UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
JULIANA DO CARMO PÚBLIO
INFLUÊNCIA DE DIFERENTES ESPESSANTES NO
CLAREAMENTO DENTAL CASEIRO: UM ESTUDO CLÍNICO
RANDOMIZADO
INFLUENCE OF DIFFERENT THICKENERS AT-HOME
TOOTH BLEACHING: A RANDOMIZED CLINICAL TRIAL
STUDY
Piracicaba 2017
JULIANA DO CARMO PÚBLIO
INFLUÊNCIA DE DIFERENTES ESPESSANTES NO
CLAREAMENTO DENTAL CASEIRO: UM ESTUDO CLÍNICO
RANDOMIZADO
INFLUENCE OF DIFFERENT THICKENERS AT-HOME
TOOTH BLEACHING: A RANDOMIZED CLINICAL TRIAL
STUDY
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Clínica Odontológica, na Área de Dentística.
Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Clinical Dentistry, in Operative Dentistry area.
Orientadora: Profa Dra Débora Alves Nunes Leite Lima
ESTE EXEMPLAR CORRESPONDE À VERSÃO FINAL DA TESE DEFENFIDA PELO ALUNO JULIANA DO CARMO PÚBLIO E ORIENTADA
PELA PROF(A). DR(A). DÉBORA ALVES NUNES LEITE LIMA.
Piracicaba 2017
Agência(s) de fomento e nº(s) de processo(s): Não se aplica. ORCID: http://orcid.org/http://orcid.org/ht
Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Odontologia de Piracicaba Marilene Girello - CRB 8/6159
Públio, Juliana do Carmo, 1984-
P96i P_O Influência de diferentes espessantes no clareamento dental caseiro: um estudo clínico randomizado ; influência de agentes espessantes no
clareamento dental / Juliana do Carmo Públio. – Piracicaba, SP : [s.n.], 2017.
P_O Orientador: Debora Alves Nunes Leite Lima.
P_ Tese (doutorado) – Universidade Estadual de Campinas, Faculdade de
Odontologia de Piracicaba.
P_O 1. Sensibilidade da dentina. 2. Dentes - Clareamento. 3. Toxicidade -
Testes. I. Lima, Debora Alves Nunes Leite,1978-. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Influence of different thickeners at-home tooth bleaching: a
randomized clinical trial study ; influence of thickeners agents on bleaching treatment
Palavras-chave em inglês:
Dentin sensitivity Teeth - Bleaching Toxicity testing
Área de concentração: Dentística
Titulação: Doutora em Clínica Odontológica Banca examinadora:
Debora Alves Nunes Leite Lima [Orientador] Cíntia Tereza Pimenta de Araújo
Alessandra Pereira de Andrade Giselle Maria Marchi Baron Flávio Henrique Baggio Aguiar
Data de defesa: 10-02-2017
DEDICATÓRIA
À Deus por tudo que me proporcionou até este momento em minha vida, guiando sempre meus passos.
Aos meus amados pais Núbia e Gilson os quais estiveram sempre ao meu lado, dando apoio e força em todos os momentos de luta, em busca de realizarmos juntos mais essa conquista em nossas vidas. Tenho por eles muito amor e enorme orgulho, sendo pais maravilhosos e presentes em minha vida. Muito Obrigada. Amo vocês!
Ao meu irmão Leonardo, sempre presente, me apoiando e ajudando com muita atenção e carinho, grande riqueza da minha vida.
AGRADECIMENTOS
À Profa. Dra. Débora Alves Nunes Leite Lima, minha querida orientadora, por toda vontade de me fazer uma pessoa e profissional melhor, pela transmissão de conhecimento, por sua competência, capacidade, paciência, e apoio em todos os momentos da minha pós-graduação. Tenho um imenso orgulho da senhora, e sou muito feliz por ter tido esta oportunidade em tê-la ao meu lado durante todos esses anos. Serei eternamente grata à Deus por tê-la me apresentado da forma mais sincera e bonita em minha vida.
À Faculdade de Odontologia de Piracicaba, na pessoa do seu Diretor Prof. Dr. Guilherme Elias Pessanha Henriques, e seu Vice-Diretor Prof. Dr. Francisco Haiter Neto onde estou tendo a oportunidade de dar um importante rumo ao crescimento profissional e científico. À Profa. Dra. Cínthia Pereira Machado Tabchoury, Coordenadora Geral do Programa de Pós-Graduação da FOP/UNICAMP.
À Profa. Dra. Karina Gonzales Silvério Ruiz Coordenadora do Curso de Pós-Graduação em Clínica Odontológica da FOP/UNICAMP.
Ao Prof. Dr. Luís Alexandre Maffei Sartini Paulillo, por toda atenção, respeito e todos seus ensinamentos que durante anos, desde a graduação, pude aprender e compreender por meio de sua habilidade em tornar a Odontologia uma profissão mágica e admirada em ser realizada com amor. Obrigada por todas as oportunidades durante minha formação.
Ao Prof. Dr. Flávio Henrique Baggio Aguiar do Departamento de Odontologia Restauradora, Área de Dentística- FOP/UNICAMP, por todo aprendizado e bons momentos durante toda a pós-graduação. Obrigada em se preocupar comigo ao longo de toda minha tese, sempre presente dando apoio nos momentos tumultuados. Grande admiração.
À Profa. Dra. Giselle Maria Marchi Baron do Departamento de Odontologia Restauradora, Área de Dentística- FOP/UNICAMP, pelo aprendizado, e sua presença serena e tranquila.
Aos Professores do Departamento de Odontologia Restauradora, Área de Dentística- FOP/UNICAMP, Prof. Dr. Luís Roberto Marcondes Martins e Prof. Dr. Marcello Gianinni, pelos ensinamentos concedidos durante a pós-Graduação.
As Professoras da Banca do meu exame de qualificação, Profa. Dra. Lúcia Trazzi Prieto, Profa. Dra. Vanessa Cavalli e a Prof. Dra. Maria Cristina Volpato, pela atenção e colaboração no aprimoramento deste estudo.
À Profa. Dra. Gláucia Mara Bovi Ambrosano, da disciplina de Bioestatística da FOP/UNICAMP, pela execução das análises deste e outros trabalhos.
Ao Anderson Catelan ex aluno de pós-graduação do Departamento de Odontologia Restauradora, Área de Dentística- FOP/UNICAMP, por todo ensinamento e atenção que sempre esteve pronto para me ajudar. Muito Obrigada por tudo.
Ao meu namorado Cleiton Pita, pessoa humana, juntos e durante anos construímos um grande e sincero amor. Meu exemplo de paciência e respeito ao próximo, luta, perseverança e determinação, ao seu lado pude aprender e viver momentos especiais. Sou feliz por tê-lo ao meu lado.
A minha grande amiga Juliana Maria, que há 22 anos de sincera amizade, esteve junto em minhas lutas e conquistas, dando apoio e me ouvindo em tudo o que eu precisava. Tenho por você um enorme carinho, amo em tê-la como amiga.
À Priscila Camondy Bertaglia, amiga linda que com seus sorrisos sinceros e abraços verdadeiros, me acolheu e deu forças, em todos os momentos de tristezas e alegrias na caminhada de pós-graduação. Obrigada por todo apoio. Você é muito querida.
À Kamila Guedes Andrade, amizade apresentada pela vida de pós-graduação que levarei para a vida toda, obrigada pelas longas conversas e boas risadas. Você é um exemplo de determinação e força de vencer na vida, demonstrando que quando desejamos algo, sempre é possível alcançá-los.
À Marília Zeczkowski, aluna de doutorado do Departamento de Odontologia Restauradora, Área de Dentística- FOP/UNICAMP, amizade que surgiu no doutorado de forma simples e singela, pude aprender o quanto é importante o respeito e sinceridade para que uma amizade cresça e faça diferença na vida, tenho um imenso carinho por você. Obrigada por tudo. À minha turma de doutorado do Departamento de Odontologia Restauradora, Área de Dentística- FOP/UNICAMP por todas as trocas de conhecimentos e aprendizados, aos meus amigos Erick, Alan, Maria do Carmo, Henrique, Tatiana, Núbia e Daniel. Muito Obrigada.
À todos meus colegas de pós-graduação mestrado e doutorado do Departamento de Odontologia Restauradora, Área de Dentística- FOP/UNICAMP, Isabel, Laura, Waldemir, Jéssica, Michele, Bruna, Maycon, Thayla, Mariana, Mari, Carol, Rodrigo, Renata, Josué e Suelen. Muito Obrigada.
Aos professores e funcionários do Departamento de Ciências Fisiológicas- Área de Farmacologia, Anestesiologia e Terapêutica- FOP/UNICAMP, principalmente aos Profs. Francisco Carlos Gropo e Maria Cristina Volpato, pela atenção e longas discussões de aprendizado em diversos momentos do meu doutorado. Muito obrigada.
Aos amigos e alunos do Departamento de Ciências Fisiológicas- Área de Farmacologia, Anestesiologia e Terapêutica- FOP/UNICAMP, Luiz Eduardo, Jonny Burga, Bruno Vilela, Bruno Nani e Josy, por todos os sorrisos quais me receberam quando estive presente no departamento, e respeito que tiveram em nossas convivências. Gosto muito de todos vocês.
Ao Adriano Luis Martins, biólogo do Departamento de Microscopia Eletrônica de Varredura da FOP/UNICAMP, por toda a paciência e didática em me ensinar à trabalhar no microscópio, sou grata por tamanho aprendizado proporcionado.
À Mônica Barnabé, ex-secretária do Departamento de Odontologia Restauradora, Área de Dentística por toda atenção e carinho, por todos os momentos de desabafos, e pela paciência em ter me escutado inúmeras vezes. Muito obrigada por sua amizade.
Aos voluntários por toda colaboração e dedicação nesse estudo. Imenso obrigada! À Drogal Manipulações por todo atendimento e colaboração durante todo esse estudo. Muito Obrigada.
E por fim, agradeço a todos que estiveram juntos no caminhar do meu doutorado, pois não podemos esquecer que conquistas não são feitas sozinhas, e sim através de apoios e presença de pessoas que nos querem bem.
RESUMO
O objetivo deste estudo clínico foi avaliar a influência do gel clareador peróxido de carbamida 10% contendo diferentes espessantes, carbopol (CPc) ou natrosol (CPn), na efetividade do clareamento, sensibilidade dental e citotoxicidade. Setenta voluntários do sexo masculino e feminino com idades entre 18 e 30 anos foram divididos aleatoriamente em dois grupos: CPc e CPn (n=35). Os géis CPc e CPn foram utilizados por quatro horas diárias, durante duas semanas. As leituras de cor dos dentes foram realizadas pelo sistema CIE Lab, com parâmetros ΔL, Δa, Δb e ΔE por meio de um espectrofotômetro de reflectância (Vita Easyshade, Vident, Brea, CA, USA) nos tempos antes do clareamento, após a primeira e segunda semana do clareamento, uma semana e um mês após o término do clareamento, nos dentes incisivo central e no canino superiores esquerdo. A avaliação da sensibilidade foi realizada por meio das escalas EVA (escala visual analógica) e descritiva antes do clareamento, durante e após uma hora do término do clareamento diário, dentro dos tempos um, três, sete, dez e quatorze dias durante o clareamento, e ainda sete e trinta dias após o clareamento. As análises de citotoxicidade “in vitro” foram realizadas por meio de teste MTT com células odontoblásticas MDPC-23, submetidos à aplicação dos agentes clareadores por 4 horas. Em Microscopia Eletrônica de Varredura (MEV), a análise qualitativa da superfície do esmalte de cada grupo foi observada por meio de réplicas. Os valores de ΔL, Δa, Δb e ΔE datados foram submetidos à análise pelo procedimento GENMOD do programa SAS. As medidas de sensibilidade dental foram analisadas pelo teste de Friedman, seguido pelo teste de Mann-Whitney (p<0,05). A distribuição normal e variâncias dos dados de viabilidade celular foram observadas após análise estatística pelos testes de Shapiro-Wilks, Levene's, análise de variância de dois fatores e Tukey foram aplicadas como análise post hoc. As análises de sensibilidade e celular foram realizadas com o pacote estatístico GraphPad® 6.0. Foi considerado nível de significância de 5% (α=0,05). Nos resultados de avaliação de cor pode-se observar que os grupos não diferiram estatisticamente em ΔL para ambos os dentes em todos os tempos. Nas análises de Δa e Δb houve diferença estatística entre os grupos CPc e CPn nos dentes incisivo e canino com maiores valores para o grupo CPc. A análise de ΔE diferiu estatisticamente entre os grupos em diferentes tempos, para os dois dentes, porém, no tempo final (Δ4) não diferiram entre si. Os tratamentos com CPc e CPn para ambos os dentes foram efetivos por apresentarem valores em ΔE≥3.3. Ambos os géis clareadores testados provocaram sensibilidade dental. Na avaliação celular, os espessantes carbopol e natrosol apresentam toxicidade similar com as células odontoblásticas MDPC-23. Nas análises em MEV, as superfícies de esmalte não apresentaram alterações como exposição prismática ou destruições pelo uso dos agentes clareadores. O clareamento com peróxido de carbamida 10% com os espessantes carbopol ou natrosol foram efetivos e ocasionaram sensibilidade dental e citotoxicidade semelhantes.
Palavras-chave: Peróxido de carbamida. Sensibilidade da dentina. Clareamento dental.
ABSTRACT
The aim of this clinical study was to evaluate the influence of bleaching gel 10% carbamide peroxide containing different carbopol (CPc) or natrosol (CPn) thickeners, on the effectiveness of bleaching, tooth sensitivity and cytotoxicity. Seventy volunteers, both male and female, between the ages of 18 to 30, were randomly divided into two groups: CPc and CPn (n = 35). The CPc and CPn gels were used four hours daily for two weeks. The color evaluation of the teeth were performed according with the CIE Lab, with ΔL, Δa, Δb and ΔE parameters by the means of a reflectance spectrophotometer (Vita Easyshade, Vident, Brea, CA, USA) at the times before bleaching, after the first and second week of bleaching, one week and one month after the end of bleaching, on the central incisor and canine upper left teeth.The tooth sensitivity assessment was performed using the VAS (Analogic visual scale)and descriptive scales before bleaching, during and one hour after the end of the daily bleaching within the times one, three, seven, ten and fourteen days during bleaching, and seven and thirty days after the end of bleaching. In vitro cytotoxicity performed were done by the MTT test with MDPC-23 odontoblastic cells, submitted to application of the bleaching agents for four hours. In Scanning Electron Microscopy (SEM), the qualitative analysis of the enamel surface of each group was observed through replicates. The values of ΔL, Δa, Δb and ΔE dated were submitted to analysis by the GENMOD procedure of the SAS program. Tooth sensitivity measurements were analyzed by the Friedman test, followed by the Mann-Whitney test (p <0.05). The normal distribution and variances of cellular viability data were observed after statistical analysis by the Shapiro-Wilks, Levene's, two-way ANOVA and Tukey tests were applied as post hoc analysis. The tooth sensitivity and cell analysis were performed with the GraphPad® 6.0 statistical package. A significance level of 5% (α=0.05) was considered. In the color evaluation results was observed that the groups did not differ statistically in ΔL for both teeth at all times. In the analyzes of Δa and Δb, there was a statistical difference between the CPc and CPn groups in the incisor and canine teeth with the highest values for the CPc group. The ΔE analysis differed statistically between the groups at different times for both teeth, but at the final time (Δ4) there were no differences between them. The treatments with CPc and CPn for both teeth were effective because they presented values in ΔE≥3.3. Both bleaching gels tested caused similar tooth sensitivity. In cell evaluation, carbopol and natrosol thickeners show similar toxicity to MDPC-23 odontoblastic cells. SEM analysis, the surfaces of the enamel showed no changes in prismatic exposure or destruction by the use of bleaching agents. Bleaching with 10% carbamide peroxide with carbopol or natrosol thickeners were effective and showed similar tooth sensitivity and citotoxicity.
Key-words: Carbamide peroxide. Tooth sensitivity. Tooth bleaching. Carboxypolymethylene.
SUMÁRIO
1 INTRODUÇÃO 12
2 ARTIGO: Influence of different thickeners at-home tooth bleaching: a 16 randomized clinical trial study
3 CONCLUSÃO 38
REFERÊNCIAS 39
APÊNDICE 1 - Detalhamento da Metodologias 42
ANEXOS
Anexo 1- Certificado- Comitê de Ética em Pesquisa 49 Anexo 2 – Registro Brasileiro de Ensaios Clínicos- ReBec 50 Anexo 3 – World Health Organization- International Clinical 51 Trials Registry Platform Anexo 4- CONSORT - Transparent Reporting of Trials 52 Anexo 5- Carta de Submissão ao Periódico- Journal of Dentistry 55
1 INTRODUÇÃO
O clareamento dental tem sido um dos tratamentos estéticos de primeira escolha na clínica odontológica, sendo considerado um tratamento conservador, que proporciona resultados estéticos satisfatórios, sem a necessidade de preparos e desgastes dentários como as coroas ou facetas (Basting et al., 2012).
As técnicas de clareamento em dentes vitais utilizam peróxido de hidrogênio e/ou peróxido de carbamida como agentes clareadores (Joiner, 2006; Joiner, 2007; Abouassi et al., 2011), ambos disponíveis na forma de gel. Na técnica de clareamento de consultório são utilizadas altas concentrações de peróxido de hidrogênio (30-40%) ou peróxido de carbamida (35-37%). Na técnica de clareamento caseiro são empregadas baixas concentrações de peróxido de hidrogênio (4-10%) ou peróxido de carbamida (10-22%) (Bernardon et al., 2010; Abouassi et al., 2011; Basting et al., 2012; Lima et al., 2014).
A eficácia do clareamento dental está relacionada com as características da estrutura dental a ser clareada e ainda com a concentração dos géis clareadores, tempo de contato do gel com as estruturas dentais. A capacidade dos radicais livres provenientes do peróxido de hidrogênio ao alcançar a dentina realizam a oxirredução dos pigmentos existentes na estrutura dental (Bernardon et al., 2010; Lima et al., 2011; Carey, 2014).
O peróxido de hidrogênio é o princípio ativo dos agentes clareadores, pode ser aplicado diretamente na superfície do dente, ou ainda ser liberado a partir da reação química do perborato de sódio ou peróxido de carbamida (Eimar et al., 2012). O peróxido de hidrogênio se dissocia em radicais livres, que oxidam moléculas orgânicas pigmentantes de cadeias longas, tornando-as em cadeitornando-as menores (grupo hidroxila) (Sulieman, 2008; Eimar et al., 2012; Soares et al., 2014). A oxidação dessas moléculas permite a eliminação destes pigmentos (Soares et al., 2014), resultando no aumento da luminosidade dental.
O peróxido de carbamida apresenta-se como uma molécula (CH6N2O3) estável, que ao
entrar em contato com a superfície do dente se dissocia em peróxido de hidrogênio (H2O2) e
ureia ((NH2)2CO). A ureia se decompõe em dióxido de carbono (CO2) e amônia (NH3), atua
como um estabilizador capaz de prolongar a vida útil do clareador, além de manter o pH do gel próximo da neutralidade. Estas características levam a uma liberação mais lenta dos radicais livres de oxigênio provenientes do peróxido de hidrogênio na estrutura dental (Navarra et al., 2014).
O peróxido de carbamida a 10% é o agente clareador com selo de certificação pela ADA (American Dental Association, 2012), considerado padrão-ouro, com eficácia comparada em
géis clareadores de baixa concentração. Além disso, estudos mostram que estes agentes clareadores em baixas concentrações quando utilizados de acordo com as recomendações dos fabricantes, ou seja respeitando o protocolo de uso, não causam alterações em células como os odontoblastos e fibroblastos (Lima et al., 2013; Almeida et al., 2015; Serraglio et al., 2016).
Com o objetivo de avaliar a ação do agentes clareadores na eficácia do clareamento dental, métodos de mensuração de cores são realizados por meio de escalas de cor e espectrofotômetro, podendo ser medidos em diferentes momentos e ao longo de todo o tratamento, permitindo acompanhar e observar a variação de cor dos dentes (Joiner 2006; Basting et al 2012; Soares et al., 2014).
As alterações em superfície de esmalte, resina composta e até mesmo a sensibilidade dental resultantes do uso dos agentes clareadores podem estar relacionados não somente ao uso do peróxido, mas também pela presença do agente espessante (Gouveia et al., 2016). Os géis clareadores contém em suas formulações um agente espessante que tem como função produzir géis cristalinos capazes de estabilizar emulsões e oferecer viscosidade às soluções. O espessante utilizado junto ao gel de peróxido de carbamida é o carbopol (polímero carboxipolimetileno) que apresenta características iônicas e baixa estabilidade de pH (Oliveira et al., 2007; Gouveia et al., 2016). Derivado do ácido carboxílico, seu pH ácido pode contribuir com a degradação da superfície do esmalte dental. Este espessante é o mais utilizado na composição dos géis clareadores caseiros; tem a função de prevenir o escoamento do agente clareador. Tal fato evita a ingestão do gel pelo paciente durante a permanência do mesmo no meio bucal (Oliveira et al., 2007).
Outro espessante disponível é o Natrosol (hidroxietilcelulose), o qual é um polímero de características não iônicas, utilizado nas indústrias farmacêuticas e de cosméticos. Tem seu uso recomendado com substâncias ácidas, por possuir pH estável (Gouveia et al., 2016). Tal característica proporciona aplicabilidade junto aos agentes clareadores (Gouveia et al., 2016). Isso pode permitir um maior controle da reação química do peróxido de carbamida nos radicais livres, causando uma penetração lenta dos radicais livres na polpa dental (Gouveia et al., 2016), podendo resultar em melhor controle da sensibilidade dentária.
Os componentes dos géis clareadores podem influenciar e promover efeitos adversos aos tecidos dentais. Entre os efeitos mais relatados temos a sensibilidade dental, que quando intensa, pode levar a suspensão do tratamento. A sensibilidade pode ocorrer em função da difusão dos agentes clareadores através do esmalte e dentina, podendo alcançar a polpa através dos túbulos dentinários, promovendo estímulos que sensibilizam os nociceptores presentes nos
túbulos dentinários, excitando-os e provocando a resposta dolorosa (Charakorn et al., 2009; Pintado- Palamino et al., 2015).
Para a avaliação da sensibilidade dental, estudos clínicos são indicados por apresentar resposta do voluntário ao tratamento proposto, porém, testes laboratoriais de avaliação de citotoxicidade são importantes mesmo em géis em baixas concentrações.
Com o propósito de avaliar a sensibilidade dental no tratamento clareador, escalas avaliadoras de dor são empregadas em estudos clínicos (Bernardon et al., 2010; de la Peña e Ratón 2014; Pintado- Palamino et al., 2015; Carlos et al., 2016). Estas escalas facilitam a mensuração da sensibilidade por meio de pontuação (Jensen et al., 1986). E dentre as escalas mais utilizadas estão: a escala analógica visual, a escala verbal de avaliação e a escala de pontuação numérica (Jensen et al., 1986; Lara-Muñoz et al., 2004).
A sensibilidade dental pode estar relacionada com a citotoxicidade do agente clareador às células, à concentração e ao número de aplicações realizadas sobre o dente. (Basting et al., 2012; Almeida et al., 2015). Testes em células odontoblásticas MDPC-23 são realizados com o objetivo de avaliar a possível citoxicidade dos clareadores em células pulpares (Lima et al., 2014; Soares et al., 2014; Almeida et al., 2015).
A literatura tem apresentado por meio de imagens em microscopia eletrônica de varredura, alterações morfológicas na superfície do esmalte decorrentes do uso de agentes clareadores (Abouassi et al., 2011; Públio et al., 2013; do Carmo Públio et al., 2013; Zeczkowski et al., 2015). Porém, muitos desses estudos avaliaram a superfície do esmalte in
vitro em diversos meios de armazenamento, como água destilada, saliva artificial ou saliva
humana previamente coletada, o que não permite uma análise real ao que ocorre na cavidade bucal (Públio et al., 2013; do Carmo Públio et al., 2013; Zeczkowski et al., 2015). Com o propósito de obter representação de alteração da superfície do esmalte, réplicas em resina epóxi são realizadas a partir de moldagens prévias dos dentes dos pacientes. Desta maneira, podendo observar a ação dos agentes clareadores, a presença da saliva, além de desafios iônicos do meio bucal no esmalte dental (Leonard et al., 2001; Navarra et al., 2014).
Diante do exposto, e com base nas análises de testes prévios do uso de agente clareador contento espessante natrosol, o presente estudo apresenta a proposta de substituir o carbopol pelo natrosol. Desta maneira, o objetivo desse estudo clínico e laboratorial foi avaliar o efeito do clareamento dental caseiro utilizando peróxido de carbamida 10% com diferentes espessantes (carbopol ou natrosol), na efetividade do clareamento (variação de cor), na
sensibilidade dental, citoxicidade e alterações morfológicas na superfície do esmalte dental resultante ao uso dos géis clareadores.
2 ARTIGO:
Influence of different thickeners in at-home tooth bleaching: a
randomized clinical trial study
Short title: Influence of thickener agents on bleaching treatment Artigo submetido ao periódico Journal of Dentistry (anexo 5)
Públio JC, Zeczkowski M, Burga-Sánchez J, Ambrosano GMB, Groppo FC, Aguiar FHB, Lima DANL.
Abstract
Objectives: To evaluate the effects of 10% carbamide peroxide (CP) with two different
thickeners, carbopol (CPc) or natrosol (CPn) on color variation, tooth sensitivity (TS) and cytotoxicity (CC).
Methods: Seventy subjects were distributed into CPc or CPn groups (n=35), in a parallel
group randomized controlled single-blind clinical trial. Bleaching gels were used by volunteers during four hours daily for two weeks. Color evaluation was performed with ΔL, Δa, Δb and ΔE by means of a reflectance spectrophotometer, before bleaching treatment (BT), first and second of BT, one week and one month after the BT ended. TS was evaluated by two pain scales, before, during and after the BT. CC was evaluated by MTT after exposure of MDPC-23 cells to the bleaching gels for four hour. Tooth epoxy replicas were made before and after of BT and analyzed on Scanning Electronic Microscope. Color changes were evaluated using the GENMOD procedure of the SAS program, TS was evaluated using Friedman test, followed by the Mann-Whitney test p<0.05. CC data were statistical analyzes by Shapiro-Wilks, Levene-s, two-way ANOVA and Tukey tests as post hoc analysis. Results: CPc and CPn showed similar color variation and TS (p≤0.05). None of the protocols affected cellular metabolism and the surface morphology of enamel. Conclusions: Bleaching gels were effective and showed similar TS. The both thickeners did not cause cytotoxicity effects.
Clinical Significance: Natrosol has been shown to be an alternative as a thickener that
can be used with bleaching gels, also tends to offer clinically satisfactory results.
Key-words: Carbamide peroxide, Tooth sensitivity, Tooth bleaching, Carboxypolymethylene,
1. Introduction
Bleaching treatment with 10% carbamide peroxide is one of the most accepted bleaching agent due to its effectiveness and biosafety in the treatment of discolored teeth and because it does not cause cellular changes[1-4]. In fact, this product got the ADA (American Dental Association) [5] Seal of Acceptance, considered the gold standard certifying.
A major concern with bleaching treatment is the adverse effects that bleaching gels can cause on tooth tissues, especially regarding to tooth sensitivity. Sensitivity to the bleaching procedure seems to be the result from the facilitated passage of hydrogen peroxide or related free radicals through the enamel and dentin achieving the pulp tissue through dentinal tubules [4,6,7].
Studies in vitro are performed in odontoblast MDPC-23 cells culture in order to evaluate the cytotoxicity of these bleaching agents [8,9]. Besides, the possible morphological changes of the enamel surface after tooth bleaching treatment is analyzed in vitro [10,11] or using the replica technique in vivo [12,13] in scanning electron microscopy .
Carbamide peroxide bleaching gel contains the thickener carbopol (carboxypolymethylene polymer) in its formulations. This product is responsible for the bleaching gel viscosity [14]. Furthermore, this thickener has the ability to keep the product in contact with dental surface prolonging the release of oxygen ions [14,15]. However, the thickener carbopol is derived from a carboxylic acid, whose acidic pH can change enamel structure, composition and morphology and so influence the tooth sensitivity.
Natrosol (hydroxyethylcellulose polymer) is a thickener, stabilizing and emulsifying agent used in cosmetic and pharmaceutical industry, which presents pH stability and can be used with acid substances such as bleaching agents [15].This can enable greater control of the chemical reaction of carbamide peroxide in free radicals, causing a slow penetration of free radical in the pulp tissue [15].Because of these characteristics, it has been suggested as an excipient on bleaching agent composition [15], demanding its in vivo evaluation, and can result in better control of the tooth sensitivity.
The control of tooth sensitivity due to bleaching gel is an important fact to be considered before the bleaching procedure to get the patient cooperation and to achieve better results. The purpose to study natrosol is to analyze its characteristics in bleaching gel in vivo, once this thickener, the natrosol, has showed promising results [15]in previous study. Therefore, the aim of this in vivo study was to compare the effects of carbopol and natrosol on the bleaching capacity, tooth sensibility and cytotoxicity. The hypothesis were: (1) the 10% carbamide
peroxide containing natrosol thickener would present an equal or better result of tooth bleaching than the commercial formulation presenting carbopol thickeners; (2) would provide lower tooth sensitivity (3) and would not be cytotoxic to MDPC-23 odontoblast cells.
2. Materials and methods
The present study was approved by the Institutional Ethics Committee (number 52007615.3.0000.5418), and described according to the Consolidated Standards of Reporting Trials (CONSORT) recommendations and based on an assumption of equivalence of treatments. The study was carried out in the clinic of the School of Dentistry from April 2016 to September 2016.
In this study two types of bleaching gel were compared. The 10% CP with carbopol (Whiteness Perfect 10%, FGM) and the 10% carbamide peroxide with natrosol (Drogal Manipulation, Piracicaba, SP, Brazil) experimental gel. The bleaching gel with natrosol was manipulated using the same basic composition of the commercial bleaching gel containing carbopol (Table 1).
Table 1. Manufactures and basic composition
Group Manufacturer Code Basic composition pH lote
CPc Whiteness Perfect 10% (FGM) CP10% Carbamide peroxide 10% Potassium nitrate 3% Sodium fluoride 0.2% Carbopol thickeners 5.82 250216 CPn Drogal Manipulation 10% CP10% Carbamide peroxide 10% Potassium nitrate 3% Sodium fluoride 0.2% Natrosol thickeners 5.86 652242
2.1 Sample size calculation
The primary outcome of this study was efficacy determined by color alteration (ΔE). This was the variable used for the determination of sample size. Considering patient as an experimental unit, the sample size was calculated to obtain a ΔE difference at the end of the study. The resulting sample size was 31 patients by group. Assuming a loss of 10% in the course of the study, the final sample size was defined as 35 participants per group. Thus, 70 patients were required. The secondary outcome was efficacy determined tooth sensitivity of 10% carbamide peroxide with carbopol or natrosol thickeners. Volunteers were randomly divided into two groups (CPc and CPn). A third person who was not involved in the intervention procedures performed the randomization process in two blocks. The allocation sequence was
placed in sealed and opaque envelopes which were only opened before the beginning of the bleaching protocol. Only the participants were unaware of the allocation group. The operator was not blinded to the procedure, due to the type of bleaching gel used, which could not be masked. Since the bleaching gels were not removed from their original packaging to avoid possible chemical changes in these products.
2.2 Patient selection
Seventy volunteers (35 men and 35 women) between 18 and 30 years old with good general and oral health - the age selected was based on adult pulp characteristics - participated in this study after signing an informed consent form with all the information regarding the risks and benefits of treatment, and additional information about the clinical trial. These volunteers fulfilled the inclusion criteria: absence of tooth sensitivity (stimulated sensitivity with air syringe); teeth with shades ranging from A2 and A3.5 reflectance spectrophotometer (Vita Easyshade, Vident, Brea, CA, USA); six maxillary and mandibular anterior teeth without caries lesions or restorations; ability to understand and respond to pain scales used. The exclusion criteria were: use of drug with anti-inflammatory, analgesic or antioxidant; dentine exposure, and with gingival recession up to 1mm; internal tooth discoloration (tetracycline stains, fluorosis, pulpless teeth); previous bleaching treatment; unable to attend follow-up appointments; dental trauma within the last six months and deleterious habits; and smokers, pregnant or breastfeeding women (Fig. 1).
Fig. 1- Flowchart of the clinical trial.
kidney stone (used medicine)(n=1) forgot to use the bleaching for 2 days (n=1)
missed 1 month trial (n=1) sore throat (used medicine) (n=1)
discontinued bleaching (n=1) missed 7day trial (n=1)
Enrollment
Randomized (n=70) Assessed for eligibility (n=128)
Allocation
Allocated to 10%CP+ carbopol (n=35) Allocated to 10%CP + natrosol (n=35)
Analysis
Analysed (n=32) Analysed (n=32)
Excluded (n=58) had canines lighter than A3 (n=21)
presence of restorations (n=7) has done bleaching before (n=12)
other reasons (n=18)
2.3 Study Intervention and Bleaching Treatments
One week before the beginning of bleaching treatment, the volunteers received a dental prophylaxis with pumice (SS White, Rio de Janeiro, RJ, Brazil) and water in a rubber cup. The volunteers were instructed to brush their teeth regularly (three times a day) using 1500 ppm fluoridated toothpaste and soft toothbrush, provided by the study investigators. Besides, the volunteers were instructed on brushing their teeth before application of bleaching gels; after the treatment time, they washed the mouth with running water and they brush their teeth after 30 minutes of finishing the daily bleaching treatment. In addition, volunteers were instructed to avoid color foods, with the aim of controlling possible changes in color analysis of the teeth throughout the study.
A full both-archs impression was obtained for each volunteer and a stone cast mold was fabricated. The bleaching trays were made in a vacuum laminator (P7- Bio-Art Dental Equipment Ltd. São Carlos, SP, Brazil) using a 1-mm-thick ethylene vinyl acetate (EVA) rubber plate (Dentalville of Brazil, Joinville, SC, Brazil). The trays were trimmed at 2 mm over the gingival margin to provide retention and stability without risking overflow bleaching gel [16].The volunteers were instructed to load the bleaching tray from second to second premolar in both arches with 10% carbamide peroxide gel with carbopol (Whiteness Perfect 10%, FGM) or 10% carbamide peroxide gel with natrosol (Drogal Manipulation, Piracicaba, SP, Brazil) and wear it for four hours daily for 14 days.
2.4 Tooth Color Shade Evaluation
A reflectance spectrophotometer (Vita Easyshade, Vident, Brea, CA, USA) was used to measure the color of the tooth, based on the CIE L*a*b* color space system, established by the Commission Internacionale de I’Eclairage (CIE; International Commission on Illumination), which allows the specification of color perceptions in three-dimensional models.
The shadewas determined using the parameter of the Easyshade device, which indicated the following values: L*, a* and b*, in which L* represents the value from 0 (black) to 100 (white) and a* and b* represents the shade, where a* is the measurement along the red-green axis and b* is the measurement along the yellow-blue axis. The shadecomparison the times of treatment (before and after) is given by the differences between the two shade (ΔE, which calculated using the formula ΔE= [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2 [17]. Three readings in each tooth were made at each time period to improve accuracy, and the arithmetic means of these readings were considered for statistical purposes.
The analysis area for shade evaluation was middle third of the buccal surface of the maxillary left central incisor and left canine. A silicone guide (Condensation Silicone Speedex- Coltene, Rio de Janeiro, RJ, Brazil) extending from maxillary canine to canine was fabricated for standardize the measurement between the different analyzed times. A window was made on the silicone guide using drill maxicut (Drill Maxicut Tungsten Carbide PM- Labordental, São Paulo, SP, Brazil) with well-formed borders.
2.5 Tooth Sensitivity Assessment
Two different pain scales were used for tooth sensitivity measurement. A visual analog scale (VAS) [18-20] which is an unidimensional measure of pain intensity that uses a 10-cm horizontal line with words “without sensitivity” at one end, and “severe sensitivity” at the opposite end. A five-point verbal rating scale (VRS) which uses adjectives to describe different levels of sensitivity (0= without sensitivity, 1=mild sensitivity, 2=moderate sensitivity, 3=considerable sensitivity, 4=severe sensitivity).
The volunteers recorded their perception of tooth sensitivity during the bleaching treatment, for three times a day (before, during and after one hour of bleaching treatment). The sensitivity measurements were done at the first, third, seventh, tenth and fourteenth days of the bleaching treatment, and at the seventh and thirtieth days after bleaching. The baseline time was used to compare the sensibility of volunteers before initial the bleaching with the after a week and after one month.
2.6 Bleaching gels pH measurement
The pH of the gels, as measured using the Orion 290A+ pH meter (Thermo Electron, Concord, CA, USA) were: CPc 5.82 and CPn 5.86.
2.7 Analysis of cytotoxicity of bleaching agent 2.7.1 Cell culture
Immortalized mouse dental papilla (MDPC-23), which present several phenotype markers for odontoblasts were used in this assay [21,22]. The cells were kept in a monolayer culture in 5% CO2 at 37°C in Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich,
USA) supplemented with antibiotics (1% penicillin 10,000 IU/ml, streptomycin 10 mg/ml - Vitrocell Embriolife, Brazil) and fetal bovine serum (10% FBS - GIBCO, Grand Island, NY, USA). The cells were collected by low speed centrifugation (300rpm) for 5min at 20°C and plated in 96 well tissue culture dishes (Product no. 3542; Costar Corp., Cambridge, MA).The
culture was replicated twice weekly by treatment with trypsin / 0.25% EDTA (1x) (GIBCO) for release of cells.
2.7.2 Test conditions
In the present study, the different concentrations of bleaching gels was based in initial pilot trial of cytotoxicity in variation of cell viability. Cells were exposed to the analyses of treatment formulations with a pH close to the physiological one and final concentration in the culture medium, as indicated below:
1. CPc bleaching: 10 mg/mL; 5 mg/ml; 2.5 mg/ml; 1.25 mg/ml; 0.63 mg/ml; 0.31 mg/ml. 2. CPn bleaching: 10 mg/ml; 5 mg/ml; 2.5 mg/ml; 1.25 mg/ml; 0.63 mg/ml; 0.31 mg/ml. 3. CPn vehicle (without carbamide peroxide): 10 mg/ml; 5 mg/ml; 2.5 mg/ml; 1.25 mg/ml; 0.63 mg/ml; 0.31 mg/ml.
4. DMSO (dimethyl sulfoxide) 0.25% - diluent used in CPc bleaching 5. PBS (phosphate-buffer saline) 0.25% -diluent used in CPn bleaching 6. Triton X-100 0.1% - negative control group, was used to kill cells.
7. DMEM culture medium - positive control group, was used to show the cellular viability. The plates (n=6), were incubated at 37°C for 4 hours (treatment time). After conclusion of the incubation, cell viability was estimated using the MTT reduction method. In this test two types of diluents was used, in the purpose to dilute the bleaching agents to make the cell treatments. The DMSO was not used to dilute the CPc and the PBS was used in the CPn. PBS was not able to diluted CPc and then the used of DMSO diluent was started.
2.7.3 Cell proliferation assay and mitochondrial function (Vybrant® MTT proliferation assay)
The proliferation capacity and metabolic function were evaluated by MTT Cell
Proliferation Assay kit (Molecular Probes Life Technologies, USA) [23]. In a Neubauer chamber, the cell counting was performed and 100μL of a solution containing approximately 5x104 cells/ml was added to each well of the tissue culture plates with 96 wells and incubated for 24h. Thereafter the wells were sold out washed twice with Phosphate-Buffer Saline (PBS- GIBCO, USA, pH 7.4), and then placed in 200 μL of MTT solution at 0.3 mg/mL in DMEM. The plates were incubated for three hours in an atmosphere of 5% CO2 at 37°C. Then the well
were washed twice with PBS and filled with 200 μL of ethanol PA. Finally, the absorbance was read in an ELISA micro-spectrophotometer (ASYS UVM340; Biochrome, England) at 570 nm. All assays were performed in triplicate in two different times.
2.8 Scanning Electron Microscope (SEM) Analysis
Four volunteers from each group were selected according with flat middle third the buccal surface of the left upper central incisor for morphological enamel analysis in SEM with epoxy resin replicas. The replicas were obtained at baseline and one day after the end of bleaching treatment. For this purpose the upper left central incisor and upper left canine were rinsed with water-spray, cleaned with cotton roller and alcohol, dried with air jets, and then an impression with silicone was taken (Express XT- 3M ESPE Sumaré, SP, Brazil).
After 24 hours, the impression was filled with epoxy resin (Buehler, Illinois, USA) and was stored at ±37°C for 5 minutes. The epoxy cast was removed from the impression, mounted on a specimen stub, and sputter-coated with gold for SEM analysis (JEOL- JSM 5600 LV, Tokyo, Japan) at a magnification of 500x and 1000x.
3. Statistical Analysis
The color data, ΔL, Δa, Δb and ΔE between initial and overtime variations were analysed using the GENMOD procedure of the SAS program considering a significance level of 5%.
The sensitivity measurements were analysed using Friedman test, followed by the Mann-Whitney test p>0.05. The sensitivity analyzes were performed with the GraphPad® 6.0 statistical package, with a significance level of 5%.
The normal distribution and variances of cellular viability data were observed after statistical analysis (Shapiro-Wilks and Levene's tests). Two-way ANOVA and Tukey's test were applied as post hoc analysis. The cellular viability analyzes were performed with the GraphPad® 6.0 statistical package, with a significance level of 5% (α=0.05).
4. Results
The characteristics of the participants included in this clinical trial are described in Table 2. Figure 1 shows the participant flow diagram in different phases of the study design.
Table 2. Demographic Features of the Participants in Each Study Group
4.1 Color Evaluation
The color measurement was done by only one operator, for all 70 volunteers, in the incisor and canine teeth at different times: before bleaching treatment (t0), 7 days after the
begging of the bleaching treatment (t1), at the end of bleaching treatment (t2), 1 week (t3) and 1
month after the bleaching treatment (t4). Tables 3 and 4, respectively, show color variation
between initial and overtime differences Δ1 (t1 – t0), Δ2 (t2 – t0), Δ3 (t3 – t0), Δ4 (t4 – t0) of ΔL, Δa,
Δb and ΔE.
The brightness variation at ΔL showed increase values overtime for incisor and canine teeth, with no statistical difference between the CPc and CPn throughout the time. However, at Δa, CPc showed significant differences from CPn at Δ3 for incisor and at Δ3 and Δ4 for canine.
Considering yellowness (Δb) significant statistical differences occurred for CPn at times Δ1, Δ2 and Δ3 for incisor and at Δ2, Δ3 and Δ4 for canine In the comparison of overall variation of color, ΔE, there were differences between CPc when compared CPn at the Δ1 and Δ3 for both teeth.
Table 3. Median (minimum/maximum value) of color measures as a function of a group and time for incisor tooth
Medians followed by different letters (within each measure vertically) show statistical differences (p≤0,05).
Feature CPc CPn
Age, mean ± SD 21.65 ± 2.72 21,88 ± 2,65
Female, n (%) 19 (54.2) 18 (51,4)
Baseline incisive color (mean ± SD, L*, a*, and b*) L*= 87.8 ± 2.9 a*= -0.36 ± 2.12 b*= 18.9 ± 3.76 L*= 87.5 ± 5.0 a*= -0.51 ± 2.71 b*= 19.5 ± 3.6
Measure Group Time
Δ1 (t1 – t0) Δ2 (t2 – t0) Δ3 (t3 – t0) Δ4 (t4 – t0) ΔL Carbopol 2.42(-11.60/7.23) a 2.62 (-3.47/6.10) a 2.88 (-9.93/8.60) a 2.92(-5.63/7.37) a Natrosol 1.44(-1.63/13.90) a 3.40(-1.57/14.17) a 2.75(-2.07/15.17) a 2.68(-1.27/14.83) a Δa Carbopol -0.90(-3.07/4.17) a -1.14(-7.63/4.10) a -1.30(-7.43/4.10) b -1.35(-7.83/3.77) a Natrosol -0.53(-3.30/0.93) a -0.88(-3.43/2.97) a -0.94(-2.57/0.07) a -0.78(-3.60/1.47) a Δb Carbopol -3.38(-8.70/1.00) b -5.32(-9.33/ -1.83) b -5.78(-9.97/-1.43) b -5.25(-12.10/-1.03) a Natrosol -2.10(-6.60/-0.60) a -3.55(-7.57/-0.40) a -3.95(-8.80/-0.80) a -5.47(-11.73/-0.60) a ΔE Carbopol 4.60(1.24/12.45) a 6.40(3.10/11.70) a 7.16(3.60/12.35) a 6.78(2.65/12.71) a Natrosol 3.32(1.15/13.99) b 5.46(2.89/14.40) a 5.20(2.06/15.49) b 6.90(2.48/15.12) a
Table 4. Median (minimum/maximum value) of color measures as a function of a group and time for canine tooth
Measure Group Time
Δ1 (t1 – t0) Δ2 (t2 – t0) Δ3 (t3 – t0) Δ4 (t4 – t0)
ΔL Carbopol 4.63(-5.50/11.77) a 5.26(-4.87/12.83) a 6.15(-16.80/13.90)a 6.10(-3.10/12.40) a
Natrosol 2.90(-1.00/12.50) a 4.65(-0.10/16.60) a 4.85(-0.20/17.40) a 4.30(-0.40/16.20) a Δa Carbopol -2.40(-12.40/0.80)a -3.25(-12.80/1.10) a -3.35(-12.00/8.00) b -3.30(-13.0/0) b
Natrosol -1.25(-5.90/1.30) a -1.80(-5.70/1.20) a -2.30-6.70/1.10) a -1.90(-7.30/3.90) a Δb Carbopol -5.65(-13.03/0.07)a -8.38(-20.80/0.07) b -9.78(-23.20/5.40) b -10.10(-21.37/-0.23)b
Natrosol -2.77(-12.13/3.27)a -5.45(-15.67/0.37) a -6.94(-17.10/1.03) a -7.58(-17.60/2.10) a
ΔE Carbopol 8.07(0/15.08) a 10.56(3.38/24.43) a 13.04(2.81/22.02) a 12.97(1.79/24.63) a
Natrosol 4.38(0.87/14.51) b 8.20(1.19/18.63) a 9.64(1.79/21.93) b 9.94(1.73/21.02) a
Medians followed by different letters (within each measure vertically) show statistical differences (p≤0,05).
4.2 Tooth Sensitivity Evaluation 4.2.1 VAS
In Fig 2, at the time before treatment, the CPc group showed a significant increase of tooth sensitivity (p <0.05) on days 10th and 14th when compared with day 1st.Measurements in periods during bleaching treatment did not present statistical differences. After treatment time showed, statistical differences on day 1st in the CPc. In the CPn group there was no statistical difference in moments before, during and after treatment time. There was no statistical difference in the comparison between the CPc and CPn groups at different times overtime (Fig. 3). CPc 0 1 2 3 4 5 1 3 7 10 14 before during after * * #
Time interval analysis (days)
S e n s it iv it y i n te n s it y ( m m ) CPn 0 1 2 3 4 5 1 3 7 10 14 before during after
Time interval analysis (days)
S e n s it iv it y i n te n s it y ( m m ) CPc/CPn - in treatment times 0 1 2 3 4 5 1 3 7 10 14 before during after * * #
Time interval analysis (days)
before during after CPc CPn S e n s it iv it y i n te n s it y ( m m )
Fig. 2- VAS values (median) of CPc and CPn sensitivity intensity in treatment times.*statistical difference in relation to overtime at the same moment (p <0.05) Friedman test, # statistical difference from the same moment at different times interval (p>0.05) Mann Whitney test.
Table 5. Median (minimum/maximum value) in mm of the graph VAS
VAS Group Time
1° 3° 7° 10° 14° before Carbopol 1(0/10) 2(0/60) 2(0/61) 2(0/62) 1,5(0/77) Natrosol 0(0/5) 0,5(0/31) 0,5(0/49) 1,5(0/46) 1,5(0/35) during Carbopol 1(0/80) 2(0/60) 3,5(0/72) 5(0/65) 2(0/80) Natrosol 0(0/14) 2,5(0/48) 0,5(0/51) 2,5(0/47) 2(0/48) after Carbopol 3(0/47) 3(0/68) 3(0/75) 3(0/79) 2(0/80) Natrosol 0(0/53) 1,5(0/50) 2(0/49) 2(0/51) 2(0/48)
(p>0.05; Mann Whitney test).
Overtime 0.0 0.5 1.0 1.5 2.0 2.5 CPc
baseline after a week
CPn
after one month
Time interval analsyis (days)
S e n s it iv it y i n te n s it y ( m m )
Fig. 3- Analysis graph of CPc and CPn sensitivity intensity on the VAS overtime.
4.2.2 VRS
In Fig. 4 the CPc group, mild, moderate and considerable sensitivity were recorded during the bleaching treatment. At the time “before”, there was a significant change in the proportion of individuals with sensitivity at times 7th, 10th and 14th days when compared with day 1st. However, at times “during” and “after” treatment there were no statistical differences overtime compared to day 1st.
In the CPn group (Fig. 4), mild and moderate sensitivities were recorded. At the moment “before” the treatment, there was a statistical difference (p <0.05) in the proportion of individuals with sensitivity from day 1st to day 3rd, which was maintained overtime. At the time “during”, the proportion of individuals with sensitivity was statistically different on days 10th and 14th from day 1st. In the “after” treatment evaluation there were no statistical differences overtime.
There was no statistical difference in the proportion of individuals with sensitivity between the CPcand CPn groups overtime and at all times when measurements were taken (Fig. 5). At the “after bleaching”, there were larger percentages of volunteers reporting sensitivities. However, after one week and at one month of bleaching, the groups did not differ statistically.
Fig. 4- Analysis graph of CPc and CPn sensitivity intensity on the VRS in treatment times.*statistical difference in relation to overtime at the same moment (p <0.05) Friedman test, # statistical difference from the same moment at different times interval (p>0.05) Mann Whitney test.
before bleaching 1 2 3 4 5 6 7 8 9 10 0 10 20 30 40 50 60 70 80 90 100 without mild moderate considerable severe * * * * * * * 1 1 l l l l l CPc CPn______ 3 CPc CPn______ 7 CPc______ 7 CPn ______ 10 CPc CPn______ 14 CPn ______CPc
Time interval analsyis
P e r c e n t a g e o f v o lu n t e e r s during bleaching 1 2 3 4 5 6 7 8 9 10 0 10 20 30 40 50 60 70 80 90 100 without mild moderate considerable severe * * l l l l l 11 l l l l l Carb Natr______ 3 Carb Natr______ 7 Carb Natr______ 7 Carb Natr______ 10 Carb Natr______ 14 Natr ______ Carb 11 l l l l l CPc CPn______ 3 CPc CPn______ 7 CPc______ 7 CPn ______ 10 CPc CPn______ 14 CPn ______CPc
Time interval analsyis
P e r c e n t a g e o f v o lu n t e e r s after bleaching 1 2 3 4 5 6 7 8 9 10 0 10 20 30 40 50 60 70 80 90 100 without mild moderate considerable severe l l l l l 11 l l l l l Carb Natr______ 3 Carb Natr______ 7 Carb Natr______ 7 Carb Natr______ 10 Carb Natr______ 14 Natr ______ Carb 1 1 l l l l l CPc CPn______ 3 CPc CPn______ 7 CPc______ 7 CPn ______ 10 CPc CPn______ 14 CPn ______CPc Time interval analsyis
P e r c e n t a g e o f v o lu n t e e r s
Before bleaching treatment
After bleaching treatment
Overtime 1 2 3 4 5 6 0 20 40 60 80 100 without mild moderate considerable severe l _______l CPc_______l CPn
after one month after a w eek
CPc CPn
_______
CPc CPn
baseline
Time interval analsyis
N u m b e r o f v o lu n te e rs
Fig. 5- Analysis graph of CPc and CPn sensitivity intensity on the VRS overtime.
In the present study there were no adverse events observed in either group.
4.3 Analysis of cytotoxicity of bleaching agent
There was no statistically significant difference (p˃0.05) compared to the cytotoxic effect between the CPc and CPn (Fig. 6), and the LD50 was 1.31 and 1.45 for the gel Test CPc and CPn, respectively. Furthermore, it was observed that both vehicle test gel as the gel diluents (Fig.7), did not affect cell viability (p˃0.05) compared to positive control (culture medium).
0 20 40 60 80 100 CPc CPn 0 1 10 Gel concentration (mg/mL) C e ll v ia b il it y %
Fig. 6- Viability of MDPC-23 cells exposed to different concentrations of two kinds of dental bleaching obtained
after 24 h incubation time. All results were normalized to a maximum value of 100% using a positive control. The data shown in the figure are mean ± SD from triplicate.
4.4 Scanning Electron Microscopy (SEM)
The SEM analysis showed no morphological changes on the enamel surface between “before” and “after” treatment periods for both bleaching agents. Photomicrographs of surfaces treated with CPc (Fig. 8-b, 8-d) showed enamel morphology with the presence of micropores and ripples, when compared to the baseline treatment (Fig. 8-a, 8-c). Similar images can be seen on surfaces treated with CPn (Fig. 9-b, 9-d) when compared to the baseline treatment (Fig. 9-a, 9-c).
Fig. 7-Effect of diluents from bleaching gel and the CPn vehicle (maximum concentration) on the viability of
MDPC-23 cells after 4h of exposure. The data shown in the figure are SD ± mean, with p˃ 0.05 from experiments performed in triplicate at two different times.
Positive control Negative control DMSO PBS CPn vehicle 0 50 100 C e ll v ia b il it y %
Fig 8. Scanning electron microscope (SEM) images of the middle third of an upper incisor treated with CPc: (a,c)
enamel before the application; (b,d) enamel after the application. Images (a, b) with x500 magnification, and images (c, d) with x1000 magnification.
Fig 9. Scanning electron microscope (SEM) images of the middle third of an upper incisor treated with CPn. (a,c)
enamel before the application; (b,d) enamel after the application. Images (a, b) with x500 magnification, and images (c, d) with x1000 magnification.
a b
c d
a b
5. Discussion
Based on the current analysis, the first hypothesis that CPn would present an equal tooth whitening when compared to CPc was accepted, because no differences were observed on ΔL and ΔE at incisor and canine color analysis at final 14 days, one week and one month after bleaching. Therefore, both bleaching treatments were effective. The CPc efficacy is proven in many studies [24-26], and the thickener change performed in the present study did not affect the CP bleaching performance. The natrosol was proposed as a thickener in CP bleaching agents and had shown promising results in the study [15].
The differences between the values of variation of tooth color between canine and incisor tooth may be related to the quantity of dentin in each teeth, which is an organic-rick substrate. The organic chromogen substances increases by continuous deposition of secondary dentin in teeth throughout life due to the physiological changes, which makes the tooth look darker [27]. The fore a greater color change is noticed on teeth that present more dentin in their composition, justifying the higher ΔL and ΔE values recorded in the canine color analyzis. The Δa analysis in incisor tooth showed difference between the groups in the time Δ3. The canine tooth showed statistical difference in Δa between the groups at Δ3 and Δ4. These values may be associated with the type of intrinsic stain and the initial teeth color they played a significant part in the final result of tooth bleaching. These values may be associated with the type of intrinsic stain and the initial teeth color they showed these final results of tooth bleaching.
The Δb analysis in incisor tooth showed statistical difference overtime between the groups, with the exception at Δ4 time. The canine tooth showed statistical difference on Δb between the groups at overtime analyzes with the exception at Δ1. In both teeth there were decreases in delta values. The reduction in parameter Δb has been reported as the most important indicator of shade change during bleaching; this variation occurs more rapidly and to greater extent than other components of CIELab system [26]. In the present study, the bleaching treatments showed efficacy in the canine, which showed increase in the Δb values. It is noteworthy in clinical studies, that dentin presents greater thickeness of organic material which contains higher pigments molecules concentration [28,29].Besides, Gerlach and others [29] showed that the younger subjects with yellowness initial tooth color exhibited the greatest mean color change post bleaching. It may be one of the reasons of the variation in the results obtained in the present study. Once the tooth containing most organic materials has a higher value variation on bleaching [28].
The thickener is added to CP bleaching gel to provide a closer contact with the tooth surface and to prolong the release of free radicals; in addition it is responsible to turn the liquid bleaching agents into gel[14,15]. The carbopol is the most used thickener in the composition of bleaching agents, although presenting characteristics of feature ionic and low stability of pH [14]. The natrosol was proposed as thickener in bleaching gels considering its large pH stability (2.0-12.0) with acidic substances or materials and non-ionic characteristics [15].
The risk caused by bleaching agents to the dentin-pulp complex depends on the ability of the peroxides of diffuse across enamel and dentin to reach the pulp tissue [30].The diffusion of the peroxides may cause damage to the pulp cells, especially to odontoblasts that underline dentin [31,32], which may result in tooth sensitivity.
Analyzing the current results, the second hypothesis that CPn would present lower tooth sensitivity throughout the bleaching treatment was rejected. In the present study no differences were observed between CPc and CPn groups concerning tooth sensitivity, independent of the sensitivity scale used, VAS or VRS. However, both groups showed significant increase in tooth sensitivity throughout the study.
The VAS and VRS scales were used to measure the TS throughout the treatment with the aim to analyze the possible different answers from the volunteers. On the VAS scale the CPc group showed significant increase on days 10th and 14th when compared with day 1st, and at the time after treatment showed statistical differences on day 1 at the same moment. On the VRS scale the groups did not show difference on statistical analyses and more sensitivity was recorded to both groups overtime of the bleaching treatments. The CPc and CPn showed difference “before” bleaching among each time when compared to time 1, although overtime these differences decreased. These differences during the time on the VAS and on the VRS may be explained by volunteers answer in the different scales. The volunteers related that the VRS is easier to answer due to the objectivity answer when compared to the VAS score.
These TS results are in agreement with those reported in the literature [27]; many studies showed that at-home bleaching frequently presents mild risk and intensity of TS, and that TS resolved during or immediately after the tooth bleaching treatment [1,3,26,27] . The TS is related to the time of the gel application on enamel, and to the concentration of the peroxide employed [24,33]. In this in vivo study the treatment protocol recommended for CPc and CPn at-home dental bleaching was four hours daily during fourteen days. This bleaching period is used and has been studied [27,32], which makes it a safe treatment. In fact, when observing the tooth sensitivity reported in the present in vivo study, none of the participants has reported any
sensitivity after one week and one month of bleaching treatments, and there was no abstention throughout the bleaching treatment.
Bleaching agents are a chemical stimulus of external origin capable of diffusing through dental structure and reach the pulp space [34] as the odontoblast cells, resulting in tooth sensitivity. The odontoblast are highly differentiated postmitotic cells, organized in a layer that underlies the dentin [34], they are responsible to produce dentin throughout the life of the tooth, besides to respond to local stimulus [35-37]. The MDPC-23 odontoblast cells in culture medium have been widely used in vitro studies to evaluate potential cytotoxic compounds and the biocompatibility of bleaching agents [9,34,38,39]. The present study showed that one application of CPc or CPn (4 hours) did not cause changes and did not kill the cells in less than 1mg/ml concentration, so the third hypothesis tested in this investigation was accepted. The CPc and CPn showed similar toxicity curves. The increase in the concentration of bleaching agents promoted reduction in the viability of odontoblasts mainly at concentrations higher than 1mg/ml. Even at the highest concentrations, the CPn vehicle was not toxic to the cells. Thus, the toxic effect is directly related to the hydrogen peroxide concentration, making HP responsible for the toxicity of the product.
The CP has approximately 3% HP, however, the HP present is capable to penetrate into enamel porosities and to diffuse through dentin, reaching the pulp tissue [34]. Studies demonstrated that CP bleaching did not cause significant effects on pulp cells, suggesting that this bleaching probably does not cause relevant alterations in dental pulp [38,39]. Moreover, many of these studies were released in vitro studies, so these results cannot be directly extrapolated to in vivo situations [8].
It is known, that dental bleaching products promote change, induce although usually temporarily, on enamel surface morphology, increasing tooth porosity [40]. Studies showed that this alteration occurred due to pH and to reactive oxidative species of the products. However, most of the studies on enamel surface analysis were performed in vitro [10,11] and frequently do not agree with in vivo observations [12,13,41]. In the present study replicates of the volunteer’s teeth treated by CPc and CPn were made. The enamel surfaces were not significantly altered (Fig.8 and 9) by any treatment. The enamel surface not showed alteration as prismatic exposition or other destruction that bleaching agent can cause. This result can be attributed to the present concentration of HP in bleaching agent be not able to change the enamel surface, besides the action of saliva in the oral cavity after removing the bleaching trays. In addition, no abrasive changes and scratches on the enamel surface were observed due to the use
of toothpaste and toothbrush. This shows the importance of use a soft toothbrushes and dentifrices that does not cause destruction of dental structures.
The bleaching agent changes the prismatic structure and the biochemical properties of the enamel, due to the loss of organic and inorganic materials [11], as carbonate and proteins from enamel [42]. The touch of saliva right after the removing of the bleaching agent, may have allowed remineralization action and the formation of the acquired pellicle [11], eliminating contact of teeth with acidic substances after bleaching.
The saliva is supersaturated with mineral, such as organic compounds calcium and phosphate, which act for the protection and maintenance of tooth structures in the dental remineralization process [43]. Besides, the saliva contains several proteins (proline-rich proteins, histatins, statherin) capable to control the deposition of minerals on teeth [11,44]. These organic components in the acquired pellicle might have acted on the enamel surface, promoting to repair of possible damages caused by bleaching agents.
In the present study the CPc and CPn were not able to change the enamel surface. Furthermore, the capacity of saliva to prevent such alterations in the dental structure, and suggests the reversibility of bleaching-related changes [45]. The 14 days regimen of CPc and CPn did not cause any effects on the morphology of enamel surface.
Bleaching agents of lower concentrations are preferred in order to minimize the effects produced by hydrogen peroxide. Based on the results of this study, the CPn bleaching treatment was an effective bleaching procedure to white teeth up. The carbopol and natrosol thickeners were not toxic to MDPC-23 odontoblasts cell. Howerer, the CPc showed the similars results when compared to CPc bleaching. The effectiveness of bleaching must be taken into consideration when choosing the safest bleaching treatment for the patient.
6. Conclusion
The CPn is a thickener that can be used in the composition of the bleaching product with safety. The CPn showed increased brightness in treatment and improving satisfaction of volunteers with dental appearance. Regard to sensitivity, both bleaching gels presented similar behavior and natrosol present in CPn did not cause toxic effects on MDPC-23 odontoblast cells.
Conflicts of interest
The authors of this manuscript certify that they have no proprietary, financial, or other personal interest of any nature or kind in any product, service, and/or company that is presented in this manuscript.
