UNIVERSIDADE FEDERAL DE SANTA CATARINA CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM ODONTOLOGIA MESTRADO EM ODONTOLOGIA
ÁREA DE CONCENTRAÇÃO: IMPLANTODONTIA
RAFAEL CURY CECATO
ANÁLISE DA VIABILIDADE E MORFOLOGIA DE QUERATINÓCITOS E FIBROBLASTOS GENGIVAIS SOB DIFERENTES MATERIAIS UTILIZADOS NA PRODUÇÃO DE
COMPONENTES PROTÉTICOS: ESTUDO IN VITRO
Dissertação de Mestrado
Orientador: Prof. Dr. Cesar Augusto Magalhães Benfatti
Florianópolis 2018
Rafael Cury Cecato
ANÁLISE DA VIABILIDADE E MORFOLOGIA DE QUERATINÓCITOS E FIBROBLASTOS GENGIVAIS SOB DIFERENTES MATERIAIS UTILIZADOS NA PRODUÇÃO DE
COMPONENTES PROTÉTICOS: ESTUDO IN VITRO
Florianópolis 2018
Dissertação apresentada para defesa do Programa de Pós-Graduação em Odontologia da Universidade Federal de Santa Catarina, Mestrado em Odontologia, Área de Concentração: Implantodontia. Orientador: Prof. Dr. Cesar Augusto Magalhães Benfatti
Cecato, Rafael Cury
Análise da viabilidade e morfologia de queratinócitos e fibroblastos gengivais sob diferentes materiais utilizados na produção de componentes protéticos: estudo in vitro / Rafael Cury Cecato ; orientador, Cesar Augusto Magalhães Benfatti, 2018.
54 p.
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde,
Programa de Pós-Graduação em Odontologia, Florianópolis, 2018.
Inclui referências.
1. Odontologia. 2. materiais dentários. 3. implante dentário. 4. citotoxicidade. 5. materiais biocompatíveis. I. Benfatti, Cesar Augusto
Magalhães. II. Universidade Federal de Santa
Catarina. Programa de Pós-Graduação em Odontologia. III. Título.
Rafael Cury Cecato
ANÁLISE DA VIABILIDADE E MORFOLOGIA DE QUERATINÓCITOS E FIBROBLASTOS GENGIVAIS SOB DIFERENTES MATERIAIS UTILIZADOS NA PRODUÇÃO DE
COMPONENTES PROTÉTICOS: ESTUDO IN VITRO
Esta dissertação foi julgada adequada para obtenção do Título de “Mestre em Odontologia – Área de concentração Implantodontia” e
aprovada em sua forma final pelo Programa de Pós-Graduação em Odontologia.
Florianópolis, 08 de outubro de 2018.
________________________ Prof.ª Dr.ª Elena Riet Correa Rivero
Coordenadora do Programa de Pós-Graduação em Odontologia
Banca Examinadora:
________________________ Prof. Dr. Cesar Augusto Magalhães Benfatti Presidente da Banca Examinadora (Orientador)
Universidade Federal de Santa Catarina
________________________ Prof. Dr. Ricardo de Souza Magini Universidade Federal de Santa Catarina
____________________________ Prof.ª Dr.ª Elizabeth Ferreira Martinez Instituto e Centro de Pesquisas São Leopoldo Mandic
Aos meus queridos pais, pelos valores transmitidos e ao constante estímulo na continuidade dos estudos.
Agradecimentos
À minha esposa, dona de meu coração, parceira e melhor amiga, pelo constante incentivo em continuar meus estudos e pela compreensão pelas horas ausentes. Estas, embora amargas, são recompensadas a cada retorno, acompanhado por um abraço apertado e pelo sorriso mais lindo que já me atrevi a cuidar.
Aos queridos filhos Gabriel e Vicente, por compreenderem, talvez não hoje, mas algum dia, que realmente o papai trabalha muito, mas ama muito seu trabalho, e só assim temos plenitude em nossa vida. Vocês estão comigo todo o tempo, em meu coração.
À minha querida irmã, a qual tenho imensurável admiração. Nossas conversas, risadas e discussões são fundamentais para, não apenas servirem de motivo para ficarmos juntos nestes dias tão corridos, mas crescermos como pessoas de caráter.
Ao querido orientador, Prof. Dr. Cesar Augusto Magalhães Benfatti, pela confiança que sempre demonstrou em mim, nas minhas ideias, nos meus “devaneios”. Você confiou em mim mesmo quando eu mesmo não acreditava. Lembro de cada palavra de incentivo. A dedicação, carinho e atenção aos seus alunos, sem exceção, é uma lição.
Ao meu instigante, inspirador e encorajador Prof. Dr. Ricardo de Souza Magini, por ser meu modelo desde o dia que enxerguei na periodontia o meu futuro. Você já era meu professor muito antes de imaginar.
À Prof.ª Dr.ª. Elizabeth Ferreira Martinez, sem a qual os ensaios deste trabalho não teriam sido possíveis. Agradeço a disponibilidade, paciência, presteza e sabedoria durante todo o trabalho, até minha defesa.
À Prof.ª Dr.ª Ariadne Cristiane Cabral da Cruz, à Prof.ª Dr.ª Cláudia Simões e à Prof.ª Dr.ª Gabriella Mercedes Peñarrieta Juanito, pelo fundamental auxílio durante o delineamento deste trabalho.
À Sra. Bianca Mittelstädt e ao Sr. Friedrich Mittelstädt, pela constante confiança e estímulo para nossa superação, sem a qual jamais faríamos diferença. Uma verdadeira liderança é caracterizada pelo exemplo, e tenho em vocês reflexo de dedicação e amor pelo trabalho.
Ao amigo e colega William de Souza Wiggers, pelos ensinamentos de engenharia e metalurgia, sempre um enorme desafio aos profissionais da área da saúde, e pelo estímulo em iniciar esta pós-graduação.
Aos amigos e colegas de trabalho, Bruno Alves Paim, Thiago Roberto Gemeli, Gabriel Westphal, Franscisco Rafael Bastini e Alex Miura, por todos os auxílios, desde o delineamento, até as interpretações dos resultados. Sua ajuda foi fundamental.
Aos queridos colegas do CEPID, uma verdadeira família, que nunca deixa ninguém na mão. Aprendi MUITO com todos e minha gratidão é eterna. Silvane Costa, pela constante paciência e preocupação em administrar a clínica, sem a qual seria impossível desenvolvermos nossas pesquisas; Suzeli Dias, parceira de atendimento, pela atenção aos nossos pacientes e dedicação às nossas aulas e trabalhos; Carolina Morch, verdadeira pesquisadora com alma de docente, por jamais negar auxílio ou esclarecimento das dúvidas (e foram muitas) durante nossos trabalhos; Gabriel Leonardo Magrin, oferecendo local para dormir ao lado da Universidade, quando seria impossível viajar de volta para casa; Edwin Ruales Carrera, pela paciência em ensinar suas excepcionais técnicas de apresentação e fotografia; Caroline Rafael, por acreditar em nosso trabalho, transformando-se em uma grande parceira; Madalena Dias, por nos mostrar que podemos ser absolutamente sérios sem perder a alegria em viver; Mariane Beatriz Sordi, pelo esmero em atender os pacientes, assim como ceder seus materiais para preparo de nossas aulas; Joaquin Lopes Chaves, pelas grandes risadas, afinal, precisamos nos divertir; Nicolás Aguilerra e Karin Gisel Apaza Bedoya, por mostrar que nossa América do Sul é linda, diversificada e composta por excepcionais odontólogos.
“O óbvio é aquilo que ninguém enxerga, até que alguém o expresse com simplicidade”.
Gibran Khalil Gibran
Resumo
O objetivo deste estudo in vitro foi avaliar a viabilidade e morfologia de fibroblastos e queratinócitos gengivais humanos, cultivados nas superfícies de titânio (Ti) (Ti6Al4V), aço inoxidável (aço) (18Cr14Ni2,5Mo) e poliéter-éter-cetona (PEEK), hipotetizando sua utilização como materiais de confecção de componentes protéticos. Foram utilizados discos de Ti (n=36), aço (n=36) e PEEK (n=36). As culturas para ensaio de viabilidade foram cultivadas nos tempos de 24h (TV1), 48h (TV2) e 72h (TV3) e avaliadas através do ensaio de MTT (colorimetric tetrazolium assay). As culturas para ensaio de morfologia e adesão celular foram cultivadas nos tempos de 24h (TM1), 48h (TM2) e 96h (TM3), examinados ao Microscópio Eletrônico de Varredura (MEV) e analisadas nas magnificações com 500X, 1000X e 2.500X. Em relação à viabilidade: os queratinócitos não apresentaram diferença estatística sobre os diferentes materiais, em todos os tempos de cultivo. A taxa de crescimento destes aumentou sobre todos os materiais, sendo mais expressivo no aço; os fibroblastos apresentaram diferença estatística superior sobre o PEEK no TV1, porém não houve diferença estatística nos demais tempos. A taxa de crescimento destes diminuiu sobre todos os materiais, sendo mais expressivo no PEEK. As análises de morfologia e adesão celular mostram aumento do número de células, adequado espraiamento e adesão em todos os tempos de cultivo (TM1, TM2 e TM3) em ambas as linhagens, sobre todos os materiais. Considerando as limitações deste estudo, todos os materiais testados são aptos para serem utilizados na fabricação de componentes protéticos para reabilitações implantossuportadas.
Palavres-chave: materiais dentários; implante dentário; citotoxicidade; materiais biocompatíveis; mucosa bucal.
Abstract
The objective of this in vitro study was to evaluate the viability and morphology of human gingival fibroblasts and keratinocytes grown on titanium (Ti) (Ti6Al4V), stainless steel (steel) (18Cr14Ni2.5Mo) and polyether-ether-ketone (PEEK) surfaces, hypothesizing their use as prosthetic components. Ti (n = 36), steel (n = 36) and PEEK (n = 36) discs were used. The cultures for viability assay were grown at 24 h (TV1), 48h (TV2) and 72h (TV3) times and evaluated by the colorimetric tetrazolium assay (MTT). The cultures for morphology and cell adhesion assays were cultured at the 24h (TM1), 48h (TM2) and 96h (TM3) times, examined by Scanning Electron Microscopy (SEM) and analyzed at magnifications with 500X, 1000X and 2,500X. Regarding the viability: the keratinocytes did not present statistical difference on the different materials, in all the times of culture. Their growth rate increased on all materials, being more expressive in steel; the fibroblasts presented a statistically superior difference on PEEK in TV1, but there was no statistical difference in the other times. The growth rate of these decreased on all materials, being more expressive in PEEK. The morphology and cell adhesion analyzes show both increase in cell numbers, adequate spreading and adhesion at all cultivation times (TM1, TM2 and TM3) in both cell lines, on all materials. Considering the limitations of this study, all materials tested are suitable for use in the manufacture of prosthetic components for implant-supported rehabilitations.
Key Words: dental materials; dental implant; cytotoxicity; biocompatible materials; oral mucosa.
LISTA DE FIGURAS
Figura 1 – Gráfico de barras indicando a viabilidade celular de queratinócitos nos diferentes materiais (controle, titânio, aço e PEEK) nos diferentes tempos de cultura (TV1, TV2 e TV3) ... 37 Figura 2 – Gráfico de barras indicando a taxa de crescimento celular de queratinócitos nos diferentes materiais (controle, titânio, aço e PEEK) entre os tempos de cultura (24h-48h e 48h-72h) ... 38 Figura 3 – Gráfico de barras indicando a viabilidade celular de fibroblastos nos diferentes materiais (controle, titânio, aço e PEEK) nos diferentes tempos de cultura (TV1, TV2 e TV3) ... 39 Figura 4 – Gráfico de barras indicando a taxa de crescimento celular de fibroblastos nos diferentes materiais (controle, titânio, aço e PEEK) entre os tempos de cultura (24h-48h e 48h-72h) .. 39 Figura 5 – Imagens de MEV de queratinócitos cultivados sobre titânio, aço e PEEK no tempo TM1. a, b, c - titânio; d, e, f - aço; g, h, i - PEEK. Magnificações de 500X, 1000X e 2.500X, respectivamente ... 40 Figura 6 – Imagens de MEV de fibroblastos cultivados sobre titânio, aço e PEEK no tempo TM1. a, b, c - titânio; d, e, f - aço; g, h, i - PEEK. Magnificações de 500X, 1000X e 2.500X, respectivamente ... 41 Figura 7 – Imagens de MEV de queratinócitos cultivados sobre titânio, aço e PEEK no tempo TM2. a, b, c - titânio; d, e, f - aço; g, h, i - PEEK. Magnificações de 500X, 1000X e 2.500X, respectivamente ... 42 Figura 8 – Imagens de MEV de fibroblastos cultivados sobre titânio, aço e PEEK no tempo TM2. a, b, c - titânio; d, e, f - aço; g, h, i - PEEK. Magnificações de 500X, 1000X e 2.500X, respectivamente ... 43
Figura 9 – Imagens de MEV de queratinócitos cultivados sobre titânio, aço e PEEK no tempo TM3. a, b, c - titânio; d, e, f - aço; g, h, i - PEEK. Magnificações de 500X, 1000X e 2.500X, respectivamente ... 44 Figura 10 – Imagens de MEV de fibroblastos cultivados sobre titânio, aço e PEEK no tempo TM3. a, b, c - titânio; d, e, f - aço; g, h, i - PEEK. Magnificações de 500X, 1000X e 2.500X, respectivamente ... 45
Lista de Abreviaturas, Símbolos e Siglas
% Percentagem
ºC Graus Celsius
µm Micrômetro(s)
MEV ou SEM Microscópio eletrônico por varredura ou
Scanning Electron Microscopy
Ti Titânio
ASTM Sociedade Americana para Testes e Materiais
ou American Society for Testing and Materials
AISI Instituto Americano de Ferro e Aço ou
American Iron and Steel Institute
ABNT Associação Brasileira de Normas Técnicas
NBR Norma brasileira
ISO Organização Internacional de Normatização ou
International Organization for Standardization
Ti6Al4V Titânio-6Alumínio-4Vanadium (ASTM F136) ou
titânio grau V
18Cr14Ni2.5Mo 18Cromo-14Níquel-2.5Molibidênio (ASTM
F138)
PEEK Poliéter-éter-cetona ou polyether-ether-ketone
NOK-SI Normal Oral Keratinocytes - Spontaneously
Immortalized
DMEM Dulbecco′s Modified Eagle′s Medium
DMSO Dimetilsulfóxido
MTT Colorimetric tetrazolium assay
TV1/TV2/TV3 Tempos de cultivo para avaliação da
viabilidade celular 24, 48 e 72 horas
TM1/TM2/TM3 Tempos de cultivo para avaliação da
morfologia celular 24, 48 e 96 horas
SUMÁRIO CAPÍTULO I ... 23 1 INTRODUÇÃO ... 25 CAPÍTULO II ... 29 2 ARTIGO EM INGLÊS ... 31 2.1 INTRODUCTION ... 32 2.2 METHODOLOGY ... 34 2.2.1 SAMPLES ... 34 2.2.2 CELL CULTURE ... 35
2.2.3 CELL VIABILITY TEST ... 35
2.2.4 CELL MORFOLOGY ... 36 2.3 ESTATISTIC ... 37 2.4 RESULTS ... 37 2.4.1 CELL VIABiLITY ... 37 2.4.2 CELL MORFOLOGY ... .40 2.5 DISCUSSION. ... 45 2.6 CONCLUSION ... 50 2.7 REFERENCES ... 51
1 INTRODUÇÃO
Nas reabilitações orais implantossuportadas, o material utilizado para fabricação dos componentes protéticos deverá não somente apresentar comportamento mecânico adequado para suportar as forças mastigatórias, mas também biocompatibilidade para que as respostas celulares dos tecidos moles (epitélio e tecido conjuntivo) permitam resultados funcionais e estéticos previsíveis.
O selamento tecidual ao redor dos componentes protéticos serve como uma vedação protetora entre o ambiente bucal e o osso peri-implantar subjacente (LAVELLE, 1981; GOULD, 1985; NARULA et al., 2012), portanto a escolha do material deve também basear-se na sua capacidade de promover a integração com o tecido conjuntivo da mucosa peri-implantar (WELANDER; ABRAHAMSSON; BERGLUNDH, 2008). Uma ótima resposta celular dos biomateriais nos tecidos moles permite uma melhor proteção de infiltração bacteriana, ausência inflamação (mucosite) nos tecidos peri-implantares e maior previsibilidade do resultado protético. Esta interação histofisiológica entre o material e tecido é dada pela composição química e características superficiais dos materiais, a qual pode ser analisada pelo equilíbrio químico e crescimento celular no seu entorno (LINDER, 1989; LINDER et al., 1989).
O titânio grau V (Ti6Al4V) tem sido pesquisado há décadas como material para o dispositivo implantável devido às suas propriedades, dentre elas a biocompatibilidade e potencial de osteointegração (LINDER, 1989; LINDER et al., 1989), considerada equivalente ao titânio grau 04 (comercialmente puro) (SHAH et al., 2016) e adequadas respostas celulares de osteoblastos, fibroblastos e macrófagos (MARKHOFF et al., 2017). Também, é amplamente utilizado para fabricação de componentes protéticos, embora não seja necessário que ocorra obrigatoriamente osteointegração nesta circunstância, e sim promoção de resposta tecidual compatível com normalidade dos tecidos peri-implantares (ABRAHAMSSON et al., 2002; GUY et al., 1993; MOON et al., 1999). A adesão ao titânio da mucosa peri-implantar foi demonstrada in vivo, com respostas celulares similares tanto em superfícies rugosas quanto lisas (ABRAHAMSSON et al., 2002). Porém, suas limitadas propriedades mecânicas nem sempre são compatíveis com as
necessidades protéticas requeridas durante as reabilitações, obrigando fabricantes a disponibilizarem componentes insatisfatórios geometricamente. Além disso, processos de degradação e diminuição da resistência à corrosão motivam que novos materiais sejam utilizados.
Diferentes materiais, além do titânio, têm sido empregados para fabricação de componentes, como zircônia, como forma de obter melhores resultados não apenas estéticos, mas também diminuir o potencial de processos inflamatórios dos tecidos circunjacentes. Apesar de estudo apresentar que a adesão tecidual é pior à liga de ouro em comparação ao titânio e à
zircônia (SAMPATANUKUL; SERICHETAPHONGSE;
PIMKHAOKHAM, 2018), em revisão sistemática, não houve diferença na performance da resposta tecidual entre componentes fabricados de titânio, liga de ouro, óxido de alumínio ou óxido de zircônia (LINKEVICIUS et al., 2009). Mais recentemente, materiais como o aço inox, com superioridade mecânica em relação ao titânio e o poliéter-éter-cetona (PEEK), têm sido estudados e utilizados como componente provisório e/ou definitivo.
PEEK é um polímero linear poliaromático semicristalino que apresenta boa combinação de resistência, rigidez, tenacidade e estabilidade (JONES; LEACH; MOORE, 1985; WILLIAMS; MCNAMARA; TURNER, 1987). Sua biocompatibilidade já é comprovada há algumas décadas, sendo testado inclusive em dispositivos implantáveis para trauma, ortopédicos e próteses para coluna (WILLIAMS; MCNAMARA; TURNER, 1987; MARYA et al., 2011; KURTZ; DEVINE, 2007. Sua estrutura química confere estabilidade a altas temperaturas (superior a 300° C), resistência a danos químicos e à radiação, compatibilidade com muitos agentes de reforço (ex. fibras de vidro e de carbono) e maior resistência se comparado a vários metais (KURTZ; DEVINE, 2007).
Como componentes protéticos, têm indicação atual para moldagem de transferência, protetores de componentes finais ou mesmo como cilindro (suporte) para próteses provisórias (PASSONI et al., 2017). Portanto, é de suma importância que o comportamento tecidual ao redor deste material seja condizente com a homeostasia tecidual, mesmo como suporte de restaurações temporárias.
O aço inox é utilizado como material para dispositivos implantáveis há décadas na área médica (BLACKWOOD; PEREIRA, 2004), principalmente na ortopedia. Sua biocompatibilidade já foi avalizada e considerada adequada para que ocorra inclusive osteointegração, desde que as condições cirúrgicas e cicatriciais (incluindo requerimento mecânico pós operatório) estejam de acordo com os limítrofes teciduais de manipulação, como aquecimento durante instrumentação e estabilidade inicial adequada (LINDER, 1989; LINDER et al., 1989).
A liga de aço inox atualmente utilizada na implantodontia para fabricação de componentes protéticos (ASTM F138 - AMERICAN SOCIETY FOR TESTING AND MATERIALS, 2013) é considerada como uma derivação refinada do aço AISI 316L (American Iron and Steel Institute), por conseguinte com melhores resultados biomecânicos (BUSS; DONATH; VICENTE, 2011; DONACHIE, 1998) e maior resistência à corrosão (DONACHIE, 1998). Uma das vantagens da utilização de liga de aço inox em comparação ao titânio grau V, material atualmente mais utilizado para este fim, é a superior resistência mecânica (ASTM F138 - AMERICAN SOCIETY FOR TESTING AND MATERIALS, 2013; DONACHIE, 1998).
Embora ambos já tenham aplicações biomédicas (KURTZ; DEVINE, 2007; BUSS; DONATH; VICENTE, 2011), o aço inox e o PEEK têm seu comportamento biológico na área da odontologia ainda pouco elucidados. Logo, estudos para análise do comportamento biológico destes materiais em tecidos moles são necessários.
Este estudo in vitro teve como objetivo avaliar a viabilidade e morfologia de fibroblastos e queratinócitos gengivais humanos, cultivados nas superfícies de titânio, aço inoxidável e PEEK, hipotetizando sua utilização como materiais de confecção de componentes protéticos.
2 ARTIGO EM INGLÊS
O artigo a seguir foi submetido na i i n i Journal of Biomedical materials research: part A. Fator de impacto: 3.373. Qualis: A1
ANALYSIS OF THE VIABILITY AND MORPHOLOGY OF KERATINOCYTES AND GINGIVAL FIBROBLASTS ON DIFFERENT MATERIALS USED IN THE PRODUCTION OF PROSTHETIC COMPONENTS: IN VITRO STUDY
R.C. Cecato1*, E.F. Martinez2, C.A.M. Benfatti1 1
Center for Education and Research on Dental Implants (CEPID), PostGraduation Program in Dentistry (PPGO), Department of Dentistry (ODT), Federal University of Santa Catarina(UFSC), Florianópolis/SC, 88040-900, Brazil
2
Institute and Research Center of São Leopoldo Mandic., Campinas/SP, 01345-477 -Brasil
*Corresponding author: R.C. Cecato, [email protected] Abstract
The objective of this in vitro study was to evaluate the viability and morphology of human gingival fibroblasts and keratinocytes grown on titanium (Ti) (Ti6A14V), stainless steel (steel) (18Cr14Ni2.5Mo) and polyether-ether-ketone (PEEK) surfaces, hypothesizing their use as prosthetic components. Ti (n = 36), steel (n = 36) and PEEK (n = 36) discs were used. The cultures for viability assay were grown at 24 h (TV1), 48h (TV2) and 72h (TV3) times and evaluated by the colorimetric tetrazolium assay (MTT). The cultures for morphology and cell adhesion assays were cultured at the 24h (TM1), 48h (TM2) and 96h (TM3) times, examined by Scanning Electron Microscopy (SEM) and analyzed at magnifications with 500X, 1000X and 2,500X. Regarding the viability: the keratinocytes did not present statistical difference on the different materials, in all the times of culture. Their growth rate increased on all materials, being more expressive in steel;
the fibroblasts presented a statistically superior difference on PEEK in TV1, but there was no statistical difference in the other times. The growth rate of these decreased on all materials, being more expressive in PEEK. The morphology and cell adhesion analyzes show both increase in cell numbers, adequate spreading and adhesion at all cultivation times (TM1, TM2 and TM3) in both cell lines, on all materials. Considering the limitations of this study, all materials tested are suitable for use in the manufacture of prosthetic components for implant-supported rehabilitations.
Key Words: dental materials; dental implant; cytotoxicity; biocompatible materials; oral mucosa.
2.1 – INTRODUCTION
In the implant oral rehabilitation, the material used to manufacture the prosthetic components should not only provide adequate mechanical behavior to withstand masticatory forces, but also biocompatibility for the cellular responses of soft tissues (epithelium and connective tissue) allow predictable functional and aesthetic results.
The sealing tissue around the prosthetic components intent as a protective seal between the oral environment and the underlying peri-implant bone1,2,3, so the choice of the material should also be based on its ability to promote integration with the connective tissue of peri-implant mucosa4. An optimal cellular response of biomaterials in the soft tissue allows a better bacterial ingress protection, absence of inflammation (mucositis) in the peri-implant tissues and improved predictability of the prosthetic result. This histophysiological interaction between the material and tissue is given by the chemical composition and surface characteristics of the materials, which can be analyzed due to the chemical balance and cellular growth in their environment5,6.
The titanium alloy grade V (Ti6Al4V) has been researched for decades as a implantable device material due to its properties, among them the biocompatibility and potential for osseointegration5,6, which is considered equivalent to the titanium grade IV (commercially pure)7, and suitable cell responses of
osteoblasts, fibroblasts and macrophages8. Also, it is widely used for the prosthetic components manufacture, though not necessarily osseointegration must occur in this circumstance, but normal promotion of tissue response of peri-implant tissues9,10,11. The titanium adhesion to the peri-implant mucosa was demonstrated in vivo, with similar cell responses in both rough and smooth surfaces9. However, their limited mechanical properties are not always compatible with the prosthetic needs required during rehabilitation, forcing manufacturers to provide geometrically unsatisfactory components. Moreover, degradation and decreased corrosion resistance motivate new materials to be used.
Different materials, besides titanium, have been used to manufacture components, such as zirconia, as a way to obtain better results, not only aesthetic but also to decrease the potential of inflammatory processes of the surrounding tissues. Although a study showed that the tissue adhesion is worse to the gold alloy in comparison to titanium and zirconia12, in a systematic review, there was no difference in the performance of the tissue response between fabricated components of titanium, gold alloy, aluminum oxide or zirconia13. More recently, other materials such as stainless steel, mechanically superior to titanium and polyether-ether-ketone (PEEK), are used as temporary and/or permanent component.
PEEK is a semi-crystalline polyaromatic linear polymer that shows good combination of strength, stiffness, toughness and stability14,15. Its biocompatibility is proven decades ago, including being tested in implantable devices for trauma, orthopedic and prostheses for the vertebral column15,16,17. Its chemical structure gives stability at high temperatures (above 300 °C/572 °F), resistance to chemicals and radiation damage, compatibility with many reinforcing agents (eg. Glass and carbon fibers) and higher resistance compared to many metals17.
As prosthetic components, they have current indication for transfer molding, final component protectors or even as cylinder (support) for temporary prostheses18. Therefore, it is extremely important that the tissue behavior around this material is consistent with the tissue homeostasis, even as support for
temporary restorations.
Stainless steel is used as material for implantable devices for decades in the medical field19, especially in orthopedics. Its biocompatibility has already been endorsed and considered adequated for osseointegration to occur, as long as the surgical and healing conditions (including mechanical postoperative requirement) are in agreement with the tissue manipulation boundaries, such as heating during instrumentation and adequate initial stability5,6.
The stainless steel alloy currently used in implantology for prosthetic components manufacturing (ASTM F13820) is considered as a refined derivation of steel AISI 316L (American Iron and Steel Institute), therefore with improved biomechanical results21,22 and higher corrosion resistance22. One of the advantages of using stainless steel alloy compared to titanium alloy (grade V), currently most used material for this purpose is the mechanical strength20,22.
Although both already have biomedical applications17,21, stainless steel and PEEK have their biological behavior in the field of dentistry still poorly understood. Therefore, studies to analyze the biological behavior of these soft tissue materials are needed. This in vitro study was conducted to evaluate viability and morphology of human gingival fibroblasts and keratinocytes, grown on the titanium, stainless steel and PEEK surfaces, hypothesizing their use as prosthetic component materials.
2.2 METHODOLOGY
2.2.1 Samples
Titanium alloy discs were made (Ti6Al14V, Ti, standard ASTM F13623, n= 36), implantable stainless steel (18Cr14Ni2,5Mo, Steel, standard ASTM F13820 and ABNT NBR ISO 5832-1:200824, n= 36) and of polyether-ether-ketone (C6H4-O-C6H4-O-C6H4-CO, PEEK, n=36), with 5 mm of diameter and 2 mm of height, metallographically prepared and supplied by the
company FGM® (Destscare/FGM-Brasil). The discs were washed and sterilized by gamma radiation. Polystyrene disks (control, n = 36 ) were used as control.
2.2.2 Cell Culture
Cell lines of keratinocytes (NOK-SI)25 and fibroblasts (third to sixth passage)26, both gingival and human, were used. These cells were used with the approval of the Ethical Committee of the University of São Paulo, Brazil (Protocol # 728/06).
Cells were grown on the different discs in Dulb o’ Modified E gl ’ Medium (DMEM) (Nuticell®, Brazil) supplemented with 10 % fetal bovine serum (Cultilab®, Brazil) and 1 % antibiotic-antimicotic solution (penicillin-streptomycin-amphotericin b) (Sigma, USA).
All procedures were performed in laminar flow cabinet to maintain sterility of materials and substances used for cell cultivation.
The cells were kept in a stove at 37 °C (98.6 °F) in a humidified atmosphere containing 95 % air and 5 % carbon dioxide.
The cultures for viability test were grown in 24 h (TV1), 48 h (TV2) and 72 h (TV3).
The cultures for morfology test and cell adhesion were grown in 24 h (TM1), 48 h (TM2) and 96 h (TM3).
2.2.3 Cell viability test
Cell cultures, in different surfaces, were tested for cell viability using the Colorimetric tetrazolium assay (MTT assay)27.
This assay evaluates the ability of metabolically active cells to reduce MTT converting the yellow tetrazolium salts (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide) to purple formazan crystals and therefore on the ability of viable cells to
cleave the tetrazole ring present in MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide) by the action of dehydrogenase enzymes present in active mitochondria, forming formazan crystals27.
In the cytotoxicity assay, cells were plated at a density of 110 cells/mm2 on different surfaces. Ten (10) μl of the MTT solution (5 mg/ml) (Sigma-Aldrich, USA), diluted in serum-free DMEM culture medium, were added to the cell cultures and incubated for 3 hours at 37 °C (98.6 °F). After this phase, 100 μl of Dimethyl sulfoxide (DMSO) (LGC, Brazil) was added and maintained for 15 minutes at room temperature.
After solubilization of the crystals, the measurement was performed in microplate reader ELX800 (Biotek Instruments, USA) at 590 nm, according to standard cultivation times (TV1, TV2 e TV3).
2.2.4 Cell morfology
Keratinocytes and fibroblasts plated on different surfaces under the same conditions described above were determined after programmed cultivation time (TM1, TM2 and TM3) with glutaraldehyde solution v/v to 2.5% in 0.1 M cacodylate buffer (pH 7,2) for 1 h at 4 °C (39.2 °F). After this time, they were washed in the same buffer solution at 0.05 M, followed by dehydration with increasing concentrations of ethyl alcohol. The samples were submitted to final drying with critical point (EM CPD 030 - LEICA, Germany), to that there was no effect of the forces occurring on surface tension and consequently sensitive shape changes. They were then assembled on aluminum brackets (stubs) and placed on a metallizer using a cathodic spray coater (208HR - Cressington Company, England) attached to thickness controller (MTM-20 Cressington High Resolution Thickness Controller, England) to receive gold-palladium coating (80/20 %) with 15 nm thick then examineted in a Scanning Electron Microscope (SEM) (JEOL JSM-6390LV, Japan) and analyzed qualitatively for cell adhesion, morphology, spreading and confluence, at 500X, 1000X and 2.500X magnifications.
2.3 – ESTATISTIC
Quantitative data were tabulated and statistically analyzed in an one-way ANOVA followed by Tukey post-test using a significance level of 5 % (Minitab 17) (Minitab Inc., USA).
2.4 – RESULTS 2.4.1- Cell viability
The viability assays showed increased mitochondrial activity, consequently cell activity growth, in both cell lines on all the materials tested, between TV1, TV2 and TV3 times.
In relation to the keratinocytes in the early times (TV1 and TV2), there was no statistically significant difference in all materials tested, but all showed statistically significant differences for less than the control. Already, in TV3 time, the cell viability on steel showed no statistically significant difference in relation to the control, as well as in comparison to titanium and PEEK. However, titanium and PEEK showed a statistically significant difference for less compared to control (Figure 01).
Figure 01 – Bar chart showing the keratinocytes cell viability in different materials (control, titanium, steel and PEEK) at different culture times (TV1, TV2 and TV3).
The growth rate of keratinocytes between culture times was also analyzed and showed an increase between the initial (24h-48h) and final (48h-72h) times. On average, there was similarity between materials between the initial times (24h-48h) (78.7% control, titanium 78.0%, steel 72.1%, PEEK 67.1%), but between
the final times the steel (187.3%) presented a higher rate than the others, followed by titanium, control and PEEK (140.6%, 115.5% and 75%, respectively) (Figure 02).
Figure 02 - Bar chart showing the cell growth rate of keratinocytes in different materials (control, titanium, steel and PEEK) between culture times (24h-48h and 48h-72h).
Regarding fibroblasts, PEEK viability showed a statistically significant difference for more in relation to the other materials in the first time (TV1), with no significant difference in relation to the control. At the second measurement, the response equalized, with no statistically significant difference between groups. However, in the last time (TV3), the cell viability on the steel showed no statistically significant difference in relation to the control, as well as in comparison to the titanium and the PEEK. However, titanium and PEEK presented a statistically significant difference for less in relation to the control (Figure 03).
Figure 03 – Bar chart showing the fibroblasts cell viability in different materials (control, titanium, steel and PEEK) at different culture times (TV1, TV2 and TV3).
The growth rate of the fibroblasts between the culture times was also analyzed and shows that, unlike keratinocytes, there was a decrease between the initial (24h-48h) and final (48h-72h) times. There was similarity between steel and control between the initial times (24h-48h) (86.1%, 82.2% respectively) with higher growth rate, followed by titanium (67.4%) and PEEK (35, 2%). However, between the final times, there was similarity between control, titanium and steel (24%, 23% and 18.9% respectively), with PEEK again with the lowest rate among the others (6.6%) (Figure 04).
Figure 04 - Bar chart showing the cell growth rate of fibroblasts in the different materials (control, titanium, steel and PEEK) between culture times (24h-48h and 48h-72h).
2.4.2 – Cell morfology
The morphology and cell adhesion analyzes show both cell number increase and adequate spreading at all cultivation times (TM1, TM2 and TM3) in both cell lines on all materials, highlighting their adhesion on all surfaces tested. It can be also seen in the images, appropriate union between the cells and consequently a positive growth response.
At TM3, all samples showed increase in the number of cells. Pictures 05 to 10 show the growth and morphology of the two cell lines (NOK-SI and fibroblasts) on the tested materials (titanium, steel and PEEK) at established culture times.
Picture 05 - MEV images of keratinocytes grown on titanium, steel and PEEK at time TM1. a, b, c - titanium; d, e, f - steel; g, h, i - PEEK. Magnification of 500X, 1000X e 2.500X, respectively.
Picture 06 - MEV images of fibroblasts grown on titanium, steel and PEEK at time TM1. a, b, c - titanium; d, e, f - steel; g, h, i - PEEK. Magnification of 500X, 1000X e 2.500X, respectively.
Picture 07 - MEV images of keratinocytes grown on titanium, steel and PEEK at time TM2. a, b, c - titanium; d, e, f - steel; g, h, i - PEEK. Magnification of 500X, 1000X e 2.500X, respectively.
Picture 08 - MEV images of fibroblasts grown on titanium, steel and PEEK at time TM2. a, b, c - titanium; d, e, f - steel; g, h, i - PEEK. Magnification of 500X, 1000X e 2.500X, respectively.
Picture 09 - MEV images of keratinocytes grown on titanium, steel and PEEK at time TM3. a, b, c - titanium; d, e, f - steel; g, h, i - PEEK. Magnification of 500X, 1000X e 2.500X, respectively.
Picture 10 - MEV images of fibroblasts grown on titanium, steel and PEEK at time TM3. a, b, c - titanium; d, e, f - steel; g, h, i - PEEK. Magnification of 500X, 1000X e 2.500X, respectively. 2.5 – DISCUSSION
It is commonly accepted that the peri-implant soft tissues have similarities with periodontal soft tissues, including the gingival, sulcular and junctional epithelium, with connective tissue just under3,28,29, including in the inflammatory response to the presence of biofilm30. However, as much as similar the gingiva and the peri-implant mucosa are, there are differences, being the underlying connective tissue structure the most striking3. In addition, an unkommon response of peri-implant mucosa around a recently placed implants could damage the prosthesis and their associated soft tissues, frustrating attempts to maintain its long term functionality and aesthetics29. Therefore, a material presenting cytotoxicity to keratinocytes and gingival fibroblasts, without adhesion, will prevent normal healing, reducing or even avoiding its protective action. Thus, it is extremely important that
the prosthetic component material to be immediately wrapped and "accepted" by the constituent cells of this tissue as soon as they are attached.
The gingival epithelial tissue is classified as stratified squamous, being the keratinocyte its main cell31. Keratinocyte cultures can be classified as immature, mature or senescent, according to their morphology, being this variation dependent on the culture time or even the age of the donator32. Its structure is divided into basale, spinosum, granulosum and corneum, being the last three originated from the first. In this, the cells are characterized by being cubic, containing tonofibrils or tonofilaments33, responsible for the formation of desmosomes or hemidesmosomes. The keratinocytes in the qualitative analysis at all times (TM1, TM2 and TM3) and on all surfaces (titanium, steel and PEEK), show regular and evident morphology and spreading. However, in TM2 and TM3 times the morphology become more characteristic. This may be an indication that this cell adhesion of all tested materials is already well accepted in the first 24 hours and it is confirmed in the following hours. It is evident the union and intimacy between the cells, through desmosomes, at all evaluated times, inherent characteristic of keratinocyte from epithelial basal layer. In some areas, this union is so evident that it is difficult to define the limits of plasma membranes, especially in the more advanced culture (TM3). In addition, the increase in concentration clearly thrives as culture time increases as well as intercellular adhesion.
Cell viability (quantitative) of keratinocytes was evaluated by the MTT assay indicated to measure cytotoxicity, proliferation or cell activation by reading mitochondrial activity27. This corroborates with the qualitative analysis (SEM), where at all times (TV1, TV2 and TV3) there was no statistically significant difference, on all the materials tested. In addition, such cell activity increased proportionally with the time of cultivation, indicating increased concentration of cell numbers.
The increase in the growth rate of keratinocytes was observed on all materials, but the growth on the steel stood out in relation to the others, which demonstrates the effectiveness of this cell line on this material. This behavior is consistent with epithelial cells, where there is a vertiginous growth at the beginning of the
cultures, decreasing in the following times (rapid turnover - high proliferation capacity for protective function).
The gingival connective tissue is known as the lamina propria and the fibroblast is its main cell. These are elongated at rest, with little cytoplasm and flattened nucleus (dark). In active, the nucleus is oval, with a higher volume of cytoplasm33.
The first step of a fibroblast in contact with the surface is adhesion to it34. To that end, they carry a scan out through cytoplasmic extensions called "filopodia", and when they find a compatible region they begin the adhesion process35. Afterwards, the cell scattering (spreading) and flattens itself, being this adaptation primordial to the cell division occurance34. Its morphology and adhesion are driven in accordance with the topography of the substrate, and its capacity to promote the focal contacts formation and the cellular cytoskeleton development are important for the material performance34,36.
In the first 24 h (TM1) it is possible to notice the formation of “filopodia” on all surfaces, but on the PEEK these extensions become more numerous and longer only in TM2 and TM3 times. Such observation possibly means the need for longer cell adhesion time in this material in comparison to the others. It is also noticed that its morphology keep up its template, which means, elongated and scattered in all analyzed times (TM1, TM2 and TM3), on all tested materials, also indicating increased concentration of cell numbers, as in keratinocytes.
An important aspect noticed in the fibroblast lineage was the response to the disc surfaces topographic characteristics, not clearly noticed in the keratinocytes. An expected response of the fibroblasts regarding topography is that they have orientation aligned with the grooves of the substrate, with up to 10º of angular variation, when the grooves are smaller than 4.0 μm36
. In this study, this analysis corroborates such proposed, but only in TM3 these characteristics is well noticed in all materials, probably by cell maturation. This finding is interesting for future guidelines in the manufacture of implant devices, so that the structural orientation of adjacent tissues is better managed.
Also, its own morphology seems to have been influenced by the grooves of the surfaces of the substrates, since in respecting this direction, they have been elongated at all times (TM1, TM2 and TM3). characteristic is intensified as the growing time increases, indicating cell maturation characteristic or even increased surface adhesion.
From the images, we can noticed that there was a similar growth in the concentration of fibroblasts between titanium and steel, whereas in PEEK, we noticed a lower concentration of fibroblasts at all times. This data is different from that found in the quantitative analysis, since the activity of these cells was significantly higher in TV1 time, and became equivalent in other times (TV2 and TV3). This fact exposes an important limitation of the MTT test, where mitochondrial activity is measured, but even existing, does not necessarily match the increase in the concentration of cell numbers in the culture37.
The fibroblast growth rate decreased over all materials. This characteristic is expected in fibroblasts, where the proliferation is lower at the beginning of the culture and grows at the end time, unlike what occurs with keratinocytes. Growth on PEEK showed the greatest decrease, reflecting the higher cellular activity on this material in the first culture time (TV1).
Titanium, including Titanium alloy grade V (Ti4Al6V23), has been used for decades as an implantable material and currently, it is chosen as a "gold standard" in dental implants7,38 and widely used as a prosthetic component. Its viability and cytotoxicity have been previously endorsed7,39,40 and are in agreement with the findings of this study.
The stainless steel (ASTM F13820), though used for decades in the medical field5,6,19, only recently had its use indicated for prosthetic components with undefined function of time, that is, to support final prosthetic restorations on implant rehabilitation. Its mechanical performance is superior to titanium, therefore, with predicates that support a greater range of clinical indications, such as angled components. However, probably because of the uniqueness of the indication, cell viability in peri-implant mucosa lineage had not been certified under conditions that simulate
prosthetic intermediate (abutment). In both qualitative and quantitative analyzes, keratinocytes as well as gingival fibroblasts showed no alterations and / or abnormalities during growth and maturation. An interesting finding is that the viability of both cell lines (keratinocytes and fibroblasts) was higher on this material compared to titanium and PEEK, being the only one that did not present significant statistical difference in relation to the control. PEEK is a thermoplastic material and as such can be converted into a variety of shapes and sizes of components by the full spectrum of manufacturing technologies such as machining or injection molding14. This versatility provides a great advantage to design, manufacture and even the cost of the components. Such benefits have been observed by the biomaterials and medical devices industry as a safe alternative for applications such as: fracture fixation plates, spinal implants, joint prostheses17 and more recently as transient prosthetic components18. Its cell cytotoxicity has previously been evaluated41,42 with appropriate results, confirming the results in this study. Its viability did not show significant statistical difference in relation to the materials tested in keratinocyte cultures, that is, equivalence between them. In fibroblast cultures, statistically significant difference was greater in TV1 time, that is, greater viability in the first 24 hours of culture and with no statistically significant difference in TV2 and TV3 times. Although it shows absence of cytotoxicity for the tested cell lines in the morphological evaluation, lower cellular concentration was observed, mainly in the first times (TM1 and TM2) in relation to the other materials.
It is important to mention that not only dental implants are made of the materials tested, but also orthopedic parts such as joint implants. However, articulated implants, although statically have a tendency to present a cellular response equivalent to dental, should work with movement (sliding) between the surfaces and, therefore, the surrounding tissue behavior, stimulated by the differentiated action of the surfaces of the material, may not be equivalent to the observed implants with fixed connections. As the union of the prosthetic implant component ambitiously motionless, a platform with frictional conical connection demonstrates to be favorable to the absence of interferences due to wear or release of biomaterial particles in these tissues.
Another important point is that all the surfaces of the sample discs were prepared by simulating the surface of single-use prosthetic intermediates on dental implants. Therefore, surface modifications such as, roughness or coating commonly used in devices for multiple uses and indications, should significantly modify cell responses. Also, as the surface topography of PEEK discs is significantly different when compared to steel and titanium, due to its non-metal peculiarity, differences in cell behavior are expected and direct comparison between all groups, in an equivalent way, is accomplished with caveats.
Through the viability and morphological analyzes executed, all the evaluated materials are consistent with the growth of cells of the tested cell lines, according to their indications, as options for medical devices manufacture. However, a qualitative analysis of the morphology only shows a two-dimensional image, and a proposal of cellular adhesion, without evidences of important cell characteristics on the surfaces under other angles. Also, there were not tests performed on cell adhesion markers, which are important to evaluate more accurately the effectiveness of adherence to each surface. Another point is, as previously stated, the viability assay (MTT) only quantifies the mitochondrial activity37, without showing the actual concentration and / or cell adhesion. Therefore, new assays with such analyzes are required for a better understanding of these properties.
2.6 – CONCLUSION
The cell viability assays of human gingival keratinocytes and fibroblasts showed viability at the times evaluated, on all surfaces tested.
The evaluation of the morphology of both cell line was considered within normality at established culture times, on all surfaces tested.
Considering the limitations of this study, all materials tested are suitable for use in the manufacture of prosthetic components for implant-supported rehabilitations.
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