Modificação em fotossensibilizador e sua aplicação na terapia fotodinâmica antimicrobiana em substrato dentinário
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Data
2016-03-31
Autores
Carrera, Emanuelle Teixeira [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O objetivo deste trabalho, dividido em dois estudos, foi avaliar: (1) in vitro o efeito da terapia fotodinâmica na viabilidade de cultura planctônica de Streptococcus mutans mediada por azul de metileno associado ou não por nitrato de sódio em combinação com Laser; e (2) avaliar in vitro o efeito da terapia fotodinâmica na viabilidade do biofilme de Streptococcus mutans mediado por azul de metileno associado ou não por nitrato de sódio, ambos em associação com Laser em comprimento de onda vermelho (660nm). No primeiro estudo, a suspensão bacteriana contendo Streptococcus mutans 1x108 UFC/mL foi preparada e 250µL foi pipetada de acordo com os grupos: L-D- (controle negativo), L-D+ (fotossensibilizador associado ou não em 6 concentrações: 0,625; 1,25; 2,5; 5; 10 e 20µM), L+D- (Laser 30J/cm2), clorexidina (controle positivo), nitrato de sódio e o grupo L+D+ (azul de metileno associado ou não ao nitrato de sódio e irradiado com Laser). Para citotoxicidade no escuro, foram acrescidos 250µL do azul de metileno associado ou não, incubado no escuro por 5 minutos. Os grupos da terapia fotodinâmica foram incubados no escuro por 5min e irradiados por Laser 30 segundos. Alíquotas de cada diluição foram cultivadas em Placa de Petri com BHI ágar e incubadas a 37° C em estufa por 48h para posterior contagem das UFC/mL. Os resultados obtidos demonstraram que não houve citotoxicidade no escuro para as diferentes concentrações utilizadas do azul de metileno associado ou não. Observou-se redução completa na viabilidade do Streptococcus mutans em fase planctônica nas concentrações entre 0,625 e 5µM para azul de metileno associado. No segundo estudo induziu-se os biofilmes sobre fragmentos de dentina bovina (3x3x2mm) e foram divididos em grupos: L-D- (controle negativo), L-D+ (fotossensibilizador associado ou não em 6 concentrações: 0,625; 1,2; 2,5; 5; 10 e 20µM), L+D- (Laser 30J/cm2), clorexidina (controle positivo), nitrato de sódio e grupo L+D+ (azul de metileno associado ou não e irradiado com Laser). Para a citotoxicidade no escuro, foram adicionados azul de metileno associado ou não, incubados no escuro por 5 minutos. Os grupos da terapia fotodinâmica foram incubados no escuro por 5 minutos e irradiados por Laser 30 segundos. Alíquotas de cada diluição foram cultivados em Placas de Petri com BHI ágar e incubados em estufa por 48h para posterior contagem de UFC/mL. Para ambos os trabalhos, os dados foram transformados em log10, analisados por ANOVA um e dois fatores e teste de Tukey p < 0,05. Os resultados mostraram reduções significativas na viabilidade no biofilme de Streptococcus mutans quando expostos ao azul de metileno associado ao nitrato de sódio, 2,13 log10 para [2,5µM] e 2,37 log10 para [5µM], comparado com o azul de metileno não associado. Pode-se concluir que em ambos os estudos a associação do fotossensibilizador quando associado ao Laser promoveram redução significativa tanto para cultura planctônica quanto para o biofilme de Streptococcus mutans quando comparado com o fotossensibilizador não associado, sendo mais eficiente que a clorexidina, podendo ser uma técnica viável para eliminar ou reduzir essas bactérias na cavidade oral.
The aim of this work, divided into two studies, was to evaluate: (1) the in vitro the effect of photodynamic therapy on the viability of Streptococcus mutans planktonic culture mediated by methylene blue associated or not by sodium nitrate in association with red Laser; and (2) the in vitro the effect of photodynamic therapy on the viability of Streptococcus mutans biofilm mediated by methylene blue associated or not by sodium nitrate in association with red Laser (660nm). In the first study the bacterial suspension containing Streptococcus mutans 1x108 CFU/mL was prepared and 250µL pipetted according to the groups: L-P- (negative control), L-P+ (photosensitizer associated or no at 6 concentrations: 0.625, 1.25, 2.5, 5, 10 and 20µM), L+P- (Laser 30J/cm2), chlorhexidine (positive control), sodium nitrate and group L+P+ (methylene blue associated or not with sodium nitrate and irradiated with Laser). For the dark cytotoxicity test, 250 µL of methylene blue associated or not, were added and incubated in the dark for 5 minutes. The groups of photodynamic therapy were incubated in the dark for 5 minutes and irradiated by red Laser during 30 seconds. Samples were incubated at 37° C during 48h for subsequent visual counting of CFU/mL. The results showed that there was no cytotoxicity in the dark for the different concentrations of methylene blue associated or not. The photodynamic therapy promoted complete reduction in the viability of Streptococcus mutans at concentrations between 0.625 and 5μM for associated methylene blue. For the second study was induced biofilms on the bovine dentin fragments (3x3x2mm) and was divided in groups: PS-L- (negative control), PS+L- (photosensitizer associated or no at 6 concentrations: 0.625, 1.25, 2.5, 5, 10 and 20µM), P-L+ (Laser 30J/cm2), chlorhexidine (positive control), sodium nitrate and group P+L+ (methylene blue associated or not with sodium nitrate and irradiated with Laser). For the dark cytotoxicity test, methylene blue associated or not were added and incubated in the dark for 5 minutes. The groups of photodynamic therapy were incubated in the dark for 5 minutes and irradiated by red Laser during 30 seconds. Samples were incubated at 37° C during 48h for subsequent visual counting of CFU/mL. For both works, data were transformed into log10, analyzed by one and two-way ANOVA and Tukey’s test at p < 0.05. The results showed significant decreases in the viability of biofilm Streptococcus mutans when exposed to associated methylene blue, 2.13 log10 to [2.5µM] and 2.37 log10 to [5µM], compared with methylene blue not associated. Can be concluded that in both works photosensitizer association, associated with red Laser caused significant reduction in both the planktonic culture as for the biofilm of Streptococcus mutans biofilm when compared with the photosensitizer not associated, being more effective than chlorhexidine, may be a viable technique to eliminate or reduce these bacteria in the oral cavity.
The aim of this work, divided into two studies, was to evaluate: (1) the in vitro the effect of photodynamic therapy on the viability of Streptococcus mutans planktonic culture mediated by methylene blue associated or not by sodium nitrate in association with red Laser; and (2) the in vitro the effect of photodynamic therapy on the viability of Streptococcus mutans biofilm mediated by methylene blue associated or not by sodium nitrate in association with red Laser (660nm). In the first study the bacterial suspension containing Streptococcus mutans 1x108 CFU/mL was prepared and 250µL pipetted according to the groups: L-P- (negative control), L-P+ (photosensitizer associated or no at 6 concentrations: 0.625, 1.25, 2.5, 5, 10 and 20µM), L+P- (Laser 30J/cm2), chlorhexidine (positive control), sodium nitrate and group L+P+ (methylene blue associated or not with sodium nitrate and irradiated with Laser). For the dark cytotoxicity test, 250 µL of methylene blue associated or not, were added and incubated in the dark for 5 minutes. The groups of photodynamic therapy were incubated in the dark for 5 minutes and irradiated by red Laser during 30 seconds. Samples were incubated at 37° C during 48h for subsequent visual counting of CFU/mL. The results showed that there was no cytotoxicity in the dark for the different concentrations of methylene blue associated or not. The photodynamic therapy promoted complete reduction in the viability of Streptococcus mutans at concentrations between 0.625 and 5μM for associated methylene blue. For the second study was induced biofilms on the bovine dentin fragments (3x3x2mm) and was divided in groups: PS-L- (negative control), PS+L- (photosensitizer associated or no at 6 concentrations: 0.625, 1.25, 2.5, 5, 10 and 20µM), P-L+ (Laser 30J/cm2), chlorhexidine (positive control), sodium nitrate and group P+L+ (methylene blue associated or not with sodium nitrate and irradiated with Laser). For the dark cytotoxicity test, methylene blue associated or not were added and incubated in the dark for 5 minutes. The groups of photodynamic therapy were incubated in the dark for 5 minutes and irradiated by red Laser during 30 seconds. Samples were incubated at 37° C during 48h for subsequent visual counting of CFU/mL. For both works, data were transformed into log10, analyzed by one and two-way ANOVA and Tukey’s test at p < 0.05. The results showed significant decreases in the viability of biofilm Streptococcus mutans when exposed to associated methylene blue, 2.13 log10 to [2.5µM] and 2.37 log10 to [5µM], compared with methylene blue not associated. Can be concluded that in both works photosensitizer association, associated with red Laser caused significant reduction in both the planktonic culture as for the biofilm of Streptococcus mutans biofilm when compared with the photosensitizer not associated, being more effective than chlorhexidine, may be a viable technique to eliminate or reduce these bacteria in the oral cavity.
Descrição
Palavras-chave
Fotoquimioterapia, Azul de Metileno, Streptococcus mutans, Biofilmes, Cárie Dentária, Photochemotherapy, Methylene blue, Biofilms, Dental caries