Efeito citotóxico de microcristais de tungstato de prata e de molibdato de prata em fibroblastos gengivais humanos cultivados em monocamada e em equivalente dermal
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Data
2017-05-12
Autores
Haro Chávez, Natali Lisette [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A associação da prata a outros compostos vem sendo estudada em uma tentativa de acentuar as propriedades antimicrobianas e reduzir a citotoxicidade deste metal quando em altas concentrações. O presente estudo investigou o efeito dos microcristais: tungstato de prata (α-Ag2WO4) e molibdato de prata (β-Ag2MoO4), no comportamento das células gengivais em monocamada e em modelo de matriz de colágeno, simulando a reparação tecidual. Para isto, fibroblastos gengivais (FGH) foram cultivados e utilizados somente entre as passagens 3 e 8 para formação de monocamada e para a confecção do equivalente dermal em matriz de colágeno em três dimensões (3D). Ambos microcristais foram utilizados na concentração fungicida mínima (CFM) capaz de matar o fungo Candida albicans (C. albicans) e foram definidas como C2: 7,81 µg/mL para tungstato de prata e 15,62 µg/mL para molibdato de prata. A partir destes valores, concentrações 10 vezes concentradas (C3) e 10 vezes diluídas (C1) foram preparadas para melhor compreender a margem de efeito dos componentes sobre as células estudadas. Células incubadas com meio de cultura na ausência de microcristais foram utilizadas como controle negativo (C-) e células incubadas com tampão de lise (TL) como controle positivo, representando 100% de morte celular. O efeito dos microcristais na morfologia, viabilidade e proliferação das células foram inicialmente avaliados e direcionaram os experimentos sequenciais. A geração de espécies reativas de oxigênio (ROS) e a análise da integridade do DNA foram experimentos mandatórios para se conhecer o real impacto dos microcristais quando em células humanas. Os resultados quantitativos e qualitativos mostraram que α-Ag2WO4 não afetou a atividade enzimática mitocondrial das células FGH cultivadas em monocamada e em matriz de colágeno, em contraste com β-Ag2MoO4. Embora, o β-Ag2MoO4 não tenha interferido na proliferação celular, este composto afetou diretamente a viabilidade dos fibroblastos durante a confecção do gel em 3D. Experimentos posteriores realizados com α-Ag2WO4 provaram que estes microcristais na concentração de C2 não danificaram o DNA. O desenvolvimento de novos materiais tem sido sempre atraente para os pesquisadores, principalmente pela possibilidade de ser utilizado para tratamento de doenças, bem como evitar a prescrição indiscriminada de antibióticos. Nossos resultados fornecem informações relevantes no que diz respeito as propriedades não citotóxicas do α-Ag2WO4 para células gengivais humanas, durante um período de tempo adequado para estimar-se o potencial de perigo.
Growing interest has been reported in combining silver with other metals to improve the antimicrobial properties, reduce silver concentration and consequently its toxicity. Herein, this study investigated the effect of microcrystals silver tungstate (α- Ag2WO4) and silver molybdate (β-Ag2MoO4) on the gingival cells and threedimensional (3D) collagen matrices behavior. For all experiments, human gingival fibroblasts cells (HGF) were used between the 3rd and 8th passage. To carry out the experiments, lowest concentrations of α-Ag2WO4 and β-Ag2MoO4 that prevents visible growth of Candida albicans (C. albicans) planktonic cells were defined as our test concentration (C2): 7,81 µg/mL for silver tungstate and 15,62 µg/mL for silver molybdate. Solutions prepared from initial MFC concentration, ten-folds diluted (C1) and ten-folds concentrated (C3), improved the knowledge about the concentration ranging effect against human cells. Complete medium (C-) was used as a negative control and lysis buffer (LB) served as positive control (C+), equating to 100% cell death. The effect of the microcrystals concentration on cell morphology, viability and proliferation of HGF cells led following experiments. Reactive oxygen species (ROS) generation and DNA integrity analyzes were mandatories to know the real impact of the microcrystals on human cells. The quantitative and qualitative results showed that α-Ag2WO4 did not affect mitochondrial enzymatic activity of HGF cells cultured in monolayer and within collagen matrices, in contrast to β-Ag2MoO4. Although, β- Ag2MoO4 did not affect cells proliferation, this compound interfered directly in cell viability during 3D gel confection. Hence, subsequent analyzes performed with α- Ag2WO4 proved that this microcrystals in C2 concentration did not injury the genomic DNA. The new materials development has always been attractive to researchers, mainly by the possibility of treating diseases as well as avoiding the indiscriminate antibiotic prescription. Our findings afford strong information about the effect of α- Ag2WO4 on cells behavior, disclosing non-cytotoxic properties against human gingival cells, during a time period adequate to measure the hazard potential.
Growing interest has been reported in combining silver with other metals to improve the antimicrobial properties, reduce silver concentration and consequently its toxicity. Herein, this study investigated the effect of microcrystals silver tungstate (α- Ag2WO4) and silver molybdate (β-Ag2MoO4) on the gingival cells and threedimensional (3D) collagen matrices behavior. For all experiments, human gingival fibroblasts cells (HGF) were used between the 3rd and 8th passage. To carry out the experiments, lowest concentrations of α-Ag2WO4 and β-Ag2MoO4 that prevents visible growth of Candida albicans (C. albicans) planktonic cells were defined as our test concentration (C2): 7,81 µg/mL for silver tungstate and 15,62 µg/mL for silver molybdate. Solutions prepared from initial MFC concentration, ten-folds diluted (C1) and ten-folds concentrated (C3), improved the knowledge about the concentration ranging effect against human cells. Complete medium (C-) was used as a negative control and lysis buffer (LB) served as positive control (C+), equating to 100% cell death. The effect of the microcrystals concentration on cell morphology, viability and proliferation of HGF cells led following experiments. Reactive oxygen species (ROS) generation and DNA integrity analyzes were mandatories to know the real impact of the microcrystals on human cells. The quantitative and qualitative results showed that α-Ag2WO4 did not affect mitochondrial enzymatic activity of HGF cells cultured in monolayer and within collagen matrices, in contrast to β-Ag2MoO4. Although, β- Ag2MoO4 did not affect cells proliferation, this compound interfered directly in cell viability during 3D gel confection. Hence, subsequent analyzes performed with α- Ag2WO4 proved that this microcrystals in C2 concentration did not injury the genomic DNA. The new materials development has always been attractive to researchers, mainly by the possibility of treating diseases as well as avoiding the indiscriminate antibiotic prescription. Our findings afford strong information about the effect of α- Ag2WO4 on cells behavior, disclosing non-cytotoxic properties against human gingival cells, during a time period adequate to measure the hazard potential.
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Palavras-chave
Nanopartículas, Fibroblastos, Toxicidade