Formação de biofilme in vitro e viabilidade de fibroblastos gengivais humanos (FMM-1) em novos materiais cerâmicos
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Data
2017-01-19
Autores
Dal Piva, Amanda Maria de Oliveira [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Este estudo avaliou a morfologia superficial de duas cerâmicas recentemente lançadas no mercado, o crescimento de biofilme heterotípico e a viabilidade de fibroblastos gengivais humanos (FMM-1) em contato com estes materiais após vitrificação ou polimento. Para isto, foram confeccionados 46 blocos (4,5x4,5x1,5 mm) de cada cerâmica: zircônia parcialmente estabilizada por ítria com alta translucidez (HT) e silicato de lítio reforçado por zircônia (SUP). Os blocos foram sinterizados/cristalizados e as superfícies dos espécimes padronizadas com aplicação de glaze (subgrupos “g”). A rugosidade superficial (Ra e RSm) foi mensurada por rugosímetro de contato (n=20) e qualitativamente por perfilometria óptica (n=2). Após esterilização, os espécimes foram contaminados para a formação de biofilme com a associação de S. mutans, S. sanguinis e C. albicans no período de 16 h (n=8) e o biofilme formado foi quantificado através da contagem de unidades formadoras de colônia (UFC) e analisado por microscopia eletrônica de varredura. A viabilidade celular a fibroblastos FMM-1 foi avaliada através do teste MTT. Cinco amostras (15x15x1,5 mm) foram confeccionadas de forma semelhante e utilizadas para determinação da energia livre de superfície (ELS) através de goniometria. Em seguida, os espécimes não-destruídos (n=26) foram resubmetidos ao protocolo de lixamento e então, polidos conforme recomendação do fabricante para avaliação das superfícies polidas (subgrupos “p”) sob todas análises em estudo. Os dados de rugosidade foram avaliados através dos testes não-paramétricos Kruskal-Wallis, Dunn e Mann-Whitney. Os dados de ELS, MTT e UFC foram avaliados por análise de variância e teste de Tukey, e os dados de MTT também foram submetidos ao teste t. Para todos os testes, foi considerado o valor de alfa = 95%. A rugosidade foi influenciada pelos materiais e tratamentos de superfícies avaliados (Ra p<0,001; RSm p=0,0002), sendo que a cerâmica HTg apresentou ranhuras, em média, mais altas e menos espaçadas que as demais. Os fatores material e superfície influenciaram a ELS (p=0,001), todos os materiais apresentam comportamento hidrofílico, mas HTp apresentou a maior ELS. A interação dos fatores superfície e micro-organismo influenciou a UFC (p=0,00), sendo que Streptococcus aderiu mais sobre superfícies glazeadas e C. albicans apresentou menor aderência para superfícies polidas. O teste MTT demonstrou citotoxicidade inicial (24 h) para as superfícies polidas, o que não se repetiu após 7 dias de contato (p=0,00). A perfilometria evidenciou a morfologia superficial mais homogênea nas amostras polidas. Exceto para HTg, as imagens de MEV evidenciaram superfícies homogêneas e os espécimes contaminados apresentaram Streptococus e C. albicans. Pode-se concluir que as superfícies glazeadas apresentam maior rugosidade e tendência a acumular biofilme. Superfícies polidas possuem elevada ELS, porém, citotoxicidade temporária.
This study evaluated the morphology of two ceramic materials recently introduced on the market, as well as the formation of heterotypic biofilm and the viability of human gingival fibroblasts (FMM-1) in contact with these materials after glazing or polishing. For this, 46 blocks (4.5x4.5x1.5 mm) of each ceramic were made: high translucency zirconia partially stabilized by yttrium (HT) and lithium silicate reinforced by zirconium (SUP). The blocks were sintered/crystallized and surfaces were standardized with glaze application (subgroup “g”). The surface roughness (Ra and RSm) was evaluated through a contact rugosimeter (n=20) and qualitatively by profilometry (n=2). After sterilization, specimens were contaminated for heterotypic biofilm formation with association of S. mutans, S. sanguinis and C. albicans during 16 h of formation (n=8) and the biofilm was quantified by counting the colony forming units (CFU/mL) and analyzed by scanning electron microscopy (SEM). The viability of FMM-1 fibroblasts was evaluated by MTT assay. 5 samples (15x15x1.5 mm) were prepared in a similar way and used to determine the surface free energy (SFE) by goniometry. Then, the non-destroyed specimens (n=26) were resubmitted to the sandpaper polishing and finished as recommended by the manufacturer to evaluate the polished surfaces (subgroup “p”) under all analyzes in study. Roughness data was evaluated using the nonparametric Kruskal-Wallis, Dunn and Mann-Whitney tests. SFE, MTT and CFU results were evaluated by ANOVA and Tukey’s test, and MTT data was also submitted to t test. For all tests, it was considered an alfa value of 95%. Roughness was influenced by materials and surface treatments (Ra p=0,001; RSm p=0,0002). HTg ceramic presented, on average, higher and less spaced grooves than others. Both material and surface factors influenced the SFE (p=0,001), all materials presented hydrophilic behavior, however HTp presented higher SFE. The interaction of surface and micro-organism factors influenced the CFU (p=0,00), Streptococcus showed more adherence on glazed surfaces and C. albicans presented less adherence on polished surfaces. MTT assay demonstrated initial (24 h) cytotoxic behavior to polished surfaces, which was not repeated within the period of 7 days. The profilometry evidenced the more homogeneous surface morphology of polished samples. Except for HTg, the SEM images showed homogeneous surfaces, and on the contaminated specimens, it was observed the presence of Streptococcus and C. albicans. It could be concluded that glazed surfaces presented a greater roughness and tend to accumulate more biofilm. Polished surfaces present high SFE, however, are temporarily cytotoxic.
This study evaluated the morphology of two ceramic materials recently introduced on the market, as well as the formation of heterotypic biofilm and the viability of human gingival fibroblasts (FMM-1) in contact with these materials after glazing or polishing. For this, 46 blocks (4.5x4.5x1.5 mm) of each ceramic were made: high translucency zirconia partially stabilized by yttrium (HT) and lithium silicate reinforced by zirconium (SUP). The blocks were sintered/crystallized and surfaces were standardized with glaze application (subgroup “g”). The surface roughness (Ra and RSm) was evaluated through a contact rugosimeter (n=20) and qualitatively by profilometry (n=2). After sterilization, specimens were contaminated for heterotypic biofilm formation with association of S. mutans, S. sanguinis and C. albicans during 16 h of formation (n=8) and the biofilm was quantified by counting the colony forming units (CFU/mL) and analyzed by scanning electron microscopy (SEM). The viability of FMM-1 fibroblasts was evaluated by MTT assay. 5 samples (15x15x1.5 mm) were prepared in a similar way and used to determine the surface free energy (SFE) by goniometry. Then, the non-destroyed specimens (n=26) were resubmitted to the sandpaper polishing and finished as recommended by the manufacturer to evaluate the polished surfaces (subgroup “p”) under all analyzes in study. Roughness data was evaluated using the nonparametric Kruskal-Wallis, Dunn and Mann-Whitney tests. SFE, MTT and CFU results were evaluated by ANOVA and Tukey’s test, and MTT data was also submitted to t test. For all tests, it was considered an alfa value of 95%. Roughness was influenced by materials and surface treatments (Ra p=0,001; RSm p=0,0002). HTg ceramic presented, on average, higher and less spaced grooves than others. Both material and surface factors influenced the SFE (p=0,001), all materials presented hydrophilic behavior, however HTp presented higher SFE. The interaction of surface and micro-organism factors influenced the CFU (p=0,00), Streptococcus showed more adherence on glazed surfaces and C. albicans presented less adherence on polished surfaces. MTT assay demonstrated initial (24 h) cytotoxic behavior to polished surfaces, which was not repeated within the period of 7 days. The profilometry evidenced the more homogeneous surface morphology of polished samples. Except for HTg, the SEM images showed homogeneous surfaces, and on the contaminated specimens, it was observed the presence of Streptococcus and C. albicans. It could be concluded that glazed surfaces presented a greater roughness and tend to accumulate more biofilm. Polished surfaces present high SFE, however, are temporarily cytotoxic.
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Palavras-chave
Cerâmica, Aderência bacteriana, Microscopia, Teste de materiais, Ceramics, Bactherial Adhesion, Microscopy, Materials testing