UNIVERSIDADE ESTADUAL DE CAMPINAS FACULDADE DE ODONTOLOGIA DE PIRACICABA
HELOISA NAVARRO PANTAROTO
AVALIAÇÃO DA ATIVIDADE FOTOCATALÍTICA E ANTIBACTERIANA DO TIO
2DEPOSITADO NA SUPERFÍCIE DO TITÂNIO COMERCIALMENTE PURO POR
MEIO DE PULVERIZAÇÃO CATÓDICA
EVALUATION OF PHOTOCALYTIC AND ANTIBACTERIAL ACTIVITY OF TiO
2ON
COMMERCIALLY PURE TITANIUM SURFACE DEPOSITED BY MAGNETRON
SPUTTERING
Piracicaba 2017
HELOISA NAVARRO PANTAROTO
AVALIAÇÃO DA ATIVIDADE FOTOCATALÍTICA E ANTIBACTERIANA DO TiO
2DEPOSITADO NA SUPERFÍCIE DO TITÂNIO COMERCIALMENTE PURO POR
MEIO DE PULVERIZAÇÃO CATÓDICA
EVALUATION OF PHOTOCALYTIC AND ANTIBACTERIAL ACTIVITY OF TiO
2ON
COMMERCIALLY PURE TITANIUM SURFACE DEPOSITED BY MAGNETRON
SPUTTERING
Dissertação apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Mestra em Clínica Odontológica, na Área de Prótese dental. Dissertation presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Master in Dental Clinic, in Dental Prosthesis area.
Orientador: Prof. Dr. Valentim Adelino Ricardo Barão
Este exemplar corresponde à versão final da dissertação defendida por Heloisa Navarro Pantaroto e orientada pelo Prof. Dr. Valentim Adelino Ricardo Barão.
Piracicaba 2017
Agência(s) de fomento e nº(s) de processo(s): FAPESP, 2015/17055-8; CAPES ORCID: http://orcid.org/http://orcid.org/00
Ficha catalográfica
Universidade Estadual de Campinas
Biblioteca da Faculdade de Odontologia de Piracicaba Marilene Girello - CRB 8/6159
Pantaroto, Heloisa Navarro,
P195a PanAvaliação da atividade fotocatalítica e antibacteriana do TiO2 depositado na
superfície do titânio comercialmente puro por meio de pulverização catódica / Heloisa Navarro Pantaroto. – Piracicaba, SP : [s.n.], 2017.
PanOrientador: Valentim Adelino Ricardo Barão.
PanDissertação (mestrado) – Universidade Estadual de Campinas, Faculdade
de Odontologia de Piracicaba.
Pan1. Titânio. 2. Fototerapia. 3. Bactérias. 4. Biofilme. I. Barão, Valentim Adelino Ricardo,1983-. II. Universidade Estadual de Campinas. Faculdade de Odontologia de Piracicaba. III. Título.
Informações para Biblioteca Digital
Título em outro idioma: Evaluation of photocatalytic and antibacterial activity of TiO2 on
commercially pure titanium surface deposited by magnetron sputtering
Palavras-chave em inglês:
Titanium Phototherapy Bacteria Biofilms
Área de concentração: Prótese Dental Titulação: Mestra em Clínica Odontológica Banca examinadora:
Valentim Adelino Ricardo Barão [Orientador] Elidiane Cipriano Rangel
Carolina Steiner Oliveira Alarcon
Data de defesa: 22-02-2017
Programa de Pós-Graduação: Clínica Odontológica
UNIVERSIDADE ESTADUAL DE CAMPINAS
Faculdade de Odontologia de Piracicaba
A Comissão Julgadora dos trabalhos de Defesa de Dissertação de Mestrado, em sessão pública realizada em 22 de Fevereiro de 2017, considerou a candidata HELOISA NAVARRO PANTAROTO aprovada.
PROF. DR. VALENTIM ADELINO RICARDO BARÃO
PROFª. DRª. ELIDIANE CIPRIANO RANGEL
PROFª. DRª. CAROLINA STEINER OLIVEIRA ALARCON
A Ata da defesa com as respectivas assinaturas dos membros encontra-se no processo de vida acadêmica do aluno.
DEDICATÓRIA
Aos meus pais Pedro e Rosemeire e aos meus irmãos João e Luciana
O apoio de vocês é fundamental para mim. Obrigada por todo amor, educação, ensinamentos, companheirismo e paciência que sempre me dedicaram, eu serei eternamente grata a tudo isso. Vocês são os melhores exemplos que eu poderia ter e agradeço a Deus todos os dias por tê-los em minha vida. Amo muito vocês!
Aos meus avós José, Mathilde e ao meu Bisavô Gonçalo
Que sempre estiveram muito presentes durante toda minha vida, e agora estão presentes em meu coração. Vocês me deixaram muitas marcas e lembranças boas, e com certeza são a minha maior saudade!
AGRADECIMENTOS
Primeiramente eu agradeço a Deus, por sempre proteger e guiar as minhas escolhas. Por todo amparo, força e coragem durante momentos difíceis. Pela minha saúde e da minha família e por todo amor e carinho que recebo de todos meus familiares e amigos, que sem dúvida me incentiva a seguir em frente. Obrigada pela minha vida, por tudo que já conquistei e por todas as pessoas incríveis que estão à minha volta!
Ao meu orientador Prof. Dr. Valentim Adelino Ricardo Barão. Muito obrigada pela oportunidade de ser sua orientada, pelos ensinamentos transmitidos e por sua disponibilidade. Sua paixão pela pesquisa é evidente e incentivadora, bem como a atenção com que trata seus alunos. Sem dúvida, um grande exemplo a ser seguido. Sinto-me privilegiada em ser sua orientada e agradecida por todos os conselhos, amizade e ótimo convívio desde a época da graduação.
À Universidade Estadual de Campinas – UNICAMP, na pessoa do Magnífico Reitor, Prof. Dr.
José Tadeu Jorge, pelo meu mestrado nesta instituição.
À Faculdade de Odontologia de Piracicaba – UNICAMP, na pessoa do seu Diretor Prof. Dr.
Guilherme Elias Pessanha Henriques.
À Coordenadora Geral da Pós-Graduação Profa. Dra. Cinthia Pereira Machado Tabchoury e a Coordenadora do Programa de Pós-Graduação em Clínica Odontológica Profa. Dra. Karina Gonzales
Silvério Ruiz.
À CAPES, pela concessão de bolsa de mestrado.
À FAPESP, pela concessão de bolsa de mestrado (Processo 2015/17055-8), que foi primordial para realização deste projeto.
Ao Laboratório de Plasmas Tecnológicos da Universidade Estadual Paulista “Júlio de
Mesquita Filho” – UNESP (Campus de Sorocaba), representado pelo Prof. Dr. Nilson Cristino da Cruz
e pela Prof. Dra. Elidiane Cipriano Rangel pela parceria no desenvolvimento deste trabalho.
Ao Laboratório de Física da Universidade Estadual Paulista “Júlio de Mesquita Filho” – UNESP
(Campus de Bauru), representado pelo Prof. Dr. José Humberto Dias da Silva pela parceria no
desenvolvimento deste trabalho.
Ao Laboratório de Física da Universidade Estadual de Campinas – UNICAMP, representado pelo Prof. Dr. Mário Antonio Bica de Moraes pela parceria no desenvolvimento deste trabalho.
Ao Laboratório de Bioquímica Oral da FOP – UNICAMP), representado pelo Prof. Dr. Jaime
Aparecido Cury pela parceria no desenvolvimento deste trabalho.
À Prof.ª Dra. Elidiane Cipriano Rangel (UNESP-Sorocaba) minha sincera gratidão por todos os ensinamentos transmitidos, pela gentileza com que sempre me recebeu. Além de toda atenção e disponibilidade para ricas discussões e esclarecimento de dúvidas durante o desenvolvimento deste estudo.
Ao Prof. Dr. Mário Bica de Moraes (UNICAMP-Campinas) pela parceria neste trabalho, disponibilidade, atenção e ótimos ensinamentos.
Ao Prof. Dr. José Humberto Dias da Silva (UNESP-Bauru) pela parceria no desenvolvimento deste estudo, pela disponibilidade, conhecimentos e atenção.
Ao Prof. Dr. Antônio Pedro Ricomini Filho por toda prontidão, gentileza e disponibilidade desde o início do desenvolvimento do projeto, sem dúvida suas ricas opiniões contribuíram muito para este estudo. Agradeço também por todos os conselhos e pela amizade conquistada neste período.
Aos docentes Profa. Dra. Altair Del Bel Cury, Profa. Dra. Célia Rizzati Barbosa, Prof. Dr.
Frederico Andrade e Silva, Prof. Dr. Marcelo Ferraz Mesquita, Prof. Dr. Mauro Antônio de Arruda Nóbilo, Prof. Dr. Rafael Leonardo Xediek Consani, Profa. Dra. Renata Cunha Matheus Rodrigues Garcia, Prof. Dr. Wander José da Silva, Prof. Dr. Wilkens Aurélio Buarque por todo conhecimento
À Sra. Eliete A. Ferreira Lima Marim, secretária do Departamento de Prótese e Periodontia da FOP-UNICAMP, pela atenção e toda gentileza. Ao Eduardo Pinez (Du), técnico do Laboratório de Prótese Total, pelo bom humor, amizade e prontidão para ajudar sempre.
Aos técnicos do Laboratório de Bioquímica Oral, Waldomiro Vieira e José Alfredo da Silva, por toda solicitude e gentileza.
Ao Nilton Francelosi pelo treinamento nos equipamentos do Laboratório de Física (UNESP – Bauru), por todos os ensinamentos, conselhos, prestatividade e amizade.
Ao Rafael Parra pela prestatividade e ajuda no manejo dos equipamentos do Laboratório de Plasmas Tecnológicos (UNESP – Sorocaba), a Jéssica Gonçalves pelo apoio na realização nas análises deste estudo e aos amigos que conquistei em Sorocaba por toda receptividade e companhia.
À minha família Pedro, Rosemeire, João e Luciana por tudo que sempre fizeram por mim, por todo amor com que me recebem e em especial pela ajuda na confecção da caixa de luz que foi essencial neste trabalho.
À Família Navarro e Família Pantaroto, em especial às famílias da minha tia Antonia, tia
Leonilde e à família dos meus padrinhos Izabel, Robson e Fabiana por todo zelo, companheirismo e
orações. O apoio e amor de vocês são e sempre serão essenciais para mim.
À todos os meus amigos do Laboratório de Prótese Total Adaias Oliveira Matos, Anna
Gabriella Camacho Presotto, Bruno Zen, Claudia Bhering, Conrado Caetano, Erika Ogawa, Gabriel Meloto, Giovana Oliveira, Guilherme Machado, Gustavo Corradini, Halina Berejuk, Isabella Marques,
Jairo Cordeiro, João Gabriel, Marina Pisani,Moisés Nogueira, Júlia Campana, Ricardo Caldas, Sales
Antonio Barbosa Junior, Vagner Reginato, Veber Bonfim muito obrigado pela boa convivência,
amizade e experiências trocadas.
Aos amigos conquistados durante a Pós Graduação, em especial Amanda Bandeira, Bruna
Ximenes, Camilla Fraga, Elis Lira, Giancarlo De la Torre, Louise Dornelas, Mariana Barbosa, Mayara Abreu, Olívia Figueredo e Victor Muñoz obrigada pela ótima amizade e companheirismo, que
tornaram estes anos mais leves.
Às amigas da graduação que estão sempre próximas: Hortência Xavier, Jéssica Camassari,
Renata Pereira e às que estão mais distantes, porém sempre em meu coração Audrey Foster, Beatriz Capelli, Cintia Maruki, Jéssica Santos, Karime Botelho, Larissa Leal, Marcela Felizardo, Rayane Ramos, Maria Giulia Pucciarelli.
Agradeço também aos meus amigos Beatriz Varela, Bruna Sbrunhera, Dante Ferrasoli, David
Ward, Erica Barbosa, Gabriel Sanches, Guilherme Dobo, Jonas Calado, Laura Grassatto, Natalia Mincev, Rodrigo Panfiett e Victor Galeazzo, por todas as alegrias vivenciadas, por entenderem os
meus momentos ausentes devido aos estudos e pela amizade de longa data e sem dúvida, muito importante pra mim.
RESUMO
A saúde peri-implantar é de suma importância para o sucesso e a longevidade dos tratamentos reabilitadores, dependendo de pilares protéticos (abutments) que proporcionem a saúde gengival. Tratamentos de superfície vem sendo estudados em abutments visando saúde periimplantar aliando propriedades antimicrobianas. O composto fotocatalítico dióxido de titânio (TiO2) tem sido incorporado em alguns tratamentos de superfície devido a sua suposta
propriedade antibacteriana, a qual ainda é incerta em biofilmes orais. Dessa forma, o objetivo deste estudo foi desenvolver um modelo de biofilme inicial multi espécies para investigar o potencial fotocatalítico e antibacteriano de filmes de TiO2 obtidos através de pulverização
catódica sobre a superfície de discos de titânio comercialmente puro (Ticp). Os grupos estudados foram: (1) cpTi polido (grupo controle); (2) A-TiO2 (fase cristalina anatase); (3)
M-TiO2 (mistura das fases cristalinas anatase e rutilo); (4) R-TiO2 (rutilo). A superfície dos discos
foram caracterizadas quanto à morfologia (microscopia eletrônica de varredura – MEV), fase cristalina (difração de raios X – DRX), composição química (espectrometria de energia dispersiva – EDS), dureza e módulo de elasticidade (nanoindentação), rugosidade (perfilometria) e energia livre de superfície (goniômetro). O potencial fotocatalítico das superfícies foi avaliado por meio da degradação do corante de Azul de Metileno (AM). A ação antibacteriana foi avaliada por meio da adesão do biofilme multi espécies (16,5 h) composto por Streptococcus sanguinis, Actinomyces naeslundii e Fusobacterium nucleatum seguido da exposição à luz UVA (1h; 2×15 W; λ = 350 nm e intensidade = 1,62 mW/cm2). A morfologia do
biofilme e a contagem de unidades formadoras de colônia (UFC) foram avaliadas. Os dados foram submetidos à análise de variância (ANOVA) e ao teste de Tukey HSD (α=0,05). Os filmes de TiO2 apresentaram espessura de ∼300 nm, dureza superior ao Ticp (p<0,05) e os grupos
M-TiO2 e R-TiO2 apresentaram módulo de elasticidade superior ao Ticp, enquanto A-TiO2 foi
similar. Quanto à rugosidade, A-TiO2 e R-TiO2 compreenderam valores inferiores ao M-TiO2 e
Ticp (p<0,05). R-TiO2 obteve menor ELS comparado aos demais grupos (p<0,05). A-TiO2 e
M-TiO2 apresentaram potencial fotocatalítico superior ao R-TiO2 (p<0,05); entretanto este
potencial não foi suficiente para promover atividade antibacteriana no biofilme oral tri-espécies (p>0,05 x controle). Este estudo traz novos esclarecimentos para o desenvolvimento de protocolos para investigar a atividade fotocatalítica do TiO2 associado ao biofilme oral.
ABSTRACT
The health of periimplant tissues is of utmost important for long-term success of prosthetic rehabilitation treatment and depends on abutments that support the gingival health. Surface treatments of abutments have been studied to promote periimplant health and antimicrobial properties. A photocatalytic compound such as titanium dioxide (TiO2) has been incorporated
into some surface treatments due to its supposed antibacterial activity, which remains uncertain on oral biofilm. In this study we developed an oral multispecies early biofilm model to investigate the photocatalytic and antimicrobial activities of TiO2 films deposited by
magnetron sputtering on commercially pure titanium (cpTi) discs. The studied groups were: (1) cpTi machined (control group); (2) A-TiO2 (anatase crystalline phase); (3) M-TiO2 (mixture
of anatase and rutile crystalline phases); (4) R-TiO2 (rutile crystalline phase). Discs’ surfaces
were characterized in terms of morphology (scanning electron microscopy - SEM), crystalline phase (X-ray diffraction analysis -XRD), chemical composition (energy dispersive spectrometry - EDS), hardness and elastic modulus (nanoindentation), roughness (perfilometer) and surface free energy (SFE) (goniometer). Photocatalytic potential was evaluated using the Methylene Blue (MB) degradation assay. The antibacterial potential was investigated by the adhesion (16.5 h) of a multi-species biofilm (16.5 h) composed by Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum followed by UVA light exposure (1h; 2×15 W; λ = 350 nm and intensity = 1.62 mW/cm2). Biofilm morphology analysis (SEM) and colony forming
units quantification (CFU) were analyzed. Data were subjected to analysis of variance (ANOVA) and Tukey HSD test (α=0.05). TiO2 films presented thickness of ∼300 nm and greater hardness
comparing to cpTi. M-TiO2 and R-TiO2 showed higher elastic modulus comparing to A-TiO2 and
cpTi surfaces (p<.05). A-TiO2 and R-TiO2 presented smaller values of roughness compared to
cpTi and M-TiO2 (p<.05). R-TiO2 presented the smallest SFE (p<.05) when compared to the
other groups. A-TiO2 and M-TiO2 films presented superior photocatalytic activity than R-TiO2
(p<0.05); however no antibacterial activity of TiO2 filmswas noted (p>0.05 vs. control). This
study brings new insights on the development of protocols for the photocatalytic activity of TiO2 in oral biofilm-associated disease.
SUMÁRIO
Introdução ... 11
Artigo:
Photocalytic activity of sputtered TiO
2films on commercially pure
titanium surface in oral multispecies biofilm
...15
Conclusão ... 43
Referências ... 44
Anexo 1:
Certificado do comitê de ética
... 47
11
1 INTRODUÇÃO
A peri-implantite é um processo inflamatório que pode acometer os tecidos moles e duros da região peri-implantar (Zitzmann et al. 2008). Sua prevalência aproxima-se de 22% segundo um recente estudo de meta-análise (Derks et al. 2015) e a colonização bacteriana apresenta-se como seu principal fator etiológico (Buser et al. 1997; Sangeeta Dhir 2013).
A adesão inicial dos micro-organismos tem impacto significante na etiopatogênese da infecção (Quirynen & Bollen 1996; Scarano et al. 2004; Elter et al. 2008), visto que a microbiota presente na superfície de implantes no primeiro mês de sua instalação se mantém semelhante mesmo após seis meses (De Boever et al. 2006). Além disso, devido ao contato com os tecidos peri-implantares, os abutments apresentam um papel importante em conservar a saúde dos implantes (Hahnel 2014).
Quando os abutments são expostos na cavidade oral, rapidamente inicia-se a formação da película salivar (Fürst et al. 2007). As moléculas presentes nesta película promovem a adesão e coagregação das espécies primárias (Kolenbrander et al. 2010; Sangeeta Dhir 2013), as quais proporcionam um ambiente favorável para a adesão dos colonizadores secundários (Fröjd et al. 2011). Interações entre as diferentes espécies de bactérias por meio de trocas metabólicas, contato físico, trocas de informações genéticas e sinalização celular permitem que os micro-organismos se multipliquem nas superfícies formando o biofilme (Kolenbrander et al. 2006).
A troca da microbiota predominantemente gram-positiva para gram-negativa leva a transição de saúde para a doença (peri-implantite) (Socransky et al. 1998). Diversos microorganismos podem estar relacionados à severidade da doença, entretanto, a associação de três gêneros de bactérias: Actinomyces, Streptococcus e Fusobacterium, têm sido considerada de grande importância no processo de colonização inicial do substrato e desenvolvimento do biofilme (Periasamy et al. 2009; Kolenbrander et al. 2010; Diaz 2012).
Actinomyces naeslundii vem sendo encontrado em maiores proporções na peri-implantite do que em áreas de implantes saudáveis (Kumar et al. 2012; da Silva et al. 2014). Fusobacterium nucleatum é um micro-organismo anaeróbio de alta prevalência em doenças peri-implantares, atuando nos processos de coadesão e coagregação, principalmente para micro-organismos peri-implantopatogênicos (Periasamy et al. 2009; Kolenbrander et al.
12 2010). Grössner-Schreiber et al. (2009) observaram in situ que 45% das bactérias identificadas na superfície de discos de titânio (Ti) com diferentes tratamentos de superfície eram Streptococcus sanguinis, o qual tem sido utilizado em modelos de adesão de bactérias devido a sua alta importância na formação de biofilme (Nakazato et al. 1989; Wolinsky et al. 1989) e por apresentar melhor adesão em superfícies cobertas com a película salivar quando comparado com outros micro-organismos (Pereira da Silva et al. 2005; Barao et al. 2015).
Tendo em vista que os abutments são elementos que estão mais expostos ao meio bucal, o controle da proliferação de micro-organismos sobre sua superfície é importante para manutenção da saúde peri-implantar e longevidade do tratamento. Ademais, as propriedades de superfície dos componentes protéticos e implantes como rugosidade, energia de superfície e composição química podem influenciar na adesão e manutenção do biofilme, e consequentemente, facilitar ou dificultar a colonização e o crescimento das espécies encontradas na cavidade oral (Teughels et al. 2006).
Os tratamentos de superfície estão sendo amplamente estudados objetivando melhorias nas superfícies de biomateriais e também em busca de superfícies antibacterianas ou que promovam menor adesão de bactérias. Para isto, compostos como o Dióxido de Titânio (TiO2) vêm sendo incorporados à alguns tratamentos de superfície, por apresentar vantagens
como estabilidade físico química, boas propriedades óticas, baixo custo, além de boa eficiência fotocatalítica (Brady et al., 1971; Okimura et al., 2001; Diebold et al., 2003). Quando exposto à luz, o TiO2 gera oxigênios reativos capazes de degradar bactérias e poluentes
orgânicos (Malato et al. 2009; Fisher et al. 2013; Synnott et al. 2013), os quais também podem promover uma superfície hidrofílica, sendo um bom candidato para aplicações em biomateriais (Lorenzetti et al. 2015).
Porém, a aplicação industrial do TiO2 têm apresentado um lento progresso, devido à
natureza polimórfica do material (Lim et al. 2014), que exibe três diferentes fases cristalinas, a anatase, o rutilo e a brookita. Dentre elas, a anatase apresenta alta atividade fotocatalítica e hidrofilicidade (Zhang et al. 2004; Hashimoto et al. 2005), o rutilo apresenta menor atividade fotocatalítica comparado a anatase, porém maior estabilidade química e dureza (Samsonov et al. 1973; Huang et al. 2006). Já a brookita é quimicamente instável e portanto, de difícil obtenção (Lee et al. 2008; Murakami et al. 2009; Arier et al. 2011).
13 A atividade fotocatalítica de filmes de anatase ajuda a degradar compostos orgânicos que estão em contato com a superfície, enquanto a hidrofilicidade pode promover uma limpeza mais eficiente (Lorenzetti et al. 2014). Levando em conta essas características, acredita-se que a incorporação de TiO2 em um tratamento de superfície poderia ser um bom
coadjuvante na prevenção de infecções bacterianas antes e durante a implantação (Lorenzetti et al. 2014).
Entretanto para gerar a formação dos oxigênios reativos responsáveis pela ação antibacteriana, é preciso promover os elétrons da banda de valência para a banda de condução do material semicondutor, mecanismo que depende do comprimento de onda da radiação incidente e do “bandgap” do material. Estudos demonstraram que a seleção da luz ideal depende do “bandgap” da estrutura cristalina obtida nos filmes de TiO2, sendo o
“bandgap” da anatase correspondente a ∼3,2 eV e do rutilo à ∼3,0 eV. De acordo com a literatura, a fonte de luz ideal para promover este processo corresponde à luz Ultravioleta (UV) (≤ 400 nm) (Landmann et al. 2012; Di Valentin et al. 2007). A fonte de luz UVA (λ = 315– 400 nm) tem sido utilizada em muitos estudos (Shiraishi et al. 2009; Choi et al. et al. 2009; Zhuang et al. 2012; Joost et al. 2015; Pleskova et al. 2016) por ser menos prejudicial aos organismos vivos quando comparada à luz UVB e UVC (Hockberger 2002; Kühn et al. 2003; Joost et al. 2015).
Para obtenção do tratamento de superfície com partículas de TiO2 existem vários
métodos como “sol-gel” (Shalini et al. 2005), “sputtering” (Mráz & Schneider 2011; Lim et al. 2014), “spray pyrolysis” (Shinde et al. 2008; Sabataityte 2006), “plasma enhanced chemical deposition (PECVD)” (Ha et al. 1996), entre outros. A pulverização catódica (do inglês: sputtering), é o método de escolha por apresentar características superiores de adesão e dureza do filme (Yaghoubi et al. 2010), uniformidade, baixo custo e alta hidrofilidade (Cao et al. 2013; Lim et al. 2014), além da possibilidade de obtenção das estruturas cristalinas anatase e rutilo, por meio de mudanças nos parâmetros de deposição como por exemplo pressão de trabalho e temperatura. Este processo ocorre dentro de uma câmara em uma atmosfera de gases (Ar e O2) a uma pressão reduzida em relação à pressão atmosférica, onde
colisões de elétrons com o gás inerte (Ar) produzem sua ionização. A colisão dos íons de argônio com o alvo presente no sistema produz a ejeção de átomos do alvo que se depositam no substrato promovendo o crescimento do filme (Pomin et al. 2011).
14 Como observado, o TiO2 pode apresentar ação antimicrobiana, porém ainda não foi
estudado seu efeito fotocatalítico em bactérias periodonto patogênicas em biofilmes multiespécie associando às fases cristalinas anatase e rutilo. Portanto, os objetivos deste estudo foram: (1) Desenvolver filmes de TiO2 nas estruturas cristalinas anatase, rutilo e
mistura (anatase + rutilo) sobre titânio comercialmente puro (Ticp) por meio de pulverização catódica; (2) Caracterizar a superfície dos discos de Ticp (superfície controle) e dos discos de Ticp revestidos com filmes de TiO2 em diferentes fases cristalinas (superfícies experimentais)
quanto à morfologia (microscopia eletrônica de varredura), composição química (espectrometria de energia dispersiva), caracterização da fase cristalina (difração de raios-x), rugosidade (perfilometria), nanodureza e módulo de elasticidade (nanoindentação), molhabilidade e energia livre de superfície (goniômetro); Avaliar as superfícies quanto ao (3) o potencial fotocatalítico dos filmes de TiO2 por meio da degradação do corante de azul de
metileno e (4) potencial antibacteriano na adesão inicial do biofilme multiespécies (16,5 h) composto por Streptococcus sanguinis, Actinomyces naeslundii e Fusobacterium nucleatum seguido da exposição à luz UVA (1h) quanto às unidades formadoras de colônia (UFC) e organização estrutural (MEV).
15 Photocalytic activity of sputtered TiO2 films on commercially pure titanium surface in oral
multispecies biofilm
Heloisa N. Pantarotoa, Antonio P. Ricomini Filhob, José H. D. Silvac , Nilton F. Azevedo Netoc,
Cortino Sukotjod, Elidiane C. Rangele, Valentim A. R. Barãoa,*.
a Department of Prosthodontics and Periodontology, Piracicaba Dental School, Univ of
Campinas (UNICAMP), Av Limeira, 901, Piracicaba, São Paulo, Brazil, 13414-903.
b Department of Physiological Science, Piracicaba Dental School, University of Campinas
(UNICAMP), Av Limeira, 901, Piracicaba, São Paulo, Brazil, 13414-903.
c Department of Physics, Univ Estadual Paulista (UNESP), Av. Eng. Luís Edmundo C. Coube,
14-01, Bauru, São Paulo, Brazil, 17033-360.
d Department of Restorative Dentistry, Univ of Illinois at Chicago (UIC), College of Dentistry,
801 S Paulina, Chicago, IL, USA, 60612.
e Laboratory of Technological Plasmas, Engineering College, Univ Estadual Paulista (UNESP),
Av Três de Março, 511, Sorocaba, São Paulo, Brazil, 18087-180.
*Corresponding author:
Av. Limeira, 901, Piracicaba, SP, Brazil 13414-903, Tel.: + 55-19-2106 5719; Fax: +55-19-2106 5218;
16 Graphical abstract
Highlights
New methodology for TiO2 activity on a multispecies early biofilm model.
TiO2 films with different crystalline phases were successfully developed.
TiO2 films improved the mechanical properties of titanium.
Anatase and mixture phases had superior photocatalytic activity. TiO2 had no significant antibacterial effect.
17 Abstract
A photocatalytic compound such as titanium dioxide (TiO2) has been incorporated into some
surfaces due to its supposed antibacterial activity, which remains unclear on oral biofilm-related disease. In this study we developed an oral multispecies early biofilm model to investigate the photocatalytic and antimicrobial activities of TiO2 films deposited by
magnetron sputtering on commercially pure titanium (cpTi) discs. The studied groups were: (1) cpTi machined (control); (2) A-TiO2 (anatase); (3) M-TiO2 (mixture of anatase and rutile); (4)
R-TiO2 (rutile). Discs’ surfaces were characterized in terms of morphology, crystalline phase,
chemical composition, hardness, elastic modulus, roughness and surface free energy (SFE). Photocatalytic potential was evaluated using the methylene blue degradation assay. The antibacterial activity was evaluated on a multispecies early biofilm (16.5 h) composed of Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nucleatum followed by UVA light exposure (1h). Early biofilm morphology analysis and colony forming units (CFU) were evaluated. All TiO2 films presented a thickness of about 300 nm and improved the
hardness and elastic modulus of cpTi surfaces (p<0.05). A-TiO2 and R-TiO2 films promoted a
slight decrease of roughness values compared to cpTi and M-TiO2 film (p<0.05). R-TiO2
presented the smallest SFE (p<0.05). A-TiO2 and M-TiO2 films presented superior
photocatalytic activity than R-TiO2 (p<0.05); no antibacterial activity of TiO2 filmswas noted
(p>0.05 vs. control). This study brings new insights on the development of protocols for the photocatalytic activity of TiO2 in oral biofilm-associated disease.
18 1. Introduction
Despite the evidence of excellent implant therapy results, peri-implantitis disease occurs [1] and is characterized as an infectious condition of the tissues and bone around osseointegrated implants presenting clinical signs of inflammation [2]. According to a recent meta-analysis study, the overall prevalence of peri-implantitis was approximately 22% [3]. One of the possible etiologies is the bacterial colonization of the implant surface and its components, such as abutments.
In the oral cavity, after placement, the abutment surfaces are immediately covered with an acquired pellicle [4] and are subjected to bacterial colonization. The genera Actinomyces and Streptococcus are the main initial colonizers and Fusobacterium is the secondary colonizer and is associated with peri-implant pathogenic biofilm [5,6].
The bacterial adherence and biofilm formation are directly influenced by the surface properties including chemical composition, surface roughness and surface free energy [7]. Hence, the development of films onto abutments and implant surfaces have been investigated as a possible way to make surfaces less prone to biofilm colonization, being interesting for the long-term success of implant therapy [8].
Furthermore, some compounds can be added to these films to promote an antibacterial action such as titanium dioxide (TiO2), which is considered a photocatalytic
compound. TiO2 occurs in two main crystalline forms: anatase and rutile [9]. When
photocatalyzed, TiO2 produces reactive oxygen species that promotes the degradation of
bacterial membranes [10]. However, this process depends on the band gap of the materials. The band gap of TiO2 corresponds to about 3.2 eV for anatase and around 3.0 eV for rutile and
it can only absorb ultraviolet light (UV) (≤400 nm) [11,12]. Among UV light sources, the UVA (λ = 315-400 nm) has been used in some studies [10,13–15] as the longer wavelength is less harmful to living organisms [10,16,17].
Several methods are used for TiO2 deposition on biomaterials such as sol-gel [18], spray
pyrolysis [19], plasma-enhanced chemical vapor deposition [20] and magnetron sputtering [21–23]. Magnetron sputtering is a widely used method due to its greater adhesion, hardness
and hydrophilicity [22,24–26] in addition to the possibility of obtaining isolated phases of anatase and rutile [27].
19 Few studies have investigated the TiO2 photocatalytic and antibacterial actions on
peri-implantitis specific microorganisms that simulate the oral environment in the implant abutment area [8,28]. Furthermore, no study has correlated the bacterial adhesion on different crystalline phases of TiO2 films. Therefore, in this study we developed TiO2 films with
different crystalline phases (anatase, rutile and a mixture of both) onto the cpTi surface using magnetron sputtering. The physical-chemical, photocatalytic degradation and antimicrobial properties using a periimplantitis-associated oral multispecies biofilm model composed of Streptococcus sanguinis, Actinomyces naeslundii and Fusobacterium nuicleatum were assessed.
2. Methodology
2.1 Experimental design
CpTi discs (grade II, American Society for Testing of Material) (MacMaster Carr, Elmhurst, IL, USA), 10 mm diameter and 2 mm thickness were randomly divided and submitted to Radiofrequency (RF) magnetron sputtering treatment to obtain a TiO2 film composed of
anatase (A-TiO2), rutile (R-TiO2) and a mixture (anatase+rutile) (M-TiO2) of crystalline
structures (experimental groups). The control group (CpTi) was not treated. The crystalline phase analysis (n=1), morphology (n=1), chemical analysis (n=1), hardness (n=1), roughness (n=5) and surface free energy (n=5) of the films were assessed. The photocatalytic activity pathway was evaluated using the methylene blue degradation technique (n=3). For the microbial assay, a three-species biofilm composed of S. sanguinis, A. naeslundii and F. nucleatum was developed onto discs for 16.5 h in a modified fluid universal medium (mFUM). Afterwards, the early biofilm was exposed to a customized UVA light apparatus for 1 h and the number of colony forming units (CFU) (n=6) was assessed and biofilm organization visualized (n=1) (Figure 1a).
20 Figure 1. a) Experimental Design. b) Microbiological assay design.
2.2 Preparation of titanium discs and surface film
CpTi discs were polished using sequential SiC grinding papers (#320, #400 and #600) (Carbimet 2, Buehler) in an automatic polisher (EcoMet/AutoMet 250 Pro, Buehler). Samples were ultrasonically cleaned in deionized water (10 min) and 70% propanol (10 min) (Sigma-Aldrich) and hot air dried [29].
TiO2 films were deposited on cpTi discs substrate by RF magnetron sputtering in a Kurt
J. Lesker sputtering chamber (model KJL—System I) using a Ti-metal target (99.999%) (Kurt J. Lesker) and Ar+O2 mixture. Before each deposition, the target was sputtered with Ar for 10
min to ensure that the target was clean during the film growth process [30]. The main deposition parameters of the films was based on previous studies [27,31]. The constant and variable parameters are summarized in Table 1.
21 Table 1. Deposition parameters of TiO2 films prepared by RF magnetron sputtering.
Constant Parameters
Target – metallic Ti (99.999%) – 7.62 cm of diameter and 0.6 cm thickness Reflected RF power – 1 to 5 W depending on deposition
Target to substrate distance: 70 mm Substrates – cpTi discs
Residual pressure of the sputtering chamber was smaller than 1×10−6 Torr.
Ar flux – 40 SCCM Variable Parameters Crystalline Phase O2 flux (SCCM) Pressure (Torr) RF Power(W) Total time deposition (min) Heater temp (°C) Film temp* (°C) Anatase 1 3.0 x 10-2 120 900 200 120 Mixture 1 1.2 x 10-2 240 660 400 288 Rutile 4 2.3 x 10-2 280 420 600 504
* The temperature on the substrate surfaces was measured by a K type (Cromel/Alumel) thermocouple.
2.3 Surface analysis
Energy dispersive spectroscopy
The elemental composition (% atomic) was obtained by EDS (JEOL JSM-6010LA) (n=1) in three different points of each surface [32].
X-ray diffraction
For assessment of the surface crystalline phases, an X-ray diffraction (XRD) (Rigaku-Ultima 2000+) was employed using Cu-Kα - λ = 1.54056 Å in a radiation operating at 40 kV and 20 mA at a continuous speed of 0.02° per second in a fixed angle 2.5° and a scan range from 15° to 80° (n=1) [32].
Scanning electron microscopy and Atomic force microscopy
Surface morphology was analyzed by SEM (JEOL IT-300/2015) (n=1). The AFM micrographs were measured in a 5 μm × 5 μm scan area in a tapping mode with a constant
22
force of 42 N/m and frequency of 320 kHz by an AFM (Park System - NX-10); Roughness average (Ra) values were obtained in three different areas (n=1).
Measurement of TiO2 film thickness
For the TiO2 film thickness measurement, the deposition was performed in half of an
amorphous silica (a-SiO2) substrate (n=1), and the step between the untreated and treated
area was measured in three different areas by a perfilometer (Dektak 150-d; Veeco).
Hardness and Elastic modulus
The hardness and elastic modulus were determined by nanoindentation analysis performed in a Hysitron Triboindenter system using a ten step partial unload function (200 to 5000 µN) applied by a Berkovick diamond indenter. For each load, ten indentations were performed (n=1) [33].
Surface roughness and Surface free energy
The surface roughness (n=5) of the samples were evaluated by topographic profiles acquired in a profilometer (Dektak 150-d; Veeco) in atmospheric conditions, based on Ra - roughness average; Rt - maximum height of the profile; Rz - average maximum height of the profile; and Rq - root mean square roughness parameters which were obtained with cut-off of 0.25 mm at 0.05 mm/s during 12 s. Three measurements were obtained in each disc and the average was calculated [34]. The surface free energy (n=5) was analyzed in a goniometer (ramé-Hart 10000; ramé-hart instrument co.) using the sessile drop method. The water (polar component) and the diiodomethane (dispersive component) contact angles were calculated using the ramé-hart DROP image Standard software (ramé-hart instrument co.) [35].
2.4 Photocatalytic Assay
The methylene blue (MB) ISO technique was used to investigate the photocatalytic degradation pathway of the TiO2 films [9,36]. The specimens were soaked in 2 mL of standard
MB (P.A.-C.I. 52.015, Synth) solution (10 µmol/L) in dark conditions (foil wrapped) for 12 h prior to the test[37] to eliminate reduction in the concentration of MB via absorption by the specimens. After that, discs were placed in a 24-well plate with 2 mL of fresh MB solution (10
23 µmol/L) in each well. The control (CpTi) and experimental (A- TiO2, M- TiO2 and R- TiO2) groups
were submitted to two experiments (n=3 per group), one in the presence of light and another under dark conditions (foil wrapped). The light source used was UVA 2×15 W (λ = 350 nm and intensity = 1.62 mW/cm2) (F15T8/Black Light Silvania) perpendicularly fixed to 7 cm to the
discs [25] in a customized apparatus.
The degradation of the MB as a function of irradiation time (15, 30, 60, 90, 120, 180 and 240 minutes) was measured spectrophotometrically (DU 800 – Beckman Coulter) by sampling the solution and returning the sample after the measurement of the solution’s absorbance at 664 nm[36]. The photocatalytic activity of the TiO2 films was calculated using
equation 1:
Photocatalytic activity (%) = [(co – c)/co] x [c1/co] × 100 (eq. 1)
where co was the concentration of the test solution of MB before irradiation, c was the
concentration of MB after UV irradiation, and c1 was the concentration of MB after the pre-
adsorption test [9].
2.5 Microbiological Assay
The control groups: I. CpTi discs in dark condition; II. CpTi discs in light presence, and test groups: III. A-TiO2; IV. M-TiO2 and V. R-TiO2 were submitted to three independent
microbiological assays (n=6) (Figure 1b).
Acquired pellicle formation
This study was approved by the Local Research and Ethics Committee (50954615.8.0000.5418/2015). To simulate the clinical oral condition in this in vitro study, whole unstimulated human saliva was obtained for 1 h per day over several days from three healthy volunteers (with their informed consent) at least 1.5 h after eating, drinking, or tooth cleaning[38]. The collected saliva was pooled and centrifuged (30 min, 4°C, 27,000 g), and the supernatant was pasteurized (60°C, 30 min) and re-centrifuged in sterile bottles. The resulting supernatant was frozen at -20°C [38]. Prior to acquired pellicle formation, discs were sterilized by gamma radiation (14.50±0.05 kGy) [39]. Each disc was placed in a well of a sterile 24-well polystyrene cell culture plate, incubated with 2 mL of saliva for 4 h at 37°C [38].
24 Biofilm Assay
Strains of S. sanguinis IAL 1832, A. naeslundii OMZ 745 and F. nucleatum OMZ 596 were grown on plates of Columbia blood agar base supplemented with 5% defibrinated blood sheep (CBA) under anaerobic incubation at 37°C for 48 h. Loopfuls of CBA-grown cells were inoculated into 9 mL of filter-sterilized fluid universal medium[40] supplemented with 67 mmol/L Sorensen's buffer, pH 7.2 ("modified fluid universal medium", mFUM) [38] and maintained by anaerobic incubation at 37°C. After 24 h, 1 mL from each tube was transferred to a new tube containing 15 mL of mFUM and incubated at 37°C for 7 h. Then, the optical density of each culture was independently adjusted to OD550 1.00 ± 0.02 and a mixture of all
strains was prepared with equal volumes of each density-adjusted culture.
The pellicle-coated discs were transferred to wells containing 1.8 mL of medium mixture of saliva (60%), mFUM (30%), horse serum (10%) and 225 μL of mixture-species inoculum. The 24-well plate was incubated anaerobically at 37°C for 16.5 h [38,41]. After 16.5 h of bacterial adhesion and organization as an early biofilm, discs were washed two times in saline solution (NaCl 0.9%) and transferred to wells containing 2 mL of NaCl 0.9% [42]. The 24-well plate was exposed to UVA light irradiation for 1 h under the same conditions as the photocatalytic test and in microaerophilic conditions due to the presence of F. nucleatum. For control, another group of CpTi discs (control surface) was submitted to the same conditions, but in the dark (foil wrapped).
Prior to this experiment, a pilot study under the same conditions was performed to determine the ideal light exposure time using only control surface (cpTi) to avoid confusing dead cell results regarding UVA light or TiO2 films. The availability of bacteria cells in the
presence of UVA light was tested at different exposure times (0, 1, 2, 3 and 4 h).
2.6 Biofilm Analysis
Colony Forming Units (CFU)
The discs were transferred to cryogenic tubes containing 3 mL of NaCl 0.9%. The tube was sonicated (7 W for 30 seconds) and from the suspension, an aliquot of 0.1 mL was 7-fold serially diluted in NaCl 0.9% and plated in the following culture media: Columbia Blood Agar (CBA) supplemented with 5% (v/v) defibrinated blood sheep for the counts of total
25 microorganism count; CBA supplemented with the antibiotics (CBA Plus) norfloxacin (1 mg/L), erythromycin (1 mg/L) and vancomycin (4 mg/L) for F. nucleatum; Mitis Salivarius Agar (MSA) for S. sanguinis and Cadmium Sulfate Fluoride Acridine Trypticase Agar (CFAT) for A. naeslundii. CBA and CBA Plus plates were incubated anaerobically at 37°C for 72 h C, while CFAT and MSA plates were incubated in an atmosphere of 10% CO2 at 3 C for 48 h. After
obtaining the counts, data were expressed as colony forming units per mL (Log10 CFU/mL).
Biofilm Organization
Additional discs were fixed in Karnovsky solution (PBS; pH 7.2), followed by dehydration in a series of ethanol washes (60%, 70%, 80%, 90% solution for 5 min and 100% for 10 min) and were then allowed to dry aseptically. Afterwards, they were gold-sputtered for observing in SEM (JEOL-JSM-5600LV) scanned at 15 kV at 500× and 5000× magnification [43].
2.7 Statistical Analysis
One-way ANOVA was used to test the influence of TiO2 on the surface properties and
number of CFU. Two-way repeated measure ANOVA was used to verify the influence of surface treatment and time on the photocatalytic activity of TiO2. Tukey HSD and Bonferroni
tests were used as post hoc techniques for multiple-comparisons when necessary. A mean difference significant at the 0.05 level was used for all tests (SPSS v.20.0, SPSS Inc.).
3 Results and Discussion
3.1 Surface Characteristics
All surfaces presented chemical composition consisting of Titanium (Ti), Oxygen (O) and Carbon (C). The TiO2 groups presented higher amounts of O and lower content of C and
Ti when compared to the control, indicating an oxide film (Table 2).
Figure 2 shows the X-Ray diffraction patterns of the cpTi and TiO2 films. The shape of
the diffraction peaks reveals that all of the contributions can be attributed to titanium (T), anatase (A) or rutile (R) phases, with the titanium crystalline phase referring to the substrate. It is important to highlight that the different samples presented different crystalline phases: anatase, mixture (anatase + rutile) and rutile, suggesting that the cpTi discs were coated with titanium oxide films with different crystalline structures.
26 Figure 2. X-Ray diffraction pattern of CpTi, A-TiO2, M-TiO2 and R-TiO2. The letters T, A and R
refers to corresponding peaks of titanium, anatase and rutile, respectively.
The morphology of substrate (cpTi) and TiO2 films grown by magnetron sputtering
clearly show different surface topographies between cpTi and TiO2 surfaces (Figure 3). Cutting
marks from the polishing process are clearly visible on the cpTi surface and, even after the sputtering treatment, these marks are still present (Figure 3), due to the thickness of TiO2 films
which ranged from 312 to 338 nm (Table 2). Figure 3a shows that TiO2 films presented rounded
particles smaller than 1 µm and arranged in agglomerates. However, no dominating orientation was discernible for any of the three tested surfaces. By means of figure 3b, it is possible to analyze different surfaces two- and three-dimensionally; A-TiO2 and M-TiO2
surfaces presented greater TiO2 coverage compared to R-TiO2, which apparently presents
smaller amount of particles (Figure 3b). A-TiO2 presented the greater Ra value followed by
R-TiO2, M-TiO2 and CpTi. This analysis conferred a different pattern compared to the Ra values
27 Figure 3. Morphology and topography analysis of CpTi, A-TiO2, M-TiO2 and R-TiO2. a)
Secondary electrons SEM micrographs obtained in a 10 mm work distance, at 10000× magnification and 20 kV electron beam (scale bar = 1 µm) and b) AFM micrographs obtained in a tapping mode with a constant force 42 N/m and frequency 320 kHz (scale bar = 1 µm).
Compared to other deposition methods, such as sol-gel, sputtered films present higher hardness and elastic modulus values [26] versus bulk materials [44]. As can be seen, TiO2 films
increased the cpTi hardness and the rutile phase presented the greatest values followed by mixture, anatase and cpTi (Table 2). Mixture and rutile phase presented higher elastic modulus compared to cpTi and anatase phase. The difference in mechanical properties between the crystalline phases can be attributed to their different structures, where the rutile phase seems to be denser than Anatase [45,46]. Furthermore higher temperatures during deposition reflects in the phase transition enhancing the crystal quality [47]. The greater mechanical performance of all TiO2 films compared to cpTi surface is an important finding, whereas
28 abutments are usually exposed to severe conditions in the oral cavity, such as in biofilm development. The biofilm removal is necessary to obtain a good prognosis throughout the long-term maintenance of the implant, with a greater abrasion resistance surface being necessary [48].
It has been reported that the surface roughness and surface free energy of an implant have an effect on the initial adherence of microorganisms [49]. In general, the mixture phase and cpTi showed slight increases in roughness compared to anatase and rutile phases (Table 2); however, the average roughness values were lower than 0.2 µm (Table 2), which is therefore suggested as a threshold surface roughness, below which bacterial adhesion cannot be reduced further [49].
There is a great relationship between the surface free energy of implant materials and microbial adherence [7], with a low surface free energy being less prone to microbial adherence [50]. The surface free energy of the studied surfaces was determined as the sum of the dispersive and polar components of the contact angle. CpTi, anatase and mixture surfaces presented a similar behavior, whereas rutile differs from them, presenting a smaller wettability. This property was measured by contact angle in which a low contact angle suggests a high surface free energy and consequently an improved wettability. The water contact angle measured on CpTi (Ɵ=81o), anatase (Ɵ=90o) and mixture (Ɵ=90o) showed an
intermediate hydrophilicity of the surfaces while rutile (Ɵ=95o) tended towards
hydrophobicity. This hydrophobic tendency of rutile can be attributed to its different structure [45].
29 Table 2. Results of thickness measurement of TiO2 films and elemental composition, hardness,
elastic modulus, roughness, surface free energy of the surfaces. Data are expressed as mean ± standard deviation.
Parameters Groups
CpTi A-TiO2 M-TiO2 R-TiO2
Elemental composition (at%)
Carbon 6.31 ± 1.02a 2.6 ± 0.79b 2.2 ± 1.12b 2.1 ± 0.89b
Oxygen 3.89 ± 0.93b 57.0 ± 1.00a 59.3 ± 0.99a 59.8 ± 0.68a
Titanium 89.87 ± 0.06a 40.2 ± 0.98b 38.4 ± 0.87b 38.1 ± 0.77b
Thickness (nm) - 338 ± 43.02a 314 ± 16.17a 312 ± 14.28a
Hardness (GPa) 4 ± 0.26d 5 ± 0.71c 7 ± 0.85b 9 ± 1.14a
Elastic modulus (GPa) 102 ± 4.23b 89 ± 6.83b 122 ± 6.72a 126 ± 24.91a
Roughness (µm)
Average (Ra) 0.17 ± 0.01a 0.15 ± 0.01b 0.17 ± 0.01a 0.15 ± 0.01b
Root mean square (Rq) 0.21 ± 0.01a 0.19 ± 0.01b 0.21 ± 0.01a 0.19 ± 0.01b
Maximum height of the profile (Rt) 1.07 ± 0.03a 1.03 ± 0.07ab 1.08 ± 0.07a 0.96 ± 0.04b
Average maximum height of the
profile (Rz) 0.83 ± 0.02a 0.75 ± 0.02ab 0.83 ± 0.06a 0.74 ± 0.04b
Surface Free Energy (mN/m)
Polar 9 ± 0.01a 5 ± 0.01b 5 ± 0.04b 4 ± 0.01b
Dispersive 36 ± 0.03ab 39 ± 0.02a 38 ± 0.01ab 33 ± 0.06b
Total 45 ± 0.03a 44 ± 0.02a 43 ± 0.01a 36 ± 0.04b
Different letters indicate significant differences among groups for each dependent variable (p<0.05, Tukey HSD test).
3.2 Photocatalytic Degradation Pathway
The photocatalytic activities of the TiO2 films were evaluated in the presence of light
and dark conditions, recording the MB degradation as a function of time. According to the data depicted in Figure 4a, TiO2 films presented a photocatalytic activity in light that increased
with time. Anatase and mixture films showed similar and greater activity compared to the rutile phase. This greater activity of the anatase phase can be attributed to its large band gap (3.2 eV), which promotes high energy to create electrons and holes and consequently to form reactive oxygen species [51,52]. Furthermore, the polymorphism presented in the mixture
30 phase can reduce the recombination of electrons and holes enhancing the photocatalytic activity [53,54]. No photocatalytic activity of TiO2 in dark conditions was noted (Figure 4b),
justifying the non-inclusion of experimental surfaces in dark conditions during the biofilm assay. The CpTi surface did not present any photocatalytic activity in either condition (Figure 4a-b).
Figure 4. Photocatalytic activity of CpTi, A-TiO2, M-TiO2 and R-TiO2 in the presence of light (a)
and in the dark (b). In (a), lower case letters represent statistical differences between exposure times within each group while capital letters denote statistical differences between groups within each exposure time (p<0.05; Bonferroni test). No statistical difference was noted in (b).
31 Considering such results, exposure to UVA light during 240 minutes (4 h) seems to be the most attractive photocatalytic performance for the TiO2 films developed herein; however,
it was necessary to check whether bacteria cells were still alive after the exposure time in control surfaces (CpTi) to avoid confusing dead cell results regarding UVA light or TiO2 films.
Therefore, the availability of bacteria cells was tested after different exposure times without TiO2 influence (Figure 5a).
Figure 5. a) Availability of bacteria cells after different UVA exposure times (purple shades) or dark times (gray shades) on CpTi surfaces. Different letters in microorganism columns show
32 statistical differences between groups (p<0.05; Tukey HSD test). (*) = no viable microorganism. b) Colony forming units (Log10 CFU/mL) of each species and total number of
microorganisms developed on surfaces after 1 h of UVA light exposure according to groups. Different letters in microorganism columns show statistical differences among groups (p<0.05; Tukey HSD test).
As can be seen, 1 h of UVA light exposure led to dead bacteria cells of F. nucleatum and S. sanguinis. UVA damage occurs following the excitation of photosensitive molecules within the cell resulting in physiological alterations, growth delay and oxidative disturbances of bacterial membranes resulting in growth inhibition [55,56]. Seeing that less than 1 h of light exposure presented a lower photocatalytic activity of the TiO2 films, it led us to submit our
antimicrobial experiment to 1 h of UVA light exposure (<10% of photocatalytic activity).
Antimicrobial activity of TiO2
In the presence of light, all surfaces exhibited similar counts of S. sanguinis, A. naeslundii, and total microorganisms (Figure 5b). These findings suggest that TiO2 films
presented no significant antibacterial effect on multi-species biofilm, even that these surfaces presented a photocatalytic activity (~10%) previously tested by MB degradation. Despite the results showing no statistical difference, the counts of F. nuclaetum presented a 90% reduction (in log scale) in the number of viable cells when comparing cpTi-light and experimental TiO2 groups. This can be attributed to the fact that the gram positive cell wall is
thicker (30–100 nm) than the gram negative cell wall (20–30 nm) [42,57]. The reduction of F. nuleatum count is an important outline as such gram-negative microbial plays an essential role in the co-aggregation events during biofilm growth and maturation [58,59].
The SEM micrographs revealed the organization of biofilms according to the material surface (Figure 6). All surfaces were covered entirely by the three-species biofilm. Suggestive images of F. nucleatum or A. naeslundii could be recognized (arrows) and S. sanguinis was arranged as multicellular aggregates (asterisk). No difference in biofilm architecture was observed between groups. A representative image of biofilm organization was obtained by a confocal laser scanning microscopy (CLSM) showing that all three species were able to grow
33 together as biofilm. The different colors observed in images are representative of S. sanguinis (green), A. naeslundii (red) and F. nucleatum (blue).
Figure 6. SEM micrographs of biofilms formed on CpTi-dark, CpTi-light, A-TiO2, M-TiO2 and
R-TiO2 at 500× and 5000× magnification. (*) = multicellular aggregates, (arrows) = Spindle-shape
rods. The representative micrograph obtained by confocal laser scanning microscopy (CLSM) shows all three-species organized on the surface after 16.5 h. The fluorescence in vitro hybridization (FISH) followed a previous protocol [32], just modified to use the probe STR405-Alexa Fluor 488 [60] instead of EUB388-Alexa Fluor 488 [32]. The micrograph was obtained with an inverted microscope (DMI 6000 CS, Leica Microsystems CMS) coupled to TCS SP5 computer-operated CLSM system (Leica Microsystems CMS).
The previously proposed findings of UV-activated TiO2 effect on cell membrane
integrity [14,25,37,61] did not corroborate with the results showed in this study. A relevant finding in most of the studies that showed TiO2 antimicrobial effectiveness did not simulate
34 the acquired pellicle on the surfaces prior to the biofilm assay [22,23,37,61,62]. Furthermore, some studies tested the TiO2 photocatalysis only in one type of isolated bacteria [37,61,63]
and other studies used longer exposure times, such as 24 h [22,23,62,63], which could not be applied in the oral cavity and in our oral biofilm model. Moreover, a study of urban waste waters found little difference between TiO2 photocatalysis and direct UVA light irradiation
[56,64] which substantiated our findings. However, the photocatalytic mechanisms on bacteria are far from being understood [65].
These findings suggest that the antibacterial action of TiO2 film needs further
investigations, and the studies must simulate the oral conditions since oral biofilm formation is a very complex process. The results gathered in this laboratory approach cannot be completely transferred to a clinical setting. Notwithstanding, numerous parameters influencing oral biofilm formation have been simulated including the human acquired pellicle on the surfaces and the use of a three-species biofilm model. In a nutshell, our findings bring new insights on the development of protocols for the photocatalytic activity of TiO2 in oral
biofilm-associated disease.
4 Conclusions
The following conclusions are drawn from this study:
• TiO2 films with different crystalline phases on cpTi were successfully developed using
magnetron sputtering.
• TiO2 films increased the cpTi hardness and elastic modulus.
• UVA light exposure for more than 1 h at the submitted conditions in this study was harmful to bacteria cells.
• The sputtered anatase, mixture and rutile-TiO2 films showed a photocatalytic activity
at the methylene blue degradation, which increased with the time. Anatase and mixture phases presented greater photocatalytic potential compared to rutile phases. • Under the present UVA illumination conditions, the photocatalytic activity was not enough to present an antibacterial action on the multi-species early biofilm model, but a reduction of 90% was noted for F. nucleatum.
35 • This study presented a new methodology to test the antimicrobial properties of TiO2
on a multi-species early biofilm model, which should be followed by future investigations.
5 Acknowledgements
The authors would like to thank the State of Sao Paulo Research Foundation (FAPESP) (grant numbers 2015/17055-8 and 2016/11470-6), and The Brazilian National Council for Scientific and Technological Development (CNPq) (grant number 442786/2014-0) for the financial support. The authors also thank the Electron Microscopy Laboratory (NAP/MEPA - ESALQ/USP), Rafael Parra and Jéssica Gonçalves for their support in the Plasma Technology Lab at Univ Estadual Paulista (UNESP), and the Brazilian Nanotechnology National Laboratory (LNNano) for the AFM facility. OMZ strains were kindly donated by Prof. Bernhard Guggenheim to Prof. Jaime A. Cury, and were stored in the collection at the Biochemistry laboratory, Department of Physiological Science.
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