe cariogenic challenge. Oper Dent May-Jun;30(3): Gonzalez Ede H, Yap AU, Hsu SC (2004) Demineralization inhibition of direct toothcolored restorative materials. Oper Dent 29(5): Cenci MS, Tenuta LM, Pereira-Cenci T, Del Bel Cury AA, ten Cate JM, Cury JA (2008) Effect of microleakage and fluoride on enamel-dentine demineralization around restorations. Caries Res 42(5): DOI: / Sidhu SK, Watson TF (1995). Resin-modified glass ionomer materials. A status report for the American Journal of Dentistry. Am J Dent 8(1): Liu Y, Hsu CY (2007) Laser-induced compositional changes on enamel: a FT-Raman study. J Dent 35(3): DOI: /j.jdent Chimello DT, Serra MC, Rodrigues-Júnior AL, Pécora JD, Corona SA (2008) Influence of Er:YAG laser on microhardness of enamel adjacent to restorations submitted to cariogenic challenge in situ. Photomed Laser Surg 26(4): DOI: /pho

43 Perito MAM, Jorge ACT, Freitas PM, Cassoni A, Rodrigues JA (in press) Cavity preparation and restorative materials influence on the prevention of secondary caries. Photomed Laser Surg.

44 Capítulo 3 Artigo aceito na revista Photomedicine and Laser Surgery Cavity preparation and restorative materials influence on the prevention of secondary caries Running Title: cavity preparation and secondary caries prevention Mario Alberto Marcondes Perito 1, Ana Carolina Tedesco Jorge 2, Patrícia Moreira de Freitas 3, Alessandra Cassoni 4, José Augusto Rodrigues 5 1- DDS, Dental Research and Graduate Studies, Division Department of Restorative Dentistry, Guarulhos University, Guarulhos, SP, Brazil. Phone: Fax: perito@prof.ung.br 2- DDS, Dental Research and Graduate Studies, Division Department of Restorative Dentistry, Guarulhos University, Guarulhos, SP, Brazil. Phone: Fax: carolinatedesco@yahoo.com.br 3- DDS, MS, PhD, Special Laboratory of Lasers in Dentistry, Department of Restorative Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil. Phone: Fax: pfreitas@usp.br 4- DDS, MS, ScD, Dental Research and Graduate Studies, Division Department of Restorative Dentistry, Guarulhos University, Guarulhos, SP, Brazil. Phone: Fax: alcassoni@gmail.com 5- DDS, MS, ScD, Dental Research and Graduate Studies, Division Department of Restorative Dentistry, Guarulhos University, Guarulhos, SP, Brazil. Phone: Fax: jrodrigues@prof.ung.br *Corresponding Author: Dr. José A. Rodrigues Department of Operative Dentistry, Guarulhos University Rua Dr. Nilo Peçanha 81, Predio U, 6 o. Andar Guarulhos, SP, Brazil, Phone: Fax: jrodrigues@prof.ung.br or guto_jar@yahoo.com.br

45 42 ABSTRACT Objective: This study evaluated in vitro the influence of cavity preparation using the Er:YAG laser and restorative materials containing fluoride on preventing caries lesions. Background: It has been suggested that cavity preparation using the Er:YAG laser has a potential for improving resistance to secondary caries on enamel. Methods: Forty unerupted human third molars teeth were used to obtain was sectioned into 72 blocks of dental enamel and distributed into 2 groups to prepare cavities measuring (1.6mmØ) with diamond burs (DB) or Er:YAG laser (LA - 6 Hz, 300 mj, 47 J/cm 2 ). After that, each group was divided into 3 subgroups and restored with a glass-ionomer cement (GI), a resin-modified glass-ionomer (RM), or a composite resin (CR). Blocks were thermalcycled and submitted to a ph challenge to develop artificial caries-like lesions. Lesions were evaluated by Knoop microhardness test. An average of 4 indentations was used. Statistical analyses were performed by ANOVA followed by Tukey s test. Results: The results (in KHN) for diamond bur cavity preparation (DB) were (GI) (±75.5); (RM) (±64.1); (CR) 39.3 (±26.5); and for Er:YAG laser cavity preparation (LA) were (GI) (±129.7); (RM) (±119.5); (CR) 96.4 (±57.4). Conclusions: There was less development of caries lesion around laser-prepared cavities than around the cavities prepared with diamond burs, however, no synergistic cariostatic effect was observed between the Er:YAG laser and glass ionomer cement. Keywords: Erbium laser, dental caries, cariostatic agents, composite resins, glass ionomer cement, fluorides, dental enamel, hardness.

46 43 INTRODUCTION A few decades ago dental caries was considered a common and unavoidable disease 1. Nowadays knowledge of the etiology and development of caries disease has allowed a reduction in caries risk and activity, by preventing and arresting caries lesions. Thus, the diagnoses of caries risk and individual treatment based on the reduction of their determinant and modulating factors are very important because there is a need for patient revert to a disease-free status by restoring the balance so that the forces tending to prevent the diseases outweigh the forces contributing to their progression. 1 One of the factors capable of moderating caries development is the presence of fluorides in the oral environment. Fluorides acts by reducing the critical ph for enamel dissolution from 5.5 to 4.5, thus enamel is able to resist a higher acid challenge 2. Fluorides may be found in the drinking water, toothpastes, mouthwash solutions, varnishes, and are also released from restorative materials. Fluoride releasing restorative materials are indicated to prevent secondary caries development in high-risk patients. 3,4,5 The potential cariostatic effect of restorative materials is described for researches that have shown high cariostatic effect of conventional glass ionomer cements, moderate cariostatic effect of glass ionomer and composite resin hybrid materials, and no cariostatic effect of composite resin materials. 3,4,6,7 On the other hand, few studies have suggested the use of lasers to modify dental enamel structure and improve its acid-resistance. The first lasers recommended for caries prevention were CO 2 laser, followed by Nd:YAG, Er:YSGG, and Er:YAG lasers. 8 Because of coincident band absorption by water and hydroxyapatite, CO 2 and erbium lasers efficiently heat the enamel surface to temperatures sufficient to inhibit acid dissolution and can prevent up to 80% of enamel dissolution in the face of an acid challenge with energy densities below the enamel ablation threshold. 9,10 Ablation is a phenomenon that occurs during laser irradiation when the laser energy is absorbed selectively by water molecules and hydrous organic components of biological tissues, causing evaporation of water and organic components and resulting in thermal effects due to the heat generated by this process, and the production of water vapor induces an increase in internal pressure within the tooth tissue, resulting in microexplosions that cause dental tissue removal. 11

47 44 Ablative parameters are used to perform cavity preparations, and although the more clinical time expensed, patients have perceived laser as more comfortable, without vibratory and auditory irritation, with a significantly reduced need for local anesthesia compared to mechanical means. 12 Among high intensity lasers, Fried et al. (1997) 9 suggested that an advantage of the Er:YAG lasers is the enamel ablation mechanism, which is primarily based on the principal absorber not in hard tissue. They reported that primary absorption in water results in water-mediated ablation, and primary absorption in the bulk of enamel rods results in melting and vaporization. 9 The absorption of the Er:YAG lasers by inorganic components (hydroxyapatite) is much lower than that of other lasers, such as CO 2 laser. 13 Thus, the absorption in water and hydrous organic components occurs rapidly before heat accumulation caused by absorption in inorganic components takes place, resulting in thermo-mechanical, explosive ablation. 13 Although the energy densities used for cavity preparation are higher than densities used for caries prevention, some heat is generated at the cavity margins during ablation, but it is not known if this heat accumulation would be enough to thermally modify the enamel and improve its acid resistance. This theory can be speculated from the results of studies that showed a tendency towards increased caries resistance after sub-ablative erbium laser irradiation 14 ; and that low energy densities of Er:YAG laser can decrease enamel solubility; as well as a clinical trial that showed that after six months, cavities prepared with Er,Cr:YSGG presented no secondary caries at the margins of the preparation sites. 15 Many studies showed the caries preventive potential of Er:YAG laser but the articles are focused on the use of sub-ablative parameters, and it is not known whether the heat accumulation during cavity preparation could provide enamel surface around cavity margins with some acid-resistance, and also whether this possible improvement in acid-resistance could act synergistically with restorative materials that release fluorides for the prevention of caries lesion development. Therefore, the aim of the present study was to investigate the effect in vitro, of cavity preparation with Er:YAG laser, on inhibiting enamel demineralization around fluoride releasing adhesive restorations.

48 45 METHODS AND MATERIALS Since this study was performed using human third molars, the research protocol was submitted to the Research Ethics Committee of the Guarulhos University and was approved in accordance with the resolution CNS# 196/96 of the National Health Committee/Health Department (Brazil). EXPERIMENTAL DESIGN The experimental units consisted of 72 dental blocks (n=12 per group) obtained from 40 unerupted human third molars. The factors under study were Method of Cavity Preparation (at two levels) and Restorative Material (at three levels) in a factorial design (Table 1). The response variable was surface microhardness in Knoop Hardness Number (KHN). Table 1- Experimental groups. Group DBGI DBRM DBRC LAGI Method of cavity preparation Diamond bur (#2292, KG Sorensen, Barueri, SP, Brazil) Diamond bur Diamond bur Er:YAG laser (Kavo Key II; Kavo, Biberach, Germany) Restorative material Glass ionomer cement (Ketac-Fil,3M/ ESPE, Seefeld, Germany) Resin modified glass ionomer (Vitremer, 3M/ESPE, St. Paul, MN, USA) Resin composite (Z250, 3M/ESPE, St. Paul, MN, USA) Glass ionomer cement LARM Er:YAG laser Resin modified glass ionomer LARC Er:YAG laser Resin composite PREPARATION OF DENTAL BLOCKS Following extractions, teeth were stored in a 0.1% Timol solution (ph 7.0) for no more than 30 days. 16 Soft-tissues were removed using periodontal curettes (HU-FRIEDY do Brasil, Rio de Janeiro- Brazil) and cleaning was performed using a slurry of pumice in a

49 46 webbed rubber cup applied with a slow-speed handpiece (Kavo do Brasil, Joinville- Brazil). The roots were removed, and the crowns were longitudinally and transversally sectioned to obtain 72 dental blocks measuring 4x4x3 mm 3 using double-faced diamond discs (#7020, KG Sorensen, São Paulo- Brazil). CAVITY PREPARATION AND RESTORATION Standardized circular cavities were prepared in the enamel blocks. Half of the samples were prepared with diamond burs. Cavities of approximately 1.6 mm in diameter and 1.6 mm deep were prepared at high speed with diamond burs No (KG Sorensen, Barueri, SP, Brazil, ) under a constant water spray coolant. The other half of the samples were irradiated using the Er:YAG laser (KaVo Key II; KaVo, Biberach, Germany) working at 2940 nm. The output power and pulse rate ranged from mj and 1 15 Hz, respectively. Working at a distance of 12 mm from the tooth surface (focused mode), a handpiece (# 2056) with a 0.63 mm spot size, and energy of 300 mj with a repetition rate of 6 Hz was used to prepare the cavities under continuous water spray (5 ml/min). The energy density was approximately 47 J/cm 2 and cavities were standardizing by visual and contact comparisons to diamond burs No used to mechanical preparation. After the cavity preparations, the blocks were randomized among the restorative material subgroups (Table 1) and the cavities in 12 blocks were restored, with one sample of each group, in one increment, according to the manufacturer s instructions. In cavities filled with Ketac-Fil, the Ketac conditioner was applied for 10 s, rinsed and dried for 10 s. Ketac-Fil was prepared within s, inserted in the cavity with a Centrix injector, protected with a mylar strip (Dentart, Polidental, São Paulo, Brazil; dimension 10x120x0.05mm 3 ) for 5 min, coated with Vitremer Finish Gloss and light-activated for 20 s by an Optilux 501 light unit (Demetron/Kerr, Danbury, CT, USA). The power density was measured by placing the light tip at the radiometer of the light unit. The light curing unit had a light tip diameter of 11 mm with an irradiance of 700 mw/cm 2. In cavities filled with Vitremer, the Primer was applied for 30 s, dried for 5 s and light-activated for 20 s. Vitremer was prepared within 45 s, inserted in the cavity with a Centrix injector, light-activated for 40 s, coated with Vitremer Finish Gloss and lightactivated for 20 s.

50 47 In cavities filled with Z-250, the 3M Scotch Bond etchant was applied for 15 s, rinsed for 10 s and air-dried. Two coats of 3M Single Bond were applied, air-dried for 5 s and light-activated for 10 s. The composite resin was inserted and light-activated for 20 s. All restored blocks were stored in 100% humidity for 24 h and then polished using the Sof-lex (3M ESPE) disks system for 15 s with each disk. THERMAL AND ACID CHALLENGE The blocks were placed into separate bags with 1 ml of deionized water and thermalcycled together for 1000 cycles in water between 5±2ºC and 55±2ºC with a dwell time of 2 min in each bath and a 15 s transfer time between baths 3. All external surfaces of each slab were coated with wax, leaving a 1.5 mm-wide margin around the restoration free of wax. The test scheme for acid challenge was designed to model a daily demineralization challenge of a 6 h and a 18 h repair (remineralization) by saliva as described by Featherstone et al. (1986) 17 and Serra & Cury (1992) 18, with the aim of simulating a high in vitro caries risk and producing artificial caries like-lesions around the restorations 2,7,19,20. The demineralization stage used an acid buffer containing 2 mmol/l Ca, 2 mmol/l PO 4, mol/l acetate at ph 4.3. The remineralization solution contained calcium and phosphate at a known degree of saturation (1.5 mmol/l Ca, 0.9 mmol/l PO 4 ), to mimic the remineralizing properties of saliva, and 50 mmol/l KCl, 20 mmol/l trihydroxymethylaminomathan buffer at ph ;18 The blocks were immersed separately in 15 ml of demineralization solution for 6 h, immersed in 15 ml of remineralization solution for 18 h, washed and immersed in demineralization solution, thereby initiating a new cycle. The ph cycles were conducted for 14 days with 10 daily cycles. In the 6 th, 7 th, 13 th, and 14 th days of the cycle, the blocks were kept only in the remineralization solution. At the end of the ph cycles, the wax was eliminated and the blocks were stored at 100% humidity until the microhardness test. MICROHARDNESS TEST The demineralization of the restored enamel blocks was assessed with a microhardness tester (PanTec, Panambra Ind. e Técnica SA, São Paulo- Brazil) and a Knoop indenter. The indentations were made keeping the long axis of the diamond instrument parallel to

51 48 the outer-leveled enamel surface, using a 25 g load applied for 5 s, and the value in micrometers of the higher diagonal was measured and automatically changed to KHN by the microhardness tester. Four measurements were made in each 100 µm around the restoration margins in the upper, left, right, and bottom sides (Figure 1). Figure 1- Location scheme for microhardness test. RESULTS The mean microhardness values and standard deviations per restorative material in each cavity preparation are presented in Table 2. Data were changed to x to obtain a normal distribution and were submitted to ANOVA considering the factorial 3X2 model to observe the factors and their interactions. There were statistical differences in the factors Restorative Materials and Method of Cavity Preparation (p< ); there was no interaction between the factors Restorative Materials and Method of Cavity preparation (p=0.3181).

52 49 Table 2- Means (standard deviation) of surface microhardness (KHN) for factors Method of Cavity Preparation (vertical), for Restorative Material (Horizontal) and for experimental subgroups. Means followed by the same lower case letters in the row indicate no statistical difference (Tukey s test, p<0.05) and different upper case letters indicate mean values that are statistically different in the column (Analysis of Variance, p<0.05) LA- Er:YAG laser A DB- Diamond Bur B GI- Ketac Fil 309.1a (129.7) (75.5) RM- Vitremer 207.3b (119.5) (64.1) CR- Z c 96.4 (57.4) 39.3 (26.5) The microhardness of enamel around cavities filled with GI showed the highest microhardness values, differing significantly from the cavities restored with RM, which showed intermediate values. The cavities filled with CR showed the lowest value (Table 2). The cavities prepared with LA showed significantly higher microhardness values than the DB cavities.

53 50 DISCUSSION Lasers with wavelengths that interact with water and hydroxyapatite allow the conservative removal of caries lesions and cavity preparation, and can also change the solubility of enamel, improving its acid resistance. 9,10,14,15 This study evaluated the development of artificial caries lesion on enamel around cavities prepared with diamond burs and Er:YAG laser, and filled with restorative materials with or without fluoride releasing properties, using a dynamic cyclic model of demineralization and remineralization, whose acid challenge was correlated to patients with high caries risk. 17 The highest development of artificial caries lesions in this study was observed in groups restored with composite resin, which had been expected because the composite resin or adhesive system used did not contain fluorides in their compositions. 3 This is consistent with reports from other studies, in which only composite resins and adhesive systems containing fluorides or antibacterial monomers are capable of showing an anticariogenic effect, which is lower than that of glass ionomers. 5,19 During acid challenges glass ionomer cements mobilize and release increased amounts of fluoride into the environment. The presence of fluorides continuously released by these restorative materials is an important feature for facilitating the re-precipitation of minerals, improving remineralization or inhibiting demineralization. 2 This is the reason for less artificial caries lesion development around cavities restored with conventional glass ionomer cement. 18,20,21 Therefore, the protection rendered by the glass ionomer cement is extended for some distance from the restoration and it is greatest in the cavity preparation area. 6 To a lesser extent than in conventional glass ionomers, the smaller concentrations of fluoride released from resin modified glass ionomer caused moderate development of artificial caries lesion, but in comparison with glass ionomer cement, it resulted in less inhibition. 4 The statistical analysis of the factor Cavity Preparation showed more artificial caries development in cavities prepared with diamond burs than in the cavities prepared with the Er:YAG laser. Numerous in vitro studies using a variety of laser wavelengths within subablative parameters have been conducted to investigate caries prevention and showed this effect. 14,22 They have shown the potential of some wavelengths to be absorbed by hydroxyapatite and water in enamel and dentin, and the conversion of the irradiated energy

54 51 into heat. This heat increase is considered to be the cause of the micro-structural and chemical changes occurring in lased enamel and dentin 22,23 and explains the increased acid resistance due to the reduction in permeability by the evaporation of organic matrix. However, the potential of the Er:YAG laser irradiation in an ablative parameter to improve the acid resistance of enamel around the cavity preparation was not totally clear, and although Chimello et al. 25 revealed that the Er:YAG laser did not differ from conventional cavity preparation, in the present study the enamel adjacent to cavities prepared with the Er:YAG laser showed less development of artificial caries lesion. 26 The mechanisms by which lasers can improve the tooth acid resistance may be due to the absorption of heat and penetration into the non-ablated enamel layers adjacent to cavity wall whose enamel was ablated during cavity preparation. 26 Thus, it can be supposed that the penetration of heat into the adjacent layers around the cavity walls may act in the same way as in the outer enamel with a reduction in organic matrix and enamel vitrification. 26 However, the present study was conducted with water cooling and it is recommended and indeed indispensable in order to avoid temperature damage to the dental pulp. 25 It is also important to point out that the present study selected safe parameters as regards temperature increase in the intrapulpal region. According to Geraldo-Martins et al. 27 the samples irradiated with the Er:YAG laser using the same parameters as the present study achieved an intrapulpal temperature increase of 1.45 (+0.64) o C. It can be also speculated that heat caused by laser is restricted to the superficial layers of the irradiated walls, which can be a limiting factor for obtaining high acid-resistance at some extended distance from the restoration margins. Although the G1 Group (GI/LA) followed by G2 Group (RM/LA) showed the highest numerical means, the interaction of factors Cavity Preparation and Restorative Materials expected to have a synergistic effect between fluoride release and the increase in the enamel acid resistance by the Er:YAG laser was not found in this study. As observed in the in vitro results, the use of Er:YAG laser in cavity preparations may be a suitable option for patients at high caries risk, by the increase in the enamel acidresistance and other advantages such as its microbial reduction potential and smear layer removal. 28 Although cavity sterilization and conditioning are obtained by Er:YAG laser irradiation, 29 some researchers showed the presence of a laser-modified layer that may adversely affect enamel and dentin bonding. This layer may obliterate enamel micropores, thus blocking the intra and interprismatic spaces, restricting resin interdiffusion into the enamel surface. 30,32 Moreover, the more acid-resistant lased surface might reduce the

55 52 effectiveness of acid etching and hybridization may be compromised. 32 In dentin hybridization, subsurface damage initiated by Er:YAG ablation may alter the subsurface under the hybrid zone and remnant denatured collagen fibrils may not resist to the forces from the polymerization shrinkage and fractures may occur, leading to microleakage. 30,31 A negative influence on marginal sealing of composite resin restorations with a total etch adhesive system after Er:YAG laser use has also been showed in microleakage studies. 30,31 However, results are poor and contradictories, other researches found no differences in the microleakage of composite resin, glass ionomer cement or resin modified glass ionomer restorations etched or prepared with Er:YAG laser. 34,35,36 Only Hadley et al. (2000) 12, in in vivo research, observed the performance of composite resin restorations in 66 cavities prepared with Er,Cr:YSGG (68,1 J/cm 2 ), in comparison with 66 cavities prepared with diamond burs. After 6 months all restorations were retained and no secondary caries was observed. In order to avoid secondary caries, not only the improvement of dental acid resistance is necessary 4 but further studies are needed as regards Er:YAG laser parameters to achieve the ideal association with adhesive systems, to produce an adequate hybrid layer to avoid the microleakage phenomena and improve dental acid-resistance. However, the use of Er:YAG laser in cavity preparation may thus be useful and effective in the prophylaxis and management of patients at high risk for dental caries, the present study showed less caries development around cavities prepared with Er:YAG laser than bur preparation but in vivo studies are necessary to confirm these in vitro results and verify the performance of the restorative materials inserted in these cavities. CONCLUSIONS The cavity preparation with Er:YAG laser may lead to an increase in the acidresistance in the enamel layers surrounding cavity walls, irrespective of the presence of fluorides in the restorative material. The high cariostatic effect was observed to conventional glass ionomer followed by the resin modified glass ionomer with a moderated cariostatic effect. The composite resin showed no cariostatic effect. No synergistic effect between glass ionomer cement and laser was observed.

56 53 DISCLOSURE article. The authors have no interest in any of the companies or products mentioned in this REFERENCES 1. Elderton, R.J. (2003). Preventive (evidence-based) approach to quality general dental care. Med. Princ. Pract. 12, (Suppl 1) Larsen, M.J. (1975). Degrees of saturation with respect to apatites in fruit juices and acidic drinks. Scand J Dent Res Rodrigues, J.A., Marchi, G.M., Serra, M.C., and Hara, A.T. (2005). Visual evaluation of in vitro cariostatic effect of restorative materials associated with dentifrices. Braz. Dent. J. 16, Dunne, S.M., Goolnik, J.S., Millar, B.J., and Seddon, R.P. (1996). Caries inhibition by a resin-modified and a conventional glass ionomer cement, in vitro. J. Dent. 24, Mukai, Y., Tomiyama, K., Shiiya, T., Kamijo, K., Fujino, F., and Teranaka, T. (2005). Formation of inhibition layers with a newly developed fluoride-releasing all-in-one adhesive. Dent. Mater. J. 24, Tantbirojn, D., Douglas, W.H., and Versluis, A. (1997). Inhibitive effect of a resinmodified glass ionomer cement on remote enamel artificial caries. Caries Res. 31, Pimenta, L.A., Fontana, U.F., Cury, J.A., Serra, M.C., Elderton, R.J. (1998). Inhibition of demineralization in vitro around amalgam restorations. Quintessence Int Jun;29(6): Harazaki, M., Hayakawa, K., Fukui, T., Isshiki, Y., and Powell, L.G. (2001). The Nd-YAG laser is useful in prevention of dental caries during orthodontic treatment. Bull. Tokyo Dent. Coll. 42, Fried, D., Zuerlein, M., Featherstone, J.D.B., Seka, W., and McCormack, S.M. (1997). IR laser ablation of dental enamel: mechanistic dependence on the primary absorber. Appl. Surf. Sci , Fried, D., Featherstone, J.D., Le, C.Q., and Fan, K. (2006). Dissolution studies of bovine dental enamel surfaces modified by high-speed scanning ablation with a lambda = 9.3- microm TEA CO(2) laser. Lasers Surg. Med. 38,

57 Aoki, A., Sasaki, K.M., Watanabe, H., and Ishikawa, I. (2000). Lasers in nonsurgical periodontal therapy. Periodontol , Hadley, J., Young, D.A., Eversole, L.R., and Gornbein, J.A. (2000). A laser-powered hydrokinetic system for caries removal and cavity preparation. J. Am. Dent. Assoc. 131, Koort, H.J., Frentzen, M. (1995). Laser effects on dental hard tissue, in: Lasers in Dentistry. Miserendino, L.J., Pick, R.M. (eds). Chicago: Quintessence, pp Apel, C., Birker, L., Meister, J., Weiss, C., Gutknecht, N. (2004). The caries-preventive potential of subablative Er:YAG and Er:YSGG laser radiation in an intraoral model: a pilot study. Photomed. Laser Surg. 22, Cecchini, R.C., Zezell, D.M., de Oliveira, E., de Freitas, P.M., and Eduardo, C. de P. (2005). Effect of Er:YAG laser on enamel acid resistance: morphological and atomic spectrometry analysis. Lasers Surg. Med. 37, Soares, L.E., Martin, A.A., Pinheiro, A.L., and Pacheco, M.T. (2004). Effects of treatment for manipulation of teeth and Er:YAG laser irradiation on dentin: a Raman spectroscopy analysis. Photomed. Laser Surg. 25, Featherstone, J.D.B, O Really, M.M., Shariati, M., and Brugler, S. (1986). Enhancement of remineralization in vitro and in vivo, in: Factors Relating to demineralization and remineralization of the teeth. Leach SA (ed.). Oxford IRL: pp Serra, M.C., Cury, J.A. (1992). The in vitro effect of glass-ionomer cement restoration on enamel subjected to a demineralization and remineralization model. Quintessence Int. 23, Okuyama, K., Nakata, T., Pereira, P.N., Kawamoto, C., Komatsu, H., and Sano, H. (2006). Prevention of artificial caries: effect of bonding agent, resin composite and topical fluoride application. Oper. Dent. 31, Mount, G.J. (1994). Glass ionomer cements: past, present and future. Oper. Dent. 19, Forsten, L. (1994). Fluoride release of glass ionomers, in: Glass-ionomers; the next generation. Hunt P. (ed.). Philadelphia: Peter Hunt pp Apfelbaum F, Mayer I, Featherstone JBD (1990) The role of HPO and CO - 3 ions in the transformation of synthetic apatites to β-ca 5 (PO 4 ) 2. J Inorg Biochem 38: Fowler BO, Kuroda S (1986) Changes in heated and in laser-irradiated human tooth enamel and their probable effects on solubility. Calcif Tissue Int 38:

58 Chimello, D.T., Serra, M.C., Rodrigues-Júnior, A.L., Pécora, J.D., Corona, S.A. (2008). Influence of Er:YAG laser on microhardness of enamel adjacent to restorations submitted to cariogenic challenge in situ. Photomed Laser Surg Apel, C., Schafer, C., and Gutknecht, N. (2003). Demineralization of Er:YAG and Er,Cr:YSGG laser-prepared enamel cavities in vitro. Caries Res. 37, Beviláqua, F.M., Zezzell, D.M., Magnani, R., Ana, P.A., and Eduardo, C.P. (2008). Fluoride uptake and acid resistance of enamel irradiated with Er:YAG laser. Photomed. Laser Surg. 23, Geraldo-Martins, V.R., Tanji, E.Y., Wetter, N.U., Nogueira R.D., and Eduardo, C.P. (2005). Intrapulpal temperature during preparation with the Er:YAG laser: an in vitro study. Photomed. Laser Surg. 23, Freitas, P.M., Navarro, R.S., Barros, J.A., Eduardo, C.P. (2007). The use of Er:YAG laser for cavity preparation: an SEM evaluation. Microsc. Res. Tech. 70, Délme, K.I.M., De Moor, R.J.G. (2007). Scanning electron microscopic evaluation of enamel and dentin surfaces after Er:YAG laser preparation and laser conditioning. Photomed. Laser Surg. 25, De Moor, R.J.G., Délme, K.I.M. (2006). Erbium lasers and adhesion to tooth structure. J. Oral Laser Applic. 6, Chimello-Sousa, D.T., de Souza, A.E., Chinelatti, M.A., Pecora, J.D., Palma-Dibb, R.G., and Milori-Corona, S.A. (2006). Influence of Er:YAG laser irradiation distance on the bond strength of a restorative system to enamel. J. Dent. 34, Ceballos, L., Toledano, M., Osorio, R., Garcia-Godoy, F., Flaitz, C., and Hicks, J. (2001). ER-YAG laser pretreatment effect on in vitro secondary caries formation around composite restorations. Am. J. Dent. 14, Corona, S.A., Borsatto, M.C., Pecora, J.D., De S.A. Rocha, R.A., Ramos, T.S., and Palma-Dibb, R.G. (2003). Assessing microleakage of different class V restorations after Er:YAG laser and bur preparation. J. Oral Rehabil. 30, Niu, W., Eto, J.N., Kimura, Y., Takeda, F.H., and Matsumoto, K. (1998). A study on microleakage after resin filling of Class V cavities prepared by Er:YAG laser. J. Clin. Laser Med. Surg. 16, Delme, K.I., Deman, P.J., Nammour, S., and De Moor, R.J. (2006). Microleakage of class V glass ionomer restorations after conventional and Er:YAG laser preparation. Photomed. Laser Surg. 24,

59 Chinelatti, M.A., Ramos, R.P., Chimelo, D.T., Corona, S.A., Pecora, J.D., and Dibb, R.G. (2006). Influence of Er:YAG laser on cavity preparation and surface treatment in microleakage of composite resin restorations. Photomed. Laser Surg. 24,

60 Capítulo 4 Artigo aceito na revista Saúde da Universidade Guarulhos Correlation between visual and superficial microhardness evaluation of artificial secondary caries Mario Alberto Marcondes Perito 1, José Augusto Rodrigues 2 1- DDS, Dental Research and Graduate Studies, Division Department of Restorative Dentistry, Guarulhos University, Guarulhos, SP, Brazil. Phone: Fax: perito@prof.ung.br 2- DDS, MS, ScD, Dental Research and Graduate Studies, Division Department of Restorative Dentistry, Guarulhos University, Guarulhos, SP, Brazil. Phone: Fax: jrodrigues@prof.ung.br *Corresponding Author: Dr. José A. Rodrigues Department of Operative Dentistry, Guarulhos University Rua Dr. Nilo Peçanha 81, Predio U, 6 o. Andar Guarulhos, SP, Brazil, Phone: Fax: jrodrigues@prof.ung.br or guto_jar@yahoo.com.br

61 58 Correlation between visual and superficial microhardness evaluation of artificial secondary caries Correlação entre avaliação visual e de microdureza superficial de cáries secundárias em esmalte Abstract This in vitro study evaluated the correlation of artificial secondary caries diagnosis on enamel between visual evaluation and superficial microhardness test. Cavities with standardized diamond burs (1.6mmØ) were prepared on thirty-six enamel blocks obtained from unerupted human third molars and were assigned into 3 groups. Each group was restored with glassionomer cement (GI), resin-modified glass-ionomer (RM), or composite resin (CR). Blocks were thermocycled and submitted to a ph challenge to develop artificial caries-like lesions. Lesions were analyzed by visual evaluation using scores and the results were submitted to Kruskal Wallis and Dunn Test. The hardness of the enamel surface surrounding the restored cavity was evaluated using Knoop microhardness test and results were submitted to ANOVA followed by Tukey s post-hoc test. Afterwards, the correlation between visual and microhardness analyses was verified by Spearman s rho nonparametric correlation test. Regarding visual analysis, no significant difference was observed between GI and RM groups, which showed less caries development than CR group. The microhardness evaluation showed significant differences among all groups with the least caries development in GI group, followed by RM and CR, respectively. The Spearman s rho coefficient of correlation demonstrated a significant weak negative correlation between the response variables. The superficial microhardness test was more sensitive to detect artificial secondary caries than visual evaluation. Key-words: dental caries, composite resin, glass ionomer cement, dental enamel, hardness, visual evaluation.

62 59 Resumo Este estudo in vitro avaliou a correlação entre a inspeção visual e a microdureza superficial no diagnóstico de lesões artificiais de cárie secundária em esmalte. Trinta e seis blocos de esmalte obtidos de terceiros molares humanos inclusos foram utilizados para a confecção de cavidades circulares padronizadas (1,6 mmø) e distribuídas em 3 sub-grupos. Cada sub-grupo foi restaurado com cimento de ionômero de vidro (GI), ionômero de vidro modificado por resina (RM), ou resina composta (CR). Os fragmentos foram termociclados e submetidos ao desenvolvimento de lesões artificiais de cárie por ciclagem de ph. As lesões foram avaliadas por inspeção visual empregando-se escores e foram avaliadas estatisticamente pelos testes de Kruskal Wallis e Dunn; e por ensaio de microdureza Knoop, que foi avaliado por ANOVA e teste de Tukey. Em seguida, a correlação entre inspeção visual e o teste de microdureza foi avaliada pelo teste não paramétrico de correlação de Spearman. Os resultados da inspeção visual não apresentaram diferença significante entre os grupos GI e RM, os quais apresentaram menor desenvolvimento de cárie do que o grupo CR. A avaliação de microdureza demonstrou diferenças significantes entre todos os grupos, sendo o menor desenvolvimento de lesão no GI seguido por RM e CR, respectivamente. O coeficiente de correlação de Spearman foi significante e demonstrou uma fraca correlação negativa entre as variáveis de resposta. O ensaio de microdureza superficial foi mais sensível para o diagnóstico da cárie secundária do que a inspeção visual. Palavras-Chave: cárie dental, resina composta, cimento de ionômero de vidro, esmalte dental, dureza, inspeção visual.

63 60 Introduction The knowledge of the etiology and development of caries disease has allowed a reduction in caries risk and activity by preventing and arresting primary and secondary caries lesions. Secondary caries should be firstly prevented by the reduction in their determinant and modulating factors to revert the patient condition from high to low risk disease status by hygiene procedures such as brushing and flossing. 1 However, the fluorides released from restorative materials may be a viable alternative to prevent secondary caries development in high-risk patients. 2,3,4 The potential cariostatic effect of restorative materials is described in researches showing high cariostatic effect of conventional glass ionomer cements, moderate cariostatic effect of glass ionomer and composite resin hybrid materials, and no cariostatic effect of composite resin materials by different analysis. 2,3,5,6 These analyses may involve less complex and cheaper methods such as visual evaluation and superficial and sub-superficial microhardness, or more difficult evaluation techniques involving expensive equipments, such as microradiography and polarized light microscopy. Since all these analyses are based on different parameters of evaluation, there is a need to verify the correlation among methods. The main objective of the present study was to evaluate the agreement between visual evaluation and superficial microhardness analysis used for the diagnosis of artificial secondary caries development.

64 61 Methods and Materials This study was performed using 20 unerupted human third molars. The research protocol was approved in accordance with the resolution CNS# 196/96 of the National Health Committee/Health Departments by the Research Ethics Committee of the Guarulhos University (Brazil). Following extractions, teeth were stored in 0.1% Timol solution (ph 7.0) for no longer than 30 days. Soft-tissues were removed using periodontal curettes (HU- FRIEDY do Brasil, Rio de Janeiro- Brazil) and teeth were cleaned using pumice slurry in a webbed rubber cup applied with a slow-speed handpiece (Kavo do Brasil, Joinville- Brazil). The crowns were longitudinally and transversally sectioned to obtain 36 dental blocks measuring 4x4x3 mm3 using double-faced diamond discs (#7020, KG Sorensen, São Paulo- Brazil). Cavity preparation and restoration The 36 enamel blocks (n=12 per group) were assigned into three subgroups according to the restorative material described in Table 3. The response variables were visual evaluation and surface microhardness expressed in Knoop Hardness Number (KHN). Table 3- Experimental groups, manufactures and composition. Group Restorative material Ingredients GI RM RC Glass ionomer cement (Ketac-Fil,3M/ ESPE, Seefeld, Germany) Resin modified glass ionomer (Vitremer, 3M/ESPE, St. Paul, MN, USA) Resin composite (Z250, 3M/ESPE, St. Paul, MN, USA) Powder: glass powder 100% Liquid: water 60-65%, polyethylene, polycarbonic acid 30-40%, tartaric acid 5-10% Primer: 2-hydroxyethyl methacrylate 45-55%, ethyl alcohol 35-45%, copolymer of itaconic and acrylic acids 10-15%. Powder: silane treated glass %, potassium persulfate < 1% Liquid: copolymer of acrylic and itaconic acids 45-50%, water 25-30%, 2-hydroxyethyl methacrylate 15 20%. Finish gloss: triethylene glycol dimethacrylate 40-60%, bisphenol a diglycidyl ether dimethacrylate (bisgma) 40 60%. Silane treated ceramic 75-85%, bisphenol a polyethylene glycol diether dimethacrylate (bisema6) 5-10%, diurethane dimethacrylate 5-10%, bisphenol a diglycidyl ether dimethacrylate (bisgma) 1-10%, triethylene glycol dimethacrylate (tegdma) <5%, water <2%.

65 62 Standardized circular cavities with 1.6 mm in diameter and 1.6 mm deep were prepared in the enamel blocks with diamond burs No (KG Sorensen, Barueri, SP, Brazil, ) at high speed under a constant water spray coolant. Afterwards, the blocks were randomly distributed to the subgroups, and were restored in one increment with each restorative material according to the manufacturers instructions. In cavities filled with Ketac-Fil, the Ketac conditioner was applied for 10 s, rinsed off and dried for 10 s. Ketac-Fil was prepared within s, inserted into the cavity with a Centrix injector, protected with a Mylar strip (Dentart, Polidental, São Paulo, Brazil) for 5 min, coated with Vitremer Finish Gloss and light-activated for 20 s with an Optilux 501 light curing unit (light tip diameter: 11 mm; irradiance: 700 mw/cm 2 ; Demetron/Kerr, Danbury, CT, USA). The power density was constantly measured by placing the light tip on the radiometer attached to the light curing unit. In cavities filled with Vitremer, the Primer was applied for 30 s, dried for 5 s and light-activated for 20 s. Vitremer was prepared within 45 s, inserted into the cavity with a Centrix injector, light-activated for 40 s, coated with Vitremer Finish Gloss and lightactivated for 20 s. In cavities filled with Z-250, the 35% phosphoric acid (Scotch Bond Etchant; 3M ESPE) was applied for 15 s, rinsed off for 10 s and the cavity was air-dried. Two coats of Adper Single Bond 2 (3M ESPE) were applied, air-dried for 5 s and light-activated for 10 s. The composite resin was inserted and light-activated for 20 s. All restored blocks were stored in 100% humidity for 24 h and were then polished using the Sof-lex (3M ESPE, St. Paul, MN, USA) disks system for 15 s with each disk. Thermal and acid challenge The restored blocks were placed into separate bags with 1 ml of deionized water and were thermocycled for 1000 cycles in water with temperature ranging from 5±2ºC to 55±2ºC with a dwell time of 2 min in each bath and 15 s-transfer time between baths. 2 The external enamel surfaces of blocks were covered with wax, leaving a 1.5 mm-wide margin around the restoration free of wax. The acid challenge was designed to simulate a daily demineralization challenge of 6 h and 18 h repair (remineralization) by saliva as described by Featherstone et al. (1986) 6 and

66 63 Serra & Cury (1992) 7, to simulate a high in vitro caries risk and to produce artificial caries like-lesions around the restorations 2,7. The demineralization stage was based on the use of an acid buffer containing 2 mmol/l Ca, 2 mmol/l PO 4, mol/l acetate at ph 4.3. The remineralization solution contained calcium and phosphate at a previously established degree of saturation (1.5 mmol/l Ca, 0.9 mmol/l PO 4 ), to mimic the remineralizing properties of saliva, and 50 mmol/l KCl, 20 mmol/l tri-hydroxymethylaminomathan buffer at ph ,7 The blocks were immersed separately in 15 ml of demineralization solution for 6 h, were immersed in 15 ml of remineralization solution for 18 h, washed and immersed in demineralization solution, thereby initiating a new cycle. The ph cycles were conducted for 14 days with 10 daily cycles. In the 6 th, 7 th, 13 th, and 14 th days of the cycle, the blocks were kept only in the remineralization solution. At the end of the ph cycles, the wax was eliminated and the blocks were stored at 100% humidity until the moment of visual evaluation and microhardness test. Visual evaluation The blocks were air-dried for 15s and standardized images were obtained from each block with a Nikon D70 digital camera with lens #105. Three calibrated examiners (Kappa>0.73) independently and blindly evaluated the images of all images projected in a dark room with approximately 100x magnification. The examiners evaluated the specimens scoring the presence and severity of caries-like lesions according to an ordinal scale ranked from 0 to 3 based on visual examination, as described in previous studies (Figure 1). 2,8 A median score was obtained from scores given by the 3 examiners for each specimen. Differences among medians were analyzed by Kruskal-Wallis and Dunn non-parametric tests. Figure 1- Scores used to visual evaluation

67 64 Microhardness test The demineralization of the restored enamel blocks was assessed with a microhardness tester (PanTec, Panambra Ind. e Técnica SA, São Paulo- Brazil) and a Knoop indenter. The indentations were made keeping the long axis of the diamond instrument parallel to the outer-leveled enamel surface, using a 25 g load applied for 5 s, and the highest diagonal length was measured in micrometers and was automatically changed to KHN. Four measurements were made on the enamel surface 100 µm far from the restoration margins in the upper, left, right, and bottom sides (Figure 2). The means of the four indentations represented the block microhardness value. The mean values of each block were analyzed by ANOVA and Tukey s post-hoc test at a pre-set alpha of Figure 2 Location of indentation in the microhardness test. Correlation between visual evaluation and microhardness test The correlation between non-parametric visual evaluation and parametric evaluation of microhardness test was evaluated by the Spearman s rho coefficient of correlation, which ranges in value from r=+1.0 for a perfect positive correlation to r=-1.0 for a perfect negative correlation. The midpoint of its range (r=0.0) corresponds to a complete lack of correlation. Values falling between r=0.0 and r=+1.0 represent a range in degrees of positive correlation, while those falling between r=0.0 and r=-1.0 represent a range in degrees of negative correlation. 9

68 65 Results The medians, minimum, and maximum scores of visual evaluation and the means of microhardness values and standard deviations per restorative material are presented in Table 1. The statistical analysis of visual data showed no differences between GI and RM groups, which in turn showed significantly less caries development than CR group (p<0.01). The microhardness data showed significant differences among groups with less caries in GI than in RM and CR, which in turn showed the highest incidence of caries (p<0.05). Table 1- Medians, minimum, and maximum of visual evaluation and the means of microhardness values and standard deviations per restorative material; Tukey s and Dunn test results. Visual Evaluation Microhardness test GI- Ketac Fil RM- Vitremer CR- Z (0-3) A (75.5) a 1 (0-3) A (64.1) b 3 (2-3) B 39.3 (26.5) c Different upper case letters indicate statistical difference (Dunn test, p<0.01); Different lower case letters indicate statistical difference (Tukey s test, p<0.05). The Spearman s rho coefficient of correlation between the response variables was statistically significant (p<0.01) but the negative correlation was considered weak (r=-0.51). Discussion This study evaluated the development of artificial caries lesion on enamel around cavities filled with restorative materials with or without fluoride release. A dynamic cyclic model of demineralization and remineralization was applied to simulate acid challenge in patients with high caries risk. 6 The highest development of artificial caries lesions in this study was observed in cavities restored with composite resin. As expected, the composite resin associated to an adhesive system deprived of fluoride in their compositions do not inhibit caries progression. 2 This is consistent with reports from other studies, in which only bioactive composite resins and adhesive systems containing fluorides or antibacterial monomers were capable of showing few cariostatic effect, which was lower than that promoted by glass ionomer cements. 4,10

69 66 In agreement with dental literature, the ionomer-based materials showed some cariostatic effect, as they mobilize and release increased amounts of fluoride into the environment during acid challenges, so enamel demineralization is prevented. Then, the presence of fluorides continuously released from ionomers is an important feature for improving enamel remineralization or inhibiting demineralization. 11 This is the reason for less artificial caries lesion development around cavities restored with conventional glass ionomer cement and moderated inhibition of resin modified glass ionomer evaluated by microhardness test. Most studies showed that the smaller fluoride concentration released from resin modified glass ionomer in comparison to that released by conventional glass ionomer cement causes moderate development of artificial caries lesion, which is generally considered less than that observed when glass ionomer cement is used. 2,3,7,11 Therefore, the visual evaluation was not able to detect the difference in caries inhibition between the group using conventional glass ionomer and that using resin modified glass ionomer. It can be supposed that the protection rendered by the glass ionomer cement is extended to some distance from the restoration and is the greatest one in the cavity preparation area. 5 Based on such assumption and on the distance of 100 µm from cavity margins stipulated for the microhardness test, the caries inhibition area provided by conventional glass ionomer could be higher than that created by the resin modified glass ionomer. Therefore, microhardness test may be considered more specific, as the visual evaluation allowed the examiners to check all enamel area free of wax around the restoration. This area was exposed to fluoride released from the glass ionomer material to the solution resulting in a general caries inhibition which was clinically similar to the resin modified glass ionomer. Thus, it can be considered that for a specific evaluation site, superficial microhardness may be required while a general evaluation of wider surrounding area may be performed by visual evaluation. This difference explains the weak agreement between visual and microhardness evaluation observed in Spearman s correlation test. The Spearman s rho correlation measures how well two variables are connected without making any assumption about the frequency distribution of the variables. The negative coefficient value observed in the present study indicates that the two evaluations are systematically inverselly related, as caries lesions visually increases while the superficial microhardness tends to decrease. However, a coefficient value closer to could have showed a perfect negative association. Another aspect that should be considered is that visual evaluation is subjective and this exam depends on the examiner expertise and calibration. The examiners in the present study were calibrated and Kappa qualified the agreement from excellent to good. In a similar

70 67 methodology, Serra induced artificial secondary caries lesion and found a good agreement between visual evaluation and sub-superficial analysis (r=-0.78; p<0.01). Visual evaluation has been associated with scores in clinical 12, epidemiological 13 studies to quantify opacities, fluorosis and white spots resulting from enamel demineralization. Also in in vitro 2,8,14, and in situ studies 15 are well accepted. When compared to other methodologies, this evaluation has some advantages, such as low cost and the possibility of the identification of differences in the cariostatic potential of restorative materials under conditions similar to clinical diagnosis conditions. 2,8 As showed in the current study, visual evaluation is simple to perform, which facilitates laboratory investigation and allows the conduction of studies in less time and at lower costs. 2,8 In addition, reproducible results have been shown between visual evaluation and microradiography and polarized light microscopy. 15 However, the use of visual evaluation needs to be cautiously inferred by the bias of the macro vision of the secondary artificial caries development by the examiner and the cariostatic effect of restorative materials close to cavity margins could not be totally observed. Then, when specific analysis of a site is required, microhardness profiles are recommended and may be used in association with visual evaluation to provide a micro and a micro response of caries development. Conclusions The superficial microhardness test was more sensitive regarding the diagnosis of artificial secondary caries development than visual evaluation, and specific analysis microhardness profiles may be recommended when a micro-site analysis is required.

71 68 References 1. Elderton RJ. Preventive (evidence-based) approach to quality general dental care. Med Princ Pract. 2003;12 Suppl 1: Rodrigues JA, Marchi GM, Serra MC, Hara AT. Visual evaluation of in vitro cariostatic effect of restorative materials associated with dentifrices. Braz Dent J. 2005;16(2): Dunne SM, Goolnik JS, Millar BJ, Seddon RP. Caries inhibition by a resin-modified and a conventional glass ionomer cement, in vitro. J Dent Jan-Mar;24(1-2): Mukai Y, Tomiyama K, Shiiya T, Kamijo K, Fujino F, Teranaka T. Formation of inhibition layers with a newly developed fluoride-releasing all-in-one adhesive. Dent Mater J Jun;24(2): Tantbirojn D, Douglas WH, Versluis A. Inhibitive effect of a resin-modified glass ionomer cement on remote enamel artificial caries. Caries Res. 1997;31(4): Featherstone, J.D.B, O Really, M.M., Shariati, M., and Brugler, S. (1986). Enhancement of remineralization in vitro and in vivo, in: Factors Relating to demineralization and remineralization of the teeth. Leach SA (ed.). Oxford IRL: pp Serra MC, Cury JA. The in vitro effect of glass-ionomer cement restoration on enamel subjected to a demineralization and remineralization model. Quintessence Int Feb;23(2): Serra MC. Análises sensorial e quantitativa do potencial cariostático de materiais restauradores contento flúor. Piracicaba, [Tese (Livre Docência) - Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas]. 9. Lowry, R. Concepts and Applications of Inferential Statistics. Chapter 3. Introduction to Linear Correlation and Regression Part 3. Accessed in 19/11/ Okuyama K, Nakata T, Pereira PN, Kawamoto C, Komatsu H, Sano H. Prevention of artificial caries: effect of bonding agent, resin composite and topical fluoride application. Oper Dent Jan-Feb;31(1): Mount GJ. Glass-ionomer cements: past, present and future. Oper Dent May- Jun;19(3): Gorelick L, Geiger AM, Gwinnet AJ. Incidence of white spot formation after bonding and banding. Am J Orthod.1982;81: Backer-Dirks O. Posteruptive changes in dental enamel. J Dent Res. 1966;45:

72 Edgar WM, Rugg-Gunn AJ, Jenkins GN, Geddes DA. Photographic and direct visual recording of experimental caries-like changes in human dental enamel. Arch Oral Biol. 1978;23: von der Fehr FR. The effect of fluorides on the caries resistance of enamel. Acta Odontol Scand. 1961;19:

73 70 4. CONCLUSÕES Com base nos trabalhos desenvolvidos, apresentados em forma de artigos, pode-se concluir que: - Na análise visual o Laser de Er:YAG não mostrou capacidade de aumentar a resitência do esmalte à cárie, apesar da observação de um número menor de lesões cariosas quando utilizado para o preparo cavitário; - Na análise de microdureza superficial o laser de Er:YAG apresentou efeito cariostático a despeito da presença de materiais restauradores contendo flúor; - O teste de microdureza superficial é mais preciso no diagnóstico do desenvolvimento de lesões de cárie secundária que a avaliação visual; Conclui-se que o Laser de Er:YAG proporcionou efeito cariostático ao redor dos preparos cavitários sendo mais evidente nas análises realizadas pelo teste de microdureza.

74 71 REFERÊNCIAS Apel C, Schafer C, Gutknecht N. Demineralization of Er:YAG and Er,Cr:YSGG laserprepared enamel cavities in vitro. Caries Res. 2003;37(1):34-7. Araujo JM; Dionísio R; Reis JIF; Santos LM. Estudo comparativo do efeitos de diferentes materiais restauradores estétuticos fluoretados no desenvolvimento de cárie dentes decíduos. Pesqui. bras. odontopediatria clín. integr. 2006;6(2): Ceballos L, Toledano M, Osorio R, Garcia-Godoy F, Flaitz C, Hicks J.ER-YAG laser pretreatment effect on in vitro secondary caries formation around composite restorations. Am J Dent. 2001;14(1):46-9. Cecchini RC, Zezell DM, de Oliveira E, de Freitas PM, Eduardo C de P. Effect of Er:YAG laser on enamel acid resistance: morphological and atomic spectrometry analysis. Lasers Surg Med. 2005;37(5): Cordeiro RCL, da Silva VL, Josgriber EB. Avaliação da forma de preparos cavitários realizados com laser, abrasão a ar e ponta diamantada. Cienc odontol. Bras 2005;8 (3) Dijkman GE, de Vries J, Lodding A, Arends J. Long-term fluoride release of visible lightactivated composites in vitro: a correlation with in situ demineralisation data. Caries Res. 1993;27(2): Ferracane JL, Mitchem JC, Adey JD. Fluoride penetration into the hybrid layer from a dentin adhesive. Am J Dent Feb;11(1):23-8. Freitas, PM. Estudo in vitro do efeito da irradiação com o laser de Er,Cr:YSGG na inibição do processo de desmineralização do esmalte dental. USP São Paulo ; p. Tese de Doutorado. Hals, E. Histology of natural secondary caries associated with silicate cement restorations in human teeth. Arch. Oral Biol., 1975; 20: Harazaki M, Hayakawa K, Fukui T, Isshiki Y, Powell LG. The Nd-YAG laser is useful in prevention of dental caries during orthodontic treatment. Bull Tokyo Dent Coll. 2001;42(2): Hibst R, Keller V. Experimental studies of the application of the Er:YAG laser on dental hard substance: I. Measurement of the ablation rate. Laser Surg Med. Cap 9, pag ; 1989). Hicks MJ, Flaitz CM, Silverstone LM. Secondary caries formation in vitro around glass ionomer restorations. Quintessence Int. 1986;17(9):

75 72 Kerber LJ, Donly KJ. Caries inhibition by fluoride-releasing primers. Am J Dent Oct;6(5): Kim JH, Kwon OW, Kim HI, Kwon YH. Acid resistance of erbium-doped yttrium aluminum garnet laser-treated and phosphoric acid-etched enamels. Angle Orthod. 2006;76(6): Klein AL, Rodrigues LK, Eduardo CP, Nobre dos Santos M, Cury JA. Caries inhibition around composite restorations by pulsed carbon dioxide laser application. Eur J Oral Sci Jun;113(3): Liu Y, Hsu CY. Laser-induced compositional changes on enamel: a FT-Raman study. J Dent. 2007;35(3): Lobo MM, Gonçalves RB, Pimenta LA, Bedran-Russo AK, Pereira PN. In vitro evaluation of caries inhibition promoted by self-etching adhesive systems containing antibacterial agents. J Biomed Mater Res B Appl Biomater Oct;75(1): Miserendino LJ & Pick RM. Laser in desntistry. Cap.11; pág , Ed Quintessence P. Co, Inc., Moi GP, Araujo FB; Barata J. Abordagem contemporânea das lesões cariosas adjacentes às restaurações na clínica odontopediátrica. Rev. Fac. Odontol. Porto Alegre 2005;46(2):5-8. Park SH, Kim KY. The anticariogenic effect of fluoride in primer, bonding agent, and composite resin in the cavosurface enamel area. Oper Dent May-Jun;22(3): Rodrigues JA, Marchi GM, Serra MC, Hara AT. Visual evaluation of in vitro cariostatic effect of restorative materials associated with dentifrices. Braz Dent J. 2005;16(2): Tantbirojn D, Douglas WH, Versluis A. Inhibitive effect of a resin-modified glass ionomer cement on remote enamel artificial caries. Caries Res. 1997;31(4): Thylstrup A. & Fejerskov O. Cariologia Clínica. 2ª ed. Ed. Santos Yamamoto HY, Sato K. Prevention of dental caries by Nd:YAG laser irradiation. J Dent Res. 1980;59:

76 73 ANEXOS ANEXO A - Certificado de Aprovação do Comitê de Ética

77 74 ANEXO B Termo de Consentimento Livre e Esclarecido Termo de Consentimento Livre e Esclarecido A influência da técnica do preparo cavitário e dos tipos de materiais restauradores na prevenção de cárie secundária A cárie secundária, lesão cariosa que se forma ao redor das restaurações, pode ser prevenida pelo uso de materiais restauradores que liberem flúor, sabe-se ainda que a aplicação de laser pode tornar o esmalte dental, mais resistente a cárie. Entretanto, o que não se sabe é se em restaurações com materiais que liberam flúor aonde a remoção da cárie foi realizada com laser o esmalte será mais resistente a cárie secundária. O objetivo deste trabalho será avaliar, in vitro, a influência da técnica do preparo cavitário e dos tipos de materiais restauradores na prevenção do desenvolvimento da ocorrência de cárie secundária.. Portanto, contamos com sua participação como doador, devido ao fato de utilizarmos 3º molares inclusos como indicação de exodontia para a realização da pesquisa. As informações contidas neste termo foram fornecidas pelo Prof. Dr. José Augusto Rodrigues (Orientador) e pela aluna Ana Carolina Tedesco Jorge, tel: (11) , R. Dr. Nilo Peçanha 67, prédio U, 6º andar, para firmar o seu consentimento livre e esclarecido, através do qual você, sujeito da pesquisa, autoriza a sua participação. Esta pesquisa consistirá em comparar, in vitro, a influência da técnica do preparo cavitário e dos tipos de materiais restauradores na prevenção de cárie secundára. Como o estudo será realizado in vitro o doador não terá nenhum desconforto ou risco, pois doará somente o dente. Assim, não está prevista nenhuma forma de ressarcimento ou garantia de tratamento Odontológico na UnG. O doador tem a liberdade de retirar seu consentimento ou se recusar a doar dentes para o estudo, a qualquer momento, conforme determinação da Resolução 196/96 do CNS do Ministério da saúde, sem qualquer tipo de prejuízo ou penalização. Os pesquisadores comprometem-se em resguardar todas as informações individuais acerca da pesquisa de forma sigilosa, não revelando a identidade do sujeito que doou os dentes. Conforme definido pela resolução 196/96 do conselho nacional de saúde, esta pesquisa foi submetida á comissão de Ética e pesquisa da UnG, e aprovada pela mesma. Por este instrumento particular declaro, para efeitos éticos e legais, que eu, CPF:, concordo com absoluta consciência dos procedimentos a que vou me submeter para a realização da pesquisa A influência da técnica do preparo cavitário e dos tipos de materiais restauradores na prevenção de cárie secundária. Guarulhos, de de Assinatura do doador Prof.Dr. José Augusto Rodrigues