UNIVERSIDADE FEDERAL DE PELOTAS Faculdade de Odontologia
Programa de Pós-Graduação em Odontologia
Dissertação
Metacrilato piperonílico: coiniciador copolimerizável para composição de sistemas de polimerização fotoiniciada
Andressa Goicochea Moreira
Andressa Goicochea Moreira
Metacrilato piperonílico: coiniciador copolimerizável para composição de sistemas de polimerização fotoiniciada
Dissertação apresentada ao Programa de Pós-Graduação da Faculdade de Odontologia da Universidade Federal de Pelotas, como requisito parcial para obtenção do título de Mestre em Odontologia, Área de concentração Dentística.
Orientadora: Profa Dra Giana da Silveira Lima Co-Orientador: Prof. Dr. Fabricio Aulo Ogliari
Co-Orientador: Prof. Dr. Evandro Piva
Andressa Goicochea Moreira
Metacrilato piperonílico: coiniciador copolimerizável para composição de sistemas de polimerização fotoiniciada
Dissertação aprovada como requisito parcial, para obtenção do grau de Mestre em Odontologia, Área de concentração Dentística. Programa de Pós-graduação em Odontologia. Faculdade de Odontologia. Universidade Federal de Pelotas.
Data da defesa: 29 de março de 2017.
Banca examinadora:
Prof. Drª. Giana da Silveira Lima
Doutora em Odontologia (área de concentração Dentística) pela Universidade Fede-ral de Pelotas.
Prof. Dr. Neftali Lenin Villarreal Carreño
Doutor em Ciências pela Universidade Federal de São Carlos. Prof. Dr. Rafael Guerra Lund
Doutor em Odontologia (área de concentração Dentística) pela Universidade Fede-ral de Pelotas
Prof. Drª. Adriana Fernandes Silva
Doutora em Biologia Buco-Dental (área de concentração de Histologia e Embriologia Bucal pela Universidade Estadual de Campinas
Prof. Dr. Rafael Ratto de Moraes
Dedico este trabalho as pessoas que amo, a todos colegas, que contribuiram tanto
diretamente, quanto indiretamente para concretização deste.
Agradecimentos
Agradeço a Deus por iluminar todos meus passos.
À Universidade Federal de Pelotas, na pessoa do Magnífico Reitor Pedro Curi Hallal.
À Faculdade de Odontologia, nas pessoas da Excelentíssima Diretora Profª Drª Adriana Etges e Excelentíssimo Vice-Diretor Prof. Dr. Luis Eduardo Rilling da Nova Cruz.
Ao Programa de Pós-Graduação em Odontologia, na pessoa do Coorde-nador do PPGO, Prof. Dr. Rafael Ratto de Moraes.
À minha orientadora, Profª. Drª. Giana da Silveira Lima, por ter aceitado fa-zer a orientação dessa dissertação, por toda a atenção e dedicação dispensada, fazendo com que esse trabalho se tornasse concreto. Por ser um exemplo de res-ponsabilidade e profissionalismo, o qual com certeza levarei como inspiração.
Aos meus co-orientadores, Prof. Dr. Evandro Piva e Prof. Dr. Fabrício Aulo Ogliari. Sou extremamente grata pela ajuda, empenho e apoio em todos os momen-tos.
A toda a equipe dos laboratórios CDC-Bio, Microbiologia, NCTBio e aos seus professores responsáveis Evandro Piva, Rafael Lund, Adriana Silva e aos técnicos destes laboratórios Tatiana Ramos, Lisângela Ferreira, muito obrigada por deixarem utilizar estes espaços para realização dos ensaios laboratoriais de mi-nha dissertação bem como obrigada pelos auxílios prestados.
Ao colega e amigo Carlos Enrique Cuevas Suárez, não há palavras para agradecer a ajuda prestada no delineamento e execução das metodologias dessa dissertação. Levo comigo o exemplo de pessoa que és: dedicado, solícito e cordial em todos os aspectos. Obrigada.
A colega de mestrado e amiga Juliana Ribeiro obrigada pela ajuda concedi-da na realização dos testes de microbiologia, e toconcedi-da a convivência diária que sem duvidas tornaram os dias mais felizes.
A amiga Leina Nakanishi, obrigada por toda paciência e ajuda a mim dedica-dos nesses dois anos, sua ajuda nos testes desenvolvidedica-dos foram muito importantes. Obrigada pela convivência e amizade.
A Colega de mestrado e amiga Raíssa Coi de Araujo, obrigada pelos finais de semana que abriste mão para me ajudar, juntas aprendemos muitas coisas, obri-gada amiga.
Ao doutorando p0e colega Wellington Luiz O. da Rosa, agradeço pela ajuda na realização dos ensaios no laboratório NCTBio.
Aos que de início eram colegas e hoje são amigos, Katielle Brauner, José Augusto, Eliseu Munchou, Rafaella Araujo, Carine Meeires, Cristina Isolan, Li-sia Lorea, Fernanda Leal, Lucas Brondani. Agradeço pelos ótimos momentos de convivência e por todo apoio nessa jornada.
Aos alunos da iniciação Científica Peterson Boeira, Graziele lowe, Henrique Fedalto muito obrigada pela ajuda na realização dos testes laboratoriais, pela ami-zade e parceria que criamos.
Aos meus pais, Almir Mackedanz Moreira e Luciana Goicochea Moreira, que são minha base, meu porto seguro, pessoas que me mostraram qual caminho percorrer e deram todo o apoio necessário para que esse sonho se tornasse reali-dade. Agradeço a eles todo o carinho e amor a mim dedicados. Obrigado, amo vo-cês.
Ao meu namorado Guilherme de Almeida pela paciência e dedicação, nos momentos finais deste trabalho.
Aos queridos amigos, estes que não precisam ser citados, mas que sabem que fazem parte das nossas vidas, que souberam entender às vezes em que estive ausente por conta dos estudos. Em tantas datas ficamos afastados, mas quando nos reencontrávamos a amizade sempre prevaleceu. Obrigado por todo carinho.
Aos que passaram por minha vida, mas infelizmente não estão mais presen-tes lembro vocês com saudades.
Notas Preliminares
A presente Dissertação foi redigida segundo o Manual de Normas para traba-lhos acadêmicos da UFPel, adotando o nível de descrição em capítulos não
conven-cionais.Disponível no endereço eletrônico:
http://sisbi.ufpel.edu.br/arquivos/PDF/Manual_Normas_UFPel_trabalhos_acad%C3 %AAmicos.pdf
O projeto de pesquisa que originou essa dissertação foi apresentado dia 25 de agosto de 2015 e aprovado pela Banca Examinadora composta pelas Prof.ª Dr.ª Adriana Fernandes da Silva, Dr.ª Bianca Palma Santana, Dr.ª Marina da Rosa Kai-zer.
Resumo
MOREIRA, Andressa Goicocchea. Metacrilato piperonílico: coiniciador copolimerizável para composição de sistemas de polimerização fotoiniciada 2017. N˚ 54f Dissertação (Mestrado em Odontologia) - Programa de Pós-Graduação em Odontologia, Faculdade de Odontologia, Universidade Federal de Pelotas, Pelotas.
A evolução dos materiais odontológicos vem contribuindo para que se consiga al-cançar a excelência em procedimentos estéticos. Diversos fatores clínicos motivam novos estudos para desenvolvimento de materiais que consigam atingir proprieda-des ópticas, mecânicas e biológicas semelhantes ao elemento dental. Assim, esta pesquisa busca contribuir para a evolução desses materiais por meio de estudos in vitro. Portanto os objetivos deste trabalho foram realizar a síntese de uma molécula, o metacrilato piperonílico, por meio de uma rota de sintese alternativa à utilização do cloreto de metacriloila, com tocixidade conhecida, e analisar se essa moléula possui potencial de atuação como um coiniciador alternativo copolimerizável para composi-ção de sistemas de polimerizacomposi-ção radicalar fotoiniciada com tecnologia de alto de-sempenho e biocompatibilidade. Para a síntese do metacrilato piperonílico, o álcool piperonílico foi esterificado utilizando o anidrido metacrilato. O produto obtido foi pu-rificado e posteriormente submetido à ressonância magnética nuclear protônica e espectroscopia no infravermelho para confirmação da estrutura. Adesivos convenci-onais foram desenvolvidos utilizando uma resina adesiva modelo, composta por 50% de bisfenol a glicidil metacrilato (Bis-GMA), 25% de 2-hidroxietil metacrilato (HEMA) e 25% de trietilenoglicol dimetacrilato (TEGDMA). Canforoquinona (CQ) na concen-tração 1% molar foi utilizada como fotoiniciador da polimerização da resina modelo, a qual foi adicionada de diferentes coiniciadores constituindo os seguintes grupos experimentais: metacrilato piperonílico (PipeM Adesivo) e EDAB (EDAB Adesivo) constituindo sistemas binários de fotoativação. Adicionalmente um grupo com adesi-vo de referência comercial Scotch Bond Multi Purpouse - 3M (SBMP Adesiadesi-vo) foi avaliado. As propriedades físico-químicas, mecânicas, microbiologicas e biológicas do polímero formado pelos adesivos experimentais foram avaliadas utilizando as metodologias de cinética e taxa de polimerização, resistência à mini-flexão, módulo de elasticidade, sorção e solubilidade, resistência de união ao microcisalhamento
(MPa) em esmalte e dentina bovina, caracterização da camada híbrida em micros-cópio eletrônico de varredura (MEV), teste antimicrobiano de contato direto modifi-cado e avaliação da sua citotoxidade. Os resultados obtidos foram tabulados e a análise estatística realizada de acordo com o ensaio. O PipeM adesivo apresentou maior grau de conversão, menor sorção e solubilidade em água quando relacionado aos demais grupos. O grupo PipeM foi semelhante apenas ao grupo EDAB no teste de resistência de união ou microcisalhamento em dentina. Em todos os outros tes-tes, os grupos foram semelhantes (p <0,05). A porcentagem de viabilidade celular avaliada após 24h mostrou que os grupos experimentais foram semelhantes à refe-rência comercial apresentando viabilidade celular próxima de 100%. Em suma o me-tacrilato piperonílico apresentou desempenho semelhante ou superior à amina terci-ária, com a vantagem de apresentar maior biocompatibilidade em função de seu po-tencial de copolimerização, representando ser um coiniciador alternativo para com-posições fotopolimerizáveis.
Palavras-chave: biomateriais; materiais dentários; coiniciadores; fotoativação;
Abstract
MOREIRA, Andressa Goicochea. Piperonyl methacrylate: coinitiator copolymerizable for the composition of system of polymerization photoinitiated. 2017. Nº 54p. Dissertation (Master degree in Dentistry). Graduate Program in Dentistry. Federal University of Pelotas, Pelotas, 2017.
The evolution of dental materials has been contributing to their success in cosmetic procedures. Several clinical factors motivate new studies to develop materials that achieve optical, mechanical and biological properties similar to the dental element. Thus, this research seeks to contribute to the evolution of these materials through in vitro studies. Therefore, the objectives of this work are to carry out the synthesis of a molecule, piperonyl methacrylate, by means of an alternative route to the use of methacryloyl chloride, with known toxicity, and to analyze if this molecule has poten-tial of acting as an alternative copolymerizable coinitiator for the composition of radi-cal polymerization systems photoinitiated with technology of high performance and biocompatibility. For the synthesis of piperonyl methacrylate, piperonyl alcohol was esterified using methacrylic anhydride. The product obtained was purified and sub-sequently subjected to proton magnetic resonance and infrared spectroscopy to con-firm the structure. Conventional adhesives were developed using an adhesive resin model, composed of 50% bisphenol A glycidyl methacrylate (Bis-GMA), 25% 2-hydroxyethyl methacrylate (HEMA) and 25% triethyleneglycol dimethacrylate (TEGDMA). Canforquinone (CQ) at 1 mol% concentration was used as a photoinitia-tor of the polymerization of the model resin, which it was added with different co-initiators constituting the following experimental groups: piperonyl methacrylate (Pi-peM Adhesive) and EDAB (EDAB Adhesive), constituting binary photoactivation sys-tems. In addition, the Scotch Bond Multi Purpose-3M commercial reference adhesive (SBMP Adhesive) was evaluated. The physicochemical, mechanical, microbiological and biological properties of the polymer formed by the experimental adhesives were evaluated using polymerization kinetics methodologies, mini-flexural strength, modu-lus of elasticity, water sorption and solubility, enamel and dentin micro-shear bond strength (MPa) and performed the characterization of the hybrid layer under scanning electron microscope (SEM), Modified direct contact antimicrobial test and evaluation
of its cytotoxicity. The results obtained, were tabulated and the statistical analysis performed according to the assay. The PipeM adhesive showed a higher degree of conversion, lower sorption and solubility in water when related to the other groups. The PipeM group was similar only to the EDAB group in the bond strength test or dentin microcracking. In all other tests, the groups were similar (p <0.05). The percentage of cell viability evaluated after 24h showed that the experimental groups were similar to the commercial reference, presenting cell viability close to 100%. In summary, the piperonyl methacrylate presented similar or superior perfor-mance to the tertiary amine, with the advantage of presenting greater biocompatibil-ity as a function of its copolymerization potential, being an alternative co-initiator for photopolymerizable compositions.
Sumário 1 Introdução... 14 2 Capítulo... 18 3 considerações Finais... 47 Referências... 48 Apêndices... 52
1 Introdução
A busca por excêlencia em procedimentos restauradores odontológicos atrelados a técnicas minimamentes invasivas, tem feito com que se busque a melhoria dos materiais restauradores diretos utilizados em restaurações de dentes com moderada perda de estrutura tecidual que necessitam ser reabilitados, por apresentarem caracteristicas estéticas que os assemelhem à estrutura dental. As resinas compostas passaram por modificações em sua estrutura, tipo, formato e quantidade de carga inorgânica (FERRACANE et al., 1998, KAIZER et al., 2016) e modificações na parte orgânica (BARBOSA et al., 2015; FEITOSA et al., 2014; MOSZNER; SALZ, 2001). Essas alterações surgiram para melhorar as características de manipulação, propiedades mecânicas e ópticas, atendendo assim a constante demanda por estética e proporcionando propriedades físicas que as assemelhem à estrutura dental a esses materiais, com o intuito de atingir a capacidade de se manter aderidos à estrutura dentária e produzir satisfatório selamento marginal na interface dente-restauração.
A união do material restaurador à dentina ocorre através da formação da camada híbrida, resultante da infiltração de monômeros resinosos entre as fibrilas colágenas expostas em função do processo de desmineralização do substrato dentinário (NAKABAYASHI et al., 1982). Avaliações in vivo e in vitro mostram que a durabilidade da união ainda é um problema (PEUMANS et al., 2005, DE MUNCK et al., 2005, DONMEZ et al., 2005). Os principais fatores que explicam a reduzida longevidade da interface de união são: incompleta impregnação das fibrilas colágenas pelos monômeros, alta permeabilidade da interface de união, sub-polimerização dos materiais, separação de fases e a degradação pela ativação de enzimas colagenolíticas (BRESCHI et al., 2008).
No esmalte a união ocorre a partir do aumento da rugosidade superficial que se dá por meio de uma reação ácido base, que acontece quando o ácido fosfórico entra em contato com a superficie do esmalte que contém hidroxiapatita, esta é absorvida e forma a rugosidade, gerando uma área de união a qual ocorre por embricamento mecânico (VAN NOORT, 2010).
Os sistemas de fotoiniciação utilizados para a polimerização radicalar de materiais odontológicos fotopolimerizáveis usualmente são binários, constituídos por dois componentes: o fotoiniciador, que pode absorver luz diretamente, e o co-iniciador, que não absorve luz, mas atua como agente redutor. O co-iniciador doa
prótons para a estabilização do radical gerado na molécula excitada do fotoiniciador, gerando espécies reativas com tempo de vida suficiente para iniciar a polimerização (ANDRZEJEWSKA, 2001).
Canforoquinona (CQ) e amina constituem o sistema de fotoiniciação amplamente utilizado na polimerização radicalar de materiais dentários à base de metacrilatos (JAKUBIAK et al., 2003, MUSANJE; FERRACANE; SAKAGUCHI, 2009, SCHROEDER; COOK; VALLO, 2008, SCHROEDER; VALLO, 2007). A polimerização fotoiniciada ocorre pela reação em cadeia entre os radicais livres, formados pelo sistema fotoiniciador e os monômeros (JAKUBIAK et al., 2003). A CQ é um típico fotoiniciador ativado pela luz visível com absorbância entre 450-500 nm (λmax= 468 nm) (OGUNYINKA et al., 2007) e requer um agente redutor para que ocorra uma polimerização eficiente (ANDRZEJEWSKA, 2001, CHEN; FERRACANE; PRAHL, 2007). Apesar deste sistema ser comumente utilizado, a CQ apresenta uma coloração fortemente amarelada e a amina por sua vez, pode apresentar um amarelamento do material, o que pode representar um problema em restaurações estéticas (NEUMANN et al., 2006, OGUNYINKA, et al., 2007, SHIN; RAWLS, 2009).
As aminas, co-iniciadoras da polimerização ativada por luz, são doadoras de hidrogênio na polimerização radicalar (ANDRZEJEWSKA, 2001). Sua influência positiva na reação de polimerização, no grau de conversão e nas propriedades do polímero formado está bem descrita (MUSANJE; FERRACANE; SAKAGUCHI, 2009, OGLIARI et al., 2007, SHIN; RAWLS, 2009, YOSHIDA; GREENER, 1994, YOSHIDA; GREENER, 1993).
A característica nucleofílica apresentada principalmente pelas aminas terciárias as torna excelentes doadoras de prótons na polimerização radicalar (ANDRZEJEWSKA, 2001). No entanto, apesar do bom desempenho como co-iniciadoras da polimerização, as aminas apresentam potencial citotóxico e mutagênico, participam da reação, no entanto não se ligam ao polímero formado, podendo ser lixiviadas após a reação de polimerização. (ALBRECHT; STEPHENSON, 1988) Dessa forma, no intuito de se obter um material com maior biocompatibilidade, é interessante a utilização de um co-iniciador mais seguro. Alternativas para reduzir a citotoxicidade de resinas odontológicas vêm sendo testadas, como por exemplo, a substituição de amina por outro co-iniciador menos tóxico (LIU et al., 2007, SCHNEIDER et al., 2008, SHI; NIE, 2007, SHI; NIE, 2007). Neste aspecto, vários anti-oxidantes presentes nos alimentos são de grande
interesse, pois se ligam facilmente a radicais livres e não são tóxicos aos tecidos do organismo humano (AMES, 1983, JOSHI et al., 2005, KUMAGAI et al., 1991, MICALE; ZAPPALA; GRASSO, 2003). Além disso, estudos têm demonstrado que derivados das benzodioxolas tem potencial anticarcinogênico, (MICALE; ZAPPALA; GRASSO, 2002, TSENG; TSHENG; LEE, 2001) antifúngico, antibacteriano, antioxidante (TAGASHIRA et al., 1997) e radioprotetor (MATOS et al., 2004), entre outros.
Benzodioxolas são substâncias que podem ser extraídas de produtos naturais como melissa, (TAGASHIRA & OHTAKE, 1998) manjericão, canela, noz moscada entre outros, para obtenção de óleos essenciais. (HICKEY, 1948) Estes derivados das benzodioxolas são amplamente encontrados na flora brasileira (MOREIRA et al., 2007), são antioxidantes que apresentam alto sinergismo com moléculas de canforoquinona. (LIU, et al., 2007) Alguns estudos demonstraram que a substituição de amina terciária por benzodioxolas (SHI; NIE, 2007, LIU, et al., 2007, LIMA et al., 2013), em um sistema de fotoiniciação binário (canforoquinona + benzodioxola) de uma blenda modelo, não afeta as propriedades mecânicas do polímero formado, quando comparado ao sistema convencional CQ/amina.
Os sistemas adesivos são resinas de composição específica e de baixa viscosidade, que constituem íntimo contato com o substrato dental. (DE MUNCK et al., 2005) Assim, estes materiais têm a capacidade de penetrar a estrutura do substrato desmineralizado, permeando os túbulos dentinários, enfrentando desafios para a reação de polimerização, como a presença umidade, (GUO et al., 2007, HIGASHI et al., 2009) além dos desafios de biocompatibilidade. (COX et al., 1998, GOLDBERG, 2008, GUO et al., 2008).
Portanto este estudo teve como objetivo a síntese de uma nova molécula, o metacrilato piperonílico, coiniciadora e copolimerizável, para aplicação em sistemas de polimerização radicalar fotoiniciada com capacidade de proporcionar uma maior biocompatibilidade e melhorias nas propriedades físico-químicas do polímero. O sistema de fotoiniciação desenvolvido neste trabalho é de extrema importância para o mercado de produtos odontológicos nacional, mostrando a possibilidade de desenvolvermos tecnologia própria em materiais com maior biocompatibilidade.
A abordagem realizada nesse estudo é a utilização do álcool piperonílico, como material de partida, um coiniciador de eficiência já conhecida. Uma característica peculiar do álcool piperonílico é a presença em sua estrutura
molecular, de sítio com labilidade suficiente para a promoção de reações específicas.
Desta forma, foi hipotetizado que substituindo o grupamento OH presente no álcool piperonílico pela função metacrilato, seria possível a obtenção de um coiniciador, uma vez que o mesmo teria a habilidade de copolimerizar juntamente com os demais monômeros do meio.
Adicionalmente é esperado que com tal abordagem, consiga-se uma maior biocompatibilidade e uma melhoria nas propriedades físico-químicas do polímero.
2 Capítulo 1
Piperonyl methacrylate: copolymerizable coinitiator for adhesive composition
Andressa Goiochea Moreiraab, Carlos Enrique Cuevas Suárezab, Wellington Luiz Oli-veira da Rosaab, Aline Oliveira Ogliariab, Cesar Liberato Petzholdc, Evandro Pivaab, Fabrício Aulo Ogliariab, Giana da Silveira Limaab.
a Graduate Program in Dentistry, Federal University of Pelotas, Pelotas-RS, Brazil.
b Department of Restorative Dentistry, Dental School, Federal University of Pelotas,
Pelotas-RS, Brazil.
c
Organic Chemistry Department, Chemistry Institute, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
Graduate Program in Dentistry, Federal University of Pelotas R. Gonçalves Chaves 457
Pelotas, RS, Brazil 96015-560 Tel/Fax: 32256741 ext. 134
*Corresponding author:
Giana da Silveira Lima
R. Gonçalves Chaves 457, room 504. Pelotas, RS, Brazil 96015-560
Tel/Fax: 32256741 ext. 134
Artigo formatado segundo as normas do periódico Dental Materials https://www.elsevier.com/journals/dental-materials/0109-5641/guide-for-authors
Abstract
objective: This study promoted the synthesis and application of a new molecule, the piperonyl methacrylate, with potential to act as coinitiator copolymerizable substance of high biocompatibility for photoinitiated radical polymerization systems. The piper-onyl methacrylate (PipM) was synthetized through an esterification reaction using methacrylic anhydride, in basic environment with triethylamine and chloroform. The product obtained was purified through of fractional distillation and chromatographic column, later submitted to the proton nuclear magnetic resonance of hydrogen to confirm the structure obtained and characterized in Infrared. The synthesis was con-firmed and piperonyl methacrylate was successfully obtained. Methods: An experi-mental adhesive resin containing camphorquinone (CQ) as photoinitiator was formu-lated to evaluate the influence of concentration of piperonyl methacrylate coinitiator on the degree of conversion (GC). Afterwards, selected experimental groups, PipeM Adhesive (CQ and PipM), EDAB Adhesive (CQ and EDAB) and SBMP Adhesive (commercial reference) were analyzed for their physicochemical, mechanical, micro-biological and micro-biological properties trough evaluation of their polymerization kinetics, mini-flexural resistance, modulus of elasticity, sorption and solubility, micro-shear bond strength to bovine enamel and dentin, characterization of hybrid layer in scan-ning electron microscope (SEM), direct contact test and cytotoxicity. Results: the Pi-peM Adhesive presented higher degree of conversion, lower sorption and solubility in water related to other groups. The PipeM group was similar only to EDAB group in the shear bond strength test in dentin. In all other tests, all groups were similar (p <0.05). The percentage of cellular viability evaluated after 24h showed that the ex-perimental groups were similar to commercial reference presenting cellular viability close to 100%. Conclusion: the piperonyl methacrylate presented similar or superior performance to the tertiary amine, with the advantage of having a higher biocompati-bility in function of its copolymerization potential, representing an alternative reagent for photopolymerizable compositions.
1 Introduction
Photopolimerizable materials were developed to prolong the working time needed for certain dental procedures in order to optimize chair-side procedures [1]. The evolution process involving materials used in odontological procedures tend to develop resin compounds with the intention to achieve a better biocompability [2–4] with dental tissues and also to obtain mechanic and visual characteristics that re-semble to the natural element, reasons why its monomer structure and inorganic par-ticles have been changed [5,6] . The use of natural materials is a new strand in den-tal materials innovation and it can add antimicrobial characteristics and improve the biocompatibility [7,8].
Nowadays, the composite resins polymerize by absorption of light, via radical polymerization. Photoactivation of these materials is initiated by the activation of molecules called initiators. After the hemolytic rupture of the initiators, free radicals are generated, which bound to the monomeric units to initiate the polymerization pro-cess and the formation of cross-linked networks with polymers of high molecular weight and high conversion rate of (c=c) bonds. In dentistry the most used photoacti-vation system is the binary tipe II which is composed by camphorquinone as photo-activator and a tertiary amine as co-initiator [9,10].
The photoinitiating molecule (CQ) is usually composed of a carbonyl group whose electrons are on the outside. CQ produces free radicals when exposed to blue light with a wave-length of 450-500 nm [11]. However, the CQ is not able to generate a sufficient amount of free radicals to initiate the polymerization process, requiring a coinitiator reducer like a tertiary amine [12,13], which acts on the reaction as a hy-drogen donor improving the generation of free radicals. In a certain moment these crosslinking networks of polymeric chains cease their growing due to the exhaustion of the production of free radicals [14].
The amines used as coinitiators in the polymerization reaction do not bind to polymeric chains formed, resulting in their elimination through its dissolution in a liq-uid, such process is called leaching. Leaching of substances that do not react during the polymerization process, like the photoinitiators and unpolymerized monomers may have a direct relationship with the quality of mechanical and biological properties of the polymer and may generate a reduction in longevity of the restoration [15]. In addition, inflammatory processes can occur through the penetration of leachate
ma-terials into the dentinal tubules causing irritation of the pulp tissue [16,17]. So given these adversities new alternatives for the composition of these polymers have been studied. Among the different possibilities, the incorporation of natural materials would be an excellent choice as they have a considerable biological activity and enhanced biocompatibility [7,8,18,19].
Adhesive systems are specific compositions with low viscosity which favors an intimate contact with the substrates to be bonded [20]. In dentin, these materials have the ability to penetrate into the demineralized dentin tubules, facing challenges to the polymerization reaction , since the presence of moisture in the dentin can im-pair the polymerization rate [21], which can condition its biocompatibility [22].
Therefore, this study will have as a goal the synthesis of a molecule, the pip-eronyl mehacrylate, using a natural derived product as raw material, for application in photoinitiated radical polymerization systems, capable of providing greater biocom-patibility and improvements in the physicochemical properties of the polymer.
2 Materials and methods 2.1 Experimental Design
This study in vitro involved the synthesis of piperonyl methacrylate through a single-step esterification using anhydrous methacrylate in a basic environment with triethyl-amine and chloroform. The final product was identified using 1H-NMR and FTIR/ATR. The illustrative drawing is shown in Figure 1. A concentration study of piperonyl methacrylate was performed (Fig. 6) and the concentration of 25% was chosen, later this was incorporated to an experimental adhesive resin and had its performance evaluated in comparison to the coinitiator already described in the literature ethyl 4-(dimethylamino)benzoate (EDAB) and a commercial reference (Single Bond Multi Purpose, 3MESPE). The adhesives were evaluated for: polymerization kinetics, de-gree of conversion within the hybrid layer , Flexural Strength and Elastic Modulus , Microshear bond strength test (MPa), Morphology of the enamel and dentin bonded interfaces, Failure pattern analysis, Sorption and Solubility, Modified Direct Contact Test and Cytotoxicity test (Fig. 2).
2.2 Reagents
Bisphenol-A glycidyl dimethacrylate (Bis-GMA), triethyleneglycol dimethacry-late (TEGDMA), 2-hydroxyethyl methacrydimethacry-late (HEMA) and camphorquinone (CQ) were obtained from Esstech Inc. (Essington, PA, USA), . The acidic monomers 1,3- glycerol dimethacrylate phosphate (GDMA-P) was synthesized as previously de-scribed [17]. Silica nanoparticles (Aerosil 380, 7nm) were obtained from Evonik In-dustries AG Inorganic Materials (Hanau-Wolfgang, Germany). Ethyl 4-dimethylamine benzoate (EDAB), triethylamine, piperonyl alcohol (PA) and methacrylic anhydride (MA) were obtained from Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA).
2.3 Synthesis of piperonyl methacrylate
Five grams of piperonyl alcohol (0.03 mol) were dissolved in 50 mL of chlo-roform in a two-necked round bottomed flask equipped with a stirrer, thermometer and dropping funnel. After cooling at 0 °C, 7.3 mL of anhydride methacrylic (0.049 molar) and 6.8 mL of triethylamine (0.049 molar) were added dropwise into the reac-tion media. The system was kept ice-cold during the dissolureac-tion process. The mixture was kept at room temperature and stirred for 24 h. At the end of the reaction, three extractions with 150 mL of distilled water, 100 mL of 1N HCl and 100 mL of 1N Na-HCO3 were performed. The organic phase was dried with anhydrous Na2SO4, and the chloroform was completely evaporated under vacuum. Product was finally puri-fied by means of chromatographic column using silica as stationary phase and a mix-ture of ethyl acetate:hexane (1:3) as mobile phase. Pyperonyl mehacrylate was ob-tained as a colorless liquid. Final product was identified using 1H NMR and FTIR/ATR.
1
H NMR (CDCl3) ppm: 1.98 (3H, -CH3), 5.11 (2H, -CH2-O-C=O), 5.59 and 6.15 (2H, C=CH2), 5.98 (2H, -O-CH2-O-), 6.80-6.89 (3H, -benzene). FTIR/ATR (cm-1): 2930 ( C-H), 1710 ( C=O), 1633 ( C=C), 1611 ( C=C aromatic), 1445 ( C-H methylene), 1038 ( C-O).
An experimental adhesive system of total conditioning of three-steps was for-mulated according to the composition presented in Table 1. This coating resin was the basis for the application of different photoinitiation systems tested as experi-mental groups. The co-initiators were incorporated into the adhesive resin constitut-ing two experimental groups: piperonyl methacrylate (PipeM Adhesive) and tertiary amine (EDAB Adhesive) [23]. The concentration of piperonyl methacrylate used for the PipeM group was determined using a screening test (Fig. 6), where the influence of its concentration in the degree of conversion of the experimental adhesive resin model was evaluated. An experimental primer composed of HEMA 30%wt, ethanol 30%wt, water 30%wt and GDMA-P 10%wt was formulated to formulate an experi-mental three-step adhesive system. The Scotchbond Multi Purpose adhesive system was evaluated as a commercial reference.
2.6 Characterization of the materials
2.6.1 Evaluation of the degree of conversion and kinetics of polymerization.
The degree of carbon double bond conversion (GC) was evaluated by means of Fourier transform infrared spectroscopy (Prestige-21; Shimadzu, Tokyo, Japan), equipped with an attenuated total reflectance (ATR) device, composed of a diamond crystal with 45° mirror angle (Pike Technologies; Madison, WI, USA). Samples of the formulated experimental Adhesives and the SBMP Adhesive commercial material in an amount sufficient to cover a standard area of the crystal (n = 3) were placed into the ATR device. An initial reading of the unpolymerized material (monomer) was per-formed using 12 scans between 1500 and 1800cm-1, and a resolution of 4cm-1. The adhesive was photoactivated for 20s using an photopolymerization unit equipped with a LED of 1200 mW/cm2 (Radii Cal; SDI, Bayswater, Victoria, Australia) and a second reading was performed for the cured material. The distance between the tip of the LED and the sample was standardized (3 mm), connecting the photoactivation unit to a holder coupled to the spectrophotometer. IRSolution software was used for scan monitoring. The analysis was performed in environment with controlled temper-ature of 23ºC and relative humidity <60%. Degree of conversion was calculated con-sidering the intensity of the vibration of the elongation type of the carbon-carbon double bond in the frequency of 1635 cm-1. The symmetric drawing in 1610 cm-1 cor-responding to the aromatic ring was used as an internal standard.The polymerization
kinetics data were plotted and curve fitting performed by using Hill’s three parameter non-linear regression. Using these data, the polymerization rate (RP (s−1)) was calcu-lated as the DC at time t subtracted of DC at time t − 1. The coefficient of determina-tion was greater than 0.95 for all curves [8,23–25]. Statistical analysis for DC was performed using One-Way ANOVA and Tukey test. The level of significance was set at (p<0.05).
2.6.2 Degree of conversion within the hybrid layer.
The degree of conversion (DC) within the hybrid layer of adhesives was eval-uated in situ in the specimens through a Confocal Raman spectrometer (XploRA, HORIBA, Chilly Mazarin France). For the test, 3 samples were used (n = 3). Bovine dentine surfaces were exposed on the central vestibular face following by an adhe-sion protocol (details will be giving after). After 24 hours of storage in distilled water and at 37ºC, they were sectioned perpendicular to the bonded interface using a cooled diamond saw at low speed (Isomet low speed, Bhueler, Lake Bluff, IL, USA). The specimens were polished with #1200 grit-silicon carbide paper for 1 minute and stored under the same conditions for 24 hours. After this period, the specimens were placed in the spectrometer for analysis. This equipment relies on the LabSpect 6® software, which was previously calibrated following the manufacturer's recommenda-tions and used the parameters for the analysis of the specimens with 535nm laser, 1% power, 600 grid and 10x objective in beam diameter (Olympus microscope, Lon-don, UK). The spectra were obtained at the interface between restorative material and tooth, at three sites distributed at the interface of the hybrid layer and enamel surface. Further processing of the spectrum was performed using LabSpect 6®. The ratio of the double bond content of the monomer at the interface was calculated ac-cording to the following formula: DC (%) = (1 - [R cured / R uncured)) x 100, where R is the ratio of the intensities of the Aromatic and aliphatic peaks at 1635 cm-1 and 1608 cm-1. [28,29] Statistical analysis was through two-way ANOVA and Student-Newman-Keuls test (p<0.05).
2.6.3 Flexural Strength and Elastic Modulus
The flexural strength (σf) and modulus of elasticity (EF) were evaluated using a three-point bending flexural test. A total of 30 bar-shaped specimens (10×2×2mm) equally divided, n= 10 for each group (PipeM Adhesive, EDAB Adhesive, SBMP
Ad-hesive) were prepared by filling the uncured material into a stainless steel mold cov-ered with polyester strip and glass slide. Subsequently, the materials were photoacti-vated on both sides for 20s in each side. After polymerization, the bars were re-moved from the mold and polished with 600 SiC abrasive paper under refrigeration and stored in distilled water at 37 °C. After 24 hours, the dimensions of the speci-mens were measured using a digital caliper (Mitutoyo, Tokyo, Japan) and submitted to a three-point flexural test in a universal mechanical testing machine (DL500, EMIC, São José dos Pinhais, PR). The load was applied in the central region of the bar, with a distance of 8mm between the points, a speed of 0.5 mm/min until failure. The values of σf and EF were calculated according to described in previous studies [30]. Statistical analysis One-Way ANOVA The level of significance was set at (p<0.05).
2.6.4 Microshear bond strength test (MPa)
For the enamel and dentin bond strength, 60 permanent bovine incisors (n=10) were used. The roots of the teeth were cut until the cement-enamel junction. Then, plastic rings were placed over each teeth and filled using cold-curing acrylic resin exposing the labial surfaces, which were wet grounded using a 180-grit silicon carbide paper to achieve flat enamel or dentine surfaces. The specimens were then polished using 600-grit silicon carbide paper for 30s to provide an uniform smear layer. The polished surfaces were observed in an optical stereomicroscope with 40X magnification for the evaluation of cracks in the enamel. Prophylaxis with pumice, water and Robinson brushes at low speed was performed before the bonding procedures.
For enamel restorations, the surface was air-dried for 10 sec and conditioned with 37% phosphoric acid gel (3M-ESPE) for 30s. After gel application, air-spray and water spray were applicate for 30s, and then the surfaces were spray-dried until complete removal of surface moisture. The adhesive systems (PipeM Adhesive, EDAB Adhesive and SBMP Adhesive), were then applied using a microbrush (KG Sorensen) and rubbed for 20 s. Two composite resin cylinders (2x2 mm) were built upon the adhesive layer using a silicon matrix and then photoactivated for 20 s, using a photopolymerization unit equipped with a LED with an intensity of 1200 mW/cm2 (Radii Cal; SDI, Bayswater, Victoria, Australia).
For dentin restorations, the surface was cleaned with air/water jet for 20 s, and then, 37% phosphoric acid gel was applied for 15 s. The surface was then washed for 30s with air/water jet and then dried with absorbent paper. An experimental primer was applied for experimental adhesives, and for the SBMP adhesive the primer of this system was used. Both were applied with friction movements for 20s, followed by air jet for 10s. After primer application, the corresponding experimental adhesive systems (PipeM Adhesive, EDAB Adhesive and SBMP Adhesive) were applied. Restoration with composite resins were performed according to the sequence previously described for the enamel. The restoration was made from commercial composite resin (Filtek Z250, 3M ESPE). The specimens were stored in distilled water at 37 °C for 24 hours.
The shear bond strength was performed in a universal mechanical testing machine at a cross-head speed of 1mm/min. using a steel wire actuator. Samples were mounted on the test machine with the interface restoration-tooth parallel to the wire. A shear force was applied until restoration debonding. The bond strength values were calculated in MPa, considering the area of the restoration. The debonded surfaces were observed in optical stereomicroscope with 40X magnification and classified as cohesive in adhesive, cohesive in resin, cohesive in dentin, adhesive or mixed type failure. Statistical analysis was performed using One-Way ANOVA and Tukey test. This analysis was used for the dentin substrate and for the enamel substrate was used One-way ANOVA, both with The level of significance was set at (p<0.05).
2.6.5 Sorption and Solubility
Ten disk-shaped (6 x 1mm) specimens per group (n = 30) were prepared in a stainless steel mold. The specimens were photoactivated for 20s on each side. After this, the specimens were transferred to a desiccator at 37 °C and their mass monitored daily until a constant mass (m1) was obtained. Then, the diameter and thickness of each specimen were measured to calculate the volume for each one (V). Samples were immersed in distilled water at 37 °C for 7 days. At the end of the 7 days, the specimens were washed with water and dried gently for 15s, then a new measurement of the weight of the specimen was obtained (m2) mass. After obtaining the m2, the specimens were again stored in the desiccators, repeating the cycle of
the beginning of the test, until a third mass (m3) constant mass obtained. For each sample, the water absorption (WS) and solubility (SL) data were calculated using the following formulas:
Water Sorption Weight% = m2-m3 x 100 m3
Solubility Weight% = m1-m3 x 100 m3
The Statistical analysis was performed One-Way ANOVA and Tukey test. The level of significance was set at (p<0.05).
2.6.6 Morphology of the enamel and dentin bonded interfaces
The enamel and dentin surfaces were polished and the adhesive system was applied as previously described and the restoration process was performed. The specimens were embedded cross-sectionally in epoxy resin in order that the dentin-resin interfaces were visible. After 24 h, the specimens were wet-polished with 600, 1200, 1500, 2000 and 2500-grit SiC papers followed by polishing with 3, 1, 0.25 and 0.1-μm diamond suspensions. The surfaces were etched with 50% phosphoric acid solution for 5 s and deproteinized by immersion in 2.5% NaOCl solution for 10 min. The specimens were ultrasonically cleansed with distilled water, dry-stored at 37ºC for 2 h, and coated with gold. The bonded interfaces were examined with a scanning electron microscopy – SEM (SSX-550; Shimadzu, Tokyo, Japan), at 15 kV.
2.7 Cytotoxicity test
The cytotoxicity test was performed according to ISO 10993-5 (2009). A cell line from L929 mouse fibroblasts was used. The cell culture medium used was DMEM (Dulbeccos Modified Eagle Medium) supplemented with 10% fetal bovine se-rum (FBS), 2% L-glutamine, penicillin (100U / ml) and streptomycin (100mg / ml). 2x104 cells in 200μl DMEM plus 10% FBS were placed in each well of a 96-well plate. The plate was incubated in a CO2 oven with temperature and pressure control in a humid environment at 37ºC, 95% air and 5% CO2 for 24h in a way that allowed
the adhesion of the cells to the bottom of the culture plate. Specimens (6mm in di-ameter and 1mm in height) of groups (PipeM Adhesive, EDAB Adhesive and SBMP Adhesive) were photoactivated by LED with irradiance of 1200 mW / cm2 (Radii Cal; SDI, Bayswater, Victoria, Australia) (n = 6). The specimens were placed in 24-well plates with 1 ml of DMEM and stored at 37 °C at pH 7.2. After 24 h, 200 μl of the elu-ate from each specimen was transferred to the 96-well plelu-ate containing the cells pre-viously prepared. After the eluates in the test wells, the plate was incubated (37 °C, 5% CO2) for a period of 24 hours allowing the products to act on the cell monolayer. After this period, the medium was sucked and 2 ml of MTT solution were added to each test well (20 μl per well) and incubated again for 24 hours to allow metabolism of MTT. After the period, the medium was sucked and the formazan resuspended in 200μl of dimethyl sulfoxide (DMSO). The results were read in a spectrophotometer with a wavelength of 560nm, where the absorbance values of as indicator of cell via-bility. Statistical analysis was performed using One-Way ANOVA and Tukey test. The level of significance was set at (p<0.05).
2.8 Modified Direct Contact Test (mDCT)
The evaluation of antimicrobial effect was performed by the modified direct contact test. That in measuring cinematic microbial growth, by close contact between the tested microorganism and material using cultures of 96-well microplate cells. Specimens of Streptococcus mutans were individually grown overnight in Brain and Heart Infusion (BHI) under anaerobic conditions, at 37 ° C for 24 h. Colonies of mi-croorganisms were then suspended separately in BHI to make suspensions of 3 × 108CFU / mL, the turbidity of the microbial cells was adjusted using spectrophotome-try (spectrophotometer, Quimis, Brazil) at 405 nm. From this inoculum, 20 μL of bac-terial suspension was added to each well, and then evaluated. Disc samples (6x1mm) were made with PipeM adhesive, EDAB Adhesive and SBMP Adhesive. Before the test, the samples were sterilized by UV (ultraviolet) light inside a laminar flow chambre. After sterilization of the specimens, they were placed in 96-well plates, with n = 3 for each group tested. The materials were placed in wells of the n = 3 microplate, with the aid of sterile tweezers. One specimen was left in each well and the material was then inoculated with 20 μl of microbial suspension (S. mutans + BHI). The disks were incubated during 1h and 24h at 37 ° C and approximately 100% relative humidity. Microplate wells containing the same volume of bacterial
suspen-sion without test discs were also incubated as controls. Then, 180 μl of culture medi-um supplemented with 10% sucrose in each well was added and stirred (Guangzhou Mecan Trading Co., Ltd., China) for 10 min. One hundred microliters of the bacterial suspension from each well was transferred for dilution. Serial dilutions and plating were carried out in disposable Petri dishes containing BHI agar, divided into eight parts. Each plate received two drops of 20 μL per dilution. The plates were then in-cubated at 37 ° C for 24 h. After the incubation period, the colony forming units (CFU / mL) were counted [31]. For microbiological activity, data were analyzed using Sta-tistical analysis between bonding agents, using Two-way ANOVA and Student-Newman-Keuls test (p<0.05).
3 Results
The structure of the newly synthesized coinitiator was identified using using FTIR and 1H NMR spectroscopies. The FTIR spectrum of Piperonyl methacrylate is shown in Figure 4, in this spectrum, the absorption band at 3256 cm-1, corresponding to the hydroxyl group of raw material disappears, which indicates that the hydroxyl group in piperonyl alcohol reacted completely with the anhydride methacrylate. Another evidence of the formation of the new compound is the appearance of new absorption bands at 1710 cm-1 and 1633 cm-1 which corresponds to symmetric vibration stretching of the C=O and C=C of the methacrylate group respectively. In 1H NMR spectrum of Piperonyl methacrylate, distinctive signals assigned to –C=C–H (6.15 ppm, cis), –C=C–H (5.59 ppm, trans) and -CH3 (1.98 ppm) were observed, which can confirm the correct substitution of the hydroxyl group by the methacrylate group into the piperonyl alcohol structure (Fig. 5).
Concentration of Piperonyl methacrylate was chosen in a screening of concentration, through the degree of conversion, with values already presented in Figure 6. The concentration of 25% was selected according to the the Statistical analysis where was performed One-Way ANOVA and Tukey test. The level of significance was set at (p<0.05), since it showed no difference in the results of degree of conversion, when compared with higher concentrations.
The behavior of materials during the reaction, in real time, was analyzed and the Figure 7 A shows the kinetics of photopolymerization from the experimental adhesives with different co-initiators and the commercial reference. Reduced
photoactivation time was required to achieve a higher degree of conversion, when compared to the adhesive with EDAB co-initiator and to SBMP commercial reference system. The average of the degree of conversion in 60 seconds for the PipeM Adhesive group was 71.96 (±0.6) % being superior than EDAB Adhesive 55.93 (±0.6)% and to the SBMP 62.12 (±3.2)%. The system formulated with PipeM as co-initiator, obtained the higher rate of polymerization value [Rpmax (s-1)], as shown in te Figure 7 B.
The Fig. 8, presents the results of degree of conversion during 20 seconds of photoactivation, performed directly on the diamond crystal by FTIR and also the degree of conversion of the adhesives evaluated in situ, in the hybrid layer, through raman spectroscopy. The PipeM adhesive evaluated directly on the crystal of the equipment, presented better performance than the other groups with an percentual average of 62.1 (± 0.6)%. When it was applied in the hybrid layer, presented similarity with commercial reference, SBMP adhesive, that obtained a degree of conversion of 36.9 (± 3.4) and both were different from the EDAB Adhesive group, which reached an average of 24.8 (± 4.9), (p <0.05).
The results for degree of conversion (20 seconds), water sorption and solubility, flexural strength, elastic modulus, microshear bond strength to enamel and dentin for the experimental group (PipeM Adhesive, EDAB Adhesive) and commercial group (SBMP Adhesive) are contained in Table 2. PipeM Adhesive presented a higher degree of Conversion and rate of polymerization, lower water sorption and solubility than the other groups. The flexural strength and modulus of elasticity, showed in the table 2, all tested groups were similar (p <0.05).
The experimental group PipeM Adhesive was similar to the EDAB Adhesive in the dentin microshear bond strength test, and in enamel all groups presented similar performance. The Figure 9 presents the distribution of failure modes in dentin and enamel surfaces (mixed failure; adhesive failure). For all groups, predominantly Adhesive failures were observed.
SEM pictures of the bonded interfaces present the characteristics of the hybrid layer obtained with the different adhesives applied (Figure 10). No appreciable differences in homogeneity and continuity of the hybrid layer along the interfaces were detected among the tested materials. The experimental systems presented the pattern of resinous tags formation similar to the conventional systems as can be observed in the commercial reference SBMP Adhesive system.
In Fig. 11, the modified direct contact test is presented, antimicrobial effect was measured in CFU/ml (colony forming unit per millimeter in 10 logarithm). It is possible to observe that in 1h the PipeM Adhesive and SBMP Adhesive presents the same microbial growth . In 24h PipeM Adhesive showed higher microbial growth and the EDAB Adhesive was similar to bacterial control.
Fig. 12 shows the percentage of cell viability assessed after 24h. The untreated group (cell control) was considered equal to 100%. The PipeM Adhesive, PipeA Adhesive and EDAB Adhesive groups showed 98%, 97% and 99% of cell viability, respectively, and were statistically similar than SBMP Adhesive (94%) and cell control.
4 Discussion
In this work, a synthesis of a new polymerizable coinitiator and its effect on several chemical-physical and biological properties is reported. Methacrylate piper-onyl was synthetized through a single step synthetic route. Several attempts were made to perform the esterification reaction using methacryloyl chloride [32]. But the resulting product of this synthetic route resulted with impurities even after the perfor-mance of the chromatographic column. Therefore, it was decided to carry out a syn-thesis with a less reactive and less toxic amount, the methacrylic anhydride. The structure of the methacrylate piperonyl was confirmed through conventional spectro-scopic characterization techniques.
The hypothetical mechanism by which the free radicals are formatted within the structure of piperonyl alcohol (PA) was described previously [8]. According to this study, when CQ is excited, there exist three possible sites for the radical formation into the structure of CQ-PA complex. For the synthesis of methacrylate piperonyl, the chemical modification of PA involved the use of one of this actives sites (-OH radi-cal), leaving the other two available for the generation of free radicals.
. Infrared spectroscopy is used on a large scale in studies of dental materials [8,24,25]. The conversion of monomers in polymers is important to determine the properties of the polymeric materials. A high degree of conversion impairs the me-chanical properties of the polymer, affecting its durability [9] and an incomplete polymerization of monomers impairs the biological properties. In the present study, it was observed that the type of co-initiator had influence on the degree of conversion and the rate of polymerization (Fig. 7). The piperonyl methacrylate, for being a
co-initiator with the methacrylate function added to its structure, has the ability to copol-ymerize with the other monomers, being integrated in the interior of the polymer chain.It was observed in the study that it reached a degree of polymerization in 20 seconds of 62.1% (± 0.6), while the adhesive with EDAB co-initiator achieved a de-gree of conversion of 46.6% (± 2.2). The conversion dede-gree was assessed in situ (Fig. 8), simulated in bovine dentition, which allows an approximation of what hap-pens in vivo. µ-Raman Spectroscopy is a technique with excellent spatial resolution that allows the analysis of diffused radiation at different wavelengths by a sample when a beam of monochromatic radiation is present. This process reveals infor-mation of any organic and / or inorganic material or compound, allowing to qualify and quantify its chemical composition, providing a quantification of the percentage of double bonds converted to the dental substrate [28,29]. In this evaluation there was a reduction in the values of the conversion degree. The hydrophilicity present inside the hybrid layer may have been responsible for the decrease of the conversion val-ues in the polymerization reaction, because water interferes in the conversion of monomers to polymers [33].
Studies that analyze different concentrations of alternative co-initiators ob-served better mechanical properties when there was an increase in the concentration of these [8,24,34]. The binary system composed of camphorquinone and amine is widely used in the composition of photopolymerizable dental materials. Its efficacy has already been tested in vitro studies [14,35,36], when exposed to light the cam-phorquinone depends on a co-initiator, as it has a short durability to start the reac-tion, however the amount of this compound formed by camphorquinone and amine should be lower because of the yellowish coloration present in these conductors [37– 39]. Other researchers observed that the co-initiators that presented better mechani-cal properties also presented greater reactivity [19,40].
The mechanical resistance of polymer composites depends, mostly, on the proper conversion of C = C during the polymerization. The 3-D configuration and cross-linking of the polymer have a great influence on the mechanical resistance, being more mechanically resistant, the materials that have higher density of cross-links between the polymer chains [41,42]. Besides that the amount of inorganic load can vary between materials, also interfering in σf and Ef of the adhesives. These fac-tors may explain why the commercial reference SBMP adhesive, even with lower GC than PipeM, was statistically similar in the Flexural Strength and Elastic Modulus test.
The high degree of conversion and rate of polymerization can increase poly-mer crosslinking and the mechanical properties of the adhesive layer, leaving low levels of unreacted monomers [43]. The high degree of conversion and rate of polymerization can increase polymer crosslinking and the mechanical properties of the adhesive layer, leaving low levels of unreacted monomers [43]. The adhesion to enamel structure is not critical as in dentin, obtaining results predictable and little in-fluenced by the variations of technique. Adhesion to dentin is a less predictable pro-cedure than enamel because it has a complex morphology which can suffer changes according to profundity [44]. The penetration of primer and adhesive may be affected by the morphology of the dental tissue at different locations of the dentin. These characteristics may explain why in enamel the microshear bond strength test ob-tained higher values of resistance than in the dentin substrate. The result of bond strength to dentin in the Pipe M adhesive groups and the EDAB Adhesive was lower than the SBMP adhesive group, may be related to the experimental primer used, the solvents present in this has the function of keeping all the interfibrilar spaces ex-panded, through Monomers. For this to occur, the expectation is that they prevent the development of inter-peptide hydrogen bonds, for the preferential connection with the molecules of water. Incomplete evaporation of the solvent / water mixture may compromise the polymerization of the material and also allow the early degradation of the bonding interface by the presence of residual humidity. Perhaps a higher per-centage of ethanol in the experimental primer, and its correct evaporation are neces-sary to ensure that there is a correct adhesion to the structure. These facts also ex-plain the predominance of adhesive lines in the dentine structure, and it is important to note that care must be taken during the application of all steps of the adhesive procedure [45].
Sorption in polymeric materials means that the ability of these materials to ab-sorb some solvent, usually water, into the polymer network, with possible rupture of the intermolecular bonds and weakening of the material and the solubility is directly related to the degree of conversion the unconverted portion of monomers in polymers can be eliminated from polymer [46]. The effects of Absorption and hydrolytic degra-dation may cause damage to the polymer structure as volumetric changes. Altera-tions such as plastification and softening, and chemical changes such as oxidation and hydrolysis and may lead to reduction in the longevity of dental restorations, as well as their elimination within the oral cavity may affect the biocompatibility with the
medium [47]. Sorption and solubility in water were lower for the piperonyl methacry-late adhesive system. It is believed that a higher percentage of monomer conversion and a more crosslinked polymeric structure may be responsible for reducing water absorption and structure swelling since the free space within the polymer network would be smaller. Since the piperonyl methacrylate copolymerizes and integrates in the polymer chain.On the other hand, the opposite occurs with the EDAB co-initiator with lower conversion rates of the material, can lead to a higher leaching of the ma-terial when stored in water and increase solubility rates.
The substitution of ethyl-4-dimethylamino benzoate (EDAB) by alternative co-initiators in dental adhesives is a strong justification, taking into account that they re-duce the risk of toxicity during their large-scale manufacture and consequently de-crease the production of toxic waste of the industries [48]. Another point of view is that monomers that do not participate in the polymerization reaction can be leached, released from the composites into the oral cavity, reaching the pulp tissue through the dentinal canaliculi, causing damage to the pulp tissue [16,17]. Therefore, bio-compatibility is one of the most frequent limitations of adhesive procedures in deep dentin [17]. After the conclusion of the cytotoxicity test, it can be seen that the per-centage of cell viability assessed after 24h was that all adhesive groups were statisti-cally similar to the untreated (cell control) group which was considered equal to 100%, Therefore the groups had no cytotoxic effect
For the evaluation of antimicrobial action of the adhesives tested, was per-formed the modified direct contact test, because in this test the microorganism is in close contact with the tested material, from the times proposed it is possible to per-form a measurement of the microbial growth through the calculation of the colony-forming units, which evaluates the viability of the cells after the test [28]. No antimi-crobial effect was observed on the adhesive systems tested and after 24 hour of di-rect contact test, all the groups presented bacterial growth similar or superior to the bacterial control.
5. Conclusion
The piperonyl methacrylate presented similar or superior performance to the tertiary amine, with the advantage of having a higher biocompatibility as a function of its copolymerization potential, representing an alternative reagent for photopolymer-izable compositions.
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