Marcelle Danelon EFEITO DE DENTIFRÍCIOS FLUORETADOS E SUPLEMENTADOS COM NANOPARTÍCULAS DE TRIMETAFOSFATO DE SÓDIO SOBRE A DESMINERALIZAÇÃO, REMINERALIZAÇÃO E EROSÃO DENTÁRIA Araçatuba – SP 2014 Marcelle Danelon Tese apresentada à Faculdade de Odontologia da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Araçatuba, para obtenção de título de Doutor em Ciência Odontológica - Área de Concentração: Saúde Bucal da Criança. Orientador: Profº DrºAlberto Carlos Botazzo Delbem Araçatuba - SP 2014 EFEITO DE DENTIFRÍCIOS FLUORETADOS E SUPLEMENTADOS COM NANOPARTÍCULAS DE TRIMETAFOSFATO DE SÓDIO SOBRE A DESMINERALIZAÇÃO, REMINERALIZAÇÃO E EROSÃO DENTÁRIA Marcelle Danelon Catalogação-na-Publicação Serviço Técnico de Biblioteca e Documentação – FOA / UNESP Danelon, Marcelle. D179d Efeito de dentifrícios fluoretados e suplementados com nanopartículas de trimetafosfato de sódio sobre a desmineralização, remineralização e erosão dentária / Marcelle Danelon. - Araçatuba, 2014 162 f. : il. ; tab. + 1 CD-ROM Tese (Doutorado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientador: Prof. Alberto Carlos Botazzo Delbem 1. Esmalte dentário 2. Flúor 3. Fosfatos 4. Desmineralização 5. Remineralização dentária 6. Erosão dentária 7. Dentifrícios I. T. Black D27 CDD 617.645 Marcelle Danelon Dados Curriculares Marcelle Danelon Nascimento 19.06.1980 – Piracicaba- SP Filiação José Antonio Danelon Alzira Elias Arruda 2003/2009 Curso de Graduação em Odontologia pela Faculdade de Odontologia de Araçatuba, FOA-UNESP. 2004-2005 Desenvolvimento de Projeto de Iniciação Científica, com auxílio de FGM-Produtos Odontológicos. 2007/2008 Desenvolvimento de Projeto de Iniciação Científica, com auxílio do Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq. 2009/2011 2009/2011 2012/2014 Desenvolvimento de Projeto de Mestrado com auxílio da Fundação de Amparo à Pesquisa do Estado de São Paulo. Especialista em Odontopediatria pela Faculdade de Odontologia de Araçatuba, FOA-UNESP. Desenvolvimento de Projeto de Doutorado, com auxílio do Conselho Nacional de Desenvolvimento Científico e Tecnológico- CNPq. Associações CROSP - Conselho Regional de Odontologia de São Paulo. SBPqO - Sociedade Brasileira de Pesquisa Odontológica. IADR- International Association for Dental Research. Marcelle Danelon COMISSÃO EXAMINADORA TESE PARA OBTENÇÃO DO GRAU DE DOUTOR Prof. Dr. Alberto Carlos Botazzo Delbem - Orientador Professor Adjunto do Departamento de Odontologia Infantil e Social, Disciplina de Odontopediatria da Faculdade de Odontologia - Araçatuba, UNESP - Universidade Estadual Paulista Júlio de Mesquita Filho, Araçatuba. Prof. Dr. Robson Frederico Cunha - Professor Adjunto do Departamento de Odontologia Infantil e Social, Disciplina de Odontopediatria da Faculdade de Odontologia - Araçatuba, UNESP - Universidade Estadual Paulista Júlio de Mesquita Filho, Araçatuba. Profa. Dra. Fernanda Lourenção Brighenti - Professora Assistente Doutora do Departamento de Clínica Infantil, Disciplina de Odontopediatria da Faculdade de Odontologia - Araraquara, UNESP - Universidade Estadual Paulista Júlio de Mesquita Filho, Araraquara. Prof. Dr. Emerson Rodrigues de Camargo - Professor Adjunto do Departamento de Química, Disciplina de Química da Universidade Federal de São Carlos – São Carlos, UFSCar - Universidade Federal de São Carlos. Douglas Roberto Monteiro - Pós-Doutorando do Departamento de Odontologia Infantil e Social, na Faculdade de Odontologia - Araçatuba, UNESP - Universidade Estadual Paulista Júlio de Mesquita Filho, Araçatuba. "A tarefa essencial do professor é despertar a alegria de trabalhar e de conhecer." (Albert Eisntein) Marcelle Danelon Dedicatória Marcelle Danelon Dedico este trabalho, Aos meus pais ALZIRA e JOSÉ ANTONIO (In Memorian), Pela confiança que depositaram em mim... Exemplos de dedicação, honestidade, simplicidade, felicidade e amor. Agradeço por todos os momentos em que estivemos juntos e pelas palavras de conforto que sempre trouxeram segurança e tranquilidade. Nada teria acontecido se eu não tivesse o apoio constante de vocês. Muitas vezes não fisicamente, mas em pensamentos e palavras; Minha querida Mãe, você é tudo para mim! Amo vocês! “Ouve, filho meu, e aceita as minhas palavras, e se te multiplicarão os anos de vida. Provérbios 4: 10”. Marcelle Danelon Agradecimentos Especiais Marcelle Danelon A Deus, Presente em todos os momentos da minha vida, protegendo-me e guiando meus passos nesse longo caminho rumo a grandes realizações. Devo a Ele todos os momentos de alegria e sucesso até aqui conquistado. Muito obrigada pela companhia. AOS MEUS QUERIDOS AVÓS CELINA E ORACY (IN MEMORIAN), Como sinto à falta de vocês.... Gostaria que estivessem aqui comigo neste momento, mas sei que de coração estão protegendo sempre os meus passos! Agradeço todos os dias a Deus por tê-los colocado em minha vida. Meus queridos avós, se hoje me tornei uma mulher sábia, com preceitos, honesta e acima de tudo guerreira, devo a vocês dois. Mesmo com simplicidade mostraram-me que com fé podemos todas as coisas, e que sem Deus nada somos! Aos meus irmãos Fabiana, Rodrigo e Victor Hugo, Obrigada por estarem junto comigo em todos os momentos de minha vida! Vocês são muito especiais, presentes maravilhosos que Deus me deu! Guardo vocês dentro do meu coração.... Marcelle Danelon Ao meu namorado Leonardo, Encontrá-lo ao longo de minha caminhada foi o maior presente que Deus poderia ter me dado. Não tenho palavras para agradecer toda à sua compreensão nos momentos de dificuldade e todo o carinho que demonstrou por mim desde que nos encontramos... Só peço a Deus que nossas vidas possam se unir a cada dia mais. Amo você, meu amor, de todo o meu coração! “Ser profundamente amado por alguém nos dá força; Amar profundamente alguém nos dá coragem”. Lao-Tseu A família de meu namorado, Vera e José. Ao conhecer vocês fica fácil entender por que o Leonardo é tão especial e querido. Sou muito feliz por tê-los como minha segunda família. Ao meu querido Orientador, Prof. Dr. Alberto Carlos Botazzo Delbem, Agradecer é admitir que houve um momento em que se precisou de alguém; é reconhecer que ninguém jamais poderá lograr para si o dom de ser auto-suficiente. Ninguém e nada cresce sozinho; sempre é preciso um olhar de apoio, uma palavra de incentivo, um gesto de compreensão, uma atitude de amor... Obrigada pela paciência, compreensão e carinho que dedicou a mim e a este trabalho. Pela confiança que depositou em mim e pela amizade eterna... Minha imensa gratidão. Marcelle Danelon À minha querida amiga e 3ª Mãe Prof. Dra. Kikue Takebayashi Sassaki, Obrigada por estar sempre presente em minha vida, sempre acreditando em mim e por nunca deixar que eu desistisse. Posso dizer, professora, que a tenho como uma amiga, e por que não dizer também como uma terceira mãe. Agradeço pela amizade, preocupação, compreensão, conversas e muitas risadas que demos ao longo desses anos. Obrigada por me ensinar o que é ser uma professora! Amo muito a senhora. “A primeira fase do saber é amar os nossos professores.” (Erasmo) Aos meus queridos tios Oracy, Osório, Irineu, Cristina, Maria Antonia, Clara, Roseli, Cláudia, Vera e Sônia, Seus conselhos foram fundamentais para o meu crescimento. Sempre me animaram com suas palavras doces, meigas e sábias, que chegavam na hora certa. Não tenho palavras para agradecer todo amor que tiveram e têm pela minha vida. Por tudo o que já fizeram por mim, minha eterna gratidão. Minha admiração por vocês é imensa, assim como a alegria de tê-los sempre por perto. Amo vocês. Marcelle Danelon Aos meus sobrinhos queridos, Rodriguinho, Gabriel, Raphael, Amanda, Aline, Vinícius, Duda e Luizinha, Obrigada por existirem em minha vida e dar à ela um novo sentido. Amo vocês! Presentes de Deus! Às minhas grandes amigas Luciene e Lidiane, Agradeço a vocês pelo imenso amor e carinho, e por dividirem comigo todos os momentos. Muitos momentos foram de lágrimas, mas vocês, minhas grandes amigas, em nenhum instante pensaram em me abandonar; sempre me confortaram com suas palavras de ânimo, me fazendo sorrir mesmo quando tudo parecia contrário. Seremos eternamente amigas! “A única amizade que vale é aquela que nasce sem nenhum motivo”. (Van Shendel) Às minha amigas Eliana M Takeshita e Fernanda L Brighenti Vocês me ajudaram em vários momentos da minha vida. Acreditaram que eu poderia voar mais alto. Obrigada por todo conhecimento que me transmitiram. Se hoje me dedico de coração à pesquisa, posso dizer que vocês fazem parte disso. Marcelle Danelon Às minhas amigas Kéia, Dani Camara, Dani Oliveira e Carla Favretto Posso dizer que em vocês pude conhecer o que é verdadeiramente uma amizade sincera. Minhas amigas agradeço a Deus por tê-las colocado em meu caminho. Vocês foram usadas para me abençoar! Contem sempre comigo! Sempre seremos amigas! Às alunas e alunos de Mestrado, Doutorado, Pós-Doutorado e Iniciação Científica: Ana Laura, Fernanda, Jaqueline, José Antonio, Kevin, Marcela, Márjully, Mariana, Thayse, Valéria, Dani Oliveira, Dani Camara, Jackeline, Maria Daniela, Michele, Nathália, Natália, Paula, Tatiana, Remberto, Carla, Carolina S Lodi, Douglas, Vinicius, Kamila, Flávia, Thiago, Erica, Gabriel e Jéssica. A amizade e colaboração de cada um foram essenciais para a conclusão desta pesquisa. Admiro todos vocês! Aos alunos Vinicius Santos e Kamila Miranda Prado, Pela grande ajuda na realização da fase laboratorial deste trabalho. “Para cada esforço disciplinado há múltiplas recompensas.” Marcelle Danelon Ao Prof. Robson, Obrigada por todos os momentos de ensinamento com os quais me presenteou. Quero que saiba que admiro sua conduta como profissional e, se um dia, em minha vida, chegar a ter metade de seus conhecimentos e habilidades, ficarei muito feliz. Aos docentes da Disciplina de Odontopediatria da Faculdade de Odontologia de Araçatuba, UNESP, Prof. Dr. Célio Percinoto, Prof. Dr. Robson Federico Cunha, Prof. Dr. Juliano Pelim Pessan, Profª Dra. Rosângela dos Santos Nery, Profª Dra. Sandra M. H. C. Ávila de Aguiar, Profª Draª Cristiane Duque, pela agradável convivência e conhecimentos transmitidos. À Maria dos Santos Ferreira Fernandes, Pela ajuda, amizade e companheirismo nesses anos. Você é especial. Ao professor Paulo Henrique dos Santos, Obrigada por ceder gentilmente o aparelho perfilômetro, o qual foi fundamental para a análise de meu estudo sobre erosão. Marcelle Danelon Ao aluno de Doutorado da Universidade Federal de São Carlos- Francisco Nunes de Souza Neto, Obrigada pela síntese e caracterização das nanopartículas de trimetafosfato de sódio (TMP). Sua ajuda foi fundamental para a realização deste trabalho. Aos queridos voluntários desta pesquisa, Sem vocês não seria possível completar mais esta etapa. Agradeço pela enorme paciência e responsabilidade que tiveram, assim como, a amizade demonstrada, pois sei que não é fácil fazer parte de um estudo ―in situ‖. Vocês foram excelentes; que bom poder contar com amigos assim! Obrigada Rodrigo, Douglas, Valentin, José Antonio, Daniela, Daniela, Luciene, Adriana, Camila, Karina, Nathália, Juliana. “A gente não faz amigos, reconhece-os”. (Vinícius de Moares) À Faculdade de Odontologia de Araçatuba, na pessoa dos professores Drª Ana Maria Pires Soubhia, digníssima Diretora e Dr. Wilson Roberto Poi, digníssimo Vice-Diretor. Ao Curso de Pós-Graduação em Ciência Odontológica da Faculdade de Odontologia de Araçatuba-UNESP, na pessoa do coordenador Prof. Dr. Alberto Carlos Botazzo Delbem. Marcelle Danelon Aos funcionários da biblioteca da Faculdade de Odontologia de Araçatuba da UNESP, Ana Cláudia, Luzia, Ivone, Cláudio, Maria Cláudia, Luiz, Denise e Izamar pela atenção e disponibilidade com que nos recebem. À Valéria, Cristiane e Lilian da Seção de Pós-Graduação da Faculdade de Odontologia de Araçatuba-UNESP, pelo profissionalismo e atenção sempre carinhosa. Ao Frigorífico FRIBOI, em especial, ao veterinário Henrique Borges e José Francisco, que permitiram a coleta dos dentes bovinos. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq (Processo: 158463/2012-9), pela concessão de recursos que possibilitou a realização deste Curso de Doutorado. A todos aqueles que, de alguma forma contribuíram para a elaboração e conclusão deste trabalho, Minha eterna gratidão... Marcelle Danelon Escudo e Proteção Achei o amor que não me deixa só Achei a alegria que é maior Achei descanso para o meu coração Achei alívio e libertação Escudo e proteção acima da razão Em Ti eu posso confiar És liberdade pra recomeçar És esperança pra continuar És meu abrigo quando a chuva vem Eu deixo tudo e corro para Ti Escudo e proteção acima da razão Em Ti eu posso confiar Tua força me faz crer Além do que eu posso ver Em Ti eu posso confiar Eu confio, eu confio em Ti! (Diante do Trono) Epígrafe Marcelle Danelon Resumo Geral Marcelle Danelon Danelon M. Efeito de dentifrícios fluoretados e suplementados com nanopartículas de trimetafosfato de sódio sobre a desmineralização, remineralização e erosão dentária [tese]. Araçatuba: Universidade Estadual Paulista; 2014. Resumo Geral O objetivo deste estudo foi avaliar a capacidade de dentifrícios convencionais (1100 ppm F) suplementados com trimetafosfato de sódio micrométrico ou nanoparticulado (TMP; TMPnano), em reduzir a desmineralização (in vitro), promover a remineralização (in situ) e reduzir a erosão dentária (in vitro). No estudo de desmineralização, blocos de esmalte bovino (n = 96) selecionados pela dureza de superfície inicial (SHi) foram divididos em oito grupos de dentifrícios (n = 12): sem fluoreto e sem TMP (Placebo); 1100 ppm F (1100 ppm F); 1100 ppm F associado 1% TMP micrométrico e nanoparticulado (1100 1%TMP; 1100 1%TMPnano), 1100 ppm F associado a 3% TMP micrométrico e nanoparticulado (1100 3%TMP; 1100 3%TMPnano), 1110 ppm F associado a 6% TMP micrométrico e nanoparticulado (1100 6%TMP; 1100 6%TMPnano). Os blocos foram submetidos a ciclagem de pH durante cinco dias, sendo o tratamento com os respectivos dentifrícios realizados 2x/dia. A seguir, determinou-se a dureza de superfície final (SHf), perda mineral integrada (IML), diferencial da perda mineral integrada (ΔIML) e fluoreto (F) no esmalte. Os resultados foram submetidos à análise de variância seguida pelo teste Student-Newman-Keuls (p < 0,001). O grupo 1100 3%TMPnano apresentou a menor perda mineral (SHf, IML e ΔIML) seguido pelo grupo 1100 3%TMP (p < 0,001). O grupo 1100 3%TMPnano apresentou a maior concentração de F no esmalte, seguido pelo 1100 6%TMPnano (p < 0,001). Para o estudo de remineralização, blocos de esmalte bovinos (n = 192) foram selecionados pela dureza de superfície Marcelle Danelon pós-desmineralização (SH1), e divididos em quatro grupos experimentais: Placebo, 1100 ppm F, 1100 3% TMP micrométrico (1100 TMP), 1100 3% TMP nanoparticulado (1100 TMPnano). Doze voluntários utilizaram dispositivos palatinos, com quatro blocos de esmalte desmineralizados, durante três dias, sendo a escovação realizada 3x/dia. Após o período de remineralização determinou-se a porcentagem de recuperação de dureza de superfície (%SHR), recuperação da perda mineral integrada (IMLR), ΔIML e F no esmalte. Os resultados de %SHR, ΔIML e F foram submetidos à análise de variância seguida pelo teste Student-Newman- Keuls (p < 0,001), já os resultados de IMLR foram submetidos ao teste Kruskal-Wallis seguido pelo teste Student-Newman-Keuls (p < 0,001). A adição de TMP aos dentifrícios fluoretados aumentou a capacidade remineralizadora (%SHR) dos mesmos quando comparado ao grupo 1100 ppm F (p < 0,001). Maiores valores de IMLR, ΔIML e F foram encontrados no grupo 1100 TMPnano (p < 0,001). No estudo de erosão, blocos de esmalte bovinos (n = 120) foram selecionados pela dureza de superfície (SHi) e divididos em cinco grupos experimentais: Placebo, 1100 ppm F (1100 ppm F), 1100 3% TMP (1100 TMP), 1100 3% TMPnano (1100 TMPnano) e 5000 ppm F (5000 ppm F). Os grupos foram divididos sob duas condições de erosão (ERO): erosão na presença de saliva humana (ERO-SH) e erosão na presença de saliva artificial (ERO-SA). O desafio erosivo foi produzido pelo ácido cítrico 4x/dia. O fator estudado foi o tipo de dentifrício (5 tipos) e condição (2 tipos: ERO-SH, ERO-SA). Os dados de SHf, desgaste e dureza em secção longitudinal (ΔKHN) foram analisados como variáveis de resposta, e a seguir submetidos à análise de variância seguido pelo teste Student-Newman-Keuls (p < 0,001). Os valores de SHf foram maiores nos grupos tratados com 1100 TMPnano e 5000 ppm de F, diferindo no grupo placebo e 1100 ppm de F (p < 0,001). Os grupos 1100 Marcelle Danelon TMPnano e 5000 ppm de F apresentaram maior efeito protetor quando comparado com o grupo 1100 ppm F, tanto para o desgaste como ΔKHN sob as duas condições SH e SA (p < 0,001). Conclui-se que é possível melhorar a eficácia de um dentifrício contendo 1100 ppm de F através da adição de TMPnano, mostrando uma eficácia superior ao dentifrício 1100 na cérie e similar ao 5000 ppm de F na erosão, e que a presença da película adquirida não interfere na ação do TMP. Palavras-chave: Esmalte dentário, Flúor, Fosfato, Nanopartícula Desmineralização, Remineralização, Erosão Dentária, Dentifrício. Marcelle Danelon General Abstract Marcelle Danelon Danelon M. Effect of fluoride toothpastes and supplemented with nano-sized sodium trimetaphosphate on enamel demineralization and remineralization and on dental erosion [tese]. Araçatuba: Universidade Estadual Paulista; 2014. General Abstract The aim of this study was to evaluate the ability of conventional toothpaste (1100 ppm F) supplemented with micrometric or nano-sized sodium trimetaphosphate (TMP; TMPnano ), in reducing demineralization (in vitro), promote remineralization ( in situ ) and reduce erosion tooth (in vitro). In the study of demineralization of bovine enamel blocks (n = 96 ) selected by the initial surface hardness ( SHi ) were divided into eight groups of toothpaste (n = 12) without fluoride and without TMP (Placebo), 1100 ppm F (1100 ppm F) 1100 ppm F associated 1% TMP micrometric and nano- sized (1100 1%TMP; 1100 1%TMPnano), 1100 ppm F associated with 3% TMP micrometric and nano-sized (1100 3%TMP; 1100 3%TMPnano), 1110 ppm F associated with 6% TMP micrometric and nano-sized TMP (1100 6%TMP; 1100 6%TMPnano). The blocks were subjected to pH cycling for five days, and treatment with their toothpastes made 2x/day. Next, we determined the final surface hardness (SHf), integrated mineral loss (IML), differential integrated mineral loss (ΔIML) and fluoride (F) in the enamel. The results were subjected to analysis of variance followed by Student-Newman-Keuls test (p < 0.001). The group 1100 3 %TMPnano had the lowest mineral loss (SHF, IML and ΔIML) followed by 1100 3%TMP group (p < 0.001). The group 100 3%TMPnano showed a higher concentration of F in the enamel, followed by 1100 6%TMPnano (p < 0.001). For the study of remineralization of bovine enamel blocks (n = 192) were selected by the hardness of the surface after demineralization (SH1), and divided into four groups: Placebo, 1100 ppm F, 1100 3 % TMP micrometric (1100 TMP) and 1100 3% TMP nano-sized (1100 TMPnano ) . Marcelle Danelon Twelve volunteers wore palatal appliances with four blocks of demineralized enamel, for three days, and brushing held 3x/day. After the period of remineralization determined the percentage of recovery of surface hardness (%SHR), recovery of integrated mineral loss (IMLR), and F ΔIML enamel. The %SHR, ΔIML and F were subjected to analysis of variance followed by Student-Newman-Keuls test (p < 0.001), since test results IMLR were subjected to Kruskal-Wallis test followed by Student-Newman-Keuls (p < 0.001). The addition of TMP to fluoridated toothpaste increased remineralizing capacity (%SHR) when compared to the same 1100 ppm F group (p < 0.001). Higher values of IMLR, ΔIML and F were found in 1100 TMPnano group (p < 0.001). In the study of erosion of bovine enamel blocks (n = 120) were selected for surface hardness (SHi) and divided into five groups: Placebo, 1100 ppm F (1100 ppm F), 1100 3% TMP micrometric (1100 TMP), 1100 3% TMP nano-sized (1100 TMPnano) and 5000 ppm F (5000 ppm F). The groups were divided under two conditions of erosion (ERO): erosion in the presence of human saliva (ERO-HS) and erosion in the presence of artificial saliva (ERO-AS). The erosive challenge was produced by citric acid 4x/day. The factor studied was the kind of toothpaste (5 types) and condition (2 types: ERO-HS; ERO AS). The data SHF wear and toughness in longitudinal section (ΔKHN) were analyzed as response variables, and then subjected to analysis of variance followed by Student-Newman-Keuls test (p < 0.001). SHF values were greater in the groups treated with 1100 TMPnano and 5000 ppm F, differing in the placebo and 1100 ppm F (p < 0.001). 1100 TMPnano and 5000 ppm F groups had higher protective effect when compared to the 1100 ppm F group, both as to wear ΔKHN under both conditions AS and HS (p<0.001). It follows that it is possible to improve the effectiveness of a toothpaste containing 1100 ppm F by adding TMPnano showing a superior efficacy in toothpaste caries and 1100 Marcelle Danelon similar to the 5000 ppm F erosion, and the presence of the acquired pellicle not interferes with the action of TMP. Keywords: Dental enamel, Fluoride, Phosphate, Nano-sized, Demineralization, Remineralization, Tooth Erosion, Toothpaste. Marcelle Danelon Lista de Abreviaturas Marcelle Danelon LISTAS DE ABREVIATURAS ºC Graus Celsius Ca Cálcio Ca++ Íon cálcio CaF+ Íon Fluoreto de cálcio CaHPO4 0 Fosfato de cálcio neutro Ca(NO3)2.4H2O Nitrato de cálcio tetra hidratado DP Desvio padrão F Fluoreto g Grama h Hora HA Hidroxiapatita HCl Ácido clorídrico HF0 Fluoreto de hidrogênio neutro H2PO- 4 Íon fosfato dihidrogênio IML Perda mineral integrada IMLR Recuperação da perda mineral integrada KCl Cloreto de potássio kgf/mm2 Quilograma-força por milímetro quadrado KHN Unidade de dureza Knoop KOH Hidróxido de Potássio L Litro M Molar n Número de amostra Na+ Íon sódio NaF Fluoreto de sódio NaOH Hidróxido de Sódio NaH2PO4.2H2O Fosfato de sódio monobásico hidratado P Fósforo pH Potencial de Hidrogênio s Segundo SH Dureza de superfície inicial Marcelle Danelon SHi Dureza de superfície inicial SHf Dureza de superfície final SH1 Dureza de superfície pós-desmineralização SH2 Dureza de superfície pós-remineralização %SH Porcentagem de dureza de superfície FI Fluoreto iônico FT Fluoreto total %SHR Porcentagem de recuperação de dureza de superfície TISAB Tampão ajustador de força iônica total TMP Trimetafosfato de sódio mg Miligrama mL Mililitro mL/mm2 Mililitro por milímetro ao quadrado mm Milímetro mm² Milímetro quadrado mol/ L Mol por litro mmol/ L Milimol por litro mV Milivolts nm nanomicrométrico PO4 - Íon fosfato SD Desvio padrão vol% min Porcentagem de volume mineral µg Micrograma μg/mm3 Micrograma por milímetro cúbico µg F/mL Micrograma de fluoreto por mililitro µm Micrômetro ∆KHN Perda integrada de dureza de subsuperfície ΔIML Diferencial de perda mineral integrada Marcelle Danelon Lista de Tabelas Marcelle Danelon TABELA CAPÍTULO 1 Table 1. Mean (SD) surface hardness (SHf), integrated mineral loss (IML, vol%) and fluoride in enamel (F) according to groups (n = 12). Page 61 Table 2. Mean (SD) differential profile of integrated mineral loss (IML) calculated for three zones in the enamel lesions according to groups (n = 12). Page 62 TABELA CAPÍTULO 2 Table 1. Mean (SD) percentage surface hardness recovery (%SHR), integrated recovery of mineral loss (IMLR) and enamel fluoride concentrations (F) according to groups (n = 12). Page 85 Table 2. Mean (SD) differential profile of integrated mineral loss (IML) calculated for three zones in the enamel lesions according to groups (n = 12). Page 86 TABELA CAPÍTULO 3 Table 1. Mean (SD) enamel wear (µm), final hardness (SHf), integrated subsurface hardness (KHN) after erosive challenge, according to the toothpastes and type of saliva (n = 12). Page 110 Marcelle Danelon Lista de Figuras Marcelle Danelon FIGURAS CAPÍTULO 1 Figure 1. X-ray patterns of the micrometric TMP and of the nano-sized TMP after milling for 48 h. Page 63 Figure 2. Graphic of differential profile hardness as a function of depth according to the groups. : Zone A (515µm), Zone B (1550µm) e Zone C (50130µm) (n = 12). Page 63 FIGURAS CAPÍTULO 2 Figure 1. X-ray patterns of the micrometric TMP and of the nano-sized TMP after milling for 48 h. Page 87 Figure 2. Differential profile hardness as a function of depth according to the groups: Zone A (515µm), Zone B (1550µm) e Zone C (50110µm) (n = 12). Page 87 FIGURAS CAPÍTULO 3 Figure 1. X-ray patterns of the micrometric TMP and of the nano-sized TMP after milling for 48 h. Page 111 Figure 2. Cross-sectional hardness profile according to the dentifrices and type of saliva (n = 12). Page 111 Marcelle Danelon Sumário Marcelle Danelon SUMÁRIO 1 INTRODUÇÃO GERAL 2 Capítulo 1- Effectiveness of fluoride toothpaste with nano-sized trimetaphosphate on enamel demineralization: in vitro study 2.1 ABSTRACT 43 2.2 INTRODUCTION 44 2.3 MATERIALS AND METHODS 45 2.4 RESULTS 49 2.5 DISCUSSION 51 2.6 REFERENCES 54 3 Capítulo 2 - Effect of fluoride toothpaste supplemented with nano-sized trimetaphosphate on enamel remineralization: an in situ study 3.1 ABSTRACT 66 3.2 INTRODUCTION 67 3.3 MATERIALS AND METHODS 68 3.4 RESULTS 74 3.5 DISCUSSION 76 3.6 REFERENCES 78 4 Capítulo 3 - Effect of fluoride toothpaste containing nano-sized sodium trimetaphosphate on enamel erosive wear in vitro 4.1 ABSTRACT 90 4.2 INTRODUCTION 91 4.3 MATERIALS AND METHODS 93 Marcelle Danelon 4.4 RESULTS 98 4.5 DISCUSSION 99 4.6 REFERENCES 102 ANEXOS 113 Marcelle Danelon Introdução Geral Marcelle Danelon 1 Introdução Geral A utilização do fluoreto (F) como forma de controle da cárie dentária teve início na década de 50, o qual passou a ser adicionado à água de abastecimento público [Narvai, 2000]. A partir disso, várias formas foram empregadas e oferecidas à população de maneiras diversas e em diferentes concentrações: prescrição de suplementos, aplicação tópica de géis e soluções, vernizes fluoretados e ainda os dentifrícios fluoretados, veículo este, mais utilizado pela população [Van Rijkom et al., 1998; Marinho et al., 2003a; Marinho et al., 2003b; Marinho et al.,2004]. Devido à introdução no mercado de vários métodos alternativos, o F alcançou comunidades onde não havia água de abastecimento fluoretada, o que é conhecido como ―efeito halo‖ [Lima e Cury, 2001]. O principal efeito preventivo de produtos de alta concentração de F, relaciona-se à maior formação de reservatórios de F na superfície do dente na forma de fluoreto de cálcio (CaF2), o qual denomina-se de flúor fracamente ligado [Saxegaard e Rölla, 1988]. Este fica adsorvido sobre a superfície do dente, agindo como um reservatório de F disponível para atuar nos momentos de queda de pH, intervindo diretamente na dinâmica do processo des-remineralização e, dessa forma, interferindo com a progressão da lesão de cárie [Featherstone, 2000; Jardim et al., 2008]. Embora se tenha observado na maioria dos países em desenvolvimento e desenvolvidos um declínio da cárie dentária, ainda, no interior desses países existem diferenças importantes em termos da prevalência da cárie entre regiões, cidades e diferentes grupos populacionais [Antunes et al., 2004]. Apesar da existência de inúmeras fontes de F disponíveis para a população, observa-se atualmente uma tendência de polarização da doença, englobando indivíduos com Marcelle Danelon alto risco de cárie, incluindo crianças [Ministério da Saúde, 2004; Dye et al., 2007], adolescentes e pessoas que não possuem acesso à água de abastecimento fluoretada e aos serviços odontológicos [Dtmyer et al., 2011]. Narvia et al. [2000], relatam em um estudo que 25% dos indivíduos concentram aproximadamente 75% dos dentes com prevalência de cárie dentária. Outros estudos correlacionam a polarização da doença principalmente com a privação econômica [Baldani et al., 2004]. Por isso, muitos autores consideram os fatores socioeconômicos como indicadores de risco para o desenvolvimento da cárie dentária [Gillcrist et al., 2001; Sogi e Bhaskar, 2002; Baldani et al., 2004]. Outro problema, embora não seja caracterizado como uma questão de ordem pública vem acometendo principalmente crianças e adolescentes nos últimos anos, é o aparecimento da erosão dentária, que é uma alteração com uma forte correlação com o estilo e qualidade de vida [Grippo et al., 2004]. Devido ao aumento no consumo de alimentos e bebidas ácidas [Lussi et al., 2004; Gambon et al., 2012], ocorre a dissolução química dos tecidos dentais mineralizados, causando perda progressiva e irreversível da estrutura mineralizada do dente, tornando-o mais susceptível à ação de distúrbios mecânicos [Jaeggi e Lussi, 1999; Attin et al, 2000; Attin et al., 2001]. Estudos relatam um aumento na prevalência, variando de 30% a 68%, especialmente entre crianças e adolescentes [Van Rijkom H, 2002; Kazoullis et al., 2007; El Aidi H et al., 2008]. No Brasil, estudos sobre a prevalência de erosão dentária em escolares com idade entre 6-12 anos relatam 19,9% de prevalência [Mangueira et al., 2009]. Já no estudo de Rios et al. [2007], o desgaste foi maior em escolares com 6 anos de idade. Moimaz et al. [2013], avaliaram o índice de erosão dentária em 1.993 pré-escolares brasileiros, com idade entre 4-6 anos. A Marcelle Danelon idade, dentes e superfícies foram utilizados como fatores de avaliação, sendo a maior prevalência observada em crianças com idade de 6 anos (58,3%), e as superfícies oclusais as mais envolvidas. Dentre as medidas eficazes para o controle e prevenção deste tipo de alteração dentária, além da mudança dos hábitos alimentares [Millward et al., 1994; Truin et al., 2005; Sales-Peres et al., 2008], encontram-se também os agentes terapêuticos, como o F [Zero et al., 2006; Ganss et al., 2007]. A capacidade do F em inibir a desmineralização e melhorar a remineralização foi relatado por Ganss et al. [2004]. Os dentifrícios são os principais veículos de liberação de F devido à sua disponibilidade e utilização generalizada pela população [Kanapka, 1990]. Assim, sabendo-se que os mesmos se destacam dentre as formas de administração tópicas mais utilizadas pela população, e que contribuem para a redução da prevalência/incidência da cárie [Stookey et al., 2004; Newby et al., 2013; Wright et al., 2014] e da erosão dentária [Faller et al., 2011; Ganss et al., 2013], seria importante aumentar a eficácia dos mesmos contra os problemas descritos acima. Para minimizar os efeitos da cárie e erosão dentária, além do F, estudos demonstram que a suplementação de dentifrícios com sais de polifosfatos possuem a capacidade de diminuir a desmineralização dentária [Takeshita et al., 2009; Moretto et al., 2010; Delbem et al., 2012]. Dentre os sais de polifosfatos com atividade anticariogênica, o trimetafosfato de sódio (TMP) micrométrico tem-se destacado na literatura [Harris et al., 1967; Gonzalez, 1971; Larson et al., 1972; Gonzalez et al., 1973; Ständtler et al., 1996; Takeshita et al., 2009; Danelon et al., 2013a; Danelon et al., 2013b, Moretto et al., 2010; Manarelli et al., 2011, Favretto et al., 2013; Pancote et al., Marcelle Danelon 2014]. De acordo com Gonzalez et al. [1973] o TMP e o F não competem para os mesmos sítios de ligação na superfície do esmalte. Entretanto, estudo recente mostra que o TMP micrométrico pode competir com o F nos sítios de ligação na hidroxiapatita se não forem associados em uma proporção molar adequada (TMP:F) [Souza et al., 2013]. Como a adsorção dos polifosfatos ocorre rapidamente [Anbar et al., 1979] e compete com a adsorção do F, o TMP e F devem ser combinados em uma proporção adequada para que não haja competição. A adição do TMP micrométrico em dentifrícios com reduzida concentração de F, exaguatórios, géis e vernizes fluoretados, mostram um efeito contra a desmineralização, remineralização do esmalte e erosão dentária [Takeshita et al., 2009; Moretto et al., 2010; Manarelli et al., 2011; Danelon et al., 2013a; Danelon et al., 2013b; Manarelli et al., 2013; Pancote et al., 2014], cujo mecanismo da ação está relacionado com uma ação local, como descrito acima, sendo adsorvido à superfície do esmalte, causando uma redução na solubilidade da hidroxiapatita e reduzindo as trocas minerais entre o meio e o esmalte [McGaughey e Stowell, 1977, Van Dijk et al., 1980; Takeshita et al., 2011]. Takeshita et al. [2009] demostraram in vitro, que a suplementação com 1% de TMP micrométrico em dentifrício de reduzida concentração de F (500 ppm F) proporcionou um efeito semelhante à um dentifrício convencional (1100 ppm F) no processo de desmineralização do esmalte. Já a associação de 3% de TMP micrométrico em soluções dentifrícias com 1500 ppm F, mostrou semelhante remineralização quando comparado à solução dentifrícia contendo 3000 ppm F suplementados ou não com 3% de TMP. Já no estudo de Moretto et al. [2010] a associação de 3% de TMP micrométrico em um dentifrício de 500 ppm F, demonstrou que o mesmo possuiu um efeito protetor semelhante ao de 5000 Marcelle Danelon ppm F e maior efeito contra a erosão e erosão/abrasão quando comparado ao 1100 ppm F. Recentemente, um estudo in vitro mostrou que a adição de 3% de TMP micrométrico em um dentifrício com 1100 ppm F melhorou seu efeito anticárie [dados não publicados]. Entretanto, estudos clínicos mostraram que esta associação (F/TMP) não apresentou benefício contra a cárie dentária [Stephen et al., 1994; O'Mullane et al., 2007], mas segundo os próprios autores este resultado por ser devido ao erro de calibração. Apesar dos estudos in vitro [Buzalaf et al., 2010] e in situ [Roberts, 1995] serem utilizados para identificar agentes anticárie, os modelos pré-clínicos não são necessariamente preditivos da atuação clínica destes agentes [Roberts, 1995]. Assim, para melhorar a efetividade do dentifrício fluoretado (1100 ppm F) associado ao TMP e aumentar a probabilidade de se obter bons resultados clínicos, seria de grande valor na odontologia avaliar se a redução das partículas do TMP a uma escala nanométrica pode aumentar o efeito do dentifrício fluoretado quando comparado ao TMP micrométrico. Hoje em dia têm-se observado um grande crescimento nos estudos sobre nanotecnologia, através da criação de nanomateriais, os quais possuem uma escala métrica de 1-100 nm. As propriedades especiais das nanopartículas derivam de sua elevada proporção entre área superficial e seu volume. Elas também têm uma porcentagem consideravelmente mais alta de átomos em sua superfície quando comparadas com partículas maiores, o que pode torná- las mais reativas. Atualmente, a nanotecnologia passa por um rápido crescimento, com grande potencial de aplicações em odontologia, como por exemplo, em dentifrícios fluoretados [Karlinsey e Zero, 2006]. A nanotecnologia Marcelle Danelon torna-se responsável pelo desenvolvimento de estratégias bio-inspiradas na remineralização do esmalte e da erosão dentária, respectivamente, dentre outras funções, como na medicina [Hannig e Hannih, 2012]. Dessa forma podemos melhorar a ação dos dentifrícios suplementando-os com nanopartículas de sais de fosfatos. Assim, com o objetivo de otimizar o efeito dos mesmos sobre o processo de des/remineralização dentária, estudos têm analisado o impacto de fosfatos nanoparticulados no processo de remineralização do esmalte [Karlinsey e Zero, 2006]. A adição de nanopartículas de fosfato tri-cálcio em dentifrícios fluoretados reduziram o processo de desmineralização em esmalte em comparação a um dentifrício convencional (1100 ppm F) [Karlinsey et al., 2007]. Nanocompósitos contendo fosfato de cálcio amorfo (ACP), CaF2 e clorexidina têm mostrado ação na atividade metabólica do biofilme e, consequentemente, uma redução da produção de ácidos [Cheng et al., 2012]. Ainda, Segundo Xu et al. [2010], compósitos contendo nanopartículas possuem a vantagem em impedir a desmineralização dentária, por apresentarem melhores propriedades físicas e mecânicas quando comparadas a compósitos tradicionais. Para a proteção do esmalte contra a cárie e erosão dentária, um fator muito importante que deve ser mencionado é a ação da saliva humana, uma vez que a mesma apresenta a capacidade de proteger o esmalte contra os desafios erosivos, através da presença de tampões e da formação da película adquirida sobre o esmalte [Lendenmann et al., 2000; Sreebny, 2000]. Esta, por sua vez, funciona como uma barreira, que impede a difusão de ácidos a partir do biofilme bacteriano para a superfície do esmalte, fornecendo proteção contra a desmineralização [Buzalaf et al., 2010]. É importante ressaltar que a maioria dos Marcelle Danelon estudos in vitro sobre erosão dentária é realizada com a presença de saliva artificial, entretanto, a mesma não possui proteínas salivares, não permitindo assim, a adequada formação da película adquirida do esmalte, a qual tem um grande impacto sobre o processo erosivo [Buzalaf et al., 2010]. Considerando-se uma situação clínica, na qual o dentifrício é aplicado sobre a estrutura dentária que contém a película formada, o efeito do mesmo poderá ser influenciado, pois esta agirá como uma barreira impedindo a interação do F com a superfície dentária, restringindo o acesso do mesmo para a hidroxiapatita e/ou funcionará como um reservatório de F [White et al., 2012]. Sabendo-se de todas as propriedades do TMP micrométrico, bem como a ação de nanopartículas de fosfatos, seria interessante a realização de estudos que avaliem novas formulações dentifrícias contendo 1100 ppm F, o qual é classificado como um dentifrício padrão de mercado, suplementadas com TMP micrométrico ou nanoparticulado, sobre a redução da desmineralização e erosão, assim como na remineralização em lesões de cárie, principalmente quando se trata de populações com alto índice de cárie. * As referências estão no anexo Q. Marcelle Danelon Capítulo 1 42 Marcelle Danelon 2.0 Effectiveness of fluoride toothpaste with nano-sized trimetaphosphate on enamel demineralization: in vitro study Danelon M a, Pessan J.P a, Souza Neto F.N b, Camargo E.R b, Delbem A.C.B a. aAraçatuba Dental School, Univ. Estadual Paulista (UNESP) Department of Pediatric Dentistry and Public Health Rua José Bonifácio 1193 Araçatuba, SP - Cep 16015-050 – Brazil bLIEC-Department of Chemistry, Federal University of São Carlos (UFSCar), 13565-905, São Carlos/São Paulo, Brazil Alberto Carlos Botazzo Delbem São Paulo State University – UNESP Department of Pediatric Dentistry Rua José Bonifácio 1193, Araçatuba Cep 16015-050 (Brazil). Tel. +55 18 3636 3235 Fax +55 18 3636 3332 Email: adelbem@foa.unesp.br * De acordo com as instruções aos autores do periódico Acta Biomaterialia (Anexo A) mailto:adelbem@foa.unesp.br 43 Marcelle Danelon 2.1 ABSTRACT The aim of this study was to evaluate conventional toothpastes containing 1100 ppm F associated or not with different concentrations of micrometric or nano- sized TMP on enamel demineralization, using a pH cycling model. Bovine enamel blocks (4 mm x 4 mm, n = 96) selected by initial surface hardness (SHi) were allocated into eight groups (n = 12), according to the test toothpastes: without fluoride and TMP (Placebo), 1100 ppm F (1100 ppm F), 1100 ppm F plus micrometric or nano-sized TMP at concentrations of 1% (1100 1%TMP; 1100 1%TMPnano), 3% (1100 3%TMP; 1100 3%TMPnano), and 6% (1100 6%TMP; 1100 6%TMPnano). Blocks were treated 2x/day with slurries of toothpastes and submitted to pH cycling for five days. Next, final surface hardness (SHf), integrated mineral loss (IML), differential profile of integrated mineral loss (ΔIML) and enamel fluoride (F) concentrations were determined. The results were subjected to ANOVA followed by Student-Newman-Keuls test (p < 0.001). Blocks treated with 1100 3%TMPnano showed significantly lower mineral loss (SHf, IML and ΔIML), followed by 1100 3%TMP group (p < 0.001). The 1100 3%TMPnano group showed significantly higher enamel F concentration followed by 1100 6%TMPnano (p < 0.001). It was concluded that supplementation of conventional toothpaste with 3%TMPnano produce a synergistic effect on the inhibition of enamel demineralization when compared to its counterpart without TMP and micrometric TMP. Keywords: Demineralization, Phosphates, Toothpastes, Nano-sized. 44 Marcelle Danelon 2.2 Introduction The prevention and treatment of early caries lesions, especially in patients at high risk are constant challenges in dentistry. In recent years, several studies with nano particles have been conducted attempting to reduce mineral loss, which have been shown to have unique remineralization properties [1-4]. Conversely, studies have been conducted to enhance the effects of fluoride (F) products against dental caries, among which the use of inorganic phosphates have been proven to produce a synergistic effect [5-8]. This has later prompted to studies assessing the impact of nano-sized phosphates addition to F toothpastes on the process of enamel remineralization [9-10]. The addition of micrometric sodium trimetaphosphate (TMP) to low-fluoride toothpastes (500 ppm F) has been studied in recent years, significantly enhancing the effects of the product against demineralization [5,11]. The addition of 3% TMP to a toothpaste with 1100 ppm F improves its anticarie effect [unpublished data], which is in line with a previous in situ study [12]. Surprisingly, however, a clinical trial showed that the association between TMP and F did not produce any additional effect against dental caries [13-14]. Clinical models and methodologies have certain limitations, which may cause the non-observation of positive results [13]. Therefore given the scarcity of studies assessing the effects of TMP added to F toothpastes on the dynamics of dental caries and considering the promising effects of nanoparticles, it would be interesting to assess whether the addition of nano-sized TMP to a conventional F toothpaste would further enhance the effects of micrometric TMP. Therefore, the purpose of the present study was to evaluate conventional toothpastes containing 1100 ppm F associated or not with different concentrations 45 Marcelle Danelon of micrometric or nano-sized TMP on enamel demineralization, using a pH cycling model. The null hypothesis was that fluoride toothpaste associated to nano-sized TMP would present the same ability to reduce enamel demineralization when compared to its counterpart without TMP and micrometric TMP. 2.3 Materials and Methods Experimental design Enamel blocks (4 mm × 4 mm, n = 96) were obtained from bovine incisors; enamel surfaces were polished (outer enamel removed ~120 μm) and the blocks selected by initial surface hardness test (SHi; 320.0 to 380.0 kgf/mm2). Blocks were then randomly distributed into 8 groups (n = 12). The experimental toothpastes contained either concentrations of 1, 3, and 6% micrometric TMP (TMP) or nano-sized TMP (TMPnano). NaF (Merck, CAS 7681-49-4, Germany) was also added at 1100 ppm F. In addition, toothpastes without TMP and F (Placebo), as well as with 1100 ppm F (without TMP) were prepared. Blocks were subjected to pH cycling and treatment with toothpaste slurries. Next, final surface hardness (SHf), integrated mineral loss (IML), differential profile of integrated mineral loss (ΔIML) and enamel fluoride (F) concentrations were determined. Synthesis and characterization of nano-sized (TMP) particles To prepare the TMP nano-sized, 70 g of pure (micrometric) sodium trimetaphosphate (Na3O9P3, Aldrich, purity ≥ 95% CAS 7785-84-4) was ball milled using 500 g of zirconia spheres (diameter of 2 mm) in 1 L of isopropanol. After 48 h, the powder was separated from the alcoholic media and ground in a mortar. The powder crystallinity was characterized by X-ray diffraction (XRD) using a 46 Marcelle Danelon Rigaku Dmax 2500 PC difractometer in the 2 range from 10 to 80o with a scanning rate of 2o/min. The coherent crystalline domains (crystallite size) were estimated using the Scherrer equation: B B K L   cos  where L is the linear dimension of a monocrystalline nanoparticle,  is the wavelength of the incident X-ray, B is the diffraction line width of the diffraction peak, B is the Bragg angle obtained from the XRD pattern, and K is a numerical constant which value is 0.9. Toothpaste formulation and fluoride and pH assessment The toothpastes were produced with the following components: titanium dioxide, carboxymethyl cellulose, methyl p-hydroxybenzoate sodium, saccharin, mint oil, glycerin, abrasive silica, sodium lauryl sulfate and deionized water. Toothpastes containing micrometric or nano-sized TMP were prepared (Aldrich Chemistry, CAS 7785-84-4, China) at concentrations of 1, 3, and 6% micrometric TMP (TMP) or nano-sized TMP (TMPnano). To these toothpastes, NaF (Merck, CAS 7681-49-4, Germany) was added to reach a concentration of 1100 ppm F. In addition, toothpastes without TMP and F (Placebo), as well as with 1100 ppm F (without TMP) were prepared. The F concentrations [15] and pH of the all toothpastes were checked [11] prior to the beginning of the study. 47 Marcelle Danelon Toothpastes treatments and pH cycling The blocks were subjected to five pH cycles during 7 days, at constant temperature of 37 °C [16]. The blocks were kept in a demineralizing solution (DE) (6 h; 2.0 mmol/L calcium and phosphate in 75 mmol/L acetate buffer, pH 4.7; 0.04 µg F/mL, 2.2 mL/mm2) followed by their immersion in a remineralizing solution (RE) (18 h; 1.5 mmol/L calcium; 0.9 mmol/L phosphate; 150 mmol/L KCl in 0.02 mol/L cacodylic buffer, pH 7.0; 0.05 µg F/mL, 1.1 mL/mm2). The treatment consisted of a 1-min soak under agitation in 2 mL/block of toothpaste:deionized water slurries (1:3 w/w), between immersion in the DE and RE solutions (twice a day). Deionized water rinses were performed between each step. The blocks were kept in fresh remineralizing solution during the last 2 days. Hardness measurements Surface hardness was determined before (SHi) and after (SHf) pH cycling using a Micromet 5114 hardness tester (Buehler, Lake Bluff, USA and Mitutoyo Corporation, Kanagawa, Japan) and the Buehler OmniMet software (Buehler, Lake Bluff, USA) with a Knoop diamond indenter under a 25 g load for 10 s. Five indentations spaced 100 µm apart were produced in the center of the enamel block (SHi). After pH cycling, 5 indentations spaced 100 µm from the baseline indentations were produced for the determination of SHf. For cross-sectional hardness measurements (KHN), enamel blocks were longitudinally sectioned through their center and embedded in acrylic resin with the cut face exposed. The samples were then gradually polished until the total exposition of the enamel. A sequence of 14 indentations were created at different distances (5, 10, 15, 20, 25, 30, 40, 50, 70, 90, 110, 130, 220, and 330 µm) from the surface of the enamel in the central region using a Micromet 5114 hardness 48 Marcelle Danelon tester (Buehler, Lake Bluff, USA and Mitutoyo Corporation, Kanagawa, Japan) and the software Buehler OmniMet (Buehler, Lake Bluff, USA) with a Knoop diamond indenter under a 5 g load for 10 s [17]. The averages were calculated for each distance and the values converted into mineral content (vol% min. = 4.3*(√KHN) + 11.3) [18]. The integrated mineral loss (IML; % vol min. × µm) of the lesion and sound enamel was calculated using the trapezoidal rule (Graph Pad Prism, version 3.02) and subtracted from the integrated area of the hardness of the sound enamel resulting in the integrated mineral loss (IML). To analyze the patterns of demineralization, differential mineral content profiles for F and F + TMP (i.e. value of 1100 TMPs groups minus 1100 group), at each of the micrometric TMP concentrations, were determined. Also, the differential profiles were calculated for the F + nano-sized TMP and F + micrometric TMP at each TMP concentrations. These differential profiles were then integrated over three depth zones in the lesion (zone A, 5–15 μm; zone B, 15–50 μm; zone C, 50–130 μm) and underlying sound enamel to yield ΔIML values [7-8]. Analysis of the F concentration present in enamel Blocks measuring 2 mm × 2 mm (n = 96) were obtained from half of the longitudinally sectioned blocks, and were fixed to a mandrel. Self-adhesive polishing discs (diameter, 13 mm) and 400-grit silicon carbide (Buehler) were fixed to the bottom of polystyrene crystal tube (J-10; Injeplast, Sao Paulo, SP, Brazil). One layer of 50.0 ± 0.05 µm was removed from each enamel block [5,19]. A total of 0.5 mL of 0.5 mol/L HCl was added to the enamel powder retained on the polishing disc fixed to the polystyrene crystal tube. This mixture was agitated 49 Marcelle Danelon for 1 h, and 0.5 mL of 0.5 mol/L NaOH solution was added [5, 20]. For the F analysis, samples were buffered with TISAB II and analyzed with an ion-specific electrode (Orion 9609) connected to an ion analyzer (Orion 720+). A 1:1 ratio (TISAB:sample) was used. The electrodes were previously calibrated with standards containing from 0.125 to 2.00 mg F/mL under the same conditions of the samples. The results were expressed as μg/mm3. Statistical analysis For statistical analysis, SigmaPlot software version 12.0 (SigmaPlot, Systat Software Incorporation, San Jose, CA, USA) was used, and the significance limit was set at 5%. The data presented normal (Shapiro-Wilk test) and homogenous (Cochran test) distribution. Data from SHf, IML and F were submitted to one-way ANOVA followed by the StudentNewmanKeuls test. The results of ΔIML (considering % of TMP and zone) were submitted to two-way ANOVA followed by the StudentNewmanKeuls test. 2.4 Results The milling processing reduced the particle size of the TMP powders without affecting the crystalline structure of the material. The X-ray diffraction (XRD) pattern of the nano-sized TMP after 48 h of milling (Figure 1) shows broader peaks due the smaller crystallites, which could be used to estimate an average particle size of 22.7 nm. The concentration of fluoride referred to as total F (TF) and ionic F (IF) of the placebo toothpaste were 9.5 (1.1) and 9.7 (0.4) ppm F, respectively. For the toothpastes with 1100 ppm F, mean values (SD) from the groups were 1,162.0 (44.1) and 1,157.2 (16.8), ranging from 1,111.0 and 1,183.9 ppm F. Mean pH of 50 Marcelle Danelon toothpastes was 7.3 (0.3) ranging from 6.8 to 7.7. Mean (SD) SHi considering all blocks was 372.8 (0.2) kgf/mm2. No significant differences were observed among the groups after random allocation (p = 0.610). The toothpaste with 1100 ppm F (Table 1) promoted SHf 3 times harder when compared with the placebo group (p < 0.001). The addition of TMP to fluoride toothpastes lead to enamel surface with lower softening when compared to fluoride toothpaste without TMP (p < 0.001). Blocks treated with 1100 3%TMPnano showed SHf 30% higher than those treated with 1100 (p < 0.001), while this effect was around 15% with after treatment with 1100 3%TMP (p < 0.001). TMP concentrations over 3% did not promote significant reduction of surface enamel softening, regardless of the particle size. The integrated mineral loss (IML, vol%minµm) was 2 times lower in the presence of F (placebo × 1100 ppm F toothpastes). The association TMP/F reduced the IML when compared with fluoride toothpaste without TMP (p < 0.001). The 1100 3%TMPnano lead to the lowest mineral loss, which was ~ 80% smaller when compared to 1100 (p < 0.001). The 1100 3%TMP toothpaste resulted in a ~ 64% reduction of mineral loss when compared to the 1100 toothpaste. The results of F present in the enamel (Table 1) showed that the addition of 1%TMP and 3%TMP increased the F concentration in 20% and 50%, respectively, when compared to 1100 ppm F group. With TMPnano toothpastes, the increase of fluoride uptake was 90%, 160% and 100%, respectively for the concentrations of 1%, 3% and 6% compared to 1100 ppm F group. Positive and significant correlations were observed between F and SHf (Pearson's r = 0.689; p < 0.001) as well as between F and IML (Pearson's r = -0.658; p < 0.001). 51 Marcelle Danelon Differential profile hardness (Figure 2 and Table 2) show different subsurface lesion patterns. The addition of TMP lead to mineral content values greater in the middle part of the lesion (1550 µm) which was improved up to 3%TMP (p < 0.001). At 6%TMP (Figure 2a), the mineral content was lower in the zone A (515 µm) and B (1550 µm) when compared to 3%TMP (p < 0.001). The differential profile hardness from figure 2b, show the additional effect of nano- sized TMP when compared to micrometric TMP. At 1%TMPnano, the mineral content was only higher (p < 0.001) in the inner part of the lesion (zone C, 50130 µm). The addition of 3%TMPnano lead to higher mineral content (p < 0.001) being greater at zone B (p < 0.001). With 6% of TMPnano, the gain mineral occurred in the zone B and C, despite being lower than 3%TMPnano (p < 0.001). 2.5 Discussion The addition of TMP to topical fluoride products, especially toothpastes, has been shown to improve their anticaries effect [5-6]. The increase of TMP concentration up to 3% improved the anticaries effect and was positively and significantly correlated to F present in the enamel. At concentrations greater than 3% of TMP, enamel fluoride uptake and enamel hardness was significantly reduced. As the effect of TMP is related to its ability to adsorb on the enamel, the TMP:F molar ratio has a strong influence on the resulting anticaries effect [5, 21- 22]. Thus, the strength of chemical bonding of TMP to enamel becomes greater than that of F in high TMP concentrations [22]. This reduces the F uptake and increases enamel demineralization (Table 1). Even though, fluoride toothpaste containing 6% TMP showed better anticaries effect than 1100 ppm F toothpaste. With regards to 1100 ppm F toothpaste, the high amount of F in enamel can 52 Marcelle Danelon explain its anticarie effect in relation to placebo group. An increase in F concentration is correlated with increased Ca concentration, indicating a close association between Ca and F ions [23-25]. To toothpastes containing TMP, probably its adsorption on enamel lead to formation of a barrier causing a reduction of acid diffusion, avoiding enamel demineralization in high degree [6,22]. Nano-sized TMP reduced the integrated mineral loss (IML, vol%µm) in 20% compared to micrometric TMP, which suggests an increased capacity of adsorption of nano-sized TMP to enamel. TMP has been shown to enhance incorporation of Ca [5,6,26] ions as well as F ions in the enamel [5,6,22,26]. The effect of TMP probably is retain charged ions of CaF+ and Ca2+ by replacing Na+ from cyclic structure [27]. The nano-sized TMP adsorbed on enamel seems to be more reactive and retain a greater amount of Ca++ and CaF+ in its negatively charged structure. At acidic pH these linkages are broken, releasing Ca++ and CaF+, which can further take part in a series of events that ultimately lead to the formation of species (CaHPO4 0 and HF0) that have a higher diffusion coefficient into the enamel [28]. These results can be explained due to Properties of nanoparticles, such as their high ratio of surface area to volume, as well as a high percentage of atoms on the surface compared to larger particles, which makes them more reactive. The mechanism above seems to explain why fluoride toothpaste with nano-sized 3% TMP reduced the mineral loss in ~ 44% compared to its micrometric counterpart, mainly in the depth of 1550 µm (Figure 2 and Table 1). The impact of this effect can be observed in the amount of F present in the enamel when nano-sized TMP is used (Table 1), since there is an increase of ~ 75% on enamel F uptake when compared to micrometric TMP. An important 53 Marcelle Danelon factor that promotes such effects is the ability of TMP to remain bound to enamel for a longer period than other polyphosphates. The current study shows that TMP affects the processes of enamel de- and re-mineralization, especially in the deeper regions of the enamel lesion. While in the outer part of the lesion (5 µm) only a small additional effect was produced by the addition of TMP to the toothpastes, a greater effect was observed in the depth of the lesion (mainly at 1550 µm), which is consistent with previous findings [6-8, 22]. Recently studies have shown that TMP inhibits remineralization in the outer layer [6] as well as reduces the precipitation of CaF2 and firmly bound fluoride [7- 8, 26-27]. Thus, the lower mineral content in the outer enamel (515 µm) observed with nano-sized TMP can be explained by: (1) its higher reactivity and retaining of Ca++ and CaF+ retention in its structure; (2) the reduction of F precipitation in the outer enamel and (3) the reduction of mineral content. Probably, this phenomenon reduces the obstruction of the pores of the enamel surface facilitating neutral species diffusion (CaHPO4 0 and HF0) into the enamel, mainly during the remineralization phase in the inner part of the lesion. Since the adsorption of polyphosphates occurs rapidly [29], which is followed by the adsorption of F; TMP and F must be combined in an appropriate molar proportion. Combining 500 ppm F with micrometric TMP observed a better efficacy when compared to toothpaste with 1100 ppm F with a molar proportion of 1.2 to 3.7 [5]. With conventional F concentration in the formulation (1100 ppm F) the equilibrium adsorption of TMP and F reaches a maximum effect with 3%TMP (TMP/F: 1.7), unlike what happens with 500 ppm F. For TMP concentrations above 3% (TMP/F: 2.5, 3.4 and 5.1), the bonding strength of hydroxyapatite with TMP reduces the link sites of F on enamel decreasing the synergistic anti-caries 54 Marcelle Danelon effect between them. When the fluoride concentration is raised to 3000 ppm F, the bond strength of the F to enamel is greater than TMP at 3% with no improvement in anticarie effect [6]. On the basis of the findings of this in vitro study, the addition of nano-sized of TMP at 3% to toothpaste with a concentration of 1100 ppm F has promoted greater inhibition of enamel demineralization. Therefore the null hypothesis is rejected Acknowledgments We thank the technicians of the laboratory of Pediatric Dentistry of the Araçatuba Dental School, UNESP, and to Mrs. Maria dos Santos Fernandes for laboratorial assistance. This study was supported by CNPq (Process: 158463/2012-9), FAPESP through the CEPID/CDMF and the INCTMN. 2.6 References [1] Li L, Pan HH, Tao JH, Xu XR, Mao CY, Gu XH, et al. Repair of enamel by using hydroxyapatite nanoparticles as the building blocks. J Mater Chem 2008; 18:4079-4084. [2] Roveri N, Battistella E, Foltran I, Foresti, E, Iafisco M, Lelli M, et al. Synthetic biomimetic carbonate-hydroxyapatite nanocrystals for enamel remineralization. Adv Mater Res 2008;47-50:821-824. [3] Hannig M, Hannig C: Nanomaterials in preventive dentistry. Nat Nanotechnol 2010;5(8):565-569. 55 Marcelle Danelon [4] Comar LP, Souza BM, Gracindo LF, Buzalaf MA, Magalhães AC. Impact of Experimental Nano-HAP Pastes on Bovine Enamel and Dentin Submitted to a pH Cycling Model. Braz Dent J 2013;24(3):273-278. [5] Takeshita EM, Castro LP, Sassaki KT, Delbem, ACB. In vitro evaluation of dentifrice with low fluoride content supplemented with trimetaphosphate. Caries Res 2009;43(1):50-56. [6] Takeshita EM, Exterkate RA, Delbem AC, ten Cate JM. Evaluation of different fluoride concentrations supplemented with trimetaphosphate on enamel de- and remineralization in vitro. Caries Res 2011;45(5):494-497. [7] Danelon, M, Takeshita, EM, Sassaki, KT, Delbem, ACB. In situ evaluation of a low fluoride concentration gel with sodium trimetaphosphate in enamel re- mineralization. Am J Dent 2013a;26(1):15-20. [8] Danelon M, Takeshita EM, Peixoto LC, Sassaki KT, Delbem AC. Effect of fluoride gels supplemented with sodium trimetaphosphate in reducing demineralization. Clin Oral Investig. 2013b; DOI 10.1007/s00784-013-1102-4. [9] Karlinsey RL, Fontana MR, Gonzalez-Cabezas C, Haider A, et al. Antiomicrobial and Anticariogenic Effect of a Unique Nanomaterial on Human Enamel. Caries Res 2007;41:330. [10] Huang SB, Gao SS and Yu HY. Effect of nano-hydroxyapatite concentration on remineralization of initial enamel lesion in vitro. Biomed Mater 2009;4(3):034104 (6pp). [11] Moretto MJ, Magalhaes AC, Sassaki KT, Delbem AC, Martinhon CC. Effect of different fluoride concentrations of experimental dentifrices on enamel erosion and abrasion. Caries Res 2010;44(2):135-140. 56 Marcelle Danelon [12] Schafer F. Evaluation of the anticaries benefit of fluoride toothpastes using an enamel insert model. Caries Res 1989; 23(2):81-86. [13] Stephen KW, Chestnutt IG, Jacobson APM, McCall DR, Chesters RK, Huntington E, et al. The effect of NaF and SMFP toothpastes on 3-years' caries increments in adolescents. Int Dent J 1994;44(1):287-295. [14] O'Mullane DM, Kavanagh D, Ellwood RP, Chesters RK, Schafer F, Huntington E, et al. A three-year clinical trial of a combination of trimetaphosphate and sodium fluoride in silica toothpastes. Journal of Dental Research 1997;76(11):1776-17781. [15] Delbem ACB, Sassaki KT, Vieira AE, Rodrigues E, Bergamaschi M, Stock SR, et al. Comparison of methods for evaluating mineral loss: hardness versus synchrotron microcomputed tomography. Caries Res 2009;43(5):359-365. [16] 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(6):514-520. [17] Delbem AC, Danelon M, Sassaki KT, Vieira AE, Takeshita EM, Brighenti FL, Rodrigues E. Effect of rinsing with water immediately after neutral gel and foam fluoride topical application on enamel remineralization: An in situ study. Arch Oral Biol 2010;55(11):913-918. [18] Kielbassa AM, Wrbas KT, Schulte-Mönting J, Hellwig E. Correlation of transversal microradiography and microhardness on in situ-induced demineralization in irradiated and nonirradiated human dental enamel. Arch Oral Biol 1999;44(3):243-251. [19] Weatherell JA, Robinson C, Strong M, Nakagaki, H. Micro-sampling by abrasion. Caries Res 1985;19(2):97-102. 57 Marcelle Danelon [20] Alves KM, Pessan JP, Brighenti FL, Franco KS, Oliveira FA, Buzalaf MA, Sassaki KT, Delbem AC. In vitro evaluation of the effectiveness of acidic fluoride dentifrices. Caries Res 2007;41(4):263-268. [21] Manarelli MM, Vieira AE, Matheus AA, Sassaki KT, et al. Effect of mouth rinses with fluoride and trimetaphosphate on enamel erosion. an in vitro study. Caries Res 2011;45(6):506-509. [22] Favretto CO, Danelon M, Castilho FCN, Vieira AEM, Delbem ACB. In vitro evaluation of the effect of mouth rinse with trimetaphosphate on enamel demineralization. Caries Res 2013;47(5):532-538. [23] Whitford GM, Wasdin JL, Schafer TE et al. Plaque fluoride concentrations are dependent on plaque calcium concentrations. Caries Res 2002;36(4):256-265. [24] Pessan JP, Sicca CM, de Souza TS, da Silva SM, Whitford GM, Buzalaf MA. Fluoride concentrations in dental plaque and saliva after the use of a fluoride toothpaste preceded by a calcium lactate rinse. Eur J Oral Sci 2006;114(6):489- 493. [25] Pessan JP, Silva SMB, Lauris JRP, Sampaio FC, Whitford GM, Buzalaf MA. Fluoride uptake by plaque from water and from toothpaste. J Dent Res 2008;87(5):461-465. [26] Souza JA, Amaral JG, Moraes JC, Sassaki KT, Delbem AC. Effect of sodium trimetaphosphate on hydroxyapatite solubility: an in vitro study. Braz Dent J 2013;24(3):235-40. [27] Manarelli, MM, Delbem ACB, Lima TMT, Castilho FCN, Pessan JP. Effect of fluoride varnish supplemented with sodium trimetaphosphate on enamel erosion and abrasion. Am J Dent 2013; In press. 58 Marcelle Danelon [28] Cochrane HJ, Saranathan S, Cai F, Cross KJ, Reynolds EC. Enamel subsurface lesion remineralisation with casein phosphopeptide stabilised solutions of calcium, phosphate and fluoride. Caries Res 2008;42(2):88-97. [29] Anbar M, Farley EP, Denson DD, Maloney KR. Localized fluoride release from fluorine-carrying polyphosphonates. J Dent Res 1979;58(3):1134-1145. 59 Marcelle Danelon Table legend Table 1. Mean (SD) surface hardness (SHf), integrated mineral loss (IML, vol%) and fluoride in enamel (F) according to groups (n = 12). Table 2. Mean (SD) differential profile of integrated mineral loss (IML) calculated for three zones in the enamel lesions according to groups (n = 12). 60 Marcelle Danelon Figure legends Figure 1. X-ray patterns of the micrometric TMP and of the nano-sized TMP after milling for 48 h. Figure 2. Graphic of differential profile hardness as a function of depth according to the groups. : Zone A (515µm), Zone B (1550µm) e Zone C (50130µm) (n = 12). 61 Marcelle Danelon Table 1. Mean values (SD) of surface hardness (SHf), integrated mineral loss (IML, vol%) and fluoride in enamel (F) according to groups (n = 12) Groups SHf (kgf/mm2) IML, vol%µm F (µg/mm3) Placebo 73.7a (8.0) 1,637.8a (203.4) 0.4a (0.1) 1100 ppm F 241.0b (8.7) 791.0b (138.7) 1.0b (0.2) 1100 1%TMP 254.9c (7.1) 587.9c (83.4) 1.2b,d (0.2) 1100 1%TMPnano 280.3d (5.5) 604.2c (40.0) 1.9c (0.6) 1100 3%TMP 276.6d (4.4) 287.1d (89.4) 1.5d (0.3) 1100 3%TMPnano 311.5e (4.4) 161.5e (25.6) 2.6e (0.7) 1100 6%TMP 223.2f (4.5) 548.3c (100.3) 1.0b (0.4) 1100 6%TMPnano 261.4g (9.9) 387.8f (41.1) 2.0c (0.4) Different superscript lowercase letters indicate statistical significance in each row (1- way ANOVA, Student-Newman Keuls test, p < 0.001). 62 Marcelle Danelon Table 2. Mean (SD) of differential profile of integrated mineral loss (IML) calculated for three zones in the enamel lesions according to groups (n = 12) Groups IMLa, vol%µm Zone A (515 µm) Zone B (1550 µm) Zone C (50130 µm) 1%TMP 31.1a,A (8.9) 138.2a,B (49.2) 46.0a,A (4.9) 3%TMP 84.4b,A (17.5) 323.7b,B (57.5) 79.6b,A (14.8) 6%TMP 20.3a,A (7.6) 195.3a,B (49.7) 73.3b,C (14.6) 1%TMPnano -92.4a,A (7.9) -18.2a,B (5.4) 26.2a,C (8.8) 3%TMPnano 21.5b,A (6.5) 107.9b,B (25.2) 25.1a,A (5.4) 6%TMPnano -11.2c,A (6.1) 49.1c,B (10.3) 2.6b,C (2.4) Distinct superscript capital letters indicate the differences between zones A, B and C in each line (StudentNewmanKeuls test, p < 0.001). Values denote means with SD in parentheses. Distinct superscript lowercase letters in the first three rows indicate statistical significance in each column considering 1%TMP, 3%TMP and 6%TMP groups (StudentNewmanKeuls test, p <0.001). Distinct superscript lowercase letters in the next three rows indicate differences between groups in each columns considering 1%TMPnano, 3%TMPnano and 6%TMPnano groups (StudentNewmanKeuls test, p < 0.001). aΔIML: positive values denote higher integrated mineral content and vice versa. 63 Marcelle Danelon Figure 1. X-ray patterns of the micrometric TMP and of the nano- sized TMP after milling for 48 h. Figure 2. Graphic of differential profile hardness as a function of depth according to the groups. : Zone A (515µm), Zone B (1550µm) e Zone C (50130µm) (n = 12). (ANOVA, Student-Newman Keuls, p < 0.001). 64 Marcelle Danelon Capítulo 2 65 Marcelle Danelon 3.0 Effect of fluoride toothpaste supplemented with nano-sized trimetaphosphate on enamel remineralization: an in situ study Danelon M a, Pessan J.P a, Souza Neto F.N b, Camargo E.R b, Delbem A.C.B a. 1 Corresponding author: Alberto Carlos Botazzo Delbem 1) Short title: In situ effect of F toothpaste with nano-sized trimetaphosphate on remineralization. 2) Key-words: Toothpaste, fluoride, phosphates, in situ, nano-sized 3) Number of words in the abstract: 248 4) Number of words in the abstract and the text: 3.309 5) Number of tables: 2 6) Number of cited references: 30 aAraçatuba Dental School, Univ. Estadual Paulista (UNESP) Department of Pediatric Dentistry and Public Health Rua José Bonifácio 1193 Araçatuba, SP - Cep 16015-050 – Brazil bLIEC-Department of Chemistry, Federal University of São Carlos (UFSCar), 13565-905, São Carlos/São Paulo, Brazil Alberto Carlos Botazzo Delbem São Paulo State University – UNESP Department of Pediatric Dentistry Rua José Bonifácio 1193, Araçatuba Cep 16015-050 (Brazil). Tel. +55 18 3636 3235 Fax +55 18 3636 3332 Email: delbem@foa.unesp.br De acordo com as instruções aos autores do periódico Journal of Dental Research (Anexo B) mailto:delbem@foa.unesp.br* 66 Marcelle Danelon 3.1 Abstract The aim of this in situ study was to evaluate the effect of fluoride toothpaste supplemented with nano-sized sodium trimetaphosphate (TMP) on enamel remineralization. This blind and cross-over study was performed in 4 phases of 3 days each. Twelve subjects used palatal appliances containing four bovine enamel blocks with artificial caries lesions. Volunteers were randomly assigned into the following treatment groups: Placebo (without F and TMP); 1100 ppm F without TMP (1100), or supplemented with 3% micrometric TMP (1100 TMP) or nano-sized TMP (1100 TMPnano). Volunteers were instructed to brush their natural teeth with the palatal appliances in the mouth during 1 minute (3 times/day), so that blocks were treated with natural slurries of toothpastes. After each phase, the percentage of surface hardness recovery (%SHR), integrated recovery mineral loss (IMLR) and differential profile of integrated mineral loss (IML) in enamel lesions were calculated. F in enamel was also determined. The results were analyzed by ANOVA and Student-Newman-Keuls tests (p < 0.05). Enamel surface became 20% harder when treated with 1100 TMPnano in comparison with 1100 (p < 0.001). Also, 1100 TMP nano showed capacity to reduce the lesion body (IMLR; IML) 66% higher when compared with 1100 TMP (p < 0.001). Enamel F uptake in the 1100 TMPnano group was 2-fold higher when compared to its counterpart without TMP (p < 0.001). It was concluded that the addition of 3% TMPnano to a conventional toothpaste was able to promote an additional remineralizing effect of artificial caries lesions. Keywords: Toothpastes, Tooth remineralization, Phosphates, Nano-sized. 67 Marcelle Danelon 3.2 Introduction The regular use of fluoride (F) toothpaste has been associated with a decline in dental caries in both developed and developing countries (Bratthall et al., 1996; Marinho et al., 2004). The greatest advantage when compared to alternative forms of topical application is the regular delivery of F (Mellberg et al., 1985), associated to the removal or disruption of the dental biofilm (Pessan et al., 2011). Given the limited effect of fluoride on the dynamics of dental caries, attempts to enhance the anticaries properties of F dentifrices have been made, among which the use of inorganic phosphates have been the most studied over recent years (Takeshita et al., 2009, 2011; Danelon et al., 2013a, 2013b). Sodium trimetaphosphate (TMP) seems to be the most effective polyphosphate against dental caries. The addition of micrometric (regular sized) TMP to low-F fluoride toothpastes has been shown to be more effective than its counterpart without TMP on enamel demineralization (Takeshita et al., 2009; Moretto et al., 2010). It has been suggested that TMP is adsorbed to enamel surface reducing enamel demineralization (Henry and Navia, 1969, Gonzalez, 1971; Gonzalez et al., 1973), reducing hydroxyapatite solubility (Mcgaughey and Stowell, 1977; Manarelli et al., 2013; Favretto et al., 2013) and mineral exchange. In recent years, several studies with nano-sized compounds other than fluoride have also been conducted, aiming to produce formulations that are effective in reducing mineral loss, as well as in enhancing mineral gain (Roveri et al., 2008; Hannig and Hannig, 2012; Comar et al., 2013). In this sense, studies have also analyzed the impact of nano-sized phosphates added to fluoride toothpastes in the process of enamel remineralization (Karlinsey et al., 2007; Huang et al., 2009). The addition of nano-sized tri-calcium phosphate fluoride 68 Marcelle Danelon toothpastes reduced the enamel demineralization process compared to a conventional toothpaste. Also, nanocomposites ACP, CaF2 and chlorhexidine have shown action in the metabolic activity of the biofilm and consequently reducing acid production (Cheng et al., 2012). Knowing that conventional toothpastes (1100 ppm F) are used by the population, especially those with high caries development it would be interesting to assess the association between nano-sized TMP with a F-toothpaste in order to verify their effectiveness in remineralizing pre-existing caries lesions when compared to counterparts with micrometric TMP or without TMP. Thus, the purpose of the study was to evaluate the remineralizing effects of conventional toothpastes (1100 ppm F) associated or not with micrometric or nano-sized 3%TMP, using an in situ model and artificially demineralized bovine enamel blocks. The null hypothesis was that fluoride toothpaste associated to nano-sized TMP would present the same ability to remineralization the enamel when compared to fluoride toothpaste (1100 ppm F). 3.3 MATERIAL AND METHODS Experimental Design This study was previously approved by the Human Ethical Committee (Protocol: 17888413.1.0000.5420). This was a blind and cross-over in situ study performed in four phases of 3 days each (Afonso et al., 2013) A sample size of twelve volunteers was calculated considering α-error level of 5%, β-error level of 20% (www.dssresearch.com). Volunteers aged 20-30 years, who were in good general and oral health (Delbem et al., 2005), presented normal salivary flow (Rios et al., 69 Marcelle Danelon 2006), and did not violate the exclusion criteria (use of any form of medication likely to interfere with salivary secretion, use of fixed or removable orthodontic appliances, pregnancy or breastfeeding, smoker, or systemic illness), were included in the study. All participants read and signed informed consent statements prior to study initiation. Enamel blocks (4 mm × 4 mm, n = 192) from bovine incisive teeth were sequentially polished and selected through surface hardness test (SH: range of 370.0 up 377.0 kgf/mm2; p = 0.080). The blocks were demineralized and submitted to post demineralization surface hardness (SH1). Based on the percentage of surface hardness loss (post demineralization) the enamel blocks were divided into four treatments groups: Placebo (no F and TMP); 1100 ppm F (1100); 1100 ppm F and 3% micrometric TMP (1100 TMP); and 1100 ppm F and 3% nano-sized TMP sized (1100 TMPnano). After each experimental period, surface hardness (SH2) was again assessed to calculate the percentage of surface hardness recovery (%SHR). The blocks were sectioned and to perform cross-sectional hardness test to calculate the integrated recovery of mineral loss (IMLR) and the differential profiles of integrated mineral loss (IML). Fluoride (F) content in enamel was also determined. Synthesis and characterization of nano-sized (TMP) particles To prepare the TMP nano-sized, 70 g of pure (micrometric) sodium trimetaphosphate (Na3O9P3, Aldrich, purity ≥ 95% CAS 7785-84-4) was ball milled using 500 g of zirconia spheres (diameter of 2 mm) in 1 L of isopropanol. After 48 h, the powers were separated from the alcoholic media and ground in a mortar. The powder crystallinity were characterized by X-ray diffraction (XRD) using a Rigaku Dmax 2500 PC difractometer in the 2 range from 10 to 80o with a 70 Marcelle Danelon scanning rate of 2o/min. The coherent crystalline domains (crystallite size) were estimated using the Scherrer equation: B B K L   cos  where L is the linear dimension of a monocrystalline nanoparticle,  is the wavelength of the incident X-ray, B is the diffraction line width of the diffraction peak, B is the Bragg angle obtained from the XRD pattern, and K is a numerical constant which value is 0.9. Toothpaste formulation and fluoride and pH assessment The toothpastes were produced with the following components: titanium dioxide, carboxymethyl cellulose, methyl p-hydroxybenzoate sodium, saccharin, mint oil, glycerin, abrasive silica, sodium lauryl sulfate and deionized water. Toothpastes containing micrometric or nano-sized TMP were prepared (Aldrich Chemistry, CAS 7785-84-4, China) at concentration of 3% micrometric TMP (TMP) or nano-sized TMP (TMPnano). To these toothpastes, NaF (Merck, CAS 7681-49-4, Germany) was added to reach a concentration of 1100 ppm F. In addition, toothpastes without TMP and F (Placebo), as well as with 1100 ppm F (without TMP) were prepared. The F concentrations (Delbem et al., 2009) and pH (Moretto et al., 2010) of all the toothpastes were checked. The mean (SD) concentration of total F (TF) and ionic fluoride (IF) (n = 3) were: placebo  9.5 (1.1) 9.7 and (0.4), 1100 ppm F  1162.0 (44.1) and 1157.2 (16.8), 1100 TMP  1162.0 (44.1) and 1157.2 (16.8), 71 Marcelle Danelon and 1100 TMPnano  1162.0 (44.1) and 1157.2 (16.8). The pH value from the groups was 7.3 (0.3) ranging from 6.8 to 7.7 Subsurface enamel demineralization All surfaces of each specimen, except the enamel surface, were coated with acid resistant varnish and subsurface enamel demineralization (Spiguel et al., 2010; Danelon et al., 2013a) was produced by immersing each enamel block in 32 mL of a solution with 1.3 mmol/L Ca, and 0.78 mmol/L P in 0.05 mol/L acetate buffer, pH 5.0; 0.03 ppm F, for 16 hours at 37°C (Queiroz et al., 2008). Mean (SD) of surface hardness after demineralization (SH1) was 63.1 KHN (2.7), and the means varied between 61.4 and 63.7 (p = 0.093). Palatal appliance preparation and treatments The oral appliance was prepared in acrylic resin (Jet - Articles Classic Odontológico, São Paulo) in accordance with Danelon et al. (2013a). Twelve volunteers wearing acrylic palatal appliance with four demineralized enamel bovine blocks were subjected to four phases of 3 days each with 7 days washout period among experimental phases (Afonso et al., 2013). The treatments with the toothpastes were performed 3 times per day inside the mouth, during the volunteers' habitual oral hygiene routine. The volunteers were oriented initially brushing their natural teeth and following to conduct three brushing strokes in each row of enamel blocks on the oral appliance, with the natural slurry (saliva/tootphaste) formed. During 7-day pre-experimental period and washout periods, the volunteers brushed their teeth with non-fluoridated toothpaste. The volunteers received all the instructions previously. After the 3-day experimental 72 Marcelle Danelon period, the blocks were removed from the appliance, cleaned using gauze and deionized water. Microhardness Analysis The enamel surface hardness was determined before (SH1) and after each phase (SH2) using a a Micromet 5114 hardness tester (Buehler, Lake Bluff, USA and Mitutoyo Corporation, Kanagawa, Japan) and the software Buehler OmniMet (Buehler, Lake Bluff, USA) with a Knoop diamond indenter under a 25 g load for 10 s. Five indentations, spaced 100 µm from each other, were made in the center of the enamel block. After each phase, five indentations were made spaced 100 µm from the baseline indentations for determination enamel surface hardness (SH2). The-recovery percentage of surface hardness (%SHR) was calculated [%SHR = 100 (SH2 – SH1)/SH1]. For the cross-sectional hardness measurements, the enamel blocks were longitudinally sectioned through their center and embedded in acrylic resin with the cut face exposed. The samples were then gradually polished until the total exposition of the enamel. One sequence of 14 indentations at different distances (5, 10, 15, 20, 25, 30, 40, 50, 70, 90, 110, 130, 220 and 330 µm) from the surface of the enamel were created in the central region, spaced 100 μm apart. This was achieved using a using a a Micromet 5114 hardness tester (Buehler, Lake Bluff, USA and Mitutoyo Corporation, Kanagawa, Japan) and the software Buehler OmniMet (Buehler, Lake Bluff, USA) with a Knoop diamond indenter under a 5 g load for 10 s (Delbem et al., 2010). The averages were calculated for each distance and the values converted into mineral content (vol% min. = 4.3*(√KHN) + 11.3) (Kielbassa et al., 1999). The integrated mineral loss (IML; vol% min × µm) of the lesion and sound enamel was calculated using the trapezoidal rule (Graph 73 Marcelle Danelon Pad Prism, version 3.02) and subtracted from the integrated area of the hardness of the sound enamel. These values were subtracted from the integrated area of the post demineralized enamel resulting in the integrated recovery of mineral loss (IMLR). To analyze the patterns of remineralization, differential mineral content profiles were calculated by subtracting the mineral values of each group at each depth from of the Placebo group (i.e., 1100 ppm F, 1100 TMP and 1100 TMPnano groups values minus the Placebo group). These differential profiles were then integrated over three depth zones in the lesion (zone A, 5–15 μm; zone B, 15–50 μm; zone C, 50–110 μm) and underlying sound enamel to yield ΔIML values (Danelon et al., 2013a, Danelon et al., 2013b). Analysis of the F concentration present in enamel Blocks measuring 2 mm × 2 mm (n = 192) were obtained from half of the longitudinally sectioned blocks, and were fixed to a mandrel. Self-adhesive polishing discs (diameter, 13 mm) and 400-grit silicon carbide (Buehler) were fixed to the bottom of polystyrene crystal tube (J-10; Injeplast, Sao Paulo, SP, Brazil). One layer of 50.0 ± 0.05 µm was removed from each enamel block (Weatherell et al., 1985; Takeshita et al., 2009). A total of 0.5 mL of 0.5 mol/L HCl was added to the enamel powder retained on the polishing disc fixed to the polystyrene crystal tube. This mixture was then agitated for 1 h, and then, 0.5 mL of 0.5 mol/L NaOH solution was added (Alves et al., 2007; Takeshita et al., 2009). For the F analysis, a specific electrode (Orion 9609) was connected to an ion analyzer (Orion 720+) and TISAB II. A 1:1 ratio (TISAB:sample) was used. The electrodes were previously calibrated with standards containing from 0.125 to 74 Marcelle Danelon 2.00 mg F/mL under the same conditions of the samples. The results were expressed as μg/mm3. Statistical Analysis Analyses were performed using the Sigma Plotm (version 12.0) and the level of statistical significance was established at 5%. The statistical power was calculated considering all the differences among groups of each primary outcome. The variables %SHR and F (log transformation) showed normal (Shapiro-Wilk) and homogeneous (Cochran test) distributions. One-way ANOVA was then performed, followed by the Student-Newman-Keuls. The IMLR data showed heterogeneous distribution and it were submitted to Kruskal-Wallis followed by the Student-Newman-Keuls. The IML values were submitted to two-way ANOVA followed by the Student-Newman-Keuls. Pearson’s correlation coefficients between F present in enamel with %SHR and IMLR were also calculated. 3.4 Results The milling processing reduced the particle size of the TMP powder without affecting the crystalline structure of the material. The X-ray diffraction (XRD) pattern of the nano-sized TMP after 48 h of milling (Figure 1) shows broader peaks due the smaller crystallites, which could be used to estimate an average particle size of 22.7 nm. The addition of TMP to fluoride toothpastes increased the %SHR in 10% (Table 1) when compared to fluoride toothpaste without TMP (p < 0.001). With nano-sized TMP, the enamel surface became 20% harder than 1100 (p < 0.001). In addition, the capacity to reduce the lesion body (IMLR) was ~ 20 higher with the addition of micrometric TMP and ~ 43% higher with the TMPnano compared with 75 Marcelle Danelon the 1100 ppm F group (p < 0.001). The statistical power (α = 0.05) calculated for %SHR and IMLR was 1.00. The integrated values from differential mineral profile showed different mineral recovery patterns (Figure 2 and Table 2). The addition of micrometric TMP did not improve the remineralization capacity of fluoride toothpaste in the outer enamel (zone A, 5–15 µm, p = 0.109) as well as in the inner enamel (zone C, 50–110 µm, p = 0.679) when compared to 1100 ppm F group, but an effect was observed in the middle of lesion (zone B, 15–50 µm) with mineral recovery of 36% (p < 0.001) for the same comparison. The 1100 3% TMPnano significantly increased mineral recovery (Figure 2 and Table 2) in all zones when compared to all other groups (p < 0.001). The additional effect of nano-sized TMP on enamel remineralization (dotted line, Figure 1) was 66% with a more pronounced effect at deeper regions of the lesion (considering all the extension). The statistical power (α = 0.05) calculated was 1.00 for IML analysis (considering group vs. zone). The addition of TMP at both particle sizes significantly increased enamel F concentrations (Table 1) when compared to the 1100 group (p < 0.001). With the nano-sized TMP, the increase was ~ 150% higher when compared to 1100 group (p < 0.001). The statistical power (α = 0.05) calculated was 1.00 for F analysis. Enamel F concentration was positively correlated with %SHR (Pearson’s r = 0.894, p< 0.001) and enamel F with IMLR (Pearson’s r = 0.896, p < 0.001). 76 Marcelle Danelon 3.5 Discussion The present study evaluated the remineralization potential of a toothpaste with 1100 ppm F supplemented with 3% of TMP nano-sized, comparing it to a standard toothpaste (1100 ppm F) using an in situ model. Previous studies have shown the effect of TMP in preventing enamel demineralization and promote remineralization when added to fluoridated formulations for topical application (Takeshita et al., 2009, 2011; Danelon et al., 2013a,b). Recently, in an in vitro study showed that the addition of 3% of micrometric TMP to a 1100 ppm F toothpaste improves its anticaries effect [data not published]. However, the clinical trials showed that improvement was not enough to bring some benefice against dental caries (Stephen et al., 1994; O'Mullane et al., 2007). Clinical models and methodologies have certain limitations, which may cause the non- observation of positive results [Stephen et al., 1994].The present study showed nano-sized TMP increases the probability of obtaining good clinical outcomes. As the effect of TMP depends on its ability to adsorb to enamel (McGaughey and Stowell, 1977; van Dijk et al., 1980) as well as retaining fluoride and calcium in its structure (Danelon et al., 2013b; Manarelli et al., 2014), the option to reduce the size of particles appears to be more effective than increasing the concentration of phosphate in the formulation. The nano-sized TMP adsorbed on enamel seems to be more reactive and retain a greater amount of Ca++ and CaF+ in its negatively charged structure. At acidic pH these linkages are broken, releasing Ca++ and CaF+, which can further take part in a series of events that ultimately lead to the formation of species (CaHPO4 0 and HF0) that have a higher diffusion coefficient into the enamel [28]. These results can be explained due to Properties of nanoparticles, such as their high ratio of surface area to volume, as 77 Marcelle Danelon well as a high percentage of atoms on the surface compared to larger particles, which makes them more reactive. The mechanism above seems to explain why fluoride toothpaste with nano-sized 3% TMP reduced the mineral loss in ~ 44% compared to its micrometric counterpart, mainly in the depth of 1550 µm (Figure 2 and Table 1). The impact of this effect can be observed in the amount of F present in the enamel when nano-sized TMP is used (Table 1), since there is an increase of ~ 75% on enamel F uptake when compared to micrometric TMP. An important factor that promotes such effects is the ability of TMP to remain bound to enamel for a longer period than other polyphosphates. The present data showed that the higher remineralization rate was associated with higher enamel fluoride uptake. This means reducing the deposition of calcium fluoride (CaF2) in the enamel and increase retention of calcium (Ca++) and F on the TMP molecule adsorbed to enamel (Danelon et al., 2013b; Manarelli et al., 2014). A greater availability of Ca++ and CaF+ may lead to the formation of CaHPO4 0 and HF0, which are known to have a much higher diffusion coefficient into the enamel when compared to charged species (Cochrane et al., 2008). The impact of this can be observed in the mineral content (IMLR) of the enamel when nano-sized TMP is used (Table 1). ΔIML values observed in this study confirm previous findings that TMP reduces mineral loss deep in the enamel (Table 2, Figure 2) (Takeshita et al., 2011; Danelon et al., 2013a, Danelon et al., 2013b). Recently studies have shown that TMP inhibits remineralization in the outer layers of tooth enamel (Takeshita et al., 2011) as well as reduces the precipitation of CaF2 and firmly bound fluoride (Danelon et al., 2013b; Souza et al., 2013; Manarelli et al., 2014). It could be speculated that this phenomenon reduces the obstruction of the pores of the 78 Marcelle Danelon enamel surface facilitating the neutral species diffusion (CaHPO4 0 and HF0) into the enamel, increasing the remineralization in the zone B (15–50 µm) of the lesion, but not in the deeper of the lesion (zone C, 50–110 µm). This area could further reduce the mineral ion diffusion to the region deeper lesion (zone C) not allowing a mineral recovery throughout the body of the lesion. In the presence of nano-sized TMP, there is a higher gain of mineral in the deeper part of the lesion (zone C). This can be explained by its higher reactivity and retaining of Ca++ and CaF+ in its structure that leads to reducing the precipitation of F in the outer enamel and consequent lower obstruction of the pores of the enamel surface. This phenomenon may facilitate the diffusion of ions carrying the process of remineralization occurs throughout the body of the lesion and in a greater degree at the deepest part of the lesion. It is likely that this effect can produce better clinical outcomes to those observed previously (Stephen et al., 1994; O'Mullane et al., 2007). Hence, the toothpaste with 1100 ppm F associated with nano-sized TMP should be a matter of clinical trials. Based on the above, it was concluded that the addition of nano-sized TMP to a conventional toothpaste promoted a significantly higher remineralizing effect when compared to a toothpaste of same F concentration, without TMP, so that the null hypothesis could be rejected. The effects of nano-sized TMP should also be assessed on enamel demineralization, in order to confirm the existence of additional or synergistic effects, prior to clinical trials. Acknowledgments We thank the volunteers for their participation and the technicians of the laboratory of Pediatric Dentistry of the Araçatuba Dental School, UNESP, and to 79 Marcelle Danelon Mrs. Maria dos Santos Fernandes for laboratorial assistance. This study was supported by CNPq (Process: 158463/2012-9), FAPESP through the CEPID/CDMF and the INCTMN. 3.6 References Afonso RL, Pessan JP, Igreja BB, Cantagallo CF, Danelon M, Delbem ACB. In situ protocol for the determination of dose response effect of low-fluoride dentifrices on enamel remineralization (2013). J Appl Oral Sci 21:525-532. Bratthall D, Hansel-Petersson G,Sundberg H. Reasons for the caries decline: What do theexperts believe (1996)? Eur J Oral Sci 104:416-422. Cheng L, Weir MD, Xu HHK, Kraigsley AM, et al. Antibacterial and physical properties of calcium–phosphate and calcium–fluoride nanocomposites with Chlorhexidine (2012). Dent Mater 28:573-583. Comar LP, Souza BM, Gracindo LF, Buzalaf MA, Magalhães AC. Impact of experimental nano-HAP pastes on bovine enamel and dentin submitted to a pH cycling model (2013). Brazilian Dental Journal 24: 273-278. Danelon, M, Takeshita, EM, Sassaki, KT, Delbem, ACB. In situ evaluation of a low fluoride concentration gel with sodium trimetaphosphate in enamel re- mineralization (2013a). Am J Dent 26:15-20. Danelon M, Takeshita EM, Peixoto LC, Sassaki KT, Delbem AC. Effect of fluoride gels supplemented with sodium trimetaphosphate in reducing demineralization (2013b). Clin Oral Investig DOI 10.1007/s00784-013-1102-1104. Delbem AC, Bergamaschi M, Rodrigues E, Sassaki KT, Vieira AE, Missel EM. Anticaries effect of dentifrices with calcium citrate and sodium trimetaphosphate (2012). J App Oral Sci 20:94-98. http://www.ncbi.nlm.nih.gov/pubmed?term=Comar%20LP%5BAuthor%5D&cauthor=true&cauthor_uid=23969919 http://www.ncbi.nlm.nih.gov/pubmed?term=Souza%20BM%5BAuthor%5D&cauthor=true&cauthor_uid=23969919 http://www.ncbi.nlm.nih.gov/pubmed?term=Gracindo%20LF%5BAuthor%5D&cauthor=true&cauthor_uid=23969919 http://www.ncbi.nlm.nih.gov/pubmed?term=Buzalaf%20MA%5BAuthor%5D&cauthor=true&cauthor_uid=23969919 http://www.ncbi.nlm.nih.gov/pubmed?term=Magalh%C3%A3es%20AC%5BAuthor%5D&c