UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
EFEITO DOS DENTIFRÍCIOS CONTENDO CÁLCIO, FOSFATO E FLUORETO
NA PREVENÇÃO DA DESMINERALIZAÇÃO DO ESMALTE BOVINONiterói 2017
UNIVERSIDADE FEDERAL FLUMINENSE FACULDADE DE ODONTOLOGIA
EFEITO DOS DENTIFRÍCIOS CONTENDO CÁLCIO, FOSFATO E FLUORETO
NA PREVENÇÃO DA DESMINERALIZAÇÃO NO ESMALTE BOVINOSUZANA CARVALHO TEIXEIRA PINTO DE SOUZA
Dissertação apresentada à Faculdade de Odontologia da Universidade Federal Fluminense, como parte dos requisitos para obtenção do título de Mestre, pelo Programa de Pós-Graduação em Odontologia.
Área de Concentração: Clínica Odontológica.
Orientador: Profa. Dr(a) Mônica Almeida Tostes
Niterói 2017
FICHA CATALOGRÁFICA
S729 Souza, Suzana Carvalho Teixeira Pinto de
Effect of dentifrice containing fTCP and CPP-ACP and fluoride in the prevention of demineralization in bovine enamel / Suzana Carvalho Teixeira Pinto de Souza... [et al.]; orientadora: Monica Almeida Tostes. – Niterói : [s.n.], 2016.
27 f.:il.
Dissertação (Mestrado em Clínica Odontológica) – Universidade Federal Fluminense, 2017.
Bibliografia: f. 33-36.
1. Remineralização dentária. 2. Dentifrício. 3. Bovino. 4. Esmalte dentário. I. Tostes, Monica Almeida. [orient.]. II. Título.
BANCA EXAMINADORA
Prof(a). Dr(a). Mônica Almeida Tostes Instituição: Universidade Federal Fluminense
Decisão: _________________________Assinatura: ________________________
Prof. Dr. Anderson Araújo Rocha
Instituição: Universidade Federal Fluminense
Decisão: _________________________Assinatura: ________________________
Prof.(a). Dr(a). Viviane Andrade Cancio de Paula
Instituição: Universidade Salgado de Oliveira (UNIVERSO)
DEDICATÓRIA
Dedico este trabalho a minha família, aos meus pais que sempre acreditaram e me apoiaram durante toda a minha trajetória, sem nunca medir esforços para me dar uma educação de qualidade, permitindo que eu viesse a caminhar sozinha. Ao meu esposo, por caminhar ao meu lado e aos meus filhos, que são a razão pelo qual busco melhorar a cada dia.
AGRADECIMENTOS
A Deus por estar sempre comigo.
Aos meus pais, pelo amor incondicional. Pelo exemplo, apoio e constante estímulo. Aos meus irmãos pela amizade de uma vida inteira, por toda a verdade que eles emitem apenas com o olhar. Pessoas a qual tenho total admiração, carinho e respeito. Ao meu marido Daniel, meu grande incentivador nessa trajetória. Amigo, companheiro e o melhor pai que eu poderia ter escolhido para os meus pequenos.
Aos meus filhos que com toda a ternura me deram forças para continuar nos momentos em que pensei em desistir. Que me fizeram ir a lágrimas em cada partida e me acolheram com sorriso em cada retorno.
À professora Mônica Almeida Tostes, pela admirável dedicação ao ensino e pesquisa. Pela oportunidade de realizar esse trabalho me orientando nessa caminhada de forma paciente. Agradeço também pela disponibilidade de tempo e por todos os ensinamentos.
Ao professor Anderson Araújo Rocha, pela oportunidade dos ensinamentos concedidos na área de química ampliando meus horizontes.
Às professoras do departamento de odontopediatria pelos ricos ensinamentos transmitidos e por contribuir com excelência na minha formação profissional.
À doutoranda Patrícia Regina Almeida de Oliveira pela contribuição dada ao projeto. À Kaline Cassiano de Araújo pela experiência valiosa do convívio e pelo companheirismo.
Aos colegas de turma, pela convivência agradável e bons momentos compartilhados. Aos técnicos e alunos dos laboratórios LABA E NAB, pelo enorme cuidado e carinho a cada novo dia, por toda presteza e suporte.
Ao Núcleo de Água e Biomassa (NAB) da UFF, pelo apoio logístico nos ensaios. A todos que contribuíram direta e indiretamente na realização desse trabalho.
RESUMO
SOUZA SCTP. EFEITO DOS DENTIFRÍCIOS CONTENDO CÁLCIO, FOSFATO E FLUORETO NA PREVENÇÃO DA DESMINERALIZAÇÃO NO ESMALTE BOVINO [dissertação]. Niterói: Universidade Federal Fluminense, Faculdade de Odontologia; 2017.
Objetivos: A cárie dentária é uma doença multifatorial que resulta de um desequilíbrio entre fatores patológicos e de proteção. Apesar de todos os esforços para seu controle, continua sendo um problema de saúde pública. O presente trabalho teve como objetivo avaliar o potencial preventivo dos dentifrícios contendo, cálcio, fosfato e fluoreto na desmineralização do esmalte bovino, in vitro.
Metodologia: Oitenta blocos de esmalte bovino com superfícies vestibulares, lixadas e polidas, foram distribuídos em quatro grupos (N=20): G1- CPP-ACPF (MI Paste Plus); G2- Crest™ Cavity Protection; G3- Clinpro™ 5000 e G4-controle, sem tratamento. Os grupos foram submetidos a ciclagem de pH durante 10 dias, sendo 6 horas na solução desmineralizadora e 18 horas na solução remineralizadora, alternando as soluções a cada 2 horas. As soluções eram trocadas a cada 48h. Nos grupos experimentais, o dentifrício diluído em água destilada (w/w 1:3) foi aplicado por 3 minutos, uma vez ao dia, após o primeiro ciclo de desmineralização.
Análise: As alterações no esmalte foram avaliadas através do teste de Microdureza Dureza Superficial (MDS) com 50gr/15. Após os resultados, os dados foram analisados mediante teste estatístico ANOVA e Tukey (p<0,05).
Resultados: A média de MDS observada em G3 (288,0 ± 41,1) foi significantemente maior do que a encontrada nos grupos G4 (238,6 ± 37,0) e G1 (241,2 ± 36,5), mas não foi diferente da média do grupo G2 (264,5 ± 39,8). O pH, Ca2+ e Ptotal nas soluções
de remineralização não foram diferentes entre todos os grupos (p <0,05). Às 24h, a concentração de Ca2+ na solução de desmineralização foi significativamente menor
em G1-MPP. A concentração de Ca2+ aumentou em todos os grupos após 48h, exceto
para o G4-CLP.
Conclusão: O dentifrício Clinpro™ 5000 apresentou a maior prevenção de desmineralização e pode ser considerado um método preventivo em pacientes de alto risco de cárie. O efeito preventivo foi superior a Mi Paste Plus, mas não do dentifrício de uso regular, Crest com 1.100 ppmF.
Palavras-chave: Sistema de cálcio e fósforo; esmalte bovino; microdureza; remineralização.
ABSTRACT
Objective: To evaluate the ability of calcium-containing prescription-strength fluoride (F) toothpastes to prevent enamel demineralization under pH cycling conditions. Methods: Enamel bovine specimens were assigned to the following groups: Group 1 (G1) MI Paste
Plus (0.2% sodium fluoride, NaF, Recaldent™, GC Corporation Tokyo, Japan); Group 2 (G2) Crest™ Cavity Protection (0.243% NaF, Procter & Gamble); Group 3 (G3) Clinpro™ 5000 (1.1% NaF, 3M ESPE, USA); and Group 4 (G4) (no treatment). The specimens were soaked in demineralizing solution for 6 h and remineralizing solution for 18 h alternatively for 10 days. The dentifrice was prepared with deionized water in a 1:3 ratio (w/w) for three minutes daily. After cycling, enamel changes were analysed by surface microhardness (SMH). Data were analysed by one-way ANOVA and Tukey’s HSD post hoc test (α = 0.05). Results: The SMH value observed for G3 (288.0 ± 41.1) was significantly higher than that found in G4 (238.6 ± 37.0) and G1 (241.2 ± 36.5), but was not different from that of G2 (264.5 ± 39.8). The pH, Ca2+ and Ptotal in the remineralization solutions were not different among all groups (p < 0.05). At 24h, the Ca2+ concentration in the demineralization solution was significantly lower in G1-MPP. Ca2+ concentration increased in all groups after 48h, except for the G4-CLP. Conclusion: Clinpro™ 5000 demonstrated a greater protective effect against demineralization than MI Paste Plus.
1 – INTRODUÇÃO
A cárie dentária é uma doença dos tecidos duros dos dentes causadas por um desequilíbrio, ao longo do tempo, nas interações entre bactérias cariogênicas na placa dentária e carboidratos fermentáveis. As lesões de mancha branca (LMB) são o primeiro sinal clínico de cárie dentária e podem ser consideradas como fase inicial da desmineralização do esmalte (Oliveira et al., 2015).
O controle da LMB requer, primeiramente, a avaliação do risco para identificações dos fatores que podem contribuir para a formação da lesão. A instrução de higiene oral e dieta deve ser reforçada e a diminuição dos intervalos das consultas para maior controle e o uso do dentifrício contendo cálcio, fosfato e fluoreto (Johansson et al., 2010).
Ao longo dos anos, o fluoreto tem sido utilizado para a prevenção e paralização do processo de cárie e tem demonstrado a sua eficácia (Marinho et al., 2003). Entretanto a sua capacidade de promover remineralização é limitada pela disponibilidade de íons cálcio e fosfato (Reynolds, 1997; Reynolds et al., 2008). A combinação de fluoreto com uma fonte de íons cálcio e fosfato biodisponíveis foi proposto como um tratamento efetivo para os estágios iniciais da doença cárie (Uysal et al., 2010; Reynolds et al., 2008; Shen et al., 2011).
Em pacientes com alto risco de cárie, dentifrícios contento cálcio, fosfato e fluoreto em diferentes concentrações tem indicações para controle, porém os resultados ainda são contraditórios. No processo de remineralização a presença do fluoreto, do cálcio e do fosfato são indispensáveis e mantê-los disponíveis na mesma formulação é um dos maiores desafios, pois o fosfato e o cálcio acabam reagindo entre si (Karlinsey et al., 2009). Assim, no Mercado novas tecnologias têm sido lançadas com o objetivo de melhor eficácia desses produtos.
Dentre estes produtos, a caseína fosfopéptido-amorfo fosfato de cálcio (CPP-ACP) está disponível como um produto dental profissional (GC tooth mousse; 10% (w/w) CPP-ACP, GC Corporation, Tóquio, Japão) que inibe a progressão da cárie e remineraliza lesões superficiais de esmalte (Reynolds, 1997; Reynolds et al., 2008). Desta maneira, oferecendo um potencial para utilização na prevenção da cárie dentária (Reynolds et al., 2008). Os fosfopéptidos de caseína - fosfato de cálcio amorfo
(CPP-ACP) têm efeitos anticariogênicos tópicos devido à sua capacidade de estabilizar cálcio e fosfato em um estado amorfo.
Os modelos de remineralização do esmalte in vitro tem sido utilizados para prever a eficácia anti-cárie do tratamento CPP-ACP, mas os resultados são ainda inconsistentes. (Souza et al., 2014; Oliveira et al., 2015, Oliveira et al., 2014; Pulido et al., 2008; Kumar et al., 2008; Ekassas, Arafa 2014; Shen et al., 2011). Em pesquisas mais recentes, a CPP-ACPF demonstrou melhores resultados em
prevenção da desmineralização (Oliveira et al., 2015, Karlinsey et al., 2009). De acordo com Kumar et al. (2008), a CPP-ACP também diminui a profundidade da lesão independente de ter ou não sido utilizada como dentifrício ou como creme tópico, e a CPP-ACP mostrou maior potencial remineralizante quando usado em combinação com um dentifrício com fluoreto do que quando utilizados sozinho. Em outros estudos, a CPP-ACPF apresenta resultados superiores ao dentifrício com 1100 e 5000 ppmF (Shen et al., 2011).
Uma exclusiva formulação tri-cálcio-fosfato (fTCP) tem sido também avaliada e mostra benefícios na remineralização do esmalte. (Vanichvatana S, Auychai P, 2013). O Clinpro™ Fosfato de Cálcio e 5000 ppm de Flúor é indicado para pacientes que apresentam risco moderado e elevado de desenvolvimento de lesões de cárie, podendo prevenir o aparecimento de novas lesões de cárie, fortalecendo o esmalte e/ou reparando lesões iniciais. A sua formulação com TCP e fluoreto de sódio a 1,1% (5000 ppm de ions fluoreto) permite maior proteção e maior disponibilidade de íons para uma melhor remineralização de lesões em profundidade (http://www.3m.com.br/3M/pt_BR/odontologia/).
Os produtos de fosfato tricálcico (fTCP), que funcionalizam o b-TCP, podem ser preparados com sílica, o que pode proporcionar oportunidades de ligação com defeitos de tecidos duros sob condição, podendo permear todo o esmalte sem reagir com o material orgânico inter-prismático, o que pode encorajar maior captação de cálcio, fosfato e flúor em lesões desmineralizadas (Karsinley et al., 2012; Ekassas, Arafa 2014). É um agente que trabalha em sinergia com flúor para criar um mineral mais forte, mais resistente aos ácidos. Tendo melhores resultados com flúor do que β-TCP ou f TCP sozinho (Karsiney et al., 2012). Estas observações são baseadas em avaliações clínicas laboratoriais e intra-orais (Elkassas, Arafa, 2014; Vanichvatana, Auychai, 2013). Além disso, nas condições experimentais da 5000ppm F, sob experiência de cárie in vivo simulada, parece inibir a desmineralização e promover a
remineralização de forma mais eficaz do que uma pasta de dentes com concentração de 1450 ppm F, ou uma pasta de dentes contendo um sistema de fosfosilicato de cálcio e sódio (Diamanti et al., 2011).
Recentemente, Elkassas D e Arafa A. (2014) avaliaram o efeito de diferentes agentes contendo cálcio, fosfato e fluoreto em um estudo in vitro utilizando quatro agentes remineralizantes: Clinpro™ verniz, ACPF, CPP-ACPF e Vanish TMXT. As alterações no esmalte foram avalidas por microdureza superficial e rugosidade de superfície em diferentes etapas – início, após desmineralização, 2 e 4 semanas após a remineralização e com desafio ácido. Os autores concluíram que os agentes remineralizantes contendo diferentes fórmulas de fosfato de cálcio e fluoreto aumentam o potencial de remineralização em comparação com a saliva artificial. Clinpro ™ verniz apresentou maior tendência de remineralização com maior resistência ao ataque ácido. Contudo, em um estudo in situ e in vitro, o Clinpro 5000 (fTCP) forneceu similar benefícios quando comparado ao dentifrício fluoretado com 1.100 ppm.F (Vanichvatana, Auychai, 2013; Scaramucci et al., 2015). Segundo Scaramucci et al., CPP-ACPF e fTCP podem ser indicados para reduzir a cárie em pacientes de alto risco e reduzir a erosão dentária em pacientes com refluxo gástrico ou outros distúrbios. No entanto, apesar do aumento dos níveis de F, que foi encontrado especialmente com o dentifrício Clinpro 5000, os produtos fluoretados com 1100 e 5000 ppmF, sem cálcio, protegem os substratos dentários contra a erosão a um grau semelhante quando comparado com o placebo.
Com base nas considerações acima, este estudo in vitro avaliou a influência do CPP-ACPF e o fTCP sobre a prevenção da desmineralização do esmalte bovino usando o teste de microdureza Knoop como método de avaliação. Em relação às superfícies do esmalte quando expostas a condição de risco de cárie elevada, a avaliação dos possíveis benefícios cariostático adicional de dentífricos contendo fluoreto em concentrações acima do padrão, parece ser particularmente útil. De outro modo, a disponibilidade de cálcio neste sistema ainda é um aspecto que deve ser investigado.
Assim, este trabalho tem como objetivo avaliar o potencial de remineralização do creme contendo CPP-ACPF e o fosfato tricálcio funcionalizado (fTCP), comparando com produtos de dentifrício de uso regular (1100 ppmF) em superfícies de esmalte bovino em estudo in vitro sob condições de ciclagem de pH. A hipótese nula a ser testada é que o dentifrício contendo cálcio e alta concentração de fluoreto
apresentará maior disponibilidade de cálcio e maior ação preventiva contra a desmineralização.
2 – METODOLOGIA
2.1. Obtenção das amostras de esmalte bovino
O presente estudo in vitro seguiu o protocolo pré-estabelecido para obtenção dos blocos de esmalte de acordo com Oliveira et al., 2015. Cerca de 100 dentes bovinos foram utilizados e mantidos em timol 0.1% até serem processados. Os dentes foram analisados, após limpos e polidos com escova Robinson e mistura de pedra-pomes fina e água destilada em baixa rotação (Kavo do Brasil S.A.). Após lavados com água destilada e secos foram escolhidos obedecendo aos seguintes critérios: ausência de trincas ou defeitos macroscópicos ou qualquer outra alteração de esmalte. Em seguida, as coroas dentárias foram seccionadas, separando-as da raiz com um disco diamantado dupla face (Diamond Wafering Blades 1/2”, diâmetro 4” x 0,012” 7 – ref. 11-4244, Buehler) em máquina Isomet 1000 (Buehler). Uma vez obtidas as 80 coroas, estas foram fixadas pela sua face distal com cera em placas de acrílico cristal de 40 mm X 40 mm X 5 mm e seccionadas no sentido mésio-distal, separando a face vestibular da lingual, com disco diamantado dupla face. Posteriormente, as faces vestibulares foram fixadas com cera nas mesmas placas de acrílico para facilitar o corte dos fragmentos no centro geométrico da superfície vestibular. Desse modo, 1 a 2 blocos de esmalte com dimensões de 4 X 4 mm foram obtidos de cada dente. Após obtidos os blocos de esmalte/dentina, estes foram fixados com cera no centro da base de acrílico cristal, de 30 mm de diâmetro por 8 mm de espessura, com a maior área plana do esmalte voltada para baixo. O lixamento da superfície de dentina foi realizada com lixa de silicone carbide de granulação 320 (Extec Corp.), com refrigeração à água, até que os blocos ficassem com espessura de 2 mm. Os blocos foram reafixados com as superfícies de esmalte voltadas para cima, de forma que ficassem visível e paralela ao bloco e a dentina. Os blocos de esmalte foram planificadas em Politriz Aromet (Aropol 2v) com lixas 600, #1000, #1200 (Arotec),
#2400 e #4000 (Presí) durante 2 minutos em velocidade baixa e polidas com disco de feltro (TEXMET C, Buehler, ref. 40-1108) e alumina 1µm (ref. 40-6530, Buehler), durante 3 minutos em velocidade alta, até obter-se uma superfície de aspecto vítreo.
2.2. Ciclagem de pH e tratamento com os produtos remineralizantes
Três agentes remineralizantes foram utilizados neste estudo, Clinpro™ 5000 (3M ESPE), Caseína Fosfopéptido Amorfo de cálcio fosfato com 900ppmF (CPP-ACPF), Dentifrício fluoretado (1.100 ppm.F) como controle positivo. No controle negativo nenhum produto foi utilizado. Na tabela 1, os produtos utilizados em cada grupo são descritos com seus princípios ativos.
Os blocos de esmalte foram submetidos a ciclagem de pH por 10 dias, a 37 ºC (Figura 1). Antes do início da ciclagem, cada bloco foi imerso em 10 mL de saliva artificial por 24 horas (0,67g/L NaCl; 0,1168g/L CaCl2; 8g/L CMC; 0,0408 g/L MgCl2; 0,96g/KCL; 1g/L C8H8O3; 24g/L C6H14O6; 964,938 ml/L H2O; 0,274 g/L KH2 PO4). Após, os blocos de esmalte foram mantidos em solução desmineralizadora por 6 horas por dia (8-10, 12-14, 16-18 h), nos demais horários, permaneceram em solução remineralizante (18 horas). A solução desmineralizante continha 2 mM Ca (Ca (NO3)2),
2 mM PO4 (KN2PO4) and 75 mM acetato com pH 4,8. A solução remineralizante
continha 1.5 mM Ca, 0.9 mM PO4, 130150 mM KCl, 0.5 mg F mL-1, 100 mM TRIS
buffer, pH 7.0). Os produtos foram aplicados uma única vez após o primeiro ciclo de desmineralização do dia. Cada produto foi diluído em 1:3 e os blocos foram imersos nesta solução por 3 minutos todos os dias após o primeiro ciclo de desmineralização. Entre as ciclagens e a aplicação dos agentes remineralizadores os blocos foram lavados por 1 min em água destilada e secos com papel absorvente. A cada 48hs as soluções foram trocadas até o término dos 10 dias. Após cada ciclo de 48 horas a concentração de Cálcio, Fosfato e o pH foram medidos como descrito a seguir (Figura 1).
Tabela 1- Princípio ativo dos produtos utilizados na pesquisa
Grupos Produtos Princípio ativo
G1 MI Paste Plus (MPP) Fluoreto de sódio 0,2%
(w/w) (900 ppmF), CPP-ACP
G2 Crest™ Cavity
Protection (DF)
(0,243% Fluoreto de sódio)
G3 Clinpro™ 5000 (CLP) Fluoreto de sódio 1,1%
(w/w) (0,63% or 5000 ppmF), tri-fosfato de cálcio.
G4 Sem tratamento (CO) Sem tratamento
2.3 Microdureza superficial (MDS)
Uma vez polido, cada bloco foi avaliado através de microdureza Knoop. A primeira avaliação foi realizada após o polimento (MDS_inicial), e a segunda, após a
ciclagem de pH (MDS_tratamento). A primeira dureza foi realizada no centro geométrico
da amostra e as demais, guardando uma distância de 50 micrômetros de distância entre elas. Dessa forma, uma fileira de 5 medidas foi obtida de cada bloco. A segunda fileira, após o tratamento, foi realizada a 100 micrômetro da primeira e na mesma distância de 50 entre elas. Posteriormente, a média de cada bloco foi obtida. Dessa forma, foi obtida a média de dureza de cada bloco e cerca de 80 blocos foram selecionados com durezas entre 278,7KHN a 396,6KHN após a MDS_inicial. Os dados
foram levados para uma planilha e, posteriormente, analisados estatisticamente.
2.4 Determinação do cálcio e fosfato (Ptotal) na solução
Os grupos experimentais foram testados para liberação de Ca2+ e Ptotal. A cada ciclo (24h e 48h), alíquotas de 0,5 mL foram retiradas e substituídas por solução “fresca”. As alíquotas das amostras foram diluídas 20 vezes em água ultrapura (resistividade> 18 M cm-1, Millipore, Bedford, MA, EUA). Na determinação de Ca2+
e Ptotal utilizou-se um instrumento de espectrometria de emissão óptica de plasma de microondas (MIP OES, 4200 MP-AES Agilent Technologies Inc., Santa Clara, CA, EUA). O gerador de plasma é baseado na excitação de microondas que pode manter um plasma de nitrogênio (5000 K). A orientação do plasma era vertical a partir de uma vista axial. A introdução da amostra ao MP foi pneumática usando um nebulizador concêntrico e câmara de pulverização ciclônica. O isolamento e a detecção da linha de emissão foi usando um monocromador Czerny-Turner sequencial e detector de carga acoplada (CCD), respectivamente.
Foi utilizado um método de calibração externo para determinação de Ca2+. Utilizaram-se soluções aquosas monoelementas contendo 1000 mg L-1 de Ca2+ e Ptotal (VHG-Labs, Manchester, NH, EUA) para preparar soluções de referência padrão. Uma curva de calibração (0,5 - 5,0 mg L-1) foi preparada com os analitos diluídos em água ultrapura. Os coeficientes de correlação obtidos foram 0,99991 (y = 814588x + 15,1) e 0,99994 (y = 419,52x - 0,4) para Ca2+ e Ptotal respectivamente. Limites de detecção (LOD = 3.10_blank a-1) resulta em 0.002 mg L-1 para Ca2+ e e 0,300 mg L-1 para Ptotal. Um teste com um método de calibração externa de correspondência de matriz mostrou que a presença de salinidade não afetou a sensibilidade da curva analítica.
2.5 Análise do pH
A medição do pH foi realizada utilizando o titulador automático Methrom 808 Titrando, utilizando o software Tiamo 2.3. O pH foi medido no final do ciclo (48 h). O eletrodo usado foi o Metrohm Unitrode com Pt 1000 (número de ordem 6.0258.000), utilizando solução aquosa de 3 mol L-1 KCl (Gehaka) como eletrólito de referência. A
calibração foi realizada utilizando os seguintes tampões aquosos de Gehaka: pH 4,01 ± 0,05 (tampão biftalato), 7,01 ± 0,05 (tampão fosfato) e 10,01 ± 0,05 (tampão bicarbonato), produzindo uma inclinação de 91% com pH (0) 7,8 a 30ºC .
Figura 1. Esquema representativo da metodologia utilizada (MPP-Mi Paste Plus; DF – dentifrício fluoretado; CLP- Clinpro; CO-controle sem tratamento
3 – ARTIGO PRODUZIDO
Title: Effect of dentifrice containing fTCP, CPP-ACP and fluoride in the prevention of demineralization in bovine enamel
Authors: aSuzana Carvalho Teixeira Pinto de Souza; bKaline Cassiano de Araújo; cJoseane Ribeiro Barbosa; dAnderson Araújo Rocha; aMônica Almeida Tostes a
Pediatric Dentistry Department, School of Dentistry, Fluminense Federal University, Rua Mário Santos Braga, no 30 Campus Valonguinho, Centro, Niterói, RJ, Brazil, CEP 24040-110
b
Undergraduate student, School of Dentistry, Fluminense Federal University, Rua Mário Santos Braga, no 30 Campus Valonguinho, Centro, Niterói, RJ, Brazil, CEP 24040-110
c
Undergraduate student, School of Chemistry, Fluminense Federal University, Outeiro de São João Batista, s/n – Campus Valonguinho, Centro, Niterói, RJ, Brazil, CEP 24040-110
dAnalytical Chemistry Institute, School of Chemistry, Fluminense Federal University,
Outeiro de São João Batista, s/n – Campus Valonguinho, Centro, Niterói, RJ, Brazil, CEP 24040-110
Corresponding author: Dr Monica Almeida Tostes – Universidade Federal
Fluminense/Faculdade de Odontologia, Rua Mário Santos Braga, no 30 Campus Valonguinho, Centro, Niterói, RJ, Brazil, CEP 24040-110; Phone: 55 21 2629-9829; e-mail: matostesuff@yahoo.com.br
Abstract
Objective: To evaluate the ability of calcium-containing prescription-strength fluoride (F-) toothpastes to prevent enamel demineralization under pH cycling conditions.
Methods: Enamel bovine specimens were assigned to the following groups: G1-MPP (MI Paste Plus, 0.2% sodium fluoride, NaF, Recaldent™, GC Corporation Tokyo, Japan); G2-FD (Crest™ Cavity Protection, 0.243% NaF, Procter & Gamble); G3-CLP (Clinpro™ 5000, 1.1% NaF, 3M ESPE, USA); and G4-CO (no treatment). The specimens were soaked in demineralizing solution for 6 h and remineralizing solution for 18 h alternatively for 10 days. The dentifrice was prepared with deionized water in
a 1:3 ratio (w/w) for three minutes daily. After cycling, enamel changes were analysed by surface microhardness (SMH). Data were analysed by one-way ANOVA and Tukey’s HSD post hoc test (α = 0.05). Results: The SMH value observed for G3-CLP (288.0 ± 41.1) was significantly higher than that found in G4-CO (238.6 ± 37.0) and G1-MPP (241.2 ± 36.5), but was not different from that of G2-FD (264.5 ± 39.8). The pH, Ca2+ and P
total in the remineralization solutions were not different among all groups
(p < 0.05). At 24h, the Ca2+ concentration in the demineralization solution was
significantly lower in G1-MPP. Ca2+ concentration increased in all groups after 48h,
except for the G4-CLP. Conclusion: Clinpro™ 5000 demonstrated a greater protective effect against demineralization, but was not different than fluoride dentifrice 1100 ppmF. Mpp did not show any benefit in this in vitro model.
Keywords: Calcium-phosphate systems; bovine enamel; microhardness;
remineralization
Introduction
White-spot lesions (WSL) are the first clinical signs of tooth caries, and can be considered as the initial stage of enamel demineralization. Dental caries is initiated by acid-producing bacteria, which cause carious lesions in the presence of fermentable carbohydrates [1]. Over the years, fluoride has been used to prevent decay or for remineralizing of WSL [2]. Fluoride is the cornerstone of the non-invasive management of non-cavitated caries lesions, but its ability to promote net remineralization is limited by the availability of calcium and phosphate ions [3,4].
The combination of fluoride and a source of bioavailable calcium and phosphate ions have been proposed as an effective treatment for the early stages of caries disease [37]. The casein phosphopeptide, amorphous calcium phosphate, is reported to have topical anticariogenic effects due to its ability to stabilize calcium and phosphate in an amorphous state. CPP not only increased fluoride incorporation into plaque, but it also increased the incorporation of fluoride into subsurface enamel and substantially increased remineralization of subsurface lesions of enamel compared with fluoride alone [4,7]. Despite this, in vitro enamel remineralization models have been widely used for prediction of the anti-caries efficacy of CPP-ACP treatment, but the results remain inconsistent[8-15]. The combination of CPP-ACP and fluoride has demonstrated better results in demineralization prevention than CPP-ACP alone [9,12,14].
Tricalcium phosphate products (functionalized β-tricalcium phosphate; fTCP) are prepared with silica, which may provide linking opportunities with hard-tissue defects under acidic conditions. Silica can permeate throughout enamel without attacking the inter-prismatic organic material, which may encourage greater calcium, phosphate, and fluoride uptake in demineralized lesions [16]. It is an agent that works in synergy with fluoride to create a stronger, more acid-resistant mineral relative to that achievable with fluoride, β-TCP, or fTCP alone [16-17]. Some initial reports have shown that Clinpro™ 5000 tooth Crème anticavity toothpaste containing fTCP, is useful for reducing WSLs [5,18]. fTCP is produced by milling TCP with sodium lauryl sulphate. This process prevents undesirable interactions between calcium and fluoride, which could render both inactive.
Based on the above considerations, this in vitro study evaluated the influence of the CPP-ACPF (MI Paste Plus- MPP) crème and fTCP on demineralization prevention of bovine enamel using the Knoop microhardness test as the evaluation method. Considering that enamel surfaces are thought to face a high caries risk situation when exposed in the cariogenic condition, the evaluation of a possible additional cariostatic benefit of toothpastes containing fluoride in concentrations above the standard, appears to be particularly useful [10,19,20]. The second objective was to evaluate the calcium and phosphate concentrations available in the remineralizing and demineralizing solutions after each cycle of 24 and 48h.
In this study, it was hypothesized that toothpastes containing a high fluoride concentration and calcium (Clinpro™ 5000) could provide additional protection against dental demineralization when compared to CPP-ACPF (MPP) and regular fluoride dentifrice (NaF, 1100 ppm F).
Material and methods
Sample preparation
Bovine incisors stored in saturated 0.1% thymol solution (pH 7.0) were used. The enamel slabs (4 x 4 mm) were prepared and mounted with sticky wax on Plexiglas
blocks. After embedding the blocks in acrylic resin, the buccal surfaces of the enamel specimens (4 x 4 x 2 mm) were ground with SiC paper (400, 600 and 1200 grits) (Struers S/A, Struer, Denmark) in order to obtain flat surfaces. The specimens were then polished using a 1 μm diamond polishing suspension with a polishing cloth (Arotec Ind & Come, Cotia, SP, Brazil). The surface hardness was measured using a 2001 MicroMet micro-hardness tester (Buehler, Lake Bluff, USA) with a Knoop type indenter, and with a static load of 50 g for 15 s. The Knoop hardness number (KHN) is calculated from the length of the indentation and the applied load. An increase in length in µm indicates a softening of the enamel due to demineralization. Five indentations separated by a distance of 100 μm were made in the central region of each block. The average of the five indentations made on each specimen was used as the SMH baseline value (SMHbaseline). After SMH measurements, 80 enamel blocks were
selected, with a Knoop hardness number (KHN) ranging from 278.8 to 396.6. The enamel slabs were distributed into four groups of 20: G1- MI Paste Plus 0.2% sodium fluoride, Recaldent™, GC Corporation Tokyo, Japan); G2 - Crest™ Cavity Protection; 0.243% NaF, Procter & Gamble); G3 -Clinpro™ 5000; 1.1% NaF, 3M ESPE, USA); and G4 placebo, no treatment (Table 1).
pH cycling and treatment with dentifrice
Before pH cycling, each enamel slab was immersed in 10 mL of artificial saliva for 24 h (0.67 g L-1 NaCl; 0.1168 g L-1 CaCl
2; 8 g L-1 CMC; 0.0408 g L-1 MgCl2; 0.96 g
L-1 KCl; 1 g L-1 C
8H8O3; 24 g L-1 C6H14O6; 964.938 mL L-1 H2O; 0.274 g L-1 KH2PO4)
(Figure 1). Following this, the enamel slabs were submitted to pH cycle for 10 days at 37°C. The specimens were immersed separately in 10 mL of the demineralizing solution (810 h, 1214 h, 1618 h), and in the remaining hours (18 h day) they were transferred to a remineralizing solution (10 mL). The demineralization stage used an acid buffer containing 2 mM Ca2+ (Ca(NO
3)2), 2 mM PO43- (KH2PO4) and 75 mM
acetate at pH 4.8. The remineralizing solution contained 1.5 mM Ca2+, 0.9 mM PO 43-,
130150 mM KCl, 0.5 mg F mL-1, 100 mM TRIS buffer, pH 7.0. Standard pH cycling
demineralization and two exposures of two hours of remineralization. After the last demineralization, the slabs were immersed in remineralizing solution for 24 h (6 h of demineralization and 18 h of remineralization). At each transfer between the different solutions, all specimens were rinsed in distilled water for 1 min before and after any solution change or dentifrice slurry application and they were wiped dry with a soft paper tower. The products were applied in a slurry at a ratio of 1:3 toothpaste: deionized water. Thus, the specimens were exposed to the dentifrice slurries once a day for three minutes after the first demineralization every day. A standardized volume (60 mL) was applied to each group. The negative control group (G4) remained unexposed to the pastes. The solutions were renewed every 48 h (five cycles of 48 h over 10 days).
Microhardness analysis
After completing 10 days of cycling, the post-treatment measurements (SMHtreated) were conducted with the same static load and time applied for the baseline measurements. Five indentations spaced 100 μm from the baseline indentations were made with a Knoop diamond indenter under a 50 g load for 15 s (Micromet 2001, Buehler, IL, USA).
Determination of calcium (Ca2+) and phosphate (P
total) in solutions
The experimental groups were tested for Ca2+ and Ptotal release. At each cycle (24h and 48h), aliquots of 0.5 mL were taken and replaced by fresh solution. The aliquots of the samples were diluted 20-fold in ultrapure water (resistivity > 18 M cm-1; Millipore, Bedford, MA, USA). A microwave plasma optical emission spectrometry instrument (MIP OES, 4200 MP-AES Agilent Technologies Inc., Santa Clara, CA, USA) was used in the determination of Ca2+ and Ptotal. The plasma generator is based on microwave excitation that can maintain a nitrogen plasma (5000 K). The orientation of
pneumatic using a concentric nebulizer and cyclonic spray chamber. Emission line isolation and detection is sequential using a Czerny-Turner monochromator and charge-coupled device (CCD) detector. Instrumental operating conditions and settings used to determine Ca2+ and Ptotal in samples are presented in Table 2.
An external calibration method was used for determination of Ca2+. Monoelement aqueous stock solutions containing 1000 mg L-1 of Ca2+ and Ptotal (VHG-Labs, Manchester, NH, USA) were used to prepare standard reference solutions. A calibration curve (0.55.0 mg L-1) was prepared with the analytes diluted in ultrapure water. The correlation coefficients obtained were 0.99991 (y = 814588x + 15.1) and 0.99994 (y = 419.52x - 0.4) for Ca2+ and Ptotal respectively. Limits of detection (LOD = 3.10_blank a-1) results in 0.002 mg 1 for Ca2+ and 0.300 mg L-1 for Ptotal. A test with a matrix-matching external calibration method showed that the presence of salinity did not affect the sensitivity of analytical curves.
The pH measurement was performed using the automatic titrator Methrom 808 Titrando, using Tiamo 2.3 software. The pH was measured at the end of cycle (48 h). The electrode used was the Metrohm Unitrode with Pt 1000 (order number 6.0258.000), using 3 mol L-1 KCl aqueous solution (Gehaka) as the reference electrolyte. The calibration was performed using the following Gehaka aqueous buffers: pH 4.01 ± 0.05 (biphthalate buffer), 7.01 ± 0.05 (phosphate buffer) and 10.01 ± 0.05 (bicarbonate buffer), yielding a slope of 91% with pH (0) 7.8 at 30oC.
Statistical analysis
The data were analysed using SPSS statistical software package for Windows, version 20.0 (IBM Corporation, New York, USA). Initially, all the data (SMHbaseline and
SMHtreated) were checked with Shapiro-Wilk’s test. Based on these preliminary
analyses, the SMHbaseline and SMHtreated data were submitted to one-way analysis of
variance and Tukey’s HSD post hoc test. Comparison before and after treatment in the same group was analysed with paired t-test. Comparison between groups was analysed with unpaired t-test. The data of pH, Ca2+ and P concentrations were
of α = 0.05.
Results
The mean SMHbaseline was not significantly different among the groups (Figure 2; ANOVA, p = 0.65). The G1-MPP, G2-FD, G3-CPL and G4-CO groups demonstrated a decrease in microhardness after pH cycling, but a significant difference was found only G4 (Paired t-test; p <0.05). Thus, mean SMHtreated was higher in G3-CLP (288.0 ± 41.1) and G2-FD (264.5 ± 39.8) than in G1-MPP (241.2 ± 36.5) and G4-CO (238.6 ± 37.0) (Figure 2). G3-CLP was significantly higher than G1 (p < 0.05) and G4-CO (p < 0.01) and G2-FD was different of the G1-CO . The other groups did not differ (p > 0.05).
The inorganic calcium availability data (mean ± SD) collected in DE solutions after 24 and 48-h period for the four groups are presented in Figure 3. The baseline calcium in this solution was 63.0 mg L-1. At 24h, the calcium available in the DE solutions was lower than 48h in the G1-MPP, G2-FD and G4-CO (paired t test; p<0.05), but no in G3-CLP. The concentration of calcium was lower in G1 when compared to G2, G3 and G4 only after 24h (unpaired t test; p<0.05). The G1 and G4 were similar (p>0.05).
The inorganic phosphate availability data (mean ± SD) collected in DE solutions after each 24 or 48-h period for the four groups are presented in Figure 4. The baseline phosphate in this solution was 64.8 mg L-1. In the 24h and 48h, the phosphate available in the DE solutions was similar to all groups (unpaired t test; p>0.05). The concentration of phosphate was lower in G1 after 48h (paired t test; p<0.05).
In the RE solution, the concentration the calcium and phosphate was similar among groups (Figure 5 and 6; p<0.05). The baseline the concentration of calcium and phosphate in this solution were 9.37 mg L-1 and 15.6 mg L-1, respectively.
The pH of the baseline solutions was 4.8 and 7.0 for demineralizing and remineralizing solutions, respectively. The data of pH was showed in Figure 7. The mean of pH was not significantly different among the groups (Kruskal Wallis; p > 0.05) and solutions.
Discussion
The purpose of this study was to evaluate CPP-ACP paste and fTCP with fluoride on demineralized enamel using surface microhardness in a pH cycling model, simulating conditions in the oral cavity. The change of the enamel was evaluated by microhardness. Microhardness analyses have been widely used to assess changes occurring in enamel after treatment with toothpaste [8,9,13,21]. Microhardness indentation measurements can provide indirect evidence of mineral loss or gain [21].
The product with 5,000 ppm sodium fluoride toothpaste containing fTCP can be used for patients at an increased risk of getting tooth decay [5]. The product is recommended for children from 6 to 16 years of age and can be applied using a thin ribbon or pea-sized amount of Clinpro™ 5,000 Anti-Cavity Toothpaste using a soft-bristled toothbrush. Teeth should then be brushed for at least two minutes, following, which the patients should expectorate [22]. Although these products with high concentrations of fluoride are not recommended for regular use, in situations of risk, such as the use of orthodontic appliances, it may be an additional treatment in the control and prevention of incipient lesions [5,23]. In addition, the Clinpro™ 5,000 Anti-Cavity Toothpaste is indicated for use as part of a professional programme for the prevention and control of dental caries [22].
In the present study, the application of fTPC-containing toothpaste (G3-CLP) resulted in a significant increase in SMH. This is evidenced by a clear dose response between G3-CLP and G4-CO; therefore, this outcome helps validate its applicability in assessing the in vitro remineralization potential of dentifrice fTCP. As observed in previous studies [18, 24], the fTPC/F combination increased the capacity of toothpaste to reduce demineralization of the enamel. These results can be explained by the synergy with fluoride to create stronger, more acid-resistant mineral relative to that achievable with fluoride or fTCP alone [24]. Another studies have also been performed whereby 5,000 ppm F plus fTCP provided significantly greater remineralization benefits [16,18,24,25]. Thus, benefices have been observed when lower concentration of fluoride plus fTCP are used [18,25,26,27].
In the cyclic model used in this study, the demineralization and remineralization solutions were changed after each cycle, so that the concentration of calcium and phosphate ions in the solutions would not affect the results. In addition, enamel
surfaces were rinsed after treatment with dentifrices, so that any treatment effect would be due to the binding of the active ingredients to the enamel. Enamel demineralization was reduced under these conditions; however, when the fTCP (G3-CLP) was employed the reduction was significantly lower.
To simulate in vivo pH cycles, previous study tested cyclic demineralization/remineralization in vitro [8-14 ] with demineralization solution at pH 4.8 and remineralization solution at pH 7 [8]. Two points should be noted. First, the 6-hour immersion in pH 4.7 demineralization solution thermodynamically favors demineralization; remineralization of enamel occurs during the 18-hour immersion in the remineralization solution at pH 7. Therefore, the fact that the enamel treated with FD toothpaste and control released greater Ca2+ at demineralizing solution (Figure 3) indicates that it could be due enamel demineralization. Second, the present study had a products containing calcium and phosphate tecnology tested under the same demineralization/ remineralization regimen. Although the Ca2+ concentration no showed difference among the groups after 48h, there was a trend of less Ca2+ concentration in DE solution in G1-MPP and G3-CLP groups. Despite of Ca2+ and Ptotal were lower DE solution, under acid condition, MPP with fluoride showed lower ability to reduce mineral loss and was similar to control (G4-CO). Probably, a greater amount of CaF in the medium, negativaly influencing the level of available fluoride [15]. On the other hand, after 48h, all groups show greater concentration the Ca2+ in DE solution when compared to 24h, except for G3-CLP group; so, the lower Ca2+ available can be relative smaller dissolution in this group. The G1-MPP produced a demineralization level similar to the 1100-ppm-fluoride toothpaste (G2-FD), but was not show a dose response (Figure 1). On the other hand, microradiography of the lesions after remineralization in situ revealed that fluoride ion alone tended to promote remineralization of the surface layer, whereas CPP ACP promoted remineralization, even in the presence of fluoride, throughout the body of the lesion. [4,7] In current study SMH is a sensitive technique for shallow lesions, and it was not provided information of lesion depth [ 28 ].
On the other hand, in this current study, bovine enamel treatment with fTCP (G3-CLP) was very sensitive to treatment, but was not different when compared to the NaF 1100 ppm F (G2-FD) group at the end of 10 days of cycling. By in situ experiments, the Clinpro™ (950 ppm) and regular fluoride dentifrices produced similar results in subsurface rehardening of white-spot lesions [7,18]. Still, the present study agrees very
well with reported findings of 500 plus fTCP and 1,100 ppmF toothpaste studies [27]. Recently, a report found no statistical differences in the remineralization of erosion lesions in bovine enamel using a 1,100, 5,000 or 5,000 ppmF plus fTCP toothpaste in vitro [17]. In addition, despite the increase in fluoride levels, which was found especially with the Clinpro™ 5000 toothpaste, all the fluoridated products protected the dental substrates against erosion to a similar degree when compared to the placebo [17]. Although in our study we also observed that the remineralization potential was not significantly different among the 5,000 ppmF plus fTCP and 1,100 ppm F dentifrices with respect to the SMH data, we observe trending that favors the 5,000 ppm F plus fTCP dentifrice over 1,100 ppm F dentifrices. In contrast, the Clinpro™ (950 ppm F) product had a low level of total calcium phosphate and failed to show enhanced remineralization over fluoride alone in an in situ study [6]. The concentration of calcium found in saliva of volunteers was 1.4 (SD = 0.2) mmol/mL and was not different from placebo [6]. In our in vitro study, the concentration of calcium and phosphate are similar in all groups; thus, the difference found between the groups could be due to the difference in concentration of the fluoride in the products.
In the present study, the application of MPP paste resulted in a decrease in SMH and was similar to placebo and G2-FD. The remineralizing of enamel with MPP has been previously studied with conflicting results [7,8,1214,19]. The conflicting results of these studies may be related to the different methods of specimen preparation, treatment period, additional application of fluoride, and different solutions for pH cycling. In this current study, to minimize differences between methodologies, all products were applied for three minutes, once a day, as per the manufacturer’s instructions of MPP, but despite this, the results were not satisfactory. This result is in accordance with Karlinsey et al. [15] and Souza et al. [8]. These results may be due to the presence of the fluoride ion in MPP that could interact with the ACP component of the casein complex, rendering both inorganic components ineffective. According to Karlinsey et al. [15], MPP gives a relatively low level of available fluoride and does not promote significant resistance to demineralization in a pH-cycling model, thus justifying the results. In current study, ratio Ca/P and pH were lower in the G1-MPP group and could justify the low performance of this product in the model studied. However, in the oral environment, only the Tooth Mousse Plus (TMP) product significantly increased salivary calcium and inorganic phosphate concentrations [6]. The TMP product increased the salivary calcium ion concentration to 35 times higher than the Clinpro™
(950ppmF) product. The TM and TMP products were both superior to 5000 ppm F, and TMP was superior to all other products in its ability to remineralize enamel subsurface lesions in situ [6]. This enhancement of remineralization by MPP products can be attributed to the level of bioavailable calcium and phosphate ions released by these products. In the oral environment, the MPP product increased the concentration of calcium, phosphate and fluoride ions in saliva, which prevents spontaneous precipitation and allows penetration of the ions deep into subsurface lesions [4,7]. In current study, the fTCP provided similar benefits to the fluoride toothpaste; however, no additional benefit of MPP was observed. The results of our study are consistent with a prior in situ study conducted by Vanichvatana and Auychai [19].
In addition, in this current study, the surfaces of the sections were rinsed, and all remnants of the products were removed immediately after the treatment application. It might be necessary to have a longer application time to be able to detect some deposition of calcium and phosphate in a remineralized lesion [5,8,11,13,26].
In summary, the extent of demineralization was evaluated using a demineralization/remineralization pH-cycling model emphasizing the prevention of lesions on bovine enamel. The products evaluated in this study included a MI Paste Plus (900 pmm F), Clinpro™ 5000 (fTCP, 5000 ppm F) and Crest™ Cavity Protection (NaF, 1100 ppm F). The principle result of the present study is the demonstration of a dose response effect of the Clinpro™ 5000 containing functionalized tricalcium phosphate and Crest™ Cavity Protection, which, under laboratory conditions, were able to inhibit caries lesions in sound bovine enamel. The prevention of lesions may be speeded up by an exogenous Ca and P supply, but the clinical significance of the remineralization should be better evaluated. In addition, the products might provide some benefits as adjunctive therapies in children and adults at higher risk of developing caries, but it does not have general recommendation [29].
Conclusion
Clinpro™ 5000 demonstrated a greater protective effect against demineralization. Clinpro™ 5000 provided similar benefits to the 11000 ppm F toothpaste, however, no additional benefit to MPP was observed when used alone in this in vitro study.
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Table 1. Experimental groups.
Groups Products Active ingredients
G1 MI Paste Plus (MPP) CPP-ACP (10 wt.%), NaF (900
ppm)
G2 Crest™ Cavity Protection
Fluoride Dentifrice (FD) NaF (1100 ppm)
G3 Clinpro™ 5000 (CLP) NaF (5000 ppm) tricalcium phosphate (fTCP)
G4 No treatment (CO) No treatment
Table 2. Agilent's 4200 MP-AES operating conditions for the determination of Ca and P in samples.
Instrument parameter Operating condition
Microwave frequencya (MHz) 2450
Microwave powera (kW) 1.0
Nebulizer/spray chamber Inert OneNeb/cyclonic
double-pass Read time (s)/number of replicates 5 / 3
Stabilization time (s) 15
Background correction Auto
Elements (Wavelength) Ca (393.366 nm) / P (213.618
nm)
Viewing position (mm)b Ca (0.0) / P (0.5)
Nebulizer Flow (L/min) Ca (0.60) / P (0.35)
a Fixed value
Figure 1. Schematic illustration of the procedure used in the pH cycling and remineralization
treatment. MPP (MI Paste Plus); FD (fluoride dentifrice 1100 ppm F); CLP (Clinpro™ 5000 ppm F) and CO (no treatment).
Figure 2. Mean of the SMHbaseline and SMHtreated values for the four groups in the study. Results are expressed as means and standard deviations (SD). Different lowercase letters indicate significant differences in enamel SMH before treatment. Different capital letters indicate significant differences in enamel SMH among the groups after treatment (ANOVA; p < 0.05) and lowercase letters among baseline groups. (G1 = MPP; G2 = FD; G3 = CLP and G4 = CO).
G1_ base line G1_ trea ted G2_ base line G2_ trea ted G3_ base line G3_ trea ted G4_ base line G4_ trea ted 0 50 100 150 200 250 300 350 S M H a a a a a A Groups B A,B C
Figure 3. Mean of the [Ca], mg/L-1] values for the four groups in the study at 24 and 48 after pH cycling in demineralizing solution. Results are expressed as means and standard deviations (SD). Different lowercase letters indicate significant differences among groups at 24h. Different capital letters indicate significant differences at 48h (unpaired t test; p < 0.05). (*) indicates differences between 24 and 48h (paired t test; p<0.05). 24 48 24 48 24 48 24 48 0 20 40 60 80 100 DE_ time [h] [C a ], m g /L ] c a b b * A* A A A G1 G2 G3 G4 * * * *
Figure 4. Mean of the [P], mg/L-1] values for the four groups in the study at 24 and 48
after pH cycling in demineralizing solution.Results are expressed as means and standard deviations (SD). Different lowercase letters indicate significant differences among groups at 24h. Different capital letters indicate significant differences at 48h (unpaired t test; p < 0.05). (*) indicates differences between 24 and 48h (paired t test; p<0.05). 24 48 24 48 24 48 24 48 0 20 40 60 80 100 DE_time [h] [P ], m g /L a A a A A a* a A * G1 G2 G3 G4
Figure 5. Mean of the [Ca], mg/L-1] values for the four groups in the study at 24 and 48 after pH cycling in remineralizing solution. Results are expressed as means and standard deviations (SD). Different lowercase letters indicate significant differences among groups at 24h. Different capital letters indicate significant differences at 48h (unpaired t test; p < 0.05). (*) Indicates differences between 24 and 48h (paired t test; p<0.05). 24 48 24 48 24 48 24 48 0 5 10 15 RE_time [h] [C a ], m g /L ] a A a A a A a A G1 G2 G3 G4
Figure 6. Mean of the [P], mg/L-1] values for the four groups in the study at 24 and 48
after pH cycling in remineralizing solution. Results are expressed as means and standard deviations (SD). Different lowercase letters indicate significant differences among groups at 24h. Different capital letters indicate significant differences at 48h (unpaired t test; p < 0.05). (*) Indicates differences between 24 and 48h (paired t test; p<0.05). 24 48 24 48 24 48 24 48 0 5 10 15 20 25 Groups [P ], m g /L G1 G2 G3 G4 a A a A a A a A
Figure 7. Mean of the pH values for the four groups in the study after pH cycling in remineralizing (RE) and demineralizing (DE) solutions. Results are expressed as means and standard deviations (SD). Similar lowercase letters indicate no significant differences in DE solutions. Similar capital letters indicate significant no differences in RE solutions. (Kruskal Wallis; p < 0.05).
G1 G2 G3 G4 G1 G2 G3 G4 0 2 4 6 8 10 Groups P h DE RE a a a a A A A A
