LILIANA CAROLINA BÁEZ QUINTERO Efeito de vernizes fluoretados suplementados com nanopartículas de Trimetafosfato de Sódio sobre a remineralização de lesões de cárie e erosão de esmalte dental in vitro ARAÇATUBA 2017 LILIANA CAROLINA BÁEZ QUINTERO Efeito de vernizes fluoretados suplementados com nanopartículas de Trimetafosfato de Sódio sobre a remineralização de lesões de cárie e erosão de esmalte dental in vitro Tese apresentada à Faculdade de Odontologia da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Araçatuba, como parte dos requisitos para obtenção do título de Doutor em Ciência Odontológica - Área de Concentração: Saúde Bucal da Criança. Orientador: Prof. Dr. Juliano Pelim Pessan Coorientadores: Prof. Tit. Alberto Carlos Botazzo Delbem Dra. Marcelle Danelon ARAÇATUBA 2017 Liliana Carolina Báez Quintero Catalogação-na-Publicação Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP Báez-Quintero, Liliana Carolina. B142e Efeito de vernizes fluoretados suplementados com nanopar- tículas de Trimetafosfato de Sódio sobre a remineralização de lesões de cárie e erosão de esmalte dental in vitro / Liliana Carolina Báez-Quintero. - Araçatuba, 2017 104 f. : il. ; tab. Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientador: Prof. Juliano Pelim Pessan Coorientador: Prof. Alberto Carlos Botazzo Delbem 1. Fluoretos tópicos 2. Polifosfatos 3. Cárie dentária 4. Erosão dentária 5. Nanopartículas I. T. Black D27 CDD 617.645 Claudio Hideo Matsumoto – CRB-8/5550 Liliana Carolina Báez Quintero DADOS CURRICULARES Liliana Carolina Báez Quintero Nascimento 22.12.1979- Soatá-Boyaca-Colômbia Filiação Ana Helda Quintero Parra Luis Carlos Báez Suescun 1997/2002 Curso de Graduação em Odontologia pela Faculdade de Odontologia da Universidade Nacional de Colômbia 2004-2008 Desenvolvimento do projeto de Mestrado em Saúde Pública, Faculdade de Medicina, Universidade Nacional de Colômbia. 2014-2017 Desenvolvimento de Projeto de Doutorado, com auxilio do Programa de Apoio a Estudantes de Doutorado do Exterior PAEDEX/AUIP/PROPG, na Faculdade de Odontologia de Araçatuba-UNESP. 2015-2016 Especialização em Odontopediatría pela Faculdade de Odontologia de Araçatuba- UNESP. Associações CROSP – Conselho Regional de Odontologia de São Paulo. SBPqO – Sociedade Brasileira de Pesquisa IADR – International Association for Dental Research. Liliana Carolina Báez Quintero COMISSÃO EXAMINADORA TESE PARA OBTENÇÃO DO GRAU DE DOUTOR Prof. Dr Juliano Pelim Pessan- Orientador, Professor Adjunto do Departamento de Departamento de Odontologia Infantil e Social, Disciplina de Odontopediatria da Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista Júlio de Mesquita Filho-UNESP. Prof. Dr. Célio Percinoto- Professor Titular do Departamento de Departamento de Odontologia Infantil e Social, Disciplina de Odontopediatria, da Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista Júlio de Mesquita Filho-UNESP. Dr. Douglas Roberto Monteiro- Doutor, Departamento de Odontologia Infantil e Social, Disciplina de Odontopediatria, da Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista Júlio de Mesquita Filho-UNESP. Dra. Cleide Cristina Rodrigues Martinhon- Doutora em Odontologia, com ênfase em Odontopediatría pela Universidade de São Paulo, USP, Brasil. Prática particular. Prof. Heitor Marques Honório- Professor Associado do Departamento de Odontopediatria, Ortodontia e Saúde Coletiva, Disciplina de Metodologia de Pesquisa e Estatística, Faculdade de Odontologia de Bauru, Universidade de São Paulo. Liliana Carolina Báez Quintero DEDICATÓRIA Dedico este trabalho a meu pai Carlos (in memoriam) e a minha mãe Helda, que sempre me acompanham e porque me ensinaram a não desistir de meus sonhos apesar das dificuldades. Aos meus irmãos Rocio, Andrés, César e Ana, por seu apoio incondicional e pela confiança que sempre me dão. Vocês são meu porto seguro. Às minhas sobrinhas Maria José e Gabriela, por me ajudar a descobrir o amor pela odontopediatría. Vocês foram as primeiras que acreditaram em mim. Liliana Carolina Báez Quintero AGRADECIMENTOS ESPECIAIS A Deus, por que todos os dias derrama bênçãos na minha vida, ele foi que me trouxe a Araçatuba, e continuará guiando meu camino. Nada é por acaso. À minha família, que, desde Colômbia, me acompanha, celebra minhas pequenas conquistas e me ajuda a levantar nas decepções e derrotas. Vocês são o melhor presente que a vida me deu. Ao Professor Juliano Pelim Pessan, por sua amizade, paciência e orientação durante estes anos em Araçatuba, desde minha chegada sempre se preocupou para que tudo estivesse bem comigo. Não tenho palavras para agradecer tudo o que você faz por mim e com certeza esse é o início de uma grande amizade e de novos trabalhos de pesquisa juntos. Ao Professor Célio Percinoto, quando conheci o senhor no ano 2009 não imaginei que teria a oportunidade de ser sua aluna, agradeço a Deus por isso. Obrigada por seus conselhos e palavras para que eu não desistisse e continuasse caminhando, ainda que tivesse dificuldades. Você terá toda minha admiração e gratidão para sempre. Ao Professor Alberto Carlos Botazzo Delbem, Por toda a ajuda no desenvolvimento deste projeto de pesquisa, por sua disponibilidade e paciência para nos ensinar e resolver todos nosos problemas no laboratorio. Ao Professor Robson Federico Cunha, pelos ensinamentos e experiências compartilhadas tanto na clínica como em sala de aula, e nos trasmitir todo seu conhecimiento e experiência na área da odontopediatria. Liliana Carolina Báez Quintero Às Professoras, Cristiane Duque e Sandra Ávila Aguiar e Rosangela Nery, pelo conhecimento transmitido, pelas orientações constantes e pela convivência diária durante a Especialização, “Bebê Clínica” e Clínicas de graduação. Agradeço imensamente pelo carinho e amizade. À Marcelle Danelon, por sua amizade e ajuda constante nas atividades do laboratorio. Sempre parava o que estava fazendo para me ajudar e incentivar a continuar com palavras carinhosas de apoio. À Professora Daniela Rios, por sua ajuda e orientação na elaboração do capitulo 2 deste trabalho, suas observações e corientações foram esenciais para obter o resultado. A Marina Emi Nagata e Thayse Hosida, vocês são as irmãs que Araçatuba me deu, neste tempo foi criado um laço de amizade e fraternidade que vai ser para a vida toda. É dificil pensar que vou estar longe, mas tenham certeza que onde quer que eu esteja vocês estarão em meu coração e sempre torcendo para que tudo dê certo em suas vidas. A Carla Favretto e Marjully Eduardo Rodrigues, nunca tinha convivido com tantas mulheres juntas. Vocês, com seus temperamentos fortes, me ensinaram a ter paciência e escutar. Tambem estarão para sempre em meu coração. Vocês são outro presente de Araçatuba. Obrigada por sua amizade sincera e por se preocuparem para tudo estivesse bem comigo. A Carla Mendes, Malu e Marina, minha pequena familia, vocês me alegram com cada um de seus triunfos, me ensinaram a ser irmã e tia. Não tenho como expressar todo o carinho que eu sinto por vocês. Vou sentir muita saudade. Liliana Carolina Báez Quintero A Malena Castro e Diego Mardegan, vocês tornaram o trabalho no laboratorio mais leve, com o sorriso vocês conseguiam amenizar meu estresse, sem vocês não teria conseguido fazer estes três capitulos. Podem contar com minha amizade e apoio sempre. Aos meus amigos Pilar Rodriguez, Rosse Falcon e Rodrigo Oliveria, vocês me acolheram quando eu pensei que ficaria sozinha, e rapidamente surgiu uma amizade desinterasada que permanece até agora, a pesar da distância. Obrigada, por fazer parte de minha vida. Aos meus amigos Pilar Rodriguez, Rosse Falcon e Rodrigo Oliveria, vocês me acolheram quando eu pensei que ficaria sozinha, e rapidamente surgiu uma amizade desinterasada que permanece até agora, a pesar da distância. Obrigada, por fazer parte de minha vida. A Karina Caiaffa, José Antonio, Giovanna Coclete, Christinne Men e Thamires Cavazana, vocês são pessoas especiacias pelo seu jeito de ser, sempre prestes a ajudar e dar o melhor de vocês. Vou sentir saudade do cinema, do café, os almoços, sorvetes e muito de nossos papos sobre a vida, as energias e planos futuros. Liliana Carolina Báez Quintero AGRADECIMENTOS À Universidade Estadual Paulista, Pela bolsa do Programa de Apoio a Estudantes de Doutorado do Exterior PAEDEX/AUIP/PROPG, na Faculdade de Odontologia de Araçatuba-UNESP. A Maria Eduarda e Mariana (PIBIC Junior), ensinar vocês as ativides no laboratorio foi um desafio, obrigada pela paciencia para enteder meu “portunhol’, e o apoio na elaboração deste projeto. Aos meus amigos de departamento, Jackeline Amaral, Mayra Paiva, Douglas Monteiro, Kelly Aida, Renan Fernandes, Luhana Garcia, Nayara Gonçalves, Jorge Cuellar, Laís Arias, Gabriela Fernandes, Caio Sampaio, Lenara Chaves, Ana Paula, Kevin Bruce, Tatiana Uemera, Remberto, Michele Manarelli, Héitor Ceolin, Karine Prado, Vanessa, Jaqueline Canova, Danielle Camara, Isabela Catazone, Silvio, Isabel Salama, Emili Vicenti, Suéllen Priscila, Leonardo Morais, Priscila Toledo, Gabriel Nunes e Jessi, pelos momentos compartilhados, companhia, sufocos, sorrisos, ajudas, conselhos e por fazer parte da minha caminhada por Araçatuba. Ao Professor Emerson Rodrigues de Camargo, pela sintesis das nanoparticulas de TMP. Aos funcionários do departamento de Odontopediatria, Luiz, Mário e Ricardo, pela ajuda e atenção Às funcionárias da seção de pós-graduação Valéria, Cristiane e Lilian, pelo profissionalismo, nos orientando e sempre estar prestes a nos ajudar. Ao Frigorífico JBS, que permitiu a coleta dos dentes bovinos. A SS White Produtos Odontológicos (Rio de Janeiro, RJ, Brasil), pela manupulação dos vernizes experimentais. Liliana Carolina Báez Quintero “Ninguém caminha sem aprender a caminhar, sem aprender a fazer o caminho caminhando, refazendo e retocando o sonho pelo qual se pôs a caminhar”. Paulo Freire Liliana Carolina Báez Quintero Báez-Quintero LC. Efeito de vernizes fluoretados suplementados com nanopartículas de Trimetafosfato de Sódio sobre a remineralização de lesões de cárie e erosão de esmalte dental in vitro. 2017. 104 f. Tese (Doutorado), Universidade Estadual Paulista, Araçatuba, 2017. RESUMO O presente estudo avaliou o efeito de vernizes fluoretados contendo nanopartículas de trimetafosfato de sódio (TMP) sobre a remineralização de lesões de cárie e erosão de esmalte dental in vitro. Assim como o padrão de liberação de F e TMP a partir destas formulações. Os vernizes testados foram: Placebo (sem flúor ou TMP, controle negativo), 2,5% NaF, 5% NaF (controle positivo), 5% NaF + 5% TMP microparticulado, 5% NaF + 2,5% TMP nanoparticulado, 5% NaF + 5% TMP nanoparticulado, além de uma formulação comercial (Duraphat), doravante denominados PLA, 2.5%F, 5%F, 5%F+5%TMPmicro, 5%F+2,5%TMPnano, 5%F+5%TMPnano e Duraphat, respectivamente. Na 1ª fase, lesões de cárie artificiais foram induzidas em blocos de esmalte bovino (n=168), os quais foram selecionados por dureza de superfície (DS). Os blocos receberam uma aplicação dos vernizes supracitados, permanecendo em contato durante 6 h. Metade dos blocos (n=12/grupo) foi utilizada para determinação da concentração de CaF2 e FA formado após o tratamento com os vernizes. A outra metade foi submetida um modelo de ciclagem de pH (6 dias). Os blocos foram analisados quanto a porcentagem de recuperação de dureza de superfície (%RDS), dureza em secção longitudinal (ΔKHN), CaF2 e FA retidos após a ciclagem de pH. Os vernizes contendo TMP promoveram %RDS significativamente maior em comparação ao verniz contendo 5%F, sem diferenças significativas entre os vernizes com TMP. Um padrão semelhante foi observado para ΔKHN, embora os valores obtidos para 5%F+5%TMPnano tenham sido 25% menores que os obtidos para 5%F+5%TMPmicro. As maiores concentrações de CaF2 foram promovidas pelos vernizes 5%F, 5%F+5%TMPmicro e Duraphat. A 2ª fase avaliou o efeito protetor dos vernizes PLA, 5%F, 5%F+5%TMPmicro, 5%F+2,5%TMPnano e 5%F+5%TMPnano sobre a erosão de esmalte bovino. Os espécimes (n=8), selecionados por DS, receberam uma única aplicação dos vernizes, permanecendo em contato durante 6 h. Em seguida, os vernizes Liliana Carolina Báez Quintero foram removidos e os blocos, submetidos a quatro desafios erosivos individuais (1 minuto, ácido cítrico, 0,75%, pH = 3,5, sob agitação), sendo analisados por DS após cada desafio. Em geral, observou-se a maior porcentagem de alteração de DS para PLA, seguido de 5%F, 5%F+5%TMPmicro e ambos os vernizes contendo TMPnano, sem diferenças significativas entre 5%F+2,5%TMPnano e 5%F+5%TMPnano. Por fim, a 3ª fase avaliou o padrão de liberação de flúor e fosfato dos vernizes PLA, 2.5%F, 5%F, 5%F+5%TMPmicro, 5%F+2,5%TMPnano, 5%F+5%TMPnano e Duraphat ao longo de 24 h, em um modelo de ciclagem de pH. Os vernizes foram aplicados em tiras de poliéster (n=8/grupo), as quais foram alternadamente imersas em soluções remineralizadora e desmineralizadora aos 30, 60, 90, 120, 180, 240, 300, 360, 420, 540, 600, 720, 780, 960, 1200 e 1440 min após a primeira imersão. As soluções foram analisadas quanto às concentrações de flúor e fosfato. Os vernizes contendo TMP apresentaram um padrão crescente exponencial quanto a liberação cumulativa de flúor até 6 horas, atingindo um platô nos tempos seguintes. Em acréscimo, os vernizes liberaram maiores quantidades de flúor quando imersos em solução desmineralizadora. De forma geral, os vernizes contendo TMPnano liberaram quantidades significativamente maiores de flúor em comparação ao TMPmicro. Os resultados da 1ª fase permitem concluir que a adição de TMP a vernizes fluoretados aumenta significativamente seu potencial remineralizador em lesões de cárie artificiais, com um efeito adicional com o uso de TMPnano em relação ao TMPmicro, embora este acréscimo não seja estatisticamente significativo. Quanto ao efeito contra desafios erosivos (2ª fase), um padrão semelhante foi observado, tendo o maior efeito protetor sido observado para os vernizes contendo TMPnano, o qual foi significativamente maior que os demais grupos. O maior efeito dos vernizes suplementados com TMP parece estar relacionado à maior liberação de flúor destes (3ª fase), especialmente TMPmicro, associada à liberação constante de TMP a partir destas formulações. Palavras-chave: Fluoretos tópicos. Polifosfatos. Cárie dentária. Erosão dentária. Nanopartículas. Liliana Carolina Báez Quintero Báez-Quintero LC. Effect of fluoride varnishes supplemented with nano- sized sodium trimetaphosphate on the remineralization of artificial caries lesions and enamel erosion in vitro. 2017. 104 f. Tese (Doutorado), Universidade Estadual Paulista, Araçatuba, 2017. ABSTRACT The present study evaluated the effect of fluoride varnishes containing nano- sized sodium trimetaphosphate (TMP) on the remineralization of artificial caries lesions and erosion of dental enamel in vitro. As well as the pattern of F and TMP release from these formulations. The varnishes tested were: Placebo (without fluoride or TMP, negative control), 2.5% NaF, 5% NaF (positive control), 5% NaF + 5% TMP micrometric, 5% NaF + 2.5% nano-sized TMP, 5% NaF + 5% nano-sized TMP, besides a commercial formulation (Duraphat), hereafter referred to as PLA, 2.5%F, 5%F, 5%F+5%TMPmicro, 5%F+2.5%TMPnano, 5%F+5%TMPnano and Duraphat, respectively. In the first phase, artificial caries lesions were induced on bovine enamel blocks (n=168), which were selected by surface hardness (SH). Blocks received a single application of the aforementioned varnishes, remaining in contact for 6 h. Half of the blocks (n=12/group) were used to determine the concentration of CaF2 and FA fluoride formed on the specimens after the treatment with the varnishes. The other half was subjected to a pH cycling model (6 days). The blocks were analyzed for the percentage of SH recovery (%SHR), cross-sectional hardness (ΔKHN), CaF2 and FA retained after pH cycling. The varnishes containing TMP promoted significantly higher %SHR compared to 5%F, without significant differences among the TMP-containing varnishes. A similar pattern was observed for ΔKHN, although the values obtained for 5%F+5%TMPnano were 25% lower than those obtained for 5%F+5%TMPmicro. The highest CaF2 concentrations were promoted by 5%F, 5%F+5%TMPmicro and Duraphat varnishes. The second phase evaluated the protective effect of PLA, 5%F, 5%F+5%TMPmicro, 5%F+2.5%TMPnano and 5%F+5%TMPnano varnishes on initial erosion of bovine enamel. Specimens (n=8), selected by SH, received a single application of the varnishes, remaining in contact for 6 h. Varnishes were then removed and the blocks, submitted to four individual erosive challenges Liliana Carolina Báez Quintero (1 minute, citric acid, 0.75%, pH = 3.5, under stirring) and analyzed by SH after each challenge. Overall, the highest percentage of SH change was observed for PLA, followed by 5%F, 5%F+5%TMPmicro and both varnishes containing TMPnano, with no significant differences between 5%F+2.5%TMPnano, 5%F+5%TMPnano. Finally, the third phase evaluated the pattern of fluoride and phosphate release from PLA, 2.5%F, 5%F, 5%F+5%TMPmicro, 5%F+2.5%TMPnano, 5%F+5%TMPnano and Duraphat varnishes over 24 h, in a pH cycling model. The varnishes were applied on polyester sheets (n=8/group), which were alternately immersed in remineralizing and demineralizing solutions at 30, 60, 90, 120, 180, 240, 300, 360, 420, 540, 600, 720 , 780, 960, 1200 and 1440 min after the first immersion. The solutions were analyzed for fluoride and phosphate concentrations. The varnishes containing TMP promoted an increasing, exponential pattern for the cumulative fluoride release up to 6 hours, reaching a plateau afterwards. In addition, higher quantities of fluoride were released when varnishes were immersed in the demineralizing solution. In general, varnishes containing TMPnano released significantly higher amount of fluoride compared to TMPmicro. The results of the 1st phase allow to conclude that the addition of TMP to fluoride varnishes significantly increases its remineralizing potential in artificial caries lesions, with an additional effect with the use of TMPnano in relation to TMPmicro, this increment was not statistically significant. As for the effect against erosive challenges (2nd phase), a similar pattern was observed, with the highest protective effect observed for both varnishes containing TMPnano, which was significantly higher than the other groups. The higher effect of varnishes supplemented with TMP seems to be related to the higher fluoride release from these products (3rd phase), especially TMPmicro, associated to the constant release of TMP from these formulations. Key words: Topical fluorides. Polyphosphates. Dental caries. Tooth erosion. Nanoparticles. Liliana Carolina Báez Quintero LISTA DE TABELAS CAPÍTULO 1 Table 1. Percentage of surface hardness recovery (%SHR) and integrated loss of subsurface hardness (ΔKNH) according to the varnishes ………………………………………………………………………. 43 Table 2. CaF2 and firmly bound fluoride 6 h after varnish application and after pH-cycling according to groups ………………………………….. 44 CAPÍTULO 3 Table 1. Fluoride released from the varnishes at each individual time point and cumulative release as a function of time and varnish composition ……………………………………………………………………. 74 Table 2. Phosphate released from the varnishes at each individual time point and cumulative release as a function of time and varnish composition …………………………………...……………………………….. 75 Liliana Carolina Báez Quintero LISTA DE FIGURAS CAPÍTULO 1 Figure 1. Differential hardness profile as a function of depth according to the groups ………...………………………………………………………. 45 CAPÍTULO 2 Figure 1. Mean percentage of surface hardness change according to the varnishes applied and the time of exposure to citric acid. Upper- and lower-case letters indicate significant differences among the varnishes (at each individual time point) and among the times after exposure to acid (within each varnish), respectively. Two-way, repeated-measures ANOVA and Student-Newman-Keuls test (p<0.05, n=8). Bars indicate SD ……………………...………………………………………………………. 57 CAPÍTULO 3 Figure 1. Time-course fluoride release from the varnishes into remineralizing and demineralizing solutions over 24 h. Vertical bars represent standard error of means, while the arrows indicate immersion in the demineralizing solution. A: fluoride release determined at each point. B: cumulative release …………………………………………………. 72 Figure 2. Time-course phosphate release from the varnishes into remineralizing and demineralizing solutions over 24 h. Vertical bars represent standard error of means, while the arrows indicate immersion in the demineralizing solution. A: phosphate release determined at each point. B: cumulative release …………………………………………………. 73 Liliana Carolina Báez Quintero SUMÁRIO 1. INTRODUÇÃO GERAL ............................................................................. 19 2. CAPÍTULO 1 2.1 Abstract .......................................................................................... 2.2 Introduction ………............................................................................ 2.3 Materials and Methods ….................................................................. 2.4 Results .............................................................................................. 2.5 Discussion ........................................................................................ 2.6 Acknowledgements ….........……………………….…………….…….. 2.7 References ……….…………………………….…...…………………... 26 27 28 32 33 38 39 3. CAPÍTULO 2 3.1 Abstract .......................................................................................... 3.2 Introduction ……................................................................................ 3.3 Material and Methods …................................................................... 3.4 Results .............................................................................................. 3.5 Discussion ........................................................................................ 3.6 Acknowledgements ……….……………..……………………………... 3.7 References ……………………………….……………………………… 48 49 49 52 52 54 55 4. CAPÍTULO 3 4.1 Abstract .......................................................................................... 4.2 Introduction ……….…....................................................................... 4.3 Materials and Methods ….................................................................. 4.4 Results .............................................................................................. 4.5 Discussion ........................................................................................ 4.6 Conclusions ……..……………………………...……………………….. 4.7 Acknowledgements …………………………………………………….. 4.8 References ………………….…………………….……………….….… 60 61 62 64 65 69 69 70 5. CONSIDERAÇÕES FINAIS ……………………...…………………….……... 76 6. ANEXOS …......……………………………………………………………….… 79 Liliana Carolina Báez Quintero 1. Introdução Geral __________________________________________________________________________ 20 Liliana Carolina Báez Quintero 1. Introdução Geral A cárie dentária é uma doença biofilme-dependente, resultado de um processo dinâmico de desmineralização e remineralização do esmalte dental decorrente de flutuações no PH [Whitford et al., 2002; Vogel, 2011]. A causa principal é o metabolismo microbiano de carboidratos em biofilmes presentes sobre as superfícies dentárias, o que pode levar à perda mineral ao longo do tempo e, consequentemente, à formação da cavidade [Kidd e Fejerskov, 2004]. Este processo é passível de intervenção, enfatizando a necessidade de diagnostico e tratamento precoce das lesões iniciais, bem como o adequado controle de fatores moduladores, como higiene bucal, dieta, fluxo salivar e exposição ao fluoreto [Kidd, 2011]. Apesar da redução na prevalência da cárie dentária observada em todo o mundo, esta doença continua sendo um desafio de saúde pública, uma vez que é influenciada por condições socioeconômicas, estilo de vida, acesso a serviços de saúde e a programas preventivos de saúde bucal [Petersen, 2005], tornando-a fortemente polarizada e apresentando uma distribuição bimodal [Narvai et al., 2006; Salas et al., 2015]. A redução na prevalência da carie dentária possibilitou o diagnostico de outras alterações não cariosas que afetam os tecidos dentários duros. Entre elas, destaca-se a erosão dentária, que é definida como a dissolução química do tecido dentário sem envolvimento bacteriano [West e Joiner, 2014]. O processo leva ao amolecimento da superfície dentária seguido de sua dissolução, tornando-a mais propensa aos impactos mecânicos [Wiegand e Attin, 2003; Lussi, et al.,2011]. Entre as medidas usadas para prevenir ou minimizar os efeitos da erosão envolvem orientações sobre a necessidade de reduzir a frequência de ingestão de alimentos e bebidas ácidas e a aplicação de fluoretos tópicos [Ganss, 2008; Salas et al.,2015]. O diagnóstico do desgaste dentário erosivo em estágios precoces é difícil, razão pela qual as lesões são diagnosticadas em estágio moderado ou avançado. Estudos epidemiológicos apresentam ampla variação entre os dados, o que possivelmente se deve a diferentes critérios de diagnóstico, idade dos _________________________________________________________________________ 21 Liliana Carolina Báez Quintero pacientes, dentre outros fatores. No Brasil, a prevalência entre crianças e adolescentes varia entre (20% a 78%) [Rios et al., 2007; Gurgel et al., 2011]. A terapia com fluoretos tem sido uma estratégia para o controle da cárie dentária e o desgaste dental erosivo. O flúor (F) tem a capacidade de retardar o prevenir o desenvolvimento de lesões de cárie dentária por reduzir a solubilidade do esmalte em meio ácido, inibir a obtenção e utilização de glicose por bactérias e promover a remineralização do esmalte [Buzalaf et al.,2011]. Quanto à erosão dentária, as terapias que contem só F têm promovido um efeito limitado na redução do desgaste dental, sendo efetivas quando é combinado com outros agentes protetores como o estanho e fosfatos [Lussi e Carvalho, 2015; Ganss et al., 2011]. No entanto, considera-se que a aplicação tópica de F a altas concentrações fornece minerais adicionais à superfície dos dentes (CaF2), que são dissolvidos durante exposições futuras a ácidos, o que reduz a taxa de dissolução do esmalte dental [Ganss et al., 2001; Largerweij et al.,2006]. Dentre os métodos de aplicação profissional de F, os vernizes fluoretados apresentam como principais vantagens a facilidade de aplicação, tempo de contato prolongado com a superfície dentária, segurança e boa aceitabilidade pelos pacientes [Pessan et al., 2005, 2011; Marinho et al., 2013]. Os vernizes são produtos viscosos que endurecem quando em contato com a saliva, formando uma película aderida à superfície dentária, a qual libera F para a superfície do esmalte, biofilme dental e saliva. Desta forma, os vernizes tanto reduzem a desmineralização, como aceleram o processo de remineralização do esmalte [Pessan et al., 2011]. Uma revisão sistemática recente concluiu que a redução nos índices ceo-s e CPO-S associada a seu uso foi 37% e 43% respectivamente [Marinho et al., 2013]. Alternativas para potencializar o efeito do F em veículos de uso caseiro e profissional têm sido estudadas, as quais incluem a redução do pH do veículo [Øgaard et al.,2001; Vilhena et al., 2010] e a suplementação com sais de cálcio e/ou de fosfato [Schemohorn et al., 1999 a,b]. A adição de trimetafosfato de sódio (TMP) a produtos fluoretados demonstrou aumentar significativamente seus efeitos protetores e terapêuticos contra a cárie dentaria [Takeshita et _________________________________________________________________________ 22 Liliana Carolina Báez Quintero al.,2009,2015; Danelon et al.,2013, 2014; Favretto 2013] e desgaste dental erosivo [Moretto et al., 2010; Manarelli et al., 2011; Pancote et al., 2014; Cruz et al., 2015]. Esta associação também foi avaliada para vernizes fluoretados, em uma série de estudos que demonstram um efeito sinérgico na desmineralização e remineralização do esmalte, bem como no desgaste erosivo do esmalte usando protocolos in vitro e in situ [Manarelli et al., 2013, 2014, 2015, 2017; Moretto et al., 2013]. A fim de aumentar ainda mais os efeitos preventivos e terapêuticos de dentifrícios contendo TMP, nanopartículas deste fosfato foram adicionadas a um dentifrício de 1100 ppm F, o que demonstrou reduzir significativamente a desmineralização do esmalte in vitro [Danelon et al., 2017a] e aumentar seu efeito remineralizador in situ [Danelon, 2015]. Esta formulação também se mostrou eficaz em aumentou os efeitos protetores sobre o desgaste dental erosivo quando comparada a formulações de mesma concentração de F, contendo TMP microparticulado ou sem TMP [Danelon et al., 2017b]. Considerando as vantagens anteriormente citadas da suplementação de dentifrícios fluoretados com nanopartículas de TMP, é possível que a adição de TMP nanoparticulado a vernizes fluoretados promova um aumento do potencial preventivo e terapêutico destas formulações. Assim o objetivo do presente estudo foi avaliar o efeito de vernizes fluoretados contendo nanopartículas de TMP sobre a remineralização de lesões de cárie artificiais e sobre a erosão de esmalte dental, em protocolos in vitro. Em acréscimo, este estudo objetivou avaliar o padrão de liberação de F e TMP a partir destas formulações, a fim de melhor compreender os mecanismos envolvidos na interação entre F e TMP liberados a partir destes produtos em função da concentração de TMP e tamanho da partícula adicionada. Para abordar o tema proposto, o estudo será apresentado em três capítulos distintos, conforme descrito abaixo: - Capítulo 1: “In vitro remineralization of caries-like lesions with fluoride varnishes containing nano-sized sodium trimetaphosphate” (artigo submetido ao periódico Journal of Dentistry); _________________________________________________________________________ 23 Liliana Carolina Báez Quintero - Capítulo 2: “Nano-sized sodium trimetaphosphate enhances the protective effect of fluoridated varnishes on initial enamel erosion” (artigo formatado nas normas do periódico Journal of Dentistry); - Capítulo 3: “Fluoride and phosphate release from fluoride varnishes supplemented with nano-sized sodium trimetaphosphate” (artigo formatado nas normas do periódico Caries Research). _________________________________________________________________________ 24 Liliana Carolina Báez Quintero 2. Capítulo 1 2. _________________________________________________________________________ 25 Liliana Carolina Báez Quintero In vitro remineralization of caries-like lesions with fluoride varnishes containing nano-sized sodium trimetaphosphate1 LC Báez-Quintero1, ACB Delbem1, ME Nagata1, MM Manarelli1, ER Camargo2, VT Sakai3, M Danelon1, JP Pessan1 1Department of Pediatric Dentistry and Public Health, School of Dentistry, Araçatuba, São Paulo State University (UNESP), Araçatuba, SP, Brazil 2 LIEC-Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos, SP, Brazil 3Department of Clinic and Surgery, School of Dentistry, Universidade Federal University of Alfenas (UNIFAL-MG), Alfenas, MG, Brazil Short title: F varnish containing nanosized TMP on enamel remineralization. Keywords: Fluoride varnish. Polyphosphates. Dental caries. Topical fluorides. Dental enamel. Corresponding author: Juliano Pelim Pessan School of Dentistry, Araçatuba, São Paulo State University (UNESP) Department of Pediatric Dentistry and Public Health Rua Jose Bonifacio 1193 16015-050 Araçatuba - SP - Brazil Tel: (+55) 18 3636 3314 Email: jpessan@foa.unesp.br Declaration of interest A patent was requested for a product used in the study, by the National Institute of Industrial Property - INPI/SP, on 10/17/2014 under number BR 10 2014 025902 3. 1 Artigo formatado de acordo com as normas do periódico Journal of Dentistry (Anexo B) _________________________________________________________________________ 26 Liliana Carolina Báez Quintero 2.1. Abstract Objective to evaluate in vitro the effect of fluoride varnishes supplemented with nano-sized TMP on the remineralization of caries-like lesions using bovine enamel. Materials and Methods: enamel blocks (n=168) were submitted to induction of caries-like lesions and randomly divided into 7 experimental groups (n=24/group): Placebo (no fluoride or TMP), 2.5% NaF, 5% NaF, 5% NaF + 5% micrometric TMP, 5% NaF + 2.5 or 5% nano-sized TMP, and a commercial varnish (DuraphatTM). Varnishes remained in contact with the blocks during 6 h; half of the blocks were used for analysis of loosely- (CaF2) and firmly-bound (FA) fluoride, while the remaining blocks were submitted to a pH-cycling regimen (6 days). The percentage of surface hardness recovery (%SHR), cross-sectional hardness (ΔKHN), and CaF2 and FA on/in enamel were determined. Data were analyzed by ANOVA and Student-Newman-Keuls’ test (p<0.05). Results: A dose-response relationship was observed between the fluoride concentration in the varnishes without TMP and %SHR (p<0.05). TMP- containing varnishes promoted significantly higher %SHR when compared to TMP-free products, without significant differences among varnishes with TMP. A similar pattern was found for ΔKHN, despite the values found for 5% NaF + 5% nano-sized TMP were 25% lower when compared with 5% NaF + 5% micrometric TMP. Varnishes containing 5% NaF, 5% NaF + 5% TMPmicro and Duraphat promoted the highest CaF2 concentrations compared to the other groups (p<0.05). Conclusion: the supplementation of fluoride varnishes with TMP enhances the remineralizing effect of these products, without a significant additional benefit of the use of nano-sized particles. Clinical Significance: the enhanced remineralizing effect promoted by the TMP-containing varnishes suggest that these products could be a viable alternative for the treatment of patients with non-cavitated carious lesions. The reduction in ΔKHN also indicates a higher effect on the subsurface when compared with a conventional formulation. Key words: Nanotechnology, Tooth Remineralization, fluoride varnish, pH- cycling, polyphosphate. _________________________________________________________________________ 27 Liliana Carolina Báez Quintero 2.2. Introduction Dental caries is the result of a dynamic process of de- and remineralization, caused by microbial metabolism of carbohydrates at the tooth surfaces, which can lead to mineral loss (critical pH around 5.5) over time and, subsequently, to cavity formation [1, 2]. Adequate oral hygiene, diet, salivary flow and fluoride exposure are modulating factors that should be evaluated and/or controlled in order to prevent or reverse the caries process [3]. Among these factors, the use of topical fluorides is known to directly interfere with the balance between de- and remineralization [4], being used worldwide in community-based, self-applied and professionally applied fluoride schemes. Regarding professionally applied fluoridated vehicles, fluoridated varnishes are widely recommended for the remineralization of initial caries lesions, since they are viscous products containing high concentrations of fluoride that harden upon contact with saliva, forming a thin film that adheres to the surface of the carious teeth, releasing F to the oral cavity for prolonged periods of time [5]. A recent meta-analysis evaluating the effect of fluoride varnishes on the prevention of caries in children and adolescents concluded that the reduction in the DMFS and dmfs indexes associated with their use are 43% and 37%, respectively [6]. The addition of sodium trimetaphosphate (TMP) to fluoridated products has been shown to increase their protective and therapeutic effects against dental caries. The supplementation of toothpastes at varying fluoride concentrations with TMP was shown to significantly enhance the effects against enamel demineralization [7], reduce enamel demineralization [8], promote significant changes in the dental biofilm regarding mineral composition and the production of extracellular polysaccharides [9], and to reduce the progression of caries lesions in children, in a randomized clinical trial [10]. This association has also been assessed for fluoridated varnishes, in in a series of studies demonstrating a synergistic effect on enamel demineralization and remineralization, as well as on enamel erosive wear using in vitro and in situ protocols [11-15]. In order to further enhance the effects of TMP-containing toothpaste, recent studies have demonstrated that the addition of nano-sized TMP to a _________________________________________________________________________ 28 Liliana Carolina Báez Quintero 1100 ppm F toothpaste significantly reduced enamel demineralization in vitro [16] and enhanced the remineralizing effect in situ [17] when compared to its counterpart containing micrometric TMP, what has been attributed to the greater reactivity of nano-sized TMP (due to the higher ratio of surface area to volume). This higher reactivity of nano-sized TMP was also recently shown to enhance the protective effect of low-fluoride toothpaste (250 ppm F) against enamel demineralization, achieving superior levels when compared to an 1100 ppm F formulation without TMP [18]. Considering the advantages of fluoride varnishes in the clinical practice and given that the above-mentioned effects of nano-sized TMP have not yet been tested for these formulations, the objective of this work was to evaluate in vitro the effect of fluoride varnishes supplemented with nano-sized TMP on the remineralization of caries-like lesions using bovine enamel. The null hypothesis was that fluoride varnishes supplemented with nano-sized TMP have a similar ability to promote enamel remineralization compared to their counterparts supplemented with micrometric TMP. 2.3. Materials and Methods Experimental Design Bovine enamel blocks (n = 168) were selected after surface hardness analysis (SH), and caries-like lesions were induced (SH1). The blocks were then randomly divided into 7 experimental groups (n = 24 / group) according to the varnishes to be tested: Placebo (no fluoride or TMP); 2.5% NaF; 5% NaF; 5% NaF/5% micrometric TMP; 5% NaF/5% nanosized TMP; 5% NaF/2.5% nanosized TMP, and commercial varnish (Duraphat, 5% NaF), hereafter abbreviated as PLA, 2.5%NaF, 5%NaF, 5%NaF/5%TMPmicro, 5%NaF/5%TMPnano, 5%NaF/2.5%TMPnano and Duraphat, respectively. Blocks were treated once with the varnishes and immersed in a remineralizing solution (4 h), followed by a demineralizing solution (2 h). Varnishes were then removed from the enamel surface and the blocks, subjected to a pH-cycling for 6 days. Finally, specimens were analyzed for SH (allowing the calculation of SH recovery (% SHR)), integrated area of subsurface lesion (ΔKHN), firmly- and _________________________________________________________________________ 29 Liliana Carolina Báez Quintero loosely-bound formed (6 h after the application of the varnishes) and retained in/on enamel (after pH-cycling). Synthesis and characterization of nano-sized TMP particles Commercial micrometric sodium trimetaphosphate (70 g, Na3O9P3, Aldrich Chemistry, China, purity ≥ 95% CAS 7785-84-4) were ball milled using 500 g of sintered zirconia spheres of 2 mm diameter in 1 litter of isopropanol in a polypropylene battle. After 48 h, at a grinding speed of 1200 rpm, powders were separated from the alcoholic medium, dried at 60 °C, and ground in a mortar. Histograms were constructed counting more than 100 particles from images obtained with a transmission electron microscope (Philips XL-30 FEG) that were treated using the public domain ImageJ image processing software. Powder crystallinity was characterized by X-ray diffraction (XRD) using a Rigaku Dmax 2500 PC difractometer in the 2θ range from 10 to 80º with a scanning rate of 2º/min. The coherent crystalline domains (crystallite size) were estimated using the Scherrer equation (L = Kλ / BcosθB), where L is the linear dimension of a monocrystalline nanoparticle, λ is the wavelength of the incident X-ray, B is the diffraction line width of the diffraction peak, θB is the Bragg angle obtained from the XRD pattern, and K is a numerical constant which value is 0.9. [17] Varnish formulation and determination of fluoride in products The varnishes were produced by SS White Dental Products (Rio de Janeiro, RJ, Brazil), containing the following components: colophony, ethyl cellulose, tolu balsam, beeswax, toluene sulfonamide, vanillin, saccharin and ethanol. F concentrations were 0 (negative control), 2.5% and 5% NaF (Merck, Germany). To the 5% NaF varnish (Fluorniz, SS White Dental Products, Brazil), sodium trimetaphosphate (Aldrich Chemistry, China) was added at 5% (micrometric or nanosized) or 2.5% (nanosized). F concentrations in the varnishes were determined using a fluoride ion specific electrode (9609 BN, Orion, USA) coupled to an ion analyzer (Orion 720 A+), and calibrated with standards containing 2.0–32.0 μg fluoride/mL, as previously described [19,11]. _________________________________________________________________________ 30 Liliana Carolina Báez Quintero Preparation of enamel blocks and induction of subsurface lesions Enamel blocks (4mm x 4mm) were obtained from bovine incisors, previously stored in 2% formaldehyde solution pH 7.0 for 30 days at room temperature [20]. The enamel surface of the blocks was serially polished and selected on the basis of their SH (320.0–380.0 KHN). All surfaces of each block, except the enamel surface, were coated with acid-resistant varnish (Risqué®-Brazil), and subsurface enamel demineralization was produced by immersing each enamel block in 32 mL of a solution with 1.3 mmol/L calcium nitrate tetrahydrate (Zigma), 0.78 mmol/L sodium dihydrogen phosphate monohydrate (Zigma) in 0.05 mol/L acetate buffer, pH 5.0; 0.8 mL F; for 16 h at 37 ºC [21]. Thereafter, the SH1 was determined. Treatment with the varnishes and pH-cycling The varnishes were applied with a microbrush on each block (n=24/group) only once, which were immersed in 4 mL of a remineralizing solution (1.5 mmol/L Ca, 0.9 mmol/L P, 0.15 mol/L KCl in 0.02 mol/L cacodylate buffer, 0.4 mL F, pH 7.0) during 4 h; followed by immersion in 12 mL of a demineralizing solution (2.0 mmol/L Ca and P in 0.075 mol/L acetate buffer, 0.45 mL F, pH 4.7) for 2 h. The varnishes were then gently removed with a blade and acetone and twelve blocks from each group were subjected a to pH- cycling at 37 ºC for 6 days [22, 11]. The other twelve blocks were stored for subsequent analysis of loosely- bound fluoride (CaF2) and firmly-bound fluoride (FA). Analysis of enamel hardness The hardness of the enamel surface was determined using a mircrohardness tester (Shimadzu HMV-2000), with a Knoop diamond indenter under a 25 g load for 10 s. Five indentations, separated by a distance of 100 μm, were made in the center of each block to analyze initial SH. After the induction of artificially demineralized lesions and after the pH cycling, SH was measured again (SH1 and SH2, respectively), 100 µm from the initial indentations (SH) [22]. The percentage of SH recovery (%SHR = [{SH2 - _________________________________________________________________________ 31 Liliana Carolina Báez Quintero SH1}/{SH - SH1}] x 100) was calculated. For cross-sectional hardness measurements, the blocks were sectioned at the center, and one of the halves was embedded in acrylic resin and gradually polished.) A sequence of 14 indentations was created at 5, 10, 15, 20, 25, 30, 40, 50, 70, 90, 110, 130, 220, and 330 μm from the enamel surface, in the central region of the blocks, using Micromet 5114 (Buehler, Lake Bluff, USA) and Buehler Omi Met software (Buehler, Lake Bluff, USA) with a Knoop diamond indenter under a 5 g load, for 10 s. Integrated hardness (KHN × µm) for the lesion into the sound enamel was calculated by the trapezoidal rule (GraphPad Prism, version 3.02) and subtracted from the integrated hardness for sound enamel to obtain the integrated area of the subsurface lesion in the enamel, which was named integrated loss of subsurface hardness (ΔKHN; KHN × µm) [23]. Analysis of loosely-bound fluoride (CaF2) on enamel The concentration of CaF2 on enamel was analyzed 6 h after the application of the varnishes (CaF2 formed; n=12/ group) and after the pH cycling (CaF2 retained; n=12/group). A digital caliper (Mitutoyo CD-15B, Mitutoyo Corporation, Japan) was used to measure the surface area of enamel blocks. Assessment of loosely-bound fluoride (alkali-soluble fluoride – CaF2 formed and retained) was performed following the methodology of Caslavska et al., 1975[24]. The surfaces of each specimen, except the enamel surface, were coated with wax. Specimens were then immersed in 0.5 ml of 1.0 mol/l KOH solution for 24 h, under constant agitation. The solution was neutralized and buffered with 0.5 ml of TISAB II modified with HCl 1.0 mol/l. An ion analyzer (720A+) and a combined ion-selective electrode (9609 BN), previously calibrated with the standards 0.0625, 0.125, 0.250, 0.500 and 1.0 μg fluoride/ml were used. [25] Analysis of firmly-bound fluoride (FA) in enamel After extraction of CaF2, enamel biopsy was performed, to provides data on firmly bound fluoride, also 6 h after varnish application (name “formed) and after the pH cycling (named “retained”). Blocks measuring 2 mm x 2 mm were _________________________________________________________________________ 32 Liliana Carolina Báez Quintero obtained from half of the longitudinally sectioned blocks, and fixed with adhesive glue on a mandrel for straight. Self-adhesive polishing discs (diameter, 13 mm) and 400-grit silicon carbide (Buehler) were fixed to the bottom of a polystyrene crystal tube (J-10; Injeplast, São Paulo, SP, Brazil). One layer of 57.0 ± 0.06 µm each was removed from the enamel block. To the resulting enamel powder, 500 µL of 1.0 mol/l HCl was added. The tubes were agitated for 1 h, and an aliquot of 250 µL of this solution was used for F analysis, after buffering with 250 µL of TISAB II modified with 1.0 mol/l NaOH [8, 25]. An ion-specific electrode (Orion 9609) was connected to an ion analyzer (Orion 720+) were used for the analysis. Statistical analysis Analyses were performed using the SigmaPlot software (version 12.0) and the level of statistical significance was established at 5%. The variables %SHR (log10 transformed) and ΔKHN (natural outcomes), showed normal (Shapiro-Wilk test) and homogeneous (Cochran test) distributions and were submitted to one-way ANOVA. Data for enamel fluoride concentrations (log10 transformed) showed normal (Shapiro-Wilk test) and homogeneous (Cochran test) distributions and were submitted to two-way ANOVA, considering the type of varnish and CaF2 and FA formed and retained. Student-Newman-Keuls’ test was used for multiple comparisons for all conditions above. 2.4. Results The mean (SD) fluoride concentrations (micrograms of fluoride per gram) in the varnishes were 433.6 (33.5), 10,758.4 (302.0), 21,378.8 (708.1), 20,154.0 (326.9), 20,400.2 (262.2), 19,827.8 (316.8) and 23,702.5 (1748.0), respectively for Placebo, 2.5% NaF, 5% NaF, 5% NaF + 5% TMPmicro, 5% NaF + 2.5% TMP nano, 5% NaF + 5% TMPnano and Duraphat. The mean (SD) surface hardness of all the bovine tooth blocks was 355.2 (11.6) KHN. After induction of artificial caries lesions in half of the blocks, the mean (SD) percentage of hardness loss was 85.1 (4.0) %, without significant differences among the groups (p=0.62). _________________________________________________________________________ 33 Liliana Carolina Báez Quintero A dose-response relationship was observed between the fluoride concentrations in the varnishes without TMP and %SHR (p<0.05). Groups treated with varnishes containing TMP showed significantly higher %SHR when compared to those without TMP, without significant differences among the TMP-containing varnishes (Table 1). A similar pattern was observed for ΔKHN, with the highest values obtained for the Placebo, and the lowest observed for the TMP-containing varnishes (Table 1; Figure 1). The highest concentration of CaF2 formed on enamel was found for the groups Duraphat, 5% NaF, 5% NaF + 5% TMPmicro, without significant differences among them. The 5% NaF + 2.5% TMPnano group presented similar values to the 2.5% NaF group. After pH- cycling, the concentration of CaF2 was lower for all groups, except for the Placebo group. The varnishes Duraphat, 5% NaF, 5% NaF + 5%TMPmicro and 5% NaF + 5%TMPnano had the highest values of CaF2 retained, without significant differences among them. (Table 2) No significant differences were observed for groups 5% NaF, 5% NaF + 5% TMPnano, 5% NaF + 5% TMPmicro and Duraphat, regarding firmly bound fluoride after varnish application (6h) or after pH-cycling (6 days). Groups treated with varnishes containing nano-sized TMP had lower firmly-bond F values formed in enamel than the other groups, except for the Placebo. Regarding firmly-bond fluoride retained in enamel, groups treated with varnishes containing TMPnano were not significantly different from 5%NaF, 5%NaF + 5%TMPmicro and Duraphat (Table 2). 2.5. Discussion Previous studies evaluating the effectiveness of fluoride varnishes supplemented with TMPmicro have shown a significantly greater remineralization of caries lesions in vitro [11] and in situ [12] compared to TMP-free varnishes with the same fluoride content (5% NaF). The present study confirmed the above-mentioned findings, despite the additional remineralizing effect observed for varnishes containing nano-sized TMP was not statistically significant compared with micrometric particles. Therefore, the study’s null _________________________________________________________________________ 34 Liliana Carolina Báez Quintero hypothesis was accepted. Some aspects of these outcomes need to be discussed for a better comprehension of the actual benefits of nano-sized TMP when added to varnishes. Despite the differences between TMPmicro and TMPnano were not significant, the formulation containing 5%NaF/5%TMPnano promoted a ~10% increase in %SHR in comparison with 5% NaF/5%TMPmicro (25% increase when compared with 5%NaF), indicating an additional benefit of this formulation over 5%NaF/5%TMPmicro. It is noteworthy, however, that this modest benefit was achieved using a short-term caries model, what cannot be fully extrapolated to in vivo conditions, in which the caries dynamics (de- and remineralization cycles) occur over much longer periods of time. Nonetheless, even a small benefit on enamel surface could be regarded as a positive aspect of the formulation, as it may be associated with an increased resistance to acids upon cariogenic challenges, consequently affecting ion mobility from and to the subsurface. A second – and most important – aspect of the present data refers to the additional benefit of 5%NaF/5%TMPnano in the subsurface (ΔKHN), which was 25% lower in comparison with the 5%NaF/5%TMPmicro (~40% when compared with 5%NaF). Despite not statistically significant, such effect represents a substantial reduction in the lesion body, which is the most relevant parameter when considering the nature of caries lesions (i.e., subsurface lesions). Given that dental caries is the net result of the cumulative effect of successive de- and remineralizing cycles, it might be hypothesized that the differences among the three varnishes (5%NaF, 5%NaF/5%TMPmicro and 5%NaF/5%TMPnano) in the present short-term study under in vitro testing conditions would become significant over time under clinical conditions, in which varnishes are usually applied 4 times at weekly intervals for the remineralization of white spot lesions (therefore producing a cumulative effect) and the patients are simultaneously exposed to other sources of fluoride (including toothpastes and water). In this sense, it should be noted that the dynamics of de- and re-mineralization in shallow lesions (as in the present study) is much faster when compared with larger, more porous lesions (as may be found in vivo) [26], what supports the above-mentioned hypothesis. _________________________________________________________________________ 35 Liliana Carolina Báez Quintero It has been reported that an ideal F:TMP molar ratio is paramount for achieving optimum results on enamel de- and remineralization. Considering the different particle sizes assessed in the present study and based on previous observations with toothpastes [17], it was expected that a lower concentration of nano-sized TMP would be required to promote a similar remineralizing effect to that achieved with micrometric TMP. While this was indeed achieved for %SHR and ΔKHN when considering 5%NaF/2.5%TMPnano and 5%NaF/5%TMPmicro, respectively, it was surprising that the use of higher concentrations of TMPnano (5%NaF/5%TMPnano) further enhanced the remineralizing effect of the varnish. This raises important questions on the reactivity of nano-sized TMP embedded in the varnish matrix after hardening. The smaller size of TMPnano might be associated with a higher interaction with the varnish matrix, which may result in a lower mobility from it to the oral environment (saliva and tooth surfaces). Therefore, increasing the amount of TMPnano on the formulation (from 2.5 to 5%) seems to have overcome this limitation, ultimately leading to a higher release of TMP from the varnish, thus achieving a higher remineralizing effect when compared to 5%NaF/2.5%TMPnano. The above-mentioned considerations would not be applicable to other topical formulations (such as toothpastes and gels), due to the higher mobility of active ingredients (F and TMP) within the vehicle [16-18]. One of the main advantages of professionally applied fluoridated vehicles is the formation of large quantities of CaF2, which act as a slow-release F reservoir controlled by the intraoral pH, able to interfere in the processes of de- and remineralization [27]. For the TMP-free formulations, a clear dose- response relationship between F content in the varnishes and the resulting CaF2 concentrations formed on enamel, what is in line with previous observations [11-12]. For the TMP-containing varnishes, however, the amount of CaF2 formed varied depending on the particle size (micro or nano) and concentration (2.5 or 5%), with the highest values observed for the 5%NaF/5%TMPmicro (similar to 5%NaF), followed by 5%NaF/5%TMPnano and 5%NaF/2.5%TMPnano (2- and 4-fold lower than 5%NaF, respectively). These data provide interesting information on the mechanisms of TMP-containing fluoridated varnishes on the dynamics of de- and remineralization. Given that _________________________________________________________________________ 36 Liliana Carolina Báez Quintero the highest remineralizing effect (5%NaF/2.5%TMPnano) was associated with a reduced formation of CaF2, the present study reinforces the concept that the main effect of this formulation is not related to the deposition of high quantities of loosely-bound fluoride [11]. Instead, previous observations indicate that the main effect of TMP is related to a reduction of acid diffusion into enamel and to the retention of ions on its molecule, which result in more reactive remineralizing species upon acid challenges [28,29, 17]. Differences between present CaF2 data and those obtained in a previous study using the same varnish formulations (except for TMPnano) also deserve comment. First, the present CaF2 data are, overall, substantially lower than those reported by Manarelli et al. [11]. Second, while the results of the 5%NaF/5%TMPmicro in the present study were similar to those found for 5%NaF, these were 2-fold lower when compared with 5%NaF in the above- mentioned study. Third, CaF2 data resulting from Duraphat were virtually identical to those seen for 5%NaF in the present study, but 40% lower in the study by Manarelli et al. [11]. Considering that both studies were conducted by the same research group, and that the test varnishes were produced by the same manufacturer, the differences above seem to be related to the natural resin used in the production of the varnishes. Shen and Autio-Gold [19] reported significant differences between tubes (of the same batch) of colophony-based varnishes containing 5% NaF, and that fluoride content can vary between doses dispensed from the same tube. Nonetheless, despite these differences inherent to the non-therapeutic component of the formulation, it must be emphasized that both in the study by Manarelli et al. [11] and in present work, a clear dose- response relationship was observed between fluoride content in the varnishes without TMP and the rate of enamel remineralization, and that TMP significantly enhanced this effect. This clearly indicates that differences in the natural resin used for the manufacturing produce negligible effects on the main outcomes assessed (hardness data). Similarly to the findings of CaF2 described above, the effect of TMP- containing varnishes does not seem to be related to enamel fluoride uptake (firmly-bound) in the outermost enamel layers, especially TMPnano, which promoted the lowest uptake among the varnishes containing 5%NaF (6 h after _________________________________________________________________________ 37 Liliana Carolina Báez Quintero varnish application). A different pattern, however, has been described for fluoridated toothpastes supplemented with TMPnano, what seem to be related to the different caries model used (demineralization), type of substrate (sound enamel), fluoride concentration (1100 ppm F) and frequency of use (2×/day, during 5 days) [16]. Another important aspect might be related to the differences between the two vehicles (toothpaste and varnish), what might affect ion mobility within the formulation, thus leading to different outcomes. Also, it is important to point out that the amount of enamel removed for firmly-bound fluoride determination in the present study was ~57µm, so that any possible changes in fluoride concentrations in deeper enamel layers would not, therefore, be detected by the analytical method employed. Regardless of the above, the absence of a linear correlation between firmly-bound fluoride and enamel remineralization after the use of fluoride varnishes [30] suggest that enamel fluoride uptake should not be considered as a main response variable in in vitro models assessing the remineralizing effect of such formulations. Some methodological implications related to the in vitro model used might also have impacted the results obtained. The removal of the varnishes 6 h after application could possibly underestimated the effect of the products, given that F and TMP release from the products was stable for up to 24 h after varnish application. However, the varnishes were removed in order to assess the chemical effect of the products (i.e., F and TMP release) instead its mechanical protection [31]. Under clinical conditions, the varnishes would be removed by mechanic forces (mastication or brushing), at varying times after application, opposed to in vitro conditions in which the varnishes would remain over the specimens for several days. Other in vitro limitations such as absence of self-applied and/or community-based fluoride sources commonly used by the patients, the short-term nature of the protocol and the lesion depth have already been discussed and might also have played an important role on the outcomes. Therefore, the investigation of the remineralizing potential of the test varnishes under in situ and in vivo conditions could provide important data on the real benefit of such formulations in caries prevention and control. In conclusion, fluoride and TMP present a synergistic effect on the remineralization of artificial caries lesions in vitro. Despite the differences _________________________________________________________________________ 38 Liliana Carolina Báez Quintero between micrometric and nano-sized particles of this polyphosphate were not significant, the marked reduction of the subsurface lesion (~25%) when compared with TMPmicro suggest that the addition of nano-sized TMP could be an interesting alternative to further enhance the remineralizing effect of TMP- containing fluoridated varnishes. Studies with different protocols would be instructive in this regard. 2.6. Acknowledgments This study was supported by CAPES (PROCAD, Grant #88881.068437/2014-01) and São Paulo State University (PAEDEX/AUIP/PROPG, scholarship to the first author).The authors thank to SS White Dental Products (Rio de Janeiro, Brazil) for manufacturing the varnishes. _________________________________________________________________________ 39 Liliana Carolina Báez Quintero 2.7. References [1] Fejerkov O. Concepts of dental caries and their consequences for understanding the disease. Community Dent Oral Epidemiol. 1997; 25:5-12. [2] Kidd EAM, Fejerkov O. What Constitutes Dental Caries? Histopathology of Carious Enamel and Dentin Related to the action of Cariogenic Biofilms. J Dent Res. 2004; 83(Spec Iss C):C35-C38. [3] Kidd E. The implications of the new paradigm of dental caries. J Dent. 2011; 39 Supp 2:S3-8. [4] Ten Cate JM. In vitro studies on the effects of fluoride on de-and remineralization. J Dent Res. 1990; 69(2):614–9. [5] Pessan JP, Toumba KJ, Buzalaf MA. Topical use of fluorides for caries control. Monogr Oral Sci. 2011; 22:115-32. [6] Marinho VC, Worthington HV, Walsh T, Clarkson JE. Fluoride varnishes for preventing dental caries in children and adolescents. Cochrane Database Syst Rev. 2013 Jul 11;(7):CD002279. [7] Takeshita EM, Danelon M, Castro LP, Sassaki KT, Delbem AC. Effectiveness of a Toothpaste with Low Fluoride Content Combined with Trimetaphosphate on Dental Biofilm and Enamel Demineralization in situ. Caries Res. 2015; 49: 394-400. [8] Takeshita EM, Castro LP, Sassaki KT, Delbem AC. In vitro evaluation of dentifrice with low fluoride content supplemented with trimetaphosphate. Caries Res. 2009; 43(1): 50-6. [9] Takeshita EM, Danelon M, Castro LP, Sassaki KT, Delbem AC. Effectiveness of a Toothpaste with Low Fluoride Content Combined with Trimetaphosphate on Dental Biofilm and Enamel Demineralization in situ. Caries Res. 2015; 49(4): 394-400. [10] Freire IR, Pessan JP, Amaral JG, Martinhon CCR, Cunha RF, Delbem ACB. Anticaries effect of low-fluoride dentifrices with phosphates in children: A randomized, controlled trial. Journal of Dentistry. 2016; 50: 37-42. _________________________________________________________________________ 40 Liliana Carolina Báez Quintero [11] Manarelli MM, Delbem ACB, Lima TMT, Castilho FCN, Pessan JP. In vitro Remineralizing Effect of Fluoride Varnishes Containing Sodium Trimetaphosphate. Caries Res. 2014; 48:299-305. [12] Manarelli MM, Delbem ACB, Binhardi TDR, Pessan JP. In situ Remineralizing Effect of Fluoride Varnishes Containing Sodium Trimetaphosphate. Clin Oral Invest. 2015;19: 2141-2146. [13] Manarelli MM, Delbem ACB, Báez-Quintero LC, De Morais FRN, Cunha RF, Pessan JP. Fluoride varnishes containing sodium trimetaphosphate reduce enamel demineralization in vitro. Acta Odontologica Scandinavica. 2017. DOI: 10.1080/00016357.2017.1318448 [14] Manarelli MM, Moretto MJ, Sassaki KT, Martinhon CC, Pessan JP, Delbem AC. Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion. Am J Dent. 2013; 26(6): 307-12. [15] Moretto MJ, Delbem AC, Manarelli MM, Pessan JP, Martinhon CC. Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion: an in situ/ex vivo study. J Dent. 2013; 41(12): 1302-6 [16] Danelon M, Pessan JP, Souza-Neto FN, Camargo ER, Delbem ACB. Effect of fluoride toothpaste with nano-sized trimetaphosphate on enamel demineralization: An in intro study. Archives of Oral Biology. 2017; 78: 82-87. [17] Danelon M, Pessan JP, Neto FN, de Camargo ER, Delbem AC. Effect of toothpaste with nano-sized trimetaphosphate on dental caries; In situ study. J Dent. 2015; 43(7); 806-13. [18] Souza MDB, Pessan JP, Lodi CS, Souza JAS, Camargo ER, Souza Neto FN, Delbem ACB. Toothpaste with Nanosized Trimetaphosphate Reduces Enamel Demineralization. JDR Clinical & Translational Research. 2016; XX(X). DOI: 10.1177/2380084416683913. [19] Shen C, Autio-Gold J. Assessing fluoride concentration uniformity and fluoride release from three varnishes. J Am. Dent Assoc. 2002; 133(2): 176-82. [20] Delbem AC, Cury JA. Effect of application time of APF and NaF gels on microhardness and fluoride uptake of in vitro enamel caries. Am J Dent. 2002; 15(3):169-72. _________________________________________________________________________ 41 Liliana Carolina Báez Quintero [21] Queiroz CS, Hara AT, Leme FP, Cury JA. pH-cycling models to evaluate the effect of low fluoride dentifrice on enamel de- and remineralization. Braz Dent J 2008; 19:21-27. [22] Vieira AE, Delbem AC, Sassaki KT, Rodrigues E, Cury JA, Cunha RF. Fluoride dose response in pH-cycling models using bovine enamel. Caries Res 2005; 39:514-520. [23] Danelon, M, Takeshita, EM, Sassaki, KT, Delbem, ACB. In situ evaluation of a low fluoride concentration gel with sodium trimetaphosphate in enamel remineralization. Am J Dent 2013; 26:15-20. [24] Caslavska V, Moreno EC, Brudevold F. Determination of the calcium fluoride formed from in vitro exposure of human enamel to fluoride solutions. Arch Oral Biol 1975; 20:333-339. [25] Dalpasquale G, Delbem ACB, Pessan JP, Nunes GP, Gorup LF, Neto FNS, de Camargo ER, Danelon M. Effect of the addition of nano-sized sodium hexametaphosphate to fluoride toothpastes on tooth demineralization: an in vitro study. Clin Oral Investig 2017; 21(5):1821-1827. [26] Buzalaf MA, Hannas AR, Magalhães AC, Rios D, Honório HM, Delbem AC. pH-cycling models for in vitro evaluation of the efficacy of fluoridated dentifrices for caries control: strengths and limitations. J Appl Oral Sci. 2010; 18(4):316-34. [27] Tenuta LM, Cerezetti RV, Del Bel Cury AA, Tabchoury CP, Cury JA. Fluoride release from CaF2 and enamel demineralization. J Dent Res. 2008;87(11):1032-6. [28] Pancote LP, Manarelli MM, Danelon M, Delbem AC. Effect of fluoride gels supplemented with sodium trimetaphosphate on enamel erosion and abrasion: in vitro study. Arch Oral Biol 2014;59(3):336-40. [29] Delbem AC, Bergamaschi M, Rodrigues E, Sassaki KT, Viera AE ME. Anticaries effect of dentifrices with calcium citrate and sodium trimetaphosphate on enamel de- and remineralization in vitro. J Appl Oral Sci. 2012;20:94–8. _________________________________________________________________________ 42 Liliana Carolina Báez Quintero [30] Al Dehailan L, Martinez-Mier EA, Lippert F. The effect of fluoride varnishes on caries lesions: an in vitro investigation. Clin Oral Investig. 2016; 20(7):1655- 62. [31] Magalhães AC, Kato MT, Rios D, Wiegand A, Attin T, Buzalaf MA. The effect of an experimental 4% Tif4 varnish compared to NaF varnishes and 4% TiF4 solution on dental erosion in vitro. Caries Res. 2008;42(4):269-74. _________________________________________________________________________ 43 Liliana Carolina Báez Quintero Table 1. Percentage of surface hardness recovery (%SHR) and integrated loss of subsurface hardness (ΔKNH) according to the varnishes Groups %SHR ΔKHN Placebo 14,6 (3,2) A 6876,7 (827,5) A 2,5% NaF 35,8 (3,7) B 5794,8 (752,0) B 5% NaF 41,1 (2,6) C 4400,2 (1266,6) C 5% NaF + 5% TMP micro 47,2 (5,4) D 3709,5 (929,5) D 5% NaF + 5% TMP nano 51,4 (3,2) D 2787,7 (781,9) D 5% NaF + 2,5% TMP nano 48,2 (3,3) D 3181,2 (742,3) D Duraphat 37,6 (4,8) B,C 4783,1 (1184,3) C Values are presented as means (SD). Different uppercase superscript letters indicate significant differences among the groups (Student –Newman Keuls’ method, n=12, p<0.05). %SHR data were log10-transformed for the statistical analysis. _________________________________________________________________________ 44 Liliana Carolina Báez Quintero Table 2. CaF2 and firmly bound fluoride 6 h after varnish application and after pH- cycling according to groups Groups CaF2 , µg/cm 2 Fluoride, µg/cm 2 Formed Retained Formed Retained Placebo 0.24(0.04) A,a 0.26(0.04) A,a 0.40(0.10) A,a 0.60(0.11) B,a 2,5% NaF 5.30(2.9) A,b 0.35(0.11) B,bc 0.91(0.15) A,bcd 0.73(0.11) B,bcd 5% NaF 22.84(7.7) A,cd 0.45(0.14) B,cdef 1.09(0.38) A,d 0.97(0.30) A,e 5% NaF + 5% TMP micro 25.51(8.3) A,d 0.58(0.09) B,ef 0.91(0.21) A,bcde 0.94(0.39) A,ef 5% NaF + 5% TMP nano 10.86(4.4) A,e 0.47(0.10) B,def 0.64(0.09) A,fg 0.77(0.17) A,cdefg 5% NaF + 2,5% TMP nano 5.70 (1.8) A,b 0.38(0.09) B,bcd 0.64(0.18) A,g 0.80(0.19) B,defg Duraphat 21.39(6,8) A,cd 0.59(0.15) B,f 0.96(0.28) A,cd 0.95(0.25) A,efg Values are presented as mean (SD). Different lowercase superscript letters show significant difference among groups in each analysis. Different uppercase superscript letters indicate differences between CaF2 formed and retained, as well as between fluoride formed and retained within each group. Data (log10-transformed) were submitted the Student-Newman-Keuls’ method, n =12 (p < 0.05). _________________________________________________________________________ 45 Liliana Carolina Báez Quintero Figure 1. Differential hardness profile as a function of depth according to the groups. _________________________________________________________________________ 46 Liliana Carolina Báez Quintero 3. Capítulo 2 _________________________________________________________________________ 47 Liliana Carolina Báez Quintero Sodium trimetaphosphate enhances the protective effect of fluoridated varnishes against initial enamel erosion (short communication)2 Liliana Carolina Báez-Quintero1, Alberto Carlos Botazzo Delbem1, Mariana Emi Nagata1, Marcelle Danelon1, Diego Felipe Mardegan Gonçalves1, Daniela Rios2, Juliano Pelim Pessan1 1Department of Pediatric Dentistry and Public Health, School of Dentistry, Araçatuba, São Paulo State University (UNESP), Araçatuba, SP, Brazil 2Department of Pediatric Dentistry, Orthodontics and Public Health, Bauru School of Dentistry, University of São Paulo, Bauru, SP, Brazil Short title: F varnishes containing TMP reduce enamel erosion Corresponding author: Juliano Pelim Pessan School of Dentistry, Araçatuba, São Paulo State University (UNESP) Department of Pediatric Dentistry and Public Health Rua Jose Bonifacio 1193 16015-050 Araçatuba - SP - Brazil Tel: (+55) 18 3636 3314 Email: jpessan@foa.unesp.br Declaration of interest A patent was requested for a product used in the study, by the National Institute of Industrial Property - INPI/SP, on 10/17/2014 under number BR 10 2014 025902 3. 2 Artigo formatado de acordo com as normas do periódico Journal of Dentistry (Anexo B) _________________________________________________________________________ 48 Liliana Carolina Báez Quintero 3.1. Abstract This study assessed the protective effect of fluoridated varnishes supplemented with micrometric or nanosized sodium trimetaphosphate (TMPmicro or TMPnano, respectively) on initial enamel erosion. Bovine enamel blocks were selected by surface hardness (SH) and randomly assigned (n=8/group) into the groups: Placebo (no F/TMP), 5% NaF, 5% NaF+5%TMPmicro, 5% NaF+2.5%TMPnano and 5% NaF+5%TMPnano. Blocks received a single application of the varnishes and were immersed in artificial saliva (6 h). Following, the varnishes were removed and the blocks, subjected to four individual erosive challenges (1-min, citric acid, 0.75%, pH=3.5, under agitation); SH was determined after each challenge. Data were analyzed by ANOVA and Student-Newman-Keuls’ test (p<0.05). Overall, the highest %SH loss was observed for Placebo, followed by 5% NaF, 5% NaF+5% TMPmicro and both varnishes containing TMPnano, without significant differences between 2.5% and 5% TMPnano. It was concluded that TMP increases the protective effect of fluoridated varnishes against erosive challenges, with an additional benefit from the use of TMPnano. Keywords: Fluoride varnish. Polyphosphates. Tooth erosion. Topical fluorides. Dental enamel. _________________________________________________________________________ 49 Liliana Carolina Báez Quintero 3.2. Introduction The limited action of conventional fluoride therapies on the prevention of tooth erosion has prompted to studies assessing the effects of novel therapeutic agents used in association with fluoride [1, 2]. Among the options available, sodium trimetaphosphate (TMP) has shown to promote a synergistic protective effect against enamel erosion, followed or not by abrasion by toothbrushing, when added to fluoridated toothpastes, gels, mouthrinses and varnishes, using in vitro and in situ protocols [3-6]. The use of nano-sized TMP has been shown to further enhance the protective effects of fluoridated toothpastes against enamel erosive wear using an in vitro protocol [7]. No evidence, however, is available for other topically applied fluoridated vehicles. Given the promising results obtained for fluoridated varnishes containing nano-sized TMP on enamel remineralization in an in vitro caries model (unpublished data), the present study assessed the effects of fluoridated varnishes supplemented with micrometric or nano-sized TMP on initial enamel erosion. The null hypothesis was that the effect of the fluoridated varnish would not be affected by the addition of TMP, regardless of the particle size. 3.3. Material and Methods Experimental design Bovine enamel blocks (n = 40), were selected after surface hardness analysis (SH). The blocks were then randomly divided into 5 experimental groups (n= 8 / group) according to the varnishes to be tested: Placebo (no fluoride or TMP); 5% NaF; 5% NaF/5% micrometric TMP; 5% NaF/5% nanosized TMP and 5% NaF/2.5% nanosized TMP, hereafter abbreviated as PLA, 5%NaF, 5%NaF/5%TMPmicro, 5%NaF/5%TMPnano and 5%NaF/2.5%TMPnano, respectively. Blocks were treated once with the varnishes and immersed in artificial saliva (6 h). Varnishes were then removed from the enamel surface and the blocks, subjected to individual erosive challenges (four erosive challenges/1 min each); SH was determined after each challenge. _________________________________________________________________________ 50 Liliana Carolina Báez Quintero Synthesis and characterization of nano-sized TMP particles Commercial micrometric sodium trimetaphosphate (70 g, Na3O9P3, Aldrich Chemistry, China, purity ≥ 95% CAS 7785-84-4) were ball milled using 500 g of sintered zirconia spheres of 2 mm diameter in 1 litter of isopropanol in a polypropylene battle. After 48 h, at a grinding speed of 1200 rpm, powders were separated from the alcoholic medium, dried at 60 °C, and ground in a mortar. Histograms were constructed counting more than 100 particles from images obtained with a transmission electron microscope (Philips XL-30 FEG) that were treated using the public domain ImageJ image processing software. Powder crystallinity was characterized by X-ray diffraction (XRD) using a Rigaku Dmax 2500 PC difractometer in the 2θ range from 10 to 80º with a scanning rate of 2º/min. The coherent crystalline domains (crystallite size) were estimated using the Scherrer equation (L = Kλ / BcosθB), where L is the linear dimension of a monocrystalline nanoparticle, λ is the wavelength of the incident X-ray, B is the diffraction line width of the diffraction peak, θB is the Bragg angle obtained from the XRD pattern, and K is a numerical constant which value is 0.9 [8]. Varnish formulation and determination of fluoride in products The varnishes were produced by SS White Dental Products (Rio de Janeiro, RJ, Brazil), containing the following components: colophony, ethyl cellulose, tolu balsam, beeswax, toluene sulfonamide, vanillin, saccharin and ethanol. F concentrations were 0 (negative control), 2.5% and 5% NaF (Merck, Germany). To the 5% NaF varnish, sodium trimetaphosphate (Aldrich Chemistry, China) was added at 5% (micrometric or nanosized) or 2.5% (nanosized). F concentrations in the varnishes were determined using a fluoride ion specific electrode (9609 BN, Orion, USA) coupled to an ion analyzer (Orion 720 A+), and calibrated with standards containing 2.0–32.0 μg fluoride/mL, as previously described [9,10]. _________________________________________________________________________ 51 Liliana Carolina Báez Quintero Preparation of enamel blocks Enamel blocks (4mm X 4mm) were obtained from bovine incisors and was serially polished. Surface hardness (SH) was determined in the following way: one indentation was made at a distance of 1000 μm (Knoop diamond, 500 g, 10 seconds, Shimadzu HMV-2000) [11] from the central region of the blocks’ surface, to facilitate indentation localization on subsequent measurement. At a distance of 200 µm from the right vertex of this greater indentation, five indentations, separated by a distance of 100 μm, were made (Knoop diamond, 25 g, 10 seconds, Shimadzu HMV-2000). Sample size was calculated based on a pilot study, according to which 7 blocks would be required to detect significant differences in mean percentage of surface hardness change of blocks treated with a Placebo (no F or TMP) and a 5% NaF varnish (mean difference = 5.79, standard deviation = 1.5), considering a power of 80% (α=0.05). Due to the possibility of losses during the processing of the specimens, 8 blocks were included in each group. Treatment with the varnishes and erosion cycles The varnishes were applied with a microbrush on each block only once, which were immersed in 4 mL of artificial saliva (1.5 mmol.l-1 Ca(NO3)2·4H2O; 0.9 mmol.l-1 NaH2PO4·2H2O; 150 mmol.l-1 KCl; 0.1 mol.l-1 Tris buffer; pH 7.0; unstirred, 37 ºC) for 6h. The varnishes were then gently removed with a blade and acetone [12]. The erosive challenge consisted of individually immersing each enamel block in 4 mL of citric acid (0.75%, pH=3.5; Synth, Brazil) under agitation (100 rpm) for 1 min at room temperature, followed by washing with deionized water for 20 seconds In total four erosive challenges were performed. Surface hardness (SHf) measurements were carried after each erosive challenge to calculate the percentage of surface hardness change (SHC = [(SHf - SHi) / (SHi)] ×100) after 1, 2, 3 and 4 min of challenge [13, 14]. _________________________________________________________________________ 52 Liliana Carolina Báez Quintero Statistical analysis For the statistical analysis, SigmaPlot software for Windows (version 12.0) was used and the significance limit was set at 5%. Data did not present normal (Shapiro-Wilk) and homogeneous (Cochran) distribution. For surface hardness at baseline (prior to the first acid challenge), data were submitted to Kruskal-Wallis test. Data on the percentage of surface hardness change were submitted to 2-way, repeated-measures ANOVA, considering the type of varnish and time of exposure to acid as variation factors. Student-Newman- Keuls’ test was used as the post hoc test for ANOVA. 3.4. Results The median (range) of surface hardness at baseline was 354.4 (349.3- 366.6) Kg/mm2 considering all groups, without significant differences among the groups prior the first acid challenge (H = 0.439, p=0.979). Significant differences were observed among the varnishes (p<0.001), times of exposure to acid (p<0.001), and for the interaction between these variables (p<0.001). At 1-min of exposure to acid, the highest surface hardness change was observed for Placebo, followed by 5% NaF and the TMP-containing varnishes (p<0.05), without differences among the latter regardless of the type of particle (micro or nano) or TMP concentration (2.5 or 5%). At 2-, 3- and 4-min significant differences were observed among Placebo, 5% NaF, 5% NaF/5% TMP micro and both varnishes containing TMP nano, however, without significant differences between 2.5% and 5% TMP nano. As for the times of exposure to acid, significant differences were observed among all times, for all varnishes tested. (Figure 1) 3.5. Discussion Several strategies have been investigated in order to prevent or minimize enamel erosive lesions, among which topical fluoride application at high concentrations has been shown to be beneficial, despite of a limited effect [15]. The present study demonstrated that TMP significantly increases the protective _________________________________________________________________________ 53 Liliana Carolina Báez Quintero effect of fluoridated varnishes against enamel erosion, and that the use of nanoparticles further enhances this additional benefit, in a short-term, in vitro model, thus leading to the rejection of the study’s null hypothesis. The protective effect of TMP when added to fluoride varnishes against enamel erosive wear had already been reported, in studies with in vitro and in situ protocols, but using a 2.5% NaF formulation produced with an artificial resin as the varnish base [6,12]. In the present study, the additional benefit of TMP was assessed in 5% NaF, colophony-based varnishes, as this is the most widely available formulation. Despite the effect of 5% NaF varnishes containing TMP had not yet been investigated against enamel erosion, an additional protective effect was somehow expected, considering the synergistic action of this formulation reported on the reduction of enamel demineralization and on the remineralization of artificial caries lesions under in vitro and in situ conditions [10,16,17]. In the present study, the benefit of conventional (micrometric) TMP was shown to be around 43% when compared with 5% NaF (after the last erosive challenge), and such effects further increased to 60-67% when nano-sized TMP was added to the varnishes. Although this additional protective effect of nano-sized particles over micrometric TMP (30-40%) cannot be directly extrapolated to in vivo conditions, it indicates that such formulations might be a promising alternative for preventing enamel mineral loss upon erosive challenges. In this sense, it is noteworthy that despite all test formulations demonstrated immediate (first challenge) and sustained (second to fourth challenges) protective effects, the TMP-containing varnishes (especially those supplemented with nano-sized particles) were more effective in sustaining the protective effects against surface hardness change when compared with 5% NaF. In fact, the percentage of surface hardness change promoted by the 5% NaF/5% TMPnano after the fourth acid challenge (10%) was very similar to that already seen for the 5% NaF after the first challenge (~8%), what seems to be attributed the greater reactivity of nano-sized TMP, ought to the higher ratio of surface area to volume when compared with conventional (micrometric) particles. This aspect is paramount from a clinical perspective, given that vehicles for professional application are used at much lower frequency than products for home use. _________________________________________________________________________ 54 Liliana Carolina Báez Quintero Furthermore, for enamel erosion the desirable predominant effect of fluoride- based preventive measures is the formation of an acid-resistant mechanical barrier rather than mineral precipitation. Given that the formation of CaF2 is significantly reduced for TMP-containing varnishes [10,16,17], the enhanced protective effect of these formulations can be regarded as a result of the strong interactions of TMP with tooth enamel, limiting acid diffusion [10]. This short-term erosion model provides valuable information on the effects of preventive agents against enamel softening after exposure to acidic challenges, given that surface hardness has been shown to be suitable for analyzing minor changes in surface enamel, without bulk enamel loss [18]. Nonetheless, in order to further investigate the potential application of the TMP- containing varnishes used in the present study, it would be instructive to assess the effect of these formulations in promoting the remineralization of enamel with initial erosion lesions, as well as the effects of this therapy against prolonged erosive challenges. Also, given the complex interplay among chemical, physical and behavioral aspects involved in enamel erosive wear, the assessment of the anti-erosive potential of the TMP-containing varnishes under in situ conditions, in subjects regularly using fluoridated toothpastes, could bring relevant information related to the real benefit of these varnishes for the clinical practice. To sum up, this study demonstrated that the addition of micrometric TMP to 5% NaF varnishes significantly reduces initial enamel erosion in vitro, and that nano-sized TMP further enhances the protective effect of the varnishes. 3.6 Acknowledgements This study was supported by São Paulo State University (PAEDEX/AUIP/PROPG, scholarship to the first author).The authors thank to SS White Dental Products (Rio de Janeiro, Brazil) for manufacturing the varnishes. A patent was requested for a product used in the study, by the National Institute of Industrial Property - INPI/SP, on 10/17/2014 under number BR 10 2014 025902 3. _________________________________________________________________________ 55 Liliana Carolina Báez Quintero 3.7. References [1] Huysmans MC, Young A, Ganss C. The role of fluoride in erosion therapy. Monogr Oral Sci 2014; 25:230-43. [2] Buzalaf MA, Magalhães AC, Wiegand A. Alternatives to fluoride in the prevention and treatment of dental erosion. Monogr Oral Sci 2014; 25:244-52. [3] Cruz NV, Pessan JP, Manarelli MM, Souza MD, Delbem AC. In vitro effect of low-fluoride toothpastes containing sodium trimetaphosphate on enamel erosion. Arch Oral Biol 2015; 60(9):1231-6. [4] Pancote LP, Manarelli MM, Danelon M, Delbem AC. Effect of fluoride gels supplemented with sodium trimetaphosphate on enamel erosion and abrasion: in vitro study. Arch Oral Biol 2014; 59 (3):336-40. [5] Manarelli MM, Vieira AE, Matheus AA, Sassaki KT, Delbem AC. Effect of mouth rinses with fluoride and trimetaphosphate on enamel erosion: an in vitro study. Caries Res 2011; 45(6):506-9. [6] Moretto MJ, Delbem AC, Manarelli MM, Pessan JP, Martinhon CC. Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion: an in situ/ex vivo study. J Dent. 2013; 41(12): 1302-6. [7] Danelon M, Pessan JP, Santos VRD, Chiba EK, Garcia LSG, de Camargo ER, Delbem ACB. Fluoride toothpastes containing micrometric or nano-sized sodium trimetaphosphate reduce enamel erosion in vitro. Acta Odontol Scand. 2017; 16:1-6. [8] Danelon M, Pessan JP, Neto FN, de Camargo ER, Delbem AC. Effect of toothpaste with nano-sized trimetaphosphate on dental caries; In situ study. J Dent. 2015; 43(7); 806-13. [9] Shen C, Autio-Gold J. Assessing fluoride concentration uniformity and fluoride release from three varnishes. J Am. Dent Assoc. 2002; 133(2): 176-82. [10] Manarelli MM, Delbem ACB, Lima TMT, Castilho FCN, Pessan JP. In vitro Remineralizing Effect of Fluoride Varnishes Containing Sodium Trimetaphosphate. Caries Res. 2014; 48: 299-305. _________________________________________________________________________ 56 Liliana Carolina Báez Quintero [11] Rakhmatullina E, Bossen A, Höschele C, Wang X, Beyeler B, Meier C, Lussi A. Application of the specular and diffuse reflection analysis for in vitro diagnostics of dental erosion: correlation with enamel softening, roughness, and calcium release. J Biomed Opt. 2011; 16(10):107002. [12] Manarelli MM, Moretto MJ, Sassaki KT, Martinhon CC, Pessan JP, Delbem AC. Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion. Am J Dent. 2013; 26(6):307-312. [13] Baumann T, Kozik J, Lussi A, Carvalho TS. Erosion protection conferred by whole human saliva, dialysed saliva, and artificial saliva. Sci Rep. 2016; 6:34760. [14] Baumann T, Bereiter R, Lussi A, Carvalho TS. The effect of different salivary calcium concentrations on the erosion protection conferred by the salivary pellicle. Sci Rep. 2017; 7(1):12999. [15] Wiegand A, Attin T. Influence of fluoride on the prevention of erosive lesions--a review. Oral Health Prev Dent. 2003;1(4):245-53. [16] Manarelli MM, Delbem ACB, Binhardi TDR, Pessan JP. In situ Remineralizing Effect of Fluoride Varnishes Containing Sodium Trimetaphosphate. Clin Oral Invest. 2015; 19: 2141-2146. [17] Manarelli MM, Delbem ACB, Báez-Quintero LC, De Morais FRN, Cunha RF, Pessan JP. Fluoride varnishes containing sodium trimetaphosphate reduce enamel demineralization in vitro. Acta Odontologica Scandinavica. 2017. DOI: 10.1080/00016357.2017.1318448 [18] Stenhagen KR, Hove LH, Holme B, Taxt-Lamolle S, Tveit AB. Comparing different methods to assess erosive lesion depths and progression in vitro. Caries Res. 2010; 44(6):555-61 _________________________________________________________________________ 57 Liliana Carolina Báez Quintero Figure 1. Mean percentage of surface hardness change according to the varnishes applied and the time of exposure to citric acid. Upper- and lower-case letters indicate significant differences among the varnishes (at each individual time point) and among the times after exposure to acid (within each varnish), respectively. Two-way, repeated- ANOVA. _________________________________________________________________________ 58 Liliana Carolina Báez Quintero 4. Capítulo 3 _________________________________________________________________________ 59 Liliana Carolina Báez Quintero Fluoride and phosphate release from fluoride varnishes supplemented with nano-sized sodium trimetaphosphate3 Liliana Carolina Báez-Quintero, Alberto Carlos Botazzo Delbem, Mariana Emi Nagata, Malena Morais Castro e Silva, Robson Frederico Cunha, José Antonio Santos Souza, Juliano Pelim Pessan Department of Pediatric Dentistry and Public Health, School of Dentistry, Araçatuba, São Paulo State University (UNESP), Araçatuba, SP, Brazil Short title: Fluoride and TMP release from varnishes Corresponding author: Juliano Pelim Pessan School of Dentistry, Araçatuba, São Paulo State University (UNESP) Department of Pediatric Dentistry and Public Health Rua Jose Bonifacio 1193 16015-050 Araçatuba - SP - Brazil Tel: (+55) 18 3636 3314 Email: jpessan@foa.unesp.br Declaration of interest A patent was requested for a product used in the study, by the National Institute of Industrial Property - INPI/SP, on 10/17/2014 under number BR 10 2014 025902 3. 3 Artigo formatado de acordo com as normas do periódico Caries Research (Anexo C) _________________________________________________________________________ 60 Liliana Carolina Báez Quintero 4.1. Abstract This study evaluated the amount of fluoride and phosphate released from fluoride varnishes containing micrometric or nano-sized sodium trimetaphosphate (TMP). The experimental varnishes included a placebo formulation (no fluoride or TMP), 2.5% NaF, 5% NaF, 5% NaF + 5% micrometric TMP, 5% NaF + 5% nano-sized TMP, 5%NaF + 2.5% nano-sized TMP, and commercial varnish (Duraphat, 5% NaF). Varnishes were applied on polyester sheets (n=8/group) and alternately immersed in remineralizing or demineralizing solutions at 30, 60, 90, 120, 180, 240, 300, 360, 420, 540, 600, 720, 780, 960, 1200 and 1440 min after first immersion. Fluoride and phosphate were analyzed with an ion-selective electrode and colorimetrically, respectively. Data were analyzed by 2-way, repeated measures ANOVA and Student- Newman-Keuls’ test (p<0.05). A dose-response relationship was observed between the fluoride content in the test varnishes without TMP and the amount of fluoride released. Regarding the TMP-containing varnishes, an exponential cumulative release was observed up to 6 h, reaching a plateau afterwards. Also, the amount of fluoride released from the varnishes increased when immersed in demineralizing solutions. Overall, varnishes containing TMPnano released significantly higher amounts of fluoride in comparison with TMPmicro. As for cumulative phosphate release from the varnishes, the general trend from the TMP-containing varnishes showed a constant increase up to 12 h, becoming less marked afterwards, while phosphate release remained fairly constant at low levels for the TMP-free products. Particle size and TMP concentration did not influence the pattern and amount of phosphate released from the varnishes. It was concluded fluoride release was significantly increased in TMP-containing formulations, and that the nanosized TMP further enhanced such effects, without affecting phosphate release from the varnishes. Key-words: Fluorine; Polyphosphates; Sodium Fluoride; Nano-sized. _________________________________________________________________________ 61 Liliana Carolina Báez Quintero 4.2. Introduction Fluoride varnishes are professionally applied vehicles considered as slow fluoride release products, due to their ability to adhere to tooth surfaces and to release fluoride to the oral environment for prolonged periods of time [Pessan et al., 2011]. The advantages of fluoride varnishes regarding ease of application, safety, patient’s acceptability, and clinical efficacy are the main reasons for their widespread use in individuals of all ages [Marinho et al., 2013]. In vitro and in situ data have shown that the addition of sodium trimetaphosphate (TMP) to fluoridated varnishes promote a synergistic effect on enamel remineralization [Manarelli et al., 2014, 2015], as well as on the reduction of enamel demineralization [Manarelli et al., 2017] and erosive wear [Manarelli et al., 2013; Moretto et al., 2013]. Also, recent data demonstrated that the use of nano-sized TMP further enhanced the protective and therapeutic effects of fluoridated varnishes on enamel remineralization and against erosive challenges, respectively (unpublished observations). In order to better understand the mechanisms of action of TMP- containing fluoridated varnishes, especially containing nano-sized particles, it is essential to determine the pattern of fluoride and TMP release from the varnishes over time. It has been previously reported that the amount of fluoride released from commercial varnishes are influenced by several factors, including the immersion medium [Lippert, 2014] and type of resin [Shen and Autio-Gold, 2002; Ritwik et al., 2012]. Regarding formulations containing phosphate salts, conflicting evidence is available depending on the type of phosphate, in studies showing that varnishes containing calcium glycerophosphate [Carvalho et al., 2015] or TMP [Manarelli et al., 2016] released higher and lower amount of fluoride, respectively, in comparison with varnishes without any phosphate added. Considering the above-mentioned variables that influence fluoride release fr