UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
DANIELLE FERREIRA SOBRAL DE SOUZA
CARACTERIZAÇÃO DE GÉIS CLAREADORES CASEIROS QUANTO
AS PROPRIEDADES FÍSICO-QUÍMICAS APÓS ENVELHECIMENTO
ARTIFICIAL ACELERADO E SEUS EFEITOS NO ESMALTE DENTAL
CHARACTERIZATION OF HOME BLEACHING GELS REGARDING
PHYSICAL-CHEMICAL PROPERTIES AFTER ACCELERATED
ARTIFICIAL AGING AND THEIR EFFECTS ON TOOTH ENAMEL
Piracicaba 2019
DANIELLE FERREIRA SOBRAL DE SOUZA
CARACTERIZAÇÃO DE GÉIS CLAREADORES CASEIROS QUANTO
AS PROPRIEDADES FÍSICO-QUÍMICAS APÓS ENVELHECIMENTO
ARTIFICIAL ACELERADO E SEUS EFEITOS NO ESMALTE DENTAL
CHARACTERIZATION OF HOME BLEACHING GELS REGARDING
PHYSICAL-CHEMICAL PROPERTIES AFTER ACCELERATED
ARTIFICIAL AGING AND THEIR EFFECTS ON TOOTH ENAMEL
Dissertação apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para obtenção do Título de Mestra em Clínica Odontológica, na Área de Dentística.
Dissertation presented to the Piracicaba Dental School of the University of Campinas in partial fulfilment of the requirements for the Degree of Master in Dental Clinic in Operative Dentistry Area.
Orientadora: Profª. Drª. Débora Alves Nunes Leite Lima
Este exemplar corresponde à versão final da dissertação defendida pela aluna Danielle Ferreira Sobral de Souza e orientada pela Profª. Drª. Débora Alves Nunes Leite Lima.
Piracicaba 2019
“Troque suas folhas, mas não perca suas raízes. Mude suas opiniões, mas não perca seus princípios.”
DEDICATÓRIA
Dedico este trabalho aos meus maiores amores Deus, meus pais Maria Aparecida e Flávio e meu noivo Gleyson.
AGRADECIMENTO ESPECIAL
À minha querida orientadora, Profª. Drª. Débora Alves Nunes Leite Lima, por todo o ensinamento, a confiança, o incentivo, o acolhimento e o cuidado. Obrigada pela sua orientação, pela amizade, pelas oportunidades dadas e pelo convívio diário mais que agradável, a senhora foi fundamental para a concretização deste trabalho. À senhora todo o meu respeito, carinho e admiração. Deixo aqui o meu muito obrigada!
AGRADECIMENTOS
À Universidade Estadual de Campinas – UNICAMP, na pessoa do seu Magnífico Reitor Prof. Dr. Marcelo Knobel.
À Faculdade de Odontologia de Piracicaba – FOP, na pessoa de seu Diretor Prof. Dr. Francisco Haiter Neto e seu Diretor Associado o Prof. Dr. Flávio Henrique Baggio Aguiar. À Profª. Drª. Karina Gonzales Silvério Ruiz, Coordenadora Geral do Programa de Pós-Graduação da FOP – UNICAMP.
Ao Prof. Dr. Valentim Adelino Ricardo Barão, Coordenador do Curso de Pós-Graduação em Clínica Odontológica da FOP – UNICAMP.
Aos Professores do Departamento de Odontologia Restauradora, Área de Dentística – FOP/UNICAMP; Prof. Dr. Luís Alexandre Maffei Sartini Paulillo, Prof. Dr. Luís Roberto Marcondes Martins, Profª. Drª. Giselle Maria Marchi Baron, Prof. Dr. Flávio Henrique Baggio Aguiar, Prof. Dr. Marcelo Gianinni, Profª. Drª. Vanessa Cavalli Gobbo pelos ensinamentos transmitidos durante o Mestrado e pela convivência diária agradável.
Aos funcionários da Coordenadoria de Pós-graduação da FOP – UNICAMP, Ana Paula Carone Gonzalez, Claudinéia Prata Pradellan, Érica Pinho Sinhoreti, Leandro Viganó e Raquel César, por toda atenção e informações prestadas ao longo desses anos de Mestrado.
Aos Professores da minha Banca de Qualificação a Profª. Drª. Maria Cecília Giorgi, ao Prof. Dr. Waldemir Francisco Vieira Junior e ao Prof. Dr. Henrique Heringer Vieira, obrigada pela avaliação e por todas as contribuições sugeridas para aprimoramento deste trabalho.
Aos Professores da minha graduação na Universidade Federal de Pernambuco (UFPE), a Profª. Drª Hilcia Mezzalira Teixeira, o Prof. Dr. Alexandre Batista Lopes do Nascimento e o Prof. Dr. Claudio Heliomar Vicente da Silva, pelo primeiro incentivo e por me apresentarem o caminho da pesquisa, e mesmo de longe pelo acompanhamento da minha trajetória. Obrigada pela amizade e todo carinho que os senhores tem por mim
Ao Prof. Dr. José Carlos Toledo Junior, do Departamento de Química da Universidade de São Paulo – USP Ribeirão Preto e ao aluno de Doutorado André Luís Condeles, por todos os ensinamentos e ajuda durante a realização dos testes.
Ao Prof. Dr. Francisco José Krug e a Especialista de Laboratório Fátima Patreze, do Departamento de Química Analítica, pelo auxílio na realização do teste de espectrofotometria de absorbância.
Ao Técnico de Laboratório de Materiais Dentários da FOP – UNICAMP, Marcos Blanco Cangiani (Marcão), por todo auxílio e ensinamentos na utilização dos equipamentos e pelas conversas.
Ao Técnico de Laboratório de Odontopediatria da FOP – UNICAMP, Marcelo Côrrea Maistro, pelos ensinamentos e auxílio no preparo de soluções.
Ao Técnico e Biólogo do Laboratório de Microscopia eletrônica de varredura (MEV) da FOP – UNICAMP, Adriano Luís Martins, pelos ensinamentos, conversas e auxílio durante o uso do MEV.
A amiga Jaiza Araújo e ao Bruno Vilela do Departamento de Ciências Fisiológicas – Área de Farmacologia, Anestesiologia e Terapêutica da FOP – UNICAMP, por todo auxílio durante testes do mestrado.
A todos os Docentes da Faculdade de Odontologia de Piracicaba, mas em especial ao Prof. Dr. Márcio Ajudarte Lopes, da área de Semiologia Oral, por sempre abrir as portas de sua casa, fazendo com que eu me sentisse mais perto do meu Recife.
A querida Thayla Hellen Nunes Gouveia, que no início do Mestrado foi designada pela nossa Professora a me conduzir dentro do laboratório e isso foi mais do que suficiente para que nos tornássemos grandes amigas e admiradoras uma da outra. Obrigada por todo o tempo que passamos juntas, por cada experiência sua transmitida a mim, por cada vez que eu falei: “Thayla, sabe o que eu estava pensando?”, por cada lanche da tarde, por cada risada, abraço, palavras de incentivo e Fé. Meu muito obrigada!
A querida amiga de Mestrado Joyce Lima, por ser esse presente de Deus na minha vida de Pós-graduação aqui na FOP, tiveram momentos difíceis que graças a nossa amizade foram mais fáceis de serem superados. Agradeço todo apoio e por essa parceria que deu certo, quero levar essa amizade por toda a vida independentemente de qual lugar iremos.
As queridas Bruna Guerra e Renata Pereira, vocês são pessoas iluminadas, obrigada pela amizade, apoio, compartilhamento de experiências e saberes. Rê, obrigada por ter me ensinado a fazer desenhos, a partir de agora vou levar essa ideia para frente.
A amiga Mariana Flor, obrigada pelo seu jeito doce, sincero e divertido. Agradeço a sua amizade principalmente nesses últimos meses do mestrado você alegra os dias de todos nós aqui na faculdade.
Ao amigo Rodrigo Lins, obrigada pela amizade, conversas e principalmente pela companhia nas clínicas da graduação, aprendi demais com você. Mesmo com esse seu jeito “sério”, sei o quanto você é divertido e legal.
Ao amigo Josué Pierote, obrigada pelo incentivo de sempre e pelas companhias nos domingos na FOP. Sou grata pela nossa amizade.
Aos amigos de turma de Mestrado Giovana Fontanetti, Matheus Kury, Janaína Damasceno, Priscila Regis, Marco Túlio, Enrico Ângelo, Marcela Ferretti, Larissa Orlando, Jorge Soto, Maria del Carmem e aos amigos do Doutorado Maicon Sebold, Laura Ferraz, Jéssica Theobaldo, Michelle Lima, Daylana Pacheco, Mayara Noronha, Caroline Mathias e Mayara Zaghi agradeço a amizade, as palavras de incentivo, as conversas de bancadas, as experiências trocadas, as companhias em clínicas, pré-clínicas e disciplinas, foram momentos únicos.
A todos os amigos das áreas de Estomatopatologia, Odontopediatria, Farmacologia, Materiais Dentários, Endodontia, Prótese Dental, que contribuíram para que a caminhada da Pós-graduação fosse mais leve.
Ao meu amor, melhor amigo e maior incentivador diário Gleyson Amaral, obrigada por todo carinho, cuidado, paciência, cumplicidade, reciprocidade e parceria na vida e na pós-graduação. Você é o maior presente que Deus colocou em minha vida, sou eternamente grata a você por me ter em seu coração e dividir comigo o mais nobre dos sentimentos, por formar a nossa família aqui em Piracicaba, por contribuir diariamente para que minha jornada da pós-graduação fosse mais leve, por me fazer sentir capaz principalmente nos momentos de dificuldades. Obrigada também por não medir esforços para que esse sonho se tornasse realidade, essa conquista é nossa. E agradeço ao nosso filhote canino Theo que sempre é luz e alegria até nos dias mais difíceis. Amo vocês incondicionalmente.
Aos meus pais Maria Aparecida Ferreira e Flávio Sobral, aos meus irmãos Maria da Conceição, Maria Cecília, Abílio Neto e Maria Eduarda, aos meus sobrinhos Rodrigo, João Vinicius, Maria Carolina, Isabelle, Arthur e a todos os meus familiares, por me amarem, me apoiarem e por sempre compreenderem a minha ausência nesses anos de Mestrado.
Essa conquista é nossa, pois todo o esforço é por e para vocês que são minha essência e minha vida!
Aos meus Avós Genil Soledade (in memoriam), Maria do Rosário Sobral (in
memoriam) e Abilio Ferreira (in memoriam), a partida de vocês dois (Vovó Rosário e Vovô
Abílio) agora em 2018 foi muito difícil para mim, principalmente por não poder estar com vocês nesse momento, mas quero que saibaim de onde estiverem que eu agradeço todo o amor que me deram. Não virei jornalista como a minha querida Vovó Rosário falava, mas estou caminhando para uma formação de Professora que todos vocês se orgulhariam também. Amo eternamente vocês!
A minha amiga e irmã de coração Priscilla Nascimento, obrigada por sua presença apesar da distância física que nos separa, sou eternamente grata a Deus por nossa amizade. Obrigada por sempre torcer e vibrar com cada conquista. Você é um pedacinho diário do meu Recife aqui em Piracicaba! Amo você.
Agradeço a Piracicaba, que tem sido o princípio do meu crescimento espiritual, profissional e da formação da minha nova família. Obrigada a essa cidade aconchegante e maravilhosa e as pessoas que conheci aqui.
Por fim, agradeço a todos que direta ou indiretamente contribuíram de alguma maneira. Obrigada!
RESUMO
Objetivo: Avaliar as propriedades físico-químicas de géis clareadores caseiros, a base de peróxido de carbamida (PC) e peróxido de hidrogênio (PH), após envelhecimento artificial acelerado (EAA) e seus efeitos no esmalte dental. Métodos: Foram obtidos um total de 360 blocos de dentes bovinos (4x4x3mm) com 1 e 2 mm de espessura de esmalte e dentina, respectivamente. Sendo 180 blocos para as análises de cor e rugosidade (Ra) e 180 blocos para as análises de microdureza superficial (KHN) e quantificação de cálcio (Ca) que foram divididos de acordo com o tratamento clareador (n=12): Controle; PC 10% (Whiteness Perfect, FGM - WP); PC 10% (Pola night, SDI - PN); PH 7,5%(Poladay, SDI - PD); PH 7,5% (White Class Calcium, FGM - WCC). As análises foram realizadas nos seguintes tempos de EAA: sem armazenamento, 1 mês e 3 meses. As propriedades físico-químicas dos géis quanto a: dureza, compressibilidade, elasticidade, coesividade, adesividade, perda de peso, pH e quantificação de Ca no esmalte dental foram analisados após o EAA nos tempos descritos acima. As análises de cor (ΔE, ΔL*, Δa* e Δb*), Ra e KHN foram realizadas antes e após os tratamentos. As concentrações analíticas (mg / L) de Ca foram medidas no 1º, 3º e 7º dia de tratamento em todos os tempos. Os dados de KHN, Ra e quantificação de Ca foram analisados através de modelos mistos para medidas repetidas e teste de Tukey-Kramer. Os valores de peso, dureza, compressibilidade e elasticidade foram submetidos a ANOVA two way e teste de Tukey. Os dados de cor (ΔL*, Δa*, Δb* e ΔE), coesividade e adesividade foram submetidos aos testes de Kruskal Wallis e Dunn. Sendo considerado um nível de significância de 5% para todas as análises. Resultados: Os grupos submetidos ao EAA apresentaram menor efetividade clareadora (ΔE) quando comparados aos grupos sem EAA. Menores valores de KHN e maiores de Ra foram encontrados após o tratamento clareador em todos os grupos, diferindo estatisticamente do controle (p<0,05). Maiores quantidades do mineral Ca foram encontradas no 1º dia de avaliação no grupo de géis submetidos ao EAA por 3 meses, independente do agente clareador utilizado (p<0,05). A quantificação de PH não apresentou diferença significativa entre os géis (p>0,05), mas houve diminuição significativa em todos os géis após 1 e 3 meses de EAA (p<0,05). Conclusão: O gel clareador quando incorretamente armazenado pode induzir efeitos negativos nos tecidos dentais. A temperatura e umidade influenciam de forma direta a estabilidade química dos agentes clareadores, reduzindo suas propriedades físico-químicas e seu teor de uniformidade, sendo recomendado o armazenamendo a uma temperatura de 5 ºC.
ABSTRACT
Aim: To evaluate the physical-chemical properties of home bleaching gels based on carbamide peroxide (CP) and hydrogen peroxide (HP), after accelerated artificial aging (AAA) and its effects on tooth enamel. Method: Three hundred sixty blocks of bovine teeth (4x4x3mm) with 1 and 2 mm thickness of enamel and dentin were obtained, respectively. A total of 180 blocks were analyzed for color and roughness (Ra) and the remaining 180 blocks for microhardness analysis (KHN) and quantification of calcium (Ca), which were divided according to the bleaching treatment (n = 12): Control; CP 10% (Whiteness Perfect, FGM - WP); CP 10% (Pola night, SDI - PN); HP 7.5% (Poladay, SDI - PD); HP 7.5% (White Class Calcium, FGM - WCC). Analyzes were performed without storage and with storage times of 1 month and 3 months. The physical-chemical properties of the gels as: hardness, compressibility, elasticity, cohesiveness, adhesiveness, weight loss, pH and Ca quantification in dental enamel were analyzed after AAA at the times described above. Color analyzes (ΔE, ΔL*, Δa*, and Δb*), Ra, and KHN were performed before and after treatments. The analytical Ca concentrations (mg / L) were measured on the 1st, 3rd, and 7th days of treatment at all times. The data of KHN, Ra and Ca quantification were analyzed through mixed models for repeated measurements and Tukey-Kramer test. The values of weight, hardness, compressibility and elasticity were submitted to ANOVA two way and Tukey test. The color data (ΔL *, Δa *, Δb * and ΔE), cohesiveness and adhesiveness were submitted to the Kruskal Wallis and Dunn tests. A significance level of 5% was considered for all analyzes. Results: The groups submitted to the AAA presented lower bleaching effectiveness (ΔE) when compared to the AAA without group. Lower KHN values and higher Ra values were found after bleaching treatment in all groups, differing statistically from the control (p <0.05). Higher amounts of Ca mineral were found on the 1st day of evaluation in the group of gels submitted to AAA for 3 months, regardless of the bleaching agent used (p <0.05). HP quantification not showed a significant difference between the gels (p> 0.05), but there was a significant decrease in all gels after 1 and 3 months of AAS (p <0.05). Conclusions: Incorrectly stored bleaching gel can induce negative effects on dental tissues. The temperature and humidity interfer directly on the chemical stability of bleaching agents, reducing their physical-chemical properties and their uniformity content. Therefore, it is recommended to store bleaching gels at a temperature of 5 ºC.
SUMÁRIO
1 INTRODUÇÃO ... 15
2 ARTIGO: Characterization of Home Bleaching Gels regarding Physical-chemical Properties after Accelerated Artificial Aging and their Effects on Tooth Enamel ... 18
3 CONCLUSÃO ... 49
REFERÊNCIAS* ... 50
APÊNDICES ... 52
APÊNDICE 1 - Análises dos géis clareadores ... 52
APÊNDICE 2 - Análises do esmalte dental ... 56
ANEXOS ... 68
Anexo 1 - Verificação de originalidade e prevenção de plágio por meio do software Turnitin ... 68
1 INTRODUÇÃO
O clareamento de dentes com vitalidade pulpar é um dos tratamentos estéticos mais requisitados e pode ser realizado através das seguintes técnicas: o clareamento de consultório, o qual é realizado pelo Cirurgião-dentista em ambiente clínico; o clareamento caseiro, no qual o paciente irá utilizar um gel clareador em moldeira individualizada, confeccionada anteriormente pelo profissional que também irá orientar o paciente quanto à aplicação do produto e por fim existe a técnica combinada, na qual há associação das duas técnicas descritas anteriormente; o dentista realizará o clareamento em consultório e também realizará o clareamento caseiro a fim de potencializar a velocidade do resultado clareador. Estas técnicas utilizam o peróxido de hidrogênio (PH) como agente clareador em concentrações que variam de acordo com a indicação e tempo de uso, entretanto a técnica de clareamento caseiro também pode utilizar o peróxido de carbamida como agente clareador (Lima et al. 2015; de Geus et al. 2016).
O mecanismo de ação dos géis ocorre por meio da reação de oxi-redução. O peróxido de hidrogênio se decompõe, liberando radicais livres de oxigênio que penetram através das porosidades dos prismas de esmalte para a dentina, devido ao baixo peso molecular (34,01 g / mol) destas substâncias, quebrando os pigmentos que escurecem a estrutura dental (Dahl and Pallesen 2003; Souza-Gabriel et al. 2011; Kwon and Wertz, 2015; Cintra et al. 2016; Públio et al. 2016). Contudo, os radicais provenientes do peróxido de hidrogênio são altamente instáveis e apresentam atuação inespecífica, ou seja, tanto podem reagir com as duplas ligações de carbono contidas nas moléculas cromógenas que escurecem os dentes, como também com a matriz orgânica e inorgânica do esmalte e dentina para obter estabilidade molecular, contribuindo para a redução da concentração do mineral cálcio na matriz dental (Basting et al. 2005; Cavalli et al. 2011; Alqahtani 2014).
Sendo assim, durante e/ou após o tratamento clareador os efeitos impertinentes, como as alterações da morfologia, o aumento da permeabilidade, da rugosidade superficial e a redução da microdureza, podem ser observados na estrutura dentária (Giannini et al. 2006; Sa et al. 2013; de Oliveira Lima et al. 2015; Vieira-Junior et al. 2016), porém esses efeitos podem ser mediados pela ação remineralizadora da saliva (Zeczkowski et al. 2015). Além disso, também é possível observar as alterações na composição química, como por exemplo a perda de cálcio e fósforo na estrutura do esmalte que modifica a morfologia dos cristais superficiais,
o que pode indicar danos ao componente orgânico da matriz dentária (Basting et al. 2005; Cavalli et al. 2010; Sasaki et al. 2015).
Para minimizar essa perda mineral, os fabricantes têm adicionado o cálcio aos géis clareadores à base de peróxido de hidrogênio, a fim de assegurar um agente clareador com a proposta de não causar desmineralização do tecido dental e sensibilidade durante e/ou pós tratamento. Com a adição do mineral pode ocorrer uma precipitação dos íons para a superfície dental que através das trocas iônicas que ocorrem com o gel, esses íons podem ser incorporados pelo esmalte dental minimizando os efeitos adversos causados pelo clareamento, preservando a composição química, a resistência e a morfologia dental (Cavalli et al. 2010; Borges et al. 2011; Sasaki et al. 2015).
O envelhecimento artificial acelerado (EAA) preconizado pela Agência Nacional de Vigilância Sanitária - ANVISA é conhecido como Teste de estabilidade acelerada (Resolução - nº. 1, de 29 de julho de 2005) que tem por finalidade acelerar a degradação química e/ou mudanças físicas de um produto farmacêutico em condições extremas de armazenamento. Os dados encontrados podem ser utilizados para analisar os efeitos químicos e físicos prolongados em condições não aceleradas e para avaliar o resultado de curtas exposições a condições fora daquelas estabelecidas no rótulo do produto que podem ocorrer, por exemplo, durante o transporte do mesmo.
Contudo, não há dados na literatura que apontem os possíveis efeitos desses géis clareadores com cálcio no esmalte dental clareado, quanto a microdureza, rugosidade e cor após o EAA desses géis ao longo do tempo. Também é sabido que os géis clareadores ficam comumente expostos em prateleiras de lojas de produtos odontológicos, muitas vezes sem padronização de temperatura e armazenamento. Dessa forma, estudos adicionais são indispensáveis para avaliar a efetividade clareadora e estabilidade química desses produtos, assim como conhecer os efeitos químicos e físicos do cálcio nas propriedades físico-químicas dos géis clareadores e do esmalte dental após o EAA destes géis.
Neste contexto, o objetivo desse estudo foi avaliar as propriedades físico-químicas; após o envelhecimento artificial acelerado, dos géis clareadores caseiros, a base de peróxido de carbamida e peróxido de hidrogênio com ou sem adição de cálcio e seus efeitos no esmalte dental clareado. As hipóteses nulas testadas foram 1) que o envelhecimento artificial não
afetaria as propriedades físico-químicas dos géis clareadores, 2) que o gel clareador não afetaria o conteúdo mineral e as propriedades físicas do esmalte dental após o tratamento clareador.
Esta Dissertação será apresentada no formato alternativo de acordo com o Art. 2º da Informação CCPG/001/2015, § 2°.
2 ARTIGO: Characterization of Home Bleaching Gels regarding Physical-chemical Properties after Accelerated Artificial Aging and their Effects on Tooth Enamel
Abstract
Objectives. To evaluate the physical-chemical properties of home bleaching gels based on
carbamide peroxide (CP) and hydrogen peroxide (HP), after accelerated artificial aging (AAA) and its effects on tooth enamel. Methods. Three hundred sixty blocks of bovine teeth (4 x 4 x 3 mm) were used and divided according to the bleaching treatment (n = 12): Control; CP 10% (Whiteness Perfect, FGM - WP); CP 10% (Pola night, SDI - PN); HP 7.5% (Poladay, SDI - PD); HP 7.5% (White Class Calcium, FGM - WCC). Hardness, compressibility, elasticity, cohesiveness, adhesiveness, weight loss, pH, and calcium (Ca) quantification in dental enamel were firstly analyzed without storage of the bleaching gels and after AAA at 1 and 3 months of storage. The analytical Ca concentrations (mg / L) were measured on the 1st, 3rd, and 7th days of treatment at all times. The data of KHN, Ra and Ca quantification were analyzed through mixed models for repeated measurements and Tukey-Kramer test. The values of weight, hardness, compressibility, and elasticity were submitted to ANOVA two way and Tukey test. The color data analyzes (ΔE, ΔL*, Δa*, Δb*), cohesiveness, and adhesiveness were submitted to the Kruskal Wallis and Dunn tests. A significance level of 5% was considered for all analyzes. Results: The groups submitted to the AAA presented lower bleaching effectiveness (ΔE) when compared to the control group. Lower KHN values and higher Ra values were found after bleaching treatment in all groups, differing statistically from the control (p < 0.05). Higher amounts of Ca mineral were found on the 1st day of evaluation in the group of gels submitted to AAA for 3 months, regardless of the bleaching agent used. Conclusions: Incorrectly stored bleaching gel can induce negative effects on dental tissues. Temperature and humidity interfer directly on the chemical stability of bleaching agents, reducing their physical-chemical properties and their uniformity contente.
Keywords: Dental Bleaching; calcium; hydrogen peroxide; carbamide peroxide; color.
Clinical significance: Hydrogen peroxide is an unstable oxidizing agent when stored at high temperatures. In consequence, the pH becomes more acidic and potentiates the demineralizing effect on enamel.
1. Introduction
Dental bleaching represents one of the more conservative and effective treatment for discolored teeth, once it does not need to perform wear on tooth structure [1,2]. The vital tooth bleaching process uses hydrogen peroxide (HP), either directly or indirectly via its generation in a carbamide peroxide (CP) The mechanism of action of HP occurs through the oxidation-reduction reaction, in which free oxygen radicals are produced after the HP decomposition [3,4]. Due to their low molecular weight (34.01 g / mol), they penetrate through the pores of enamel prisms to the dentin breaking the pigments that darken the tooth structure [2,3,5–7]. However, the HP radicals are highly unstable and unspecific. They can react with the double carbon bonds contained in the chromogenic molecules that darken the teeth, as well as with the organic and inorganic matrix in order to obtain stability [2,8].
In result, there is mineral loss (calcium and phosphorus) [4,9,10] that contribute to the adverse effects of the bleaching treatment, such as changes in morphology, increase in permeability and surface roughness, and decrease in microhardness [11–14]. Thus, to prevent dental tissue demineralization and sensitivity during and / or after treatment, manufacturers have added calcium to the HP bleaching gels. Although the mechanism is not well understood, it has been suggested that the addition of calcium promotes the precipitation of ions on the dental surface through ionic exchanges that occur with the gel. These ions can be incorporated by the dental enamel minimizing the adverse effects caused by the whitening, preserving the chemical composition, resistance and dental morphology [1,13,14].
The recommendation of the manufacturers on the product label is to storage the bleaching gels at a temperature between 5 and 25 °C in order to keep HP stable. However, these products are commonly exposed on shelves of dental stores without standardization of temperature and storage. According to [15,16], HP undergoes decomposition realising oxygen when exposed to high temperature. Therefore, additional studies are indispensable to evaluate the bleaching effectiveness and stability of the bleaching agents after accelerated artificial aging (AAA).
However, there are no data in the literature that point out the possible effects of these bleaching agents on the bleached tooth enamel, regarding microhardness, roughness, and color after AAA. The AAA aims to accelerate the chemical degradation and /or physical changes of a product under extreme storage conditions (temperature, humidity, oxygen, pressure, sunlight, and others) to simulate the normal aging process in a shorter time [17]. The data collected through this method can be used to evaluate the result of short product exposures to unusual conditions than those stated on the label (eg. during transport of the product), as well as to analyze the prolonged chemical and physical effects on the product when stored under non-accelerated conditions.
Therefore, the aim of this study was to evaluate in vitro the physico-mechanical properties of calcium bleaching gels submitted to AAA, and their effects on the physical-mechanical and chemical properties of bleached dental enamel. The nulls hypotheses tested were 1) that the AAA would not affect the physico-chemical properties of the bleaching gels, 2) that the bleaching gel would not affect the mineral content and physical properties of the dental enamel after the bleaching treatment.
2. Material and Methods
AAA tests are experiments conducted under pre-established conditions of temperature and humidity, representing a model of the environment climatic conditions in which the products are transported and stored during their shelf-life [17,18]. The AAA of the bleaching gels containing 7.5 % hydrogen peroxide or 10 % carbamide peroxide (Table 1) was carried out on 3 months, on the following times of analysis: without storage, after 1 month of storage, and after 3 months of storage under temperature-controlled at 40 °C ± 2 °C and relative humidity of 75 % ± 5 %.
Table 1: Bleaching treatment agents, composition and protocol of use.
Bleaching Agent Manufacture Lot number Bleaching protocol* Composition* CP 10 % - WP (Whiteness Perfect®) FGM Produtos Odontológicos, Joinville, SC, Brazil 071217 4 hours/day 10 % CP, neutralized carbopol, potassium nitrate, sodium fluoride, glycol humectant, deionized water. CP 10 % - PN (Pola Night®) SDI Limited, Bayswater, Victoria, Austrália 170335 2 hours/day 10 % CP, thickener, glycerol, water, flavoring. HP 7.5 % - PD (Pola Day®) SDI Limited, Bayswater, Victoria, Austrália 170130 45minutes/day 7.5 % HP, thickener, glycerol, water, flavoring. HP Calcium 7.5 % - WCC (White Class Calcium®) FGM Produtos Odontológicos, Joinville, SC, Brazil 121217 1 hour/day 7.5 % HP, neutralized carbopol, potassium nitrate, sodium fluoride, calcium gluconate, humectant, deionized water. *Manufacturer information; HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium
The pH was measured with a Digital pH meter PHS-3B. The bleaching gels were placed in polystyrene tubes, and then the pH meter electrode was immersed in the gel, in which was kept all the time, to not induce the formation of air bubbles in the gels sample during the measurements. The electrode was in close contact with each gel according to the recommended bleaching time of each manufacturer (measuring baseline values at 15-minutes intervals). Between measures of different bleaching gels, the electrode was washed with deionized water and dried with absorbent papers. The data obtained were tabulated and submitted to simple arithmetic mean analysis.
2.3. Weight Analysis
The gels were weighed in a high precision analytical balance (AX 220, Shimadzu, Tokyo, Japan) in their respective packages (syringes), before and after AAA on the times: without storage, with 1 and 3 months of storage (n=5).
2.4. Hydrogen peroxide quantification
The chemical analysis with the selective colorimetric method for hydrogen peroxide was performed by absorbance spectrophotometry to determine the concentration of hydrogen peroxide available in the bleaching gels (n=5).
The chemical principle of this method is based on the selective iron oxidation of the horse radish peroxidase (HRP) enzyme by hydrogen peroxide. HRP compound I HRP-Fe = O5+, (equation 1), oxidizes DCFH to DCF (equation 2) as follows:
HRP-Fe3+ + H2O2 → HRP-Fe=O5+ + H2O (1)
HRP-Fe=O5+ + DCFH → HRP-Fe3+ + DCF (2)
The quantification method was performed by addition of a hydrogen peroxide standard using an analytical curve. The absorption spectrum was recorded in the range of 300 to 800 nm
immediately before and after addition of the gel with increasing concentrations of standards. The concentration of peroxide released into the gel in the diluted solution was determined by the ratio between the linear and the angular coefficients in mol L-1.
2.5. Physical-mechanical analysis properties
The physical-mechanical properties (hardness, cohesiveness, compressibility, elasticity, adhesiveness) of the bleaching gels were evaluated from the texture profile of the formulations as recommended by several studies [19,20]. Texture analysis was used as a mean to compare the texture properties of gels before and after the AAA on the times: without storage, with 1 and 3 months of storage (n=5).
Thus, a texture analyzer (TA-XT plus - Stable Micro Systems Ltd., Surrey, UK) was used. From each bleaching gel studied, 10 g were weighed and then carefully placed in a polystyrene reservoir. The 10 mm diameter compressor (polycarbonate analytical specimen) of the texture analyzer was compressed twice inside of the different bleaching gels samples at a speed of 2 mm per second, at depth of 5 mm for a time of 15 seconds between the end of the first compression and the beginning of the second one.
2.6. Sample Preparation
Enamel/dentin blocks of 4 x 4 x 3 mm, with 1 mm of enamel and 2 mm of dentin, were obtained from the middle third of the buccal surface of bovine incisors and then stored in a 0.01 % thymol solution at 4 ºC for 30 days prior the use. The sections to obtain the blocks were performed using a low-speed water-cooled diamond saw (Isomet, Buehler Ltd, Lake Bluff, IL, USA). They were then subsequently serially ground with 600-, 1200-, 2000- and 4000-grit SiC papers (Buehler Ltd) and polished with cloths and diamond paste (1 and 0.25 µm, Buehler Ltd). All specimens were placed in an ultrasonic machine for 10 minutes (Marconi, Piracicaba, São Paulo, Brazil) to remove residual particles and smear layers. A total of 360 blocks were selected,
being 180 blocks used for color and roughness (Ra) analysis and the remaining 180 blocks for Knoop microhardness analysis (KHN) and quantification of calcium (Ca). All prepared specimens were stored in artificial saliva at 37 ºC, which was renewed every day in order to simulate the humidity and temperature of the oral environment.
2.7. Specimen-staining protocol
In order to stain the samples, they were immersed in a solution of tea produced by mixing 1.6 g of black tea (Leão alimentos e bebidas Ltda, Paraná, PR, Brazil) in 100 mL of boiled distilled water for 3 minutes and infused for 5 minutes. The solution was replaced every 24 hours for 6 days. After that, the samples were stored in artificial saliva (composition: 1.5 mmol / L Ca, 0.9 mmol / L P, mmol / L KCl, Tris buffer 0.1 mol / L) with a pH adjusted for 7.0 in an incubater at 37 °C (± 1 °C) [12,21] for 14 days, with daily solution exchange for color stabilization [22].
2.8. Bleaching procedures
Prior to the bleaching treatment, sample holding with a 2:1 acrylic resin base in the center were made. To create enough space for the specimen accommodation, block of 5 x 5 mm of addition silicone (Elite HD + normal setting- © ZermackSpA- BadiaPolesine, RO, Italy) were included on the bulkheads base before the polymerization of the resin. After the polymerization, the blocks were removed and the specimens were placed with the aid of wax (Cera pegajosa, Asfer Indst. Química Ltda, São Caetano do Sul, São Paulo, SP, Brazil), leaving only the surface of the dental enamel exposed.
The bleaching treatment was performed for 7 consecutive days for analyze of the physical-chemical properties of dental enamel after the use of artificially aged bleaching gels. The gels were individually weighed on an analytical balance and standardized on each sample (Shimadzu AUW 220 d, Kyoto, Japan) until reaching a weight of 0.01 g [14]. The gels remained
on the surface of the specimens placed in hermetically sealed containers with a relative humidity at 37 oC ± 1 to simulate the oral cavity according to the time recommendation of each
manufacturer (Table 1).
After that, the specimens were rinsed with deionized water everyday. Specificlly, in the 1st, 3rd, and 7th days, the specimens were rinsed with 3 mL with deionized water, witch was collected for subsequent chemical analysis, to quantify lost mineral content. The specimens were then dried with absorbent paper (Kleenex - Kimberly-Clark, Brazil) and stored in artificial saliva (pH = 7.0) in an incubator at 37 °C.
2.9. Rinse solution collect
On the 1st, 3rd, and 7th days, all gel samples (n = 12) from each group were removed by washing with 3 mL of deionized water in closed bottles and submerging in an ultrasonic bath for 1 minute (Marconi, Piracicaba, São Paulo, SP, Brazil) and 1 minute of vigorous stirring (Solution SizerAP56, Phoenix Luferco, Araraquara, SP, Brazil) for better gel separation from the enamel surface and homogenization of the rinse solution [14,23]. After sonicating and vigorously stirring the specimens, the specimens were removed from the rinse solution and replaced in individual bottles containing artificial saliva.
The baseline rinse solution was obtained by diluting 0.07 g of gel (which was not applied to the enamel) with 21 mL of deionized water. Immediately it was homogenized on ultrasound and then stirred in a tubes stirring, as previously described. This procedure determined the Ca concentration in the bleaching gels and served as a comparison for the loss or absorption after bleaching treatment. The solution of this rinse was collected and stored at a controlled temperature (-18 °C ± 2) to later analyse its concentrations of Ca via inductively coupled absorption spectrophotometry.
For Ca quantification (n = 12) an inductively coupled plasma optical emission spectrometer - ICP OES (ICap 7400 Duo, Thermo Scientific) was used. The technique recommends that the liquid sample be dispersed in a gas phase (argon) using a quartz nebulizer (Meinhard), form droplets. These droplets are transported to a region of high temperature plasma between 5000 to 10000 K. These droplets will undergo desolvation and form microcrystals that will result in the formation of excited atoms and ions, which will produce atomic and ionic lines. These lines are the radiations of the visible and ultraviolet regions of the electromagnetic spectrum, each line formed corresponds to the concentration of the element to be analyzed in the sample (Figure 1).
Figure 1 - Layout of ICP-OES for calcium quantification
Five solutions containing five known concentrations of Ca (0.01, 0.1, 1, 2.5 and 5 mg / L) were used to calibration the equipment and define specific atomic emission spectra the mineral, with calcium being evaluated at wavelength 393.366 nm.
The data were obtained with the aid of software according to the operational parameters: frequency of the generator at 40 MHz; radio frequency power at 1.2 KW; plasma gas flow at 12 L min-1; auxiliary gas flow at 0.5 L min-1; nebulizer gas flow at 0.6 L min-1; sample flow at mL min-1; observation height at 12 mm; pump speed at 60 RPM; integration time of 15 seconds;
and replica number of 1.
2.11. Color measurements
Color reading was performed in condition an ambient light cabine (GTI Mini Matcher MM 1e, GTI Graphic Technology Inc, Newburgh, NY, USA) in standardized daylight (n = 12). The spectral distribution was measured using a reflectance spectrophotometer (CM 700 d, Konica Minolta, Osaka, Japan) based on the CIE L* a* b* system which defined the colour of an object within a three-dimensional space of colours through specific software. The L* coordinate represents the luminosity (white–black) axis, a* represents the green–red axis, and b* represents the blue–yellow axis. Before the measurements, the spectrophotometer was calibrated using white and black reflectance standards.
The differences in the L *, a * and b* values between the initial reading and final (after treatment) were expressed in ΔL *, Δa * and Δb *, and the general colour change was calculated using the following equation: ΔE = [(L1 - L0) 2 + (a1 - a0)2 + (b1 - b0)2]1/2. The analysis was
performed at before and after the treatments (bleaching).
2.12. Surface roughness (Ra)
A mechanical profilometer (Surf test- 211, Mitutoyo, Tokyo, Japan) was used to perform three measurements with the needle passing trough the center the sample in different directions turning the sample 120º at a constant speed of 0.1 mm / second and cut-off of 0.25 mm on the enamel surface using the parameter Ra (µm). The avarage value of the three readings was considered as the final mean roughness value (Ra) (n = 12).
2.13. Knoop Microhardness analysis
Knoop microhardness (KHN) analysis was performed before and after all treatments (bleaching). For analysis of the KHN, A Microdurometer tester (HMV‑2000, Shimadzu Corporation, Tokyo, Japan) with diamond indentator was used under a 50 g for 5 seconds. Five indentations on the on the enamel surface in each sample were performed as follows: The first indentation was centrally made and the other four ones at a distance of 100 µm from the center. The mean value of the five indentations was calculated as being the KHN value for each sample (n = 12).
2.14. Statistical analysis
After exploratory analyzis using R software (R Core Team 2018, Vienna, Austria), the Ca quantification and compressibility data were transformed into logarithm and the KHN square root data to meet the assumptions of the parametric analysis. After transforming the required data, KHN, Ra, and Ca quantification were analyzed by mixed models for time-repeated measures considering factorial bleaching x storage time. Multiple comparisons were performed by the Tukey-Kramer test. The variables weight, hardness, compressibility, elasticity were analyzed by ANOVA two way and Tukey test. For color variation data (ΔL *, Δa *, Δb * and ΔE), cohesiveness and adhesiveness did not meet the assumptions of a parametric analysis and were analyzed by the Kruskal Wallis and Dunn tests. The significance level was established at 5% for all analyses.
3. Results
3.1. pH Analysis
The pH was measured at 15 minutes intervals throughout the time of bleaching gel use, at the end a arithmetic mean was presented in table 2. The pH analysis indicates an decrease of pH related to the increase of storage time being the lowers values presented in the bleaching /
treatment groups of stored for 3 months at the temperature of 40 ºC and humidity of 75 %. HP Calcium 7.5 % - WCC showed the lowers pH values at times whithout storage and 3 months being considered the bleaching gel with greater demineralizing potential when compared to the others.
Table 2. Mean values of the pH analysis as a function of treatment and storage time.
Storage time Bleaching/Treatment CP 10 % WP CP 10 % PN HP 7.5 % PD HP Calcium 7.5% WCC Without storage 6.39 7.18 6.40 6.17 1 month storage 5.73 6.76 6.19 5.79 3 month storage 5.53 6.38 5.74 5.43
HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium
3.2. Weight Analysis
The weigth results are presented in Table 3. For the weigth results, the statistical analysis demonstrated an effect of the bleaching / treatment factor (p < 0.0001) and effect of storage time factor (p < 0.0001). After 3 months storage at temperature 40 ºC, and humidity at 75 % all the groups, the weigth values differed statistically from the without storage, and 1 month values (p < 0.05), with decreasing values for each of bleaching / treatment group.
Table 3. Mean values (standard deviation) of weight (g) as a function of treatment and storage time. Storage time Bleaching/Treatment CP 10 % WP CP 10 % PN HP 7.5 % PD HP Calcium 7.5 % WCC Without storage 7.53 (0.12) Ba 7.52 (0.09) Ba 7.46 (0.07) Ca 7.59 (0.05) Aa
1 month storage 7.53 (0.12) Ba 7.50 (0.10) Ba 7.48 (0.07) Ca 7.57 (0.05) Aa 3 month storage 7.45 (0.12) Bb 7.49 (0.10) Bb 7.41 (0.06) Cb 7.54 (0.06) Ab HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium. Means followed by different letters (uppercase letters in the lines and lowercase letters in the columns vertical comparing storage time) are different (p ≤ 0.05).
3.3. Hydrogen peroxide quantification
The hydrogen peroxide quantification results did not differ statistically among factor the bleaching / treatment groups (p = 0.1522), presented table 4. However, there was a significant hydrogen peroxide decrease in all bleaching / treatment groups regardless agent bleaching present after 1 and after 3 months storage (p <0.05).
Table 4. Mean (standard deviation) of the hydrogen peroxide quantification as a function of treatment and storage time.
Storage time Bleaching/Treatment CP 10 % WP CP 10 % PN HP 7.5 % PD HP Calcium 7.5% WCC Without storage 0.26 (0.02) Aa 0.24 (0.01) Aa 0.25 (0.04) Aa 0.24 (0.02) Aa 1 month storage 0.25 (0.01) Ab 0.23 (0.01) Ab 0.23 (0.04) Ab 0.22 (0.02) Ab 3 month storage 0.17 (0.01) Ac 0.17 (0.01) Ac 0.19 (0.02) Ac 0.17 (0.01) Ac HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium. Means followed by different letters (uppercase letters in the lines and lowercase letters in the columns comparing storage time) are different (p ≤ 0.05).
3.4. Physical-mechanical properties analysis
The results of hardness, compressibility, and elasticity was presented in Table 5. For the hardness results, the statistical analysis demonstrated an effect of the bleaching / treatment groups factor (p < 0.0001), storage time factor (p < 0.0001) and interaction of the factors (p=0.0001). The results hardness of CP 10 % - WP and HP Calcium 7.5 % - WCC groups was significantly higher than CP 10 % - PN and HP 7.5 % - PD, regardless of storage time (p < 0.05). After 3 month storage all bleaching gels showed lowers values of hardness when
compared to without storage, and 1 month (p < 0.05), being the highest values found in CP 10 % - WP followed HP Calcium 7.5 % - WCC, CP 10 % - PN and HP 7.5 % - PD.
For the compressibility data the statistical analysis demonstrated an effect of the bleaching / treatment groups factor (p < 0.0001), storage time factor (p < 0.0001) and interaction of theirs factors (p < 0.0001). CP 10 % - PN and HP 7.5 % - PD showed significative decrease of compressibility when compared without storage (p < 0.05). Regardless storage time CP 10 % - WP following of HP Calcium 7.5 % - WCC presented greater compressibility than CP 10 % - PN and HP 7.5 % - PD (p < 0.05).
About elasticity data, no estatistical significance were found in storage time factor (p = 0.0785), and bleaching /treatment x storage time groups factor (p = 0.1018). However, bleaching / treatment was significance (p = < 0.0001). Higher values of elasticity were found in CP 10 % - PN and HP 7.5 % - PD when compared CP 10 % - WP followed HP Calcium 7.5 % - WCC in all storage time (p < 0.05).
Concerning Table 6, for cohesiveness data, there was no statistical difference among the groups CP 10 % - WP, CP 10 % - PN, HP 7.5 % - PD, and HP Calcium 7.5 % - WCC regardless of storage time (p > 0.05). In adhesiveness data CP 10 % - PN and HP 7.5 % - PD presented significantly less negative adhesiveness than CP 10 % - WP and HP Calcium 7.5 % - WCC groups (p < 0.05). After 3 month of storage, the adhesiveness was reduced in all groups (p < 0.05).
Table 5. Mean values (standard deviation) of the variables of physical-mechanical properties hardness, compressibility and elasticity as a function of treatment and storage time
Variable Storage time
Bleaching/Treatment CP 10 % WP CP 10 % PN HP 7.5 % PD HP Calcium 7.5 % WCC Hardness (N) Without storage 0.43 (0.04) Aa 0.20 (0.01) Ba 0.15 (0.01) Ca 0.40 (0.01) Aa 1 month storage 0.44 (0.03) Aa 0.17 (0.01) Ba 0.14 (0.01) Ba 0.43 (0.02) Aa 3 month storage 0.39 (0.02) Ab 0.12 (0.01) Cb 0.11 (0.00) Cb 0.34 (0.01) Bb Compressibility (N/mm)
Without storage 1.35 (0.21) Aa 0.71 (0.02) Ba 0.55 (0.03) Ca 1.29 (0.08) Aab 1 month storage 1.49 (0.15) Aa 0.58 (0.02) Bb 0.49 (0.01) Cab 1.43 (0.08) Aa 3 month storage 1.28 (0.09) Aa 0.45 (0.03) Bc 0.42 (0.01) Bb 1.17 (0.06) Ab Elasticity (N/s)
Without storage 0.95 (0.01) Ba 1.01 (0.03) Aa 0.97 (0.02) ABa 0.94 (0.05) Ba 1 month storage 0.97 (0.01) ABa 0.99 (0.03) Aa 1.00 (0.04) Aa 0.92 (0.02) Ba 3 month storage 0.95 (0.02) Ba 1.03 (0.01) Aa 1.02 (0.04) Aa 0.94 (0.03) Ba HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium. Means followed by different letters (uppercase letters in the lines and lowercase letters in the columns vertical comparing storage time for each variable) are different (p ≤ 0.05).
Table 6. Median values (minimum and maximum) of the variables of physical-mechanical cohesiviness and adhesiveness as a function of treatment and storage time
Variable Storage time
Bleaching/Treatment
CP 10 % WP CP 10 % PN HP 7.5 % PD HP Calcium 7.5 %
WCC Cohesiveness Without storage 0.90 (0.85; 1.04) Aa 0.89 (0.87; 0.93) Aa 0.89 (0.88; 0.91) Aa 0.88 (0.82; 0.91) Aa
1 month storage 0.87 (0.83; 0.90) Aa 0.88 (0.87; 0.90) Aa 0.88 (0.84; 0.89) Aa 0.87 (0.825 0.87) Aa 3 month storage 0.89 (0.83; 0.90) Aa 0.86 (0.84; 1.00) Aa 0.84 (0.83; 0.90) Aa 0.86 (0.85; 0.87) Aa Adhesiveness
(N/mm) Without storage -0.49 (-0.54; -0.41) Ab -0.23 (-0.23; -0.22) Bb -0.19 (-0.20; -0.18) Bb -0.46 (-0.47; -0.32) Aab 1 month storage -0.45 (-0.57; -0.44) Ab -0.19 (-0.20; -0.18) Bb -0.16 (-0.17; -0.15) Bb -0.50 (-0.53; -0.42) Ab 3 month storage -0.39 (-0.41; -0.35) Aa -0.13 (-0.14; -0.10) Ba -0.11 (-0.12; -0.11) Ba -0.38 (-0.40; -0.33) Aa HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium. Medians followed by different letters (uppercase letters in the lines and lowercase letters in the columns comparing storage time for each variable) are different (p ≤ 0.05)
3.5. Calcium Quantification
The values of the relative weight of Ca (mg / L) are presented in Table 7. HP Calcium 7.5 % - WCC presented higher Ca loss than HP 7.5 % - PD in 1 st and 3 rd day evaluation for time without storage (p < 0.001) and in 3rd day for 3 month storage. Also the mean higher was HP Calcium 7.5 % - WCC than CP 10 % - WP on the 3 rd day of evaluation whithout storage and the 1st day the 1 month storage (p < 0.001). When compared with CP 10 % - PN, HP Calcium 7.5 % - WCC presented higher Ca loss in 3 rd and 7 th day of evaluation without storage and in
7 th day for 1 month storage (p < 0.001).
Concerning CP 10 % - WP, this presented higher Ca loss than CP 10 % - PN in 7 th day evaluation in times: without storage and 1 month storage (p < 0.0001). The 3 month storage groups in 1 st day evaluation time presented higher loss of Ca when compared with without storage and 1 month storage groups (p < 0.0001).
Table 7. Mean values (standard deviation) of Ca concentration (mg / L) in rinsing water as a function of treatment, storage time, and evaluation time
Storage time Evaluation time
Bleaching/Treatment
CP 10 % WP CP 10 % PN HP 7.5 % PD HP Calcium 7.5 % WCC
Without storage
1st day 0.65 (0.16) ABa 0.66 (0.13) ABa 0.53 (0.08) Bab 0.92 (0.37) Aa 3rd day 0.25 (0.08) Cb 0.36 (0.09) BCb 0.42 (0.13) Bb 0.83 (0.18) Aa 7th day 0.68 (0.15) Aa 0.32 (0.11) Bb 0.79 (0.19) Aa 0.84 (0.39) Aa 1 month storage 1st day 0.45 (0.10) Bb 0.61 (0.20) ABa 0.78 (0.12) Aa 0.90 (0.34) Aa 3rd day #0.81 (0.26) Aa #0.56 (0.14) Aa #0.76 (0.21) Aa 0.74 (0.15) Aa 7th day 0.73 (0.12) Aa 0.42 (0.09) Ba 0.87 (0.17) Aa 1.01 (0.18) Aa 3 month storage 1st day #@1.66 (0.64) Aa #@2.18 (0.62) Aa #@2.05 (0.64) Aa #@1.51 (0.42) Aa 3rd day #1.14 (0.32) Aab #@1.41 (0.36) Ab #0.67 (0.21) Bb 1.05 (0.36) Aab 7th day 0.87 (0.42) Ab #@0.78 (0.26) Ac 0.93 (0.24) Ab 0.75 (0.20) Ab HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium. Means followed by different letters (uppercase letters in the lines and lowercase letters in the columns comparing evaluation time within each storage time) are different (p ≤ 0.05). #Differs from the without storage, under the same treatment conditions and evaluation time. (p ≤ 0.05). @Differs of 1 month of storage under the same treatment conditions and evaluation time (p ≤ 0.05).
3.6. Color
Table 8 showed the values of color variation (ΔL *, Δa *, Δb *, and ΔE). ΔL * was significantly higher values of luminosity in the groups with bleaching gel, regardless of the agent used, in all storage times when compared to control (p < 0.001). Concerning 3 months storage, CP 10 % - WP presented higher value of ΔL when compared CP 10 % - PN (p < 0.001). HP Calcium 7.5 % - WCC and CP 10 % - PN showed lowers values ΔL in 3 month storage when compared to without storage an 1 month storage (p < 0.001).
For Δa * data in without storage, all the bleaching gels groups showed statistically different compared to the control (p < 0.001). Considering storage time 1 and 3 months when comparing to control no statistical differents n Δa * values (p > 0.05).
The values Δb * showed a negative variation, with a statistically significant difference compared to the control (ρ < 0.001) for all bleaching gels groups and regardless storage time. Evaluation Δb * no difference was shown among the groups CP 10 % - WP, CP 10 % - PN, HP 7.5 % - PD, and HP Calcium 7.5 % - WCC under the same conditions storage (p > 0.05).
Concerning the ΔE values the highest averages were found in the treated groups that contained bleaching agent (CP 10 % - WP; CP 10 % -PN; HP 7.5 % - PD, and HP Calcium 7.5 %) regardless of the type, concentration, and storage time with a statistically significant difference compared to the control (ρ < 0.001).
Table 8. Median values (minimum and maximum) for measures of color variation as a function of treatment and storage time. Variable Storage time
Bleaching/Treatment
Control CP 10 % WP CP 10 % PN HP 7.5 % PD HP Calcium 7.5 % WCC
ΔL*
Without storage 0.70 (-0.19; 1.31) Ba 6.94 (5.18; 15.24) Aa 6.07 (4.17; 8.42) Aa 5.09 (3.86; 9.21) Aa 5.81 (2.91; 15.39) Aa 1 month storage 0.52 (0.10; 1.80) Ba 8.83 (0.55; 15.52) Aa 5.65 (2.51; 13.71) Aa 7.61 (3.64; 11.39) Aa 6.13 (1.64; 12.24) Aa 3 month storage 0.42 (0.18; 1.34) Ca 6.15 (2.96; 9.61) Aa 3.60 (1.53; 8.83) Bb 4.94 (2.84; 6.25) ABa 3.89 (2.52; 5.75) ABb
Δa*
Without storage -0.07 (-0.41; 0.19) Aa -0.90 (-4.89; -0.16) Ba -0.74 (-1.89; 0.03) Ba -0.64 (-2.46; -0.48) Ba -0.78 (-2.90; 0.20) Ba 1 month storage -0.12 (-0.62; 0.05) Aa -0.54 (-3.28; 1.28) Aa -0.41 (-1.14; 0.70) Aa -0.63 (-1.78; 0.56)Aa -0.44 (-1.04; 0.48) Aa 3 month storage -0.43 (-0.52; -0.21) Ab -1.14 (-2.57; -0.05) Aa -0.70 (-2.94; -0.17) Aa -0.73 (-2.00; -0.26) Aa -0.82 (-1.62; -0.09) Aa
Δb*
Without storage -0.42 (-0.87; -0.09) Aab -4.66 (-11.67; 1.10) Ba -3.55 (-6.65; -0.42) Ba -3.52 (-5.53; -2.19) Ba -3.50 (-4.91; -1.69) Ba 1 month storage -0.21 (-1.85; 0.01) Aa -4.25 (-8.11; 0.88) Ba -3.86 (-5.44; -0.95) Ba -3.11 (-4.65; -1.28) Ba -2.43 (-5.42; -1.30) Ba 3 month storage -0.69 (-0.97; -0.46) Ab -4.77 (-7.09; -2.12) Ba -2.80 (-5.15; -1.85) Ba -3.39 (-5.53; -2.47) Ba -3.48 (-4.49; -2.08) Ba ΔE Without storage 0.91 (0.46; 1.50) Ba 8.59 (5.92; 18.21) Aa 7.08 (4.85; 10.27) Aa 6.18 (4.48; 10.81) Aa 6.87 (4.10; 16.28) Aa 1 month storage 0.80 (0.22; 2.20) Ba 9.71 (2.42; 17.77) Aa 7.25 (4.99; 14.31) Aa 8.07 (3.87; 11.90) Aa 6.64 (3.75; 12.49) Aa 3 month storage 1.01 (0.59; 1.63) Ba 8.25 (4.31; 11.25) Aa 4.69 (2.53; 9.69) Ab 5.93 (4.22; 8.22) Aa 5.22 (3.26; 7.36) Aa HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium. Medians followed by different letters (uppercase letters in the lines and lowercase letters in the columns comparing storage time for each variable) are different (p ≤ 0.05).
3.7. Knoop Microhardness (KHN) and Surface roughness (Ra)
The results described in Table 9 refer to the KHN and Ra values of the specimens before and after the different treatments. There was no statistical difference among the groups regarding KHN and Ra before bleaching treatment (p > 0.05).
The microhardness values decreased, regardless of the agent used or the addition of calcium. After the bleaching treatment, all groups showed lower KHN averages when compared to the control group and evaluated: without storage, with 1 and 3 months storage (p < 0.0011). For data without storage and 1 month storage, after bleaching treatment, no there was statistical difference among CP 10 % - WP, CP 10 % - PN, HP 7.5 % - PD, and HP Calcium 7.5 % - WCC groups (p > 0.05). Concerning 3 months storage, after bleaching treatment, CP 10 % - WP, HP 7.5 % - PD, and HP Calcium 7.5 % - WCC groups presented statistical difference when compared to CP 10 % - PN group (p < 0.0001).
For CP 10% - WP results after the bleaching treatment with 3 months of storage presented lower values of KHN, differing statistically in the same group and evaluation time of without storage and 1 month (p < 0.0001). HP Calcium 7.5% - WCC group with 1 and 3 months of storage showed lower values of KHN after bleaching treatment when compared without storage to the same group and evaluation time (p < 0.0001).
About Ra results, after bleaching treatment with to CP 10 % - WP, CP 10 % - PN, HP 7.5 % - PD, and HP Calcium 7.5 % - WCC groups wihtout storage and 1 month storage had significantly greater Ra than the control groups (p < 0.001), with no significant difference between bleaching agents (p > 0.05). Among the bleaching agents stored for 3 months, the HP Calcium 7.5 % - WCC, and CP 10 % - PN groups, respectively, showed roughness higher than the CP 10 % - WP, and HP 7.5 % - PD (p < 0.0001) and all differed from the control group (p < 0.001).
CP 10 % - WP data stored for 1 and 3 months showed highest values of when compared evaluation time without storage (p < 0.001). The group treated with CP 10% - PN after 3 months storage statistically differs from the without storage and 1 month in same evaluation time (p < 0.001). HP 7.5 % - PD and HP Calcium 7.5 % - WCC groups after 3 months storage presented highest values of Ra when compared to evaluation time without storage and 1 month storage (p < 0.001).
Table 9. Mean values (standard deviation) of KHN and Ra as a function of treatment, storage time, and evaluation time.
Method Storage time
Evaluation time Bleaching/Treatment Control CP 10% WP CP 10% PN HP 7.5% PD HP Calcium 7.5% WCC Microhardness
Without storage Before 340.20 (3.60) Aa 340.23 (6.85) Aa 340.36 (4.81) Aa 340.12 (5.05) Aa 340.18 (5.66) Aa After 339.83 (3.49) Aa 298.53 (15.69) Bb 292.88 (10.52) Bb 289.48 (11.06) Bb 300.86 (9.07) Bb 1 month storage Before 340.90 (10.04) Aa 340.05 (8.13) Aa 340.11 (10.83) Aa 340.00 (8.10) Aa 340.69 (7.42) Aa After 339.43 (9.82) Aa 284.20 (8.15) Bb 281.32 (17.18) Bb 281.00 (12.32) Bb #276.48 (17.04) Bb
3 month storage Before 340.11 (6.87) Aa 340.54 (4.42) Aa 340.69 (7.01) Aa 340.44 (7.19) Aa 340.30 (5.20) Aa After 339.52 (6.76) Aa #@265.81 (23.20) Cb 294.75 (15.49) Bb 278.04 (7.44) Cb #271.10 (15.57) Cb
Roughness
Without storage Before 0.10 (0.01) Aa 0.10 (0.01) Ab 0.12 (0.01)Ab 0.11 (0.01) Ab 0.11 (0.01) Ab After 0.10 (0.01) Ba 0.15 (0.01) Aa 0.15 (0.01) Aa 0.14 (0.01) Aa 0.14 (0.01) Aa 1 month storage Before 0.11 (0.01) Aa 0.10 (0.01) Ab 0.11 (0.01) Ab 0.11 (0.01) Ab 0.11 (0.01) Ab After 0.11 (0.01) Ba #0.16 (0.01) Aa 0.14 (0.01) Aa 0.15 (0.01) Aa 0.15 (0.01) Aa
3 month storage Before 0.10 (0.01) Aa 0.11 (0.01) Ab 0.11 (0.01) Ab 0.11 (0.01) Ab 0.11 (0.01) Ab After 0.11 (0.01) Ca #0.17 (0.01) Ba #@0.18 (0.01) Aa #@0.16 (0.01) Ba #@0.19 (0.02) Aa
HP: Hydrogen Peroxide; CP: Carbamide Peroxide; WP: Whiteness Perfect; PN: Pola Night; PD: Pola Day; WCC: WhiteClass Calcium. Mean values followed by diferente lettrers (uppercase letters in the lines and lowercase letters in the columns comparing evaluation time with in each storage time ) indicate statistical differences (p ≤ 0.05). # Differs from the without storage time under the same treatment conditions,
4. Discussion
Null hypothesis 1 was rejected because the AAA affected the physico-chemical properties of the bleaching gels. The null hypothesis 2 also was rejected because the mineral content and physical properties of the dental enamel were affected after the bleaching treatment.
The AAA is used to assessed the physical, chemical, and biological degradation of the material in a short time [17,18]. In this study, the AAA test was used to simulate conditions that maybe occur during wrong product transportation or storage, outside the manufacturer recomendation (temperatures between 5 ºC and 25 °C).
HP is an oxidizing agent which action is potentiated by temperature, compromising its stability, longevity and efficiency [15,16]. Thus, subjecting the bleaching gel to AAA lead to a greater components degradation, such as loss of water and weight, HP oxidation, pH reduction, and increase of air bubble formation. The 10 % CP solution is equivalent to 3.35 % HP [2,24], although this was not observed in the HP quantification results: CP values was similar to HP, when they the former should be lower. This is occurs because HP is stable when properly stored, but it undergoes decomposition with release of oxygen when present at high temperatures and pH low [15]. This justifies the lower values found in the pH, weight, and HP quantification of the 3 months’ storage groups.
When there is HP decomposition, there is release of molecular oxygen and heat, increasing the temperature and accelerating more the decomposition [15,16]. From the results of the tests performed, it is observed a greater degradation of the bleaching gels after 1 and 3 months of AAA. Thus, it is observed that these bleaching gels groups presented greatest physical-mechanical properties changes than those without storage. The texture properties of the gels are an important parameter in optimizing and maintenance the product shape. Moreover, as it is known that physical-mechanical properties of polymeric gels can be
manipulated by changes in the concentration of the polymer used, the pH of the formulation, and the presence of additives [20].
Gel hardness is the force required to promote deformation in the gel in relation to viscosity. This expresses the gel applicability in the teeth. Adhesiveness, which an indicator for the retention time on the enamel, are directly correlated to the polymer concentration (carbopol or glycerol). It is a desirable characteristic, as a higher adhesiveness value could imply greater adhesion at the tissue surface and increase the retention time [19]. Compressibility evaluates the ability of the gel to with stand the compression exerted during the test. Cohesiveness is the work required to deform the gel during the first compression of the compressor disc, evaluation how cohesive the molecules are. Elasticity is the ability of the gel to return to it original shape [19,20,25]. It was observed that all the gels that were subjected to AAA showed a reduction in their physical-mechanical properties.
Bovine teeth present morphological characteristics and physical-chemical properties resembling from of human enamel [26]. Some authors evaluated mineral loss of calcium associated with bleaching with different methodologies [9,10,14,23,27]. However, we opted for optical emission spectrometry evaluation with inductively coupled plasma (OES). ICP-OES is considered one of the most important methods of atomic spectrometry. Its advantages are: multi elemental atomic detection (up to 60 elements in a single sample), high precision, sensitivity and speed of analysis (1 minute) and low sample consumption (2 mL) [28–30]. Thus, Ca detection was quantified in the rinse solution during the bleaching treatment. HP Calcium 7.5 % WCC showed higher values of Ca when compared to HP 7.5 % PD, in the 1st and 3rd day of bleaching no storage. In the 1st day of bleaching, the groups with 3 months storage presented higher amounts of Ca than the without storage and the 1 month groups.
HP can produce different types of reactive oxygen species during its reaction, depending on temperature and pH. Therefore, radicals, such as superoxide and hydroxyl anions, under
conditions of non-alcaline pH, may interact with the organic and inorganic enamel matrix, favoring demineralization and increasing the loss of mineral content [15,24]. This explains the presence of Ca after the bleaching treatment in the rinse solution, since calcium is one of the dental matrix constituents.
In this study, the specimens were previously stained with a black tea solution to standardize their initial color. Any reduction in the reddish-brown color shades prove the effectiveness of the bleaching method [8,21]. The color of the dental enamel was assessed according to the CIE system (L* a* b*), which quantifes the general color variation (∆E) [8,31]. The values for ΔL revealed an increase in the luminosity of all specimens treated, regardless of the type of bleaching agent used. The changes into ∆a* parameter occurred from the a* + (red) axis to the a* - (green) axis, showing less reddish-brown shades. Changes from the b* + (yellow) axis to the b* - (blue) axis were observed, showing less yellowish shade.
A thereshold of perception in colour variation occurs when ΔE >3 (ΔE = 3.7) [1,4,7]. In this present study, variation in color was hight and clinically perceptible, regardless of the bleaching agent used or storage time (∆E > 3). Therefore, different bleaching treatments were able to alter the color of the dental enamel. However, the time and conditions of the storage influenced negatively in the ∆E, possible due to HP greater degradation. It been well accepted in the literature that hydrogen peroxide has unspecific action within the dental structure [2,4,8]. The AAA was performed to simulate the conditions that often the bleaching gel is stored, marketed and / or transported by dental stores. Therefore, with the findings of this study, the demineralization of the enamel during a bleaching treatment can be attributed to the mechanism of the action of HP, pH, thickeners, and storage type.
The agents bleaching used contain thickeners a carbopol or a glycerin base. Carbopol slows down the release of hydrogen peroxide and is thus effective over a longer period of time. The formed film prevents ion exchange with the saliva in the remineralization process [2,32].
Thus, greater changes in the surface of the dental enamel are observed. The thickeners are viscosity agents that act as modulators in the bleaching oxy-reduction reaction, releasing hydrogen peroxide [9]. Due this component is acid, its may cause demineralization of the dental enamel surface and alter the morphology, changing the light reflectance pattern [9,32].
The dental enamel KHN decreased and the Ra increased in all groups when the bleaching agents were subjected to AAA or not, corroborating with studies that observed morphology changes during/after bleaching treatment [12,13,23,33]. The low KHN and high Ra values found in specimens treated with the 1 and 3 months storage gel may results from the change in the enamel morphology surface, caused by the action of the thickener and low pH of the bleaching resulting in erosive/demineralization effect.
The presence of calcium in the bleaching gel formulation altered the mineral content of tooth enamel, could be explained by the fact that there was demineralization due to low pH of bleaching gel. HP Calcium 7.5 % WCC (calcium gluconate) was expected to have a positive effect on tooth enamel, decreasing the changes on physical-mechanical properties and mineral content [34], but this was not observed. According to Furlan et al., 2017 [34] the effects of calcium gluconate are still being studied. It has been reported that calcium gluconate may have interfered with the transitional mineral exchanges that occurred during the bleaching process, resulting in a worse action of the gels [24]. Since that perhydroxyl and reactive oxygen released during bleaching could be inhibited by calcium gluconate [34].
5. Conclusions
- Incorrectly stored bleaching gel can induce negative effects on dental tissues.
- The temperature and humidity interfer directly on the chemical stability of bleaching agents, reducing their physical-chemical properties and their uniformity content. Therefore, it is recommended to store bleaching gels at a temperature of 5 ºC.
