Efeito de cianoacrilatos e adesivo tissular a base de veneno de cobra na citotoxicidade sobre fibroblastos gengivais e estresse oxidativo sobre células do biofilme de C. albicans / Effect cyanoacrylate and snake venom tissue adhesive on cytotoxicity of gingival fibroblasts and oxidative stress of C. albicans biofilms

RESUMO

A estomatite protética é uma patologia que acomete muitos usuários de prótese total. Um dos principais fatores da sua etiologia é a fácil adesão de microrganismos, como a Candida albicans, à resina acrílica, principalmente devido às características de superfície facilitadoras deste material. Por esta razão, estuda-se materiais que possam modificar as características de superfície da resina acrílica sem alterar as suas propriedades mecânicas. A investigação das propriedades desses materiais, como a biocompatibilidade e como eles influenciam as células de C. albicans, é de extrema importância para uma futura aplicação clínica. Dessa forma, a proposta desta pesquisa foi avaliar a citotoxicidade sobre fibroblastos gengivais humanos (FGH) e o estresse oxidativo causado em células de C. albicans por 6 produtos (etil-cianoacrilato convencional -ECAc; etil-cianoacrilato em forma de gel formulação a -ECAga; etil-cianoacrilato em forma de gel formulação b -ECAgb; butil-cianoacrilato - BCA; octil-cianoacrilato OCA; selante tissular a base de veneno de cobra-VC;), que quando aplicados na superfície da resina acrílica, modificam suas características de superfície, visando diminuir a formação do biofilme de C. albicans. Espécimes de resina acrílica foram confeccionados e duas camadas de cada produto foram aplicadas nas superfícies. Cada espécime foi deixado em 1, 066 mL de meio de cultura durante 24 horas para extração e utilização do meio condicionado para os dois ensaios. Para a investigação da citotoxicidade, os FGH foram cultivados em placas de 96 poços, meios condicionado de cada grupo foram depositados em cada poço onde permaneceram em contato por 24, 48 e 72 h. Após o processamento para o ensaio de MTT as placas foram analisadas em espectrofotômetro a 500 nm. Foram realizados quatro experimentos independentes em triplicata (n=12). Para a investigação de estresse oxidativo (ERO) causado em C. albicans, biofilme foi desenvolvido em placas de 96 poços durante 24 h. Então, os meios condicionados foram adicionados aos poços e a placa foi mantida em agitadora durante 1 h. Após a retirada dos meios condicionados, foi adicionado a cada poço o reagente CellRox® Deep Red e leituras foram realizadas a cada 30 min durante 3 h em fluorímetro a 640/665nm. Amostras foram coletadas e ensaio de cultura microbiológica foi executado para a realização da normalização dos valores. Foram realizados 3 experimentos independentes em triplicata (n=9). Os resultados obtidos pelo ensaio MTT foram analisados por meio de ANOVA a 2 critérios, seguido pelo teste Tukey para a comparação entre grupos (p<0,05). Para o ensaio ROS, a análise estatística realizada foi ANOVA a 1 critério, seguida, igualmente, por Tukey (p<0,05). Os resultados mostraram que todos os grupos apresentaram certo grau de citotoxicidade quando comparados ao controle, com exceção do VC (ECAcestresse oxidativo em células de C. albicans (OCA

ABSTRACT

Denture stomatitis is a disease that affects many denture wearers. One of the main factors of its cause is the easy adhesion of microorganisms, such as Candida albicans, to acrylic resin, mainly due to its permissive surface characteristics. For this reason, materials that can modify surface characteristics of acrylic resin without changing its mechanical properties are being studied. The investigation of these materials features, such as biocompatibility and how they affect Candida albicans, is of utmost importance for future clinical application. Thus, the purpose of this study was to evaluate the cytotoxicity of human gingival fibroblasts (HGF) and oxidative stress in C. albicans cells after contact with 6 experimental products (conventional ethyl-cyanoacrylate -ECAc; ethyl-cyanoacrylate in gel formulation "a" -ECAga; ethyl-cyanoacrylate in gel formulation "b" -ECAgb; butyl- cyanoacrylate - BCA; octyl-cyanoacrylate -OCA; tissue adhesive based on snake venom -VC) applied to the surface of the resin acrylic, modifying its surface characteristics, in order to decrease C. albicans biofilm formation. Acrylic resin specimens were manufactured and two layers of each product were applied to the surfaces. Each specimen was immersed in 1,066 mL of growth media for 24 hours for extraction and the resulting conditioned media were used for both tests. To investigate cytotoxicity, HGF were cultured in 96-well plates and conditioned media from each group were deposited into each well, where they remained in contact for 24, 48 and 72h. After the MTT assay processing, plates were analyzed in a spectrophotometer at 500 nm. Four independent experiments were performed in triplicate (n=12). To investigate oxidative stress (ROS), C. albicans biofilm was developed in 96-well plates for 24 h, conditioned media were added to the wells and the plate was allowed to stir for 1 h. Then, conditioned media were removed and CellRox® Deep Red reagent was added to each well. Readings were performed every 30 min for 3 h in fluorometer at 640/665nm. Samples were collected and microbiological culture test was executed to values normalization. Three independent experiments were performed in triplicate (n=9). Results obtained in MTT assay were analyzed by two-way ANOVA and Tukey's test for comparison between groups (p<0.05). for comparison between groups (p<0.05). For ROS results, one-way ANOVA was performed followed by Tukey test, as well (p<0.05). The results showed that all groups presented different degrees of cytotoxicity when compared to control group, except for VC (ECAcoxidative stress in C. albicans cells (OCA

Subject(s)

Humans; Biofilms/drug effects; Candida albicans/drug effects; Cyanoacrylates/pharmacology; Fibroblasts/drug effects; Gingiva/cytology; Snake Venoms/pharmacology; Tissue Adhesives/pharmacology; Acrylic Resins/chemistry; Analysis of Variance; Cell Survival/drug effects; Cells, Cultured; Oxidative Stress/drug effects; Reproducibility of Results; Time Factors

Candida albicans; Cell survival; Cianoacrilatos; Cyanoacrylates; Denture stomatitis; Estomatite sob prótese; Estresse oxidativo; Oxidative stress; Sobrevivência celular

ABSTRACT

Denture stomatitis is a disease that affects many denture wearers. One of the main factors of its cause is the easy adhesion of microorganisms, such as Candida albicans, to acrylic resin, mainly due to its permissive surface characteristics. For this reason, materials that can modify surface characteristics of acrylic resin without changing its mechanical properties are being studied. The investigation of these materials features, such as biocompatibility and how they affect Candida albicans, is of utmost importance for future clinical application. Thus, the purpose of this study was to evaluate the cytotoxicity of human gingival fibroblasts (HGF) and oxidative stress in C. albicans cells after contact with 6 experimental products (conventional ethyl-cyanoacrylate -ECAc; ethyl-cyanoacrylate in gel formulation "a" -ECAga; ethyl-cyanoacrylate in gel formulation "b" -ECAgb; butyl- cyanoacrylate - BCA; octyl-cyanoacrylate -OCA; tissue adhesive based on snake venom -VC) applied to the surface of the resin acrylic, modifying its surface characteristics, in order to decrease C. albicans biofilm formation. Acrylic resin specimens were manufactured and two layers of each product were applied to the surfaces. Each specimen was immersed in 1,066 mL of growth media for 24 hours for extraction and the resulting conditioned media were used for both tests. To investigate cytotoxicity, HGF were cultured in 96-well plates and conditioned media from each group were deposited into each well, where they remained in contact for 24, 48 and 72h. After the MTT assay processing, plates were analyzed in a spectrophotometer at 500 nm. Four independent experiments were performed in triplicate (n=12). To investigate oxidative stress (ROS), C. albicans biofilm was developed in 96-well plates for 24 h, conditioned media were added to the wells and the plate was allowed to stir for 1 h. Then, conditioned media were removed and CellRox® Deep Red reagent was added to each well. Readings were performed every 30 min for 3 h in fluorometer at 640/665nm. Samples were collected and microbiological culture test was executed to values normalization. Three independent experiments were performed in triplicate (n=9). Results obtained in MTT assay were analyzed by two-way ANOVA and Tukey's test for comparison between groups (p<0.05). for comparison between groups (p<0.05). For ROS results, one-way ANOVA was performed followed by Tukey test, as well (p<0.05). The results showed that all groups presented different degrees of cytotoxicity when compared to control group, except for VC (ECAcoxidative stress in C. albicans cells (OCA