Eficácia da inativação fotodinâmica mediada pela curcumina em diferentes formulações sobre biofilme e efeito nas propriedades de materiais restauradores resinosos e esmalte dental
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Data
2016-08-05
Autores
Santezi Neto, Carolina [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A formação de biofilmes em dentes e mucosas é considerada principal fator associado ao desenvolvimento de infecções bucais. Antimicrobianos tópicos, como a clorexidina (CLX), são utilizados para descontaminação de lesões cariosas, bolsas periodontais, e descontaminação bucal previamente a procedimentos cirúrgicos, porém podem promover efeitos adversos, além da possibilidade de promover resistência nos micro-organismos (MOs). Dessa forma, a busca por tratamentos antimicrobianos alternativos, como a Inativação Fotodinâmica (PDI) é muito relevante nas ciências odontológicas. Estudos prévios sugeriram que a curcumina (CUR) pode ser um promissor fotossensibilizador para PDI, porém existem ainda alguns aspectos que precisam ser mais bem avaliados. Assim, o presente estudo avaliou a eficácia da PDI mediada pela CUR para eliminação do biofilme dental e a existência de possíveis efeitos que essa terapia pode promover sobre algumas propriedades de materiais restauradores e superfície dental. O projeto foi dividido em três estudos consecutivos sendo que o Estudo 1 avaliou a eficácia da PDI utilizando diferentes formulações de CUR (líquida – DMSO a 10%; microemulsão eumulgin, hidrogel quitosana e microemulsão ácido oleico) sobre biofilmes mono-espécie de Streptococcus mutans (UA 159 ATCC 700610; Sm), Lactobacillus casei (ATCC 4646; Lc) e Candida albicans (ATCC 90028; Ca) formados in vitro. Previamente à realização dos tratamentos, a análise da taxa de fotodegradação das diferentes formulações e concentrações (20, 40, 60 e 80 µM) de CUR nos sistemas de hidrogel foi realizada e os resultados mostraram que, para o tempo de PDI proposto, as únicas formulações em hidrogel que poderiam ser utilizadas seriam a de quitosana (R2=0,989) e a de ácido oleico (R2=0,976), ambas na concentração de 80 µM. Em seguida a padronização da concentração celular de cada espécie foi estabelecida para o crescimento do biofilme e os seguintes grupos de tratamento foram testados para todas as formulações e espécies microbianas avaliadas: C-L- (CUR e luz ausentes; controle negativo), C-L+ (CUR ausente e luz presente - 18J/cm2; LED azul), C+L- (CUR presente e luz ausente) e C+L+ (CUR e luz presentes; PDI). Os dados passaram por análise descritiva e análise inferencial (ANOVA two way seguida do pós- teste Games Howell), indicando que o efeito da formulação, do tratamento e da interação entre eles foi altamente significativo (p < 0,0001) para as três espécies testadas, sendo os grupos PDI mediados pela formulação veiculada em DMSO os que evidenciaram maior ação antimicrobiana para todos os MOs avaliados. Dentre as formulações em hidrogel, aquela que promoveu efeito antimicrobiano foi a veiculada no sistema denominado hidrogel ácido oleico, sendo essa a formulação de escolha para a realização dos estudos subsequentes. O Estudo 2 avaliou o efeito da PDI mediada pela CUR veiculada no sistema denominado hidrogel ácido oleico sobre a estabilidade de cor, microdureza e rugosidade superficial de corpos de prova confeccionados em esmalte dental bovino e resinas compostas. Os grupos de tratamento avaliados foram CN (controle negativo), AcO (controle positivo) e PDI. Os dados foram analisados descritivamente e, em seguida, análise inferencial (ANOVA de medidas repetidas mista seguida por pós-teste de Sidak para as variáveis “estabilidade de cor” e “microdureza” e Games Howell para a “rugosidade superficial”) foi realizada para as três variáveis, sendo considerado para a “estabilidade de cor” somente dois momentos de leitura (imediatamente e 60 dias após os tratamentos) e para as variáveis “microdureza” e “rugosidade superficial” as leituras efetuadas anteriormente aos tratamentos e 60 dias após a realização deles. Os resultados da ANOVA mostraram que para a variável “estabilidade de cor”, os fatores “tempo”, “tratamento”, “resina” e “interação entre eles” foram significativos (p ≤ 0,021), sendo que de forma geral essa alteração foi maior nas leituras efetuadas no tempo “60 dias após os tratamentos”. Para a variável microdureza superficial, com exceção dos fatores “tempo x tratamento” e “tratamento”, todos os outros se mostraram significativos (p < 0,001), evidenciando que a PDI para as resinas TPH e AML não promoveu qualquer efeito deletério sobre essa propriedade; por outro lado, para a resina Z350, na leitura realizada no tempo “60 dias após os tratamentos”, houve redução da dureza quando comparada com os grupos CN e AcO, sugerindo efeito negativo da terapia sobre a microdureza. A ANOVA mostrou que o fator “tempo” não foi significativo (p = 0,991), tendo sido significativo somente os fatores “superfície”, “tratamento” e “superfície x tratamento” (p < 0,001). Além disso, foi possível verificar com os resultados que a rugosidade superficial das resinas TPH e Z350 não foi alterada, enquanto que a rugosidade da resina AML foi reduzida após realização dos tratamentos. Diferentemente, para os corpos de prova confeccionados em esmalte dental bovino, as três variáveis investigadas não sofreram qualquer efeito negativo frente à realização dos tratamentos, tendo sido significativo somente o fator “tempo” (p ≤ 0,002). O Estudo 3, piloto, avaliou a eficácia do protocolo de PDI previamente estabelecido sobre biofilmes multiespécies formados sobre blocos de esmalte dental bovino in situ. Para isso, após aprovação pelo comitê de ética em pesquisa desta faculdade, 7 voluntários foram selecionados, moldados para confecção de dispositivo intra-bucal e orientados quanto ao uso do aparelho para formação do biofilme (48 horas). A etapa microbiológica consistiu na remoção dos blocos de esmalte do dispositivo, realização dos tratamentos (C+L+ e C-L-) e quantificação das células viáveis em meios de cultura específicos para Sm, Lc, Candida spp e em meio inespecífico para crescimento de MOs em geral. Os dados foram tratados e a análise inferencial (MANOVA seguida de ANOVA a um fator) demonstrou que o fator “grupo de tratamento” foi altamente significativo (p < 0,001) sobre a viabilidade microbiana, sendo observado eficácia da PDI sobre Sm e MOs em geral. Desse modo, conclui-se que embora a eficácia da PDI mediada pela CUR para redução de biofilmes formados in vitro tenha sido comprovada, esta se encontra diretamente relacionada ao tipo de veículo usado para a solubilização do fotossensibilizador. Além disso, o protocolo “CUR a 80 µM veiculada em hidrogel ácido oleico associada à dose de 18J/cm2 com tempo de pré-irradiação de 5 minutos” se mostrou eficaz para a redução quantitativa de Sm e MOs em geral de biofilmes crescidos in situ e não apresentou qualquer efeito deletério na estabilidade de cor, microdureza e rugosidade superficial de esmalte dental bovino. Em contrapartida, para algumas resinas, efeitos negativos sobre essas propriedades foram observados. Assim, o uso desse protocolo parece ser seguro sobre superfícies de esmalte dental, enquanto que para resinas, uma avaliação criteriosa previamente ao uso deve ser realizada, entretanto mais investigações são necessárias para melhor compreensão dos achados.
Formation of dental biofilm is the main factor associated to development of oral infections. Although topic antimicrobials, such as chlorhexidine (CHX), have been used to decontaminate the oral surfaces, they may promote adverse effects on hard and soft tissues as well as the possibility of promoting microbial resistance. Thus, the investigation of alternative antimicrobial treatments, such the Photodynamic Inactivation (PDI), is relevant to dental sciences. Previous research suggested that the curcumin (CUR) might be a promising photosensitizer for PDI, but there are some aspects that need to be better evaluated. The aim of this study was to assess the efficacy of CUR-mediated PDI against dental biofilms and the existence of possible negative effects of PDI on the properties of composite resins and dental surface. The research was divided into 3 consecutive studies. The study 1 aimed to evaluate the PDI efficacy using different CUR formulations (liquid – DMSO (dimethylsulfoxide) at 10%; eumulgin hydrogel, chitosan hydrogel and oleic acid hydrogel) on mono-species biofilm of Streptococcus mutans (UA 159 ATCC 700610; Sm), Lactobacillus casei (ATCC 4646; Lc) and Candida albicans (ATCC 90028; Ca) formed in vitro. Prior to the execution of the treatments, the photodegradation rate of each formulation in concentrations 20, 40, 60 and 80 µM was performed and the results showed that the chitosan hydrogel (R2=0,989) and oleic acid hydrogel (R2=0,976) were able to be used in the tests, both at 80 µM. Then, cell concentration of each species was standardized in order to the grow biofilm and the following treatment groups were tested for all formulations and evaluated species: C-L- (no CUR and light; negative control), C-L+ (no CUR and with light - 18J/cm2; blue LED), C+L- (with CUR and no light) and C+L+ (with both CUR and light; PDI). Data were analyzed descriptvely and inferential analysis (two-way ANOVA followed by Games Howell test) indicated that the effect of the formulation, treatment and the interaction between them was highly significant (p < 0,0001) for the three species tested, and the PDI groups mediated by the formulation of DMSO at 10% presented the best antimicrobial activity for all evaluated microrganisms (MOs). Among the hydrogel formulations, oleic acid hydrogel promoted antimicrobial effect, thus it was selected for conducting subsequent studies. Study 2 evaluated the effect of PDI-mediated CUR carried in a system called hydrogel oleic acid on the stability of color, hardness and surface roughness of specimens made of bovine enamel and composite resins. The treatment groups evaluated were CN (negative control), AcO (positive control) and PDI. Data were analyzed descriptively and then, inferential analysis (ANOVA mixed repeated measures followed by Sidak post-test for variables "color stability" and "microhardness"; Games and Howell for "surface roughness") was performed. However, to do the analysis for the “color stability" variable, it was considered only 2 moments of color measurement (immediately and 60 days after treatment). In the same way, the measurements for "hardness" and "surface roughness" were assessed prior to treatment and 60 days after performing them. To the "color stability" variable, the ANOVA results showed that the factors "time", "treatment", "resin" and "interaction between them" were significant (p ≤ 0.021) on the color change of resins, and, in general, this change was greater in the measurements assessed in time "60 days after the treatments". For the microhardness variable, except the factors "time x treatment" and "treatment", all others were significant (p < 0,001), showing that PDI for TPH and AML resins did not cause any deleterious effect on this property. On the other hand, for the resin Z350, the measurements performed at time "60 days after the treatment", showed a reduction in hardness values compared to the values found in CN and AcO groups, suggesting that PDI promoted a negative effect on the hardness. ANOVA showed no significance on factor "time" (p = 0.991), but it showed that the factors "surface", "treatment" and "treatment x area" was significant (p < 0.001). In addition, results showed that the surface roughness of TPH and Z350 resins did not change, while the AML was reduced after both treatments. Differently, for the specimens prepared with dental enamel, the three investigated variables did not suffer any negative effect after the treatments, and the factor “times” was the only one considered significant (p ≤ 0.002). Study 3, a pilot, evaluated the effectiveness of the PDI protocol previously established on multi-species biofilms formed on bovine enamel blocks in situ. For this, after approval by the ethics committee in research of this faculty, 7 volunteers were selected and impressions were taken for making intra-oral device. They also were instructed on the use of the device for biofilm formation (48 hours). Microbiological step consisted in removal of enamel blocks of the device, application of the treatments (C+L+ and C-L-) and quantification of viable cells in specific culture media for Sm, Lc, Candida spp and unspecific media for MOs general growth. The data were processed and inferential analysis (MANOVA followed by ANOVA by a factor) showed that the factor "treatment group" was highly significant (p <0.001) on Sm and general MOs viability. Thus, in conclusion, although the efficacy of PDI mediated by CUR in order to reduce biofilms formed in vitro has been established, the efficacy is directly related to the type of vehicle used for solubilization of the photosensitizer. Furthermore, the protocol "CUR at 80 µM carried by hydrogel oleic acid associated with fluence of 18J/cm2 with pre-irradiation time of 5 minutes" was effective for the quantitative reduction of Sm and MOs in general of the biofilms which grown in situ and showed no deleterious effect on color stability, hardness and surface roughness of tooth enamel. However, for some resins, negative effects on these properties were observed. Thus, the use of this protocol appears to be safe on surfaces of enamel, while for resins, a careful evaluation prior to use should be made. Thus, more investigations are necessary in order to better understand the findings.
Formation of dental biofilm is the main factor associated to development of oral infections. Although topic antimicrobials, such as chlorhexidine (CHX), have been used to decontaminate the oral surfaces, they may promote adverse effects on hard and soft tissues as well as the possibility of promoting microbial resistance. Thus, the investigation of alternative antimicrobial treatments, such the Photodynamic Inactivation (PDI), is relevant to dental sciences. Previous research suggested that the curcumin (CUR) might be a promising photosensitizer for PDI, but there are some aspects that need to be better evaluated. The aim of this study was to assess the efficacy of CUR-mediated PDI against dental biofilms and the existence of possible negative effects of PDI on the properties of composite resins and dental surface. The research was divided into 3 consecutive studies. The study 1 aimed to evaluate the PDI efficacy using different CUR formulations (liquid – DMSO (dimethylsulfoxide) at 10%; eumulgin hydrogel, chitosan hydrogel and oleic acid hydrogel) on mono-species biofilm of Streptococcus mutans (UA 159 ATCC 700610; Sm), Lactobacillus casei (ATCC 4646; Lc) and Candida albicans (ATCC 90028; Ca) formed in vitro. Prior to the execution of the treatments, the photodegradation rate of each formulation in concentrations 20, 40, 60 and 80 µM was performed and the results showed that the chitosan hydrogel (R2=0,989) and oleic acid hydrogel (R2=0,976) were able to be used in the tests, both at 80 µM. Then, cell concentration of each species was standardized in order to the grow biofilm and the following treatment groups were tested for all formulations and evaluated species: C-L- (no CUR and light; negative control), C-L+ (no CUR and with light - 18J/cm2; blue LED), C+L- (with CUR and no light) and C+L+ (with both CUR and light; PDI). Data were analyzed descriptvely and inferential analysis (two-way ANOVA followed by Games Howell test) indicated that the effect of the formulation, treatment and the interaction between them was highly significant (p < 0,0001) for the three species tested, and the PDI groups mediated by the formulation of DMSO at 10% presented the best antimicrobial activity for all evaluated microrganisms (MOs). Among the hydrogel formulations, oleic acid hydrogel promoted antimicrobial effect, thus it was selected for conducting subsequent studies. Study 2 evaluated the effect of PDI-mediated CUR carried in a system called hydrogel oleic acid on the stability of color, hardness and surface roughness of specimens made of bovine enamel and composite resins. The treatment groups evaluated were CN (negative control), AcO (positive control) and PDI. Data were analyzed descriptively and then, inferential analysis (ANOVA mixed repeated measures followed by Sidak post-test for variables "color stability" and "microhardness"; Games and Howell for "surface roughness") was performed. However, to do the analysis for the “color stability" variable, it was considered only 2 moments of color measurement (immediately and 60 days after treatment). In the same way, the measurements for "hardness" and "surface roughness" were assessed prior to treatment and 60 days after performing them. To the "color stability" variable, the ANOVA results showed that the factors "time", "treatment", "resin" and "interaction between them" were significant (p ≤ 0.021) on the color change of resins, and, in general, this change was greater in the measurements assessed in time "60 days after the treatments". For the microhardness variable, except the factors "time x treatment" and "treatment", all others were significant (p < 0,001), showing that PDI for TPH and AML resins did not cause any deleterious effect on this property. On the other hand, for the resin Z350, the measurements performed at time "60 days after the treatment", showed a reduction in hardness values compared to the values found in CN and AcO groups, suggesting that PDI promoted a negative effect on the hardness. ANOVA showed no significance on factor "time" (p = 0.991), but it showed that the factors "surface", "treatment" and "treatment x area" was significant (p < 0.001). In addition, results showed that the surface roughness of TPH and Z350 resins did not change, while the AML was reduced after both treatments. Differently, for the specimens prepared with dental enamel, the three investigated variables did not suffer any negative effect after the treatments, and the factor “times” was the only one considered significant (p ≤ 0.002). Study 3, a pilot, evaluated the effectiveness of the PDI protocol previously established on multi-species biofilms formed on bovine enamel blocks in situ. For this, after approval by the ethics committee in research of this faculty, 7 volunteers were selected and impressions were taken for making intra-oral device. They also were instructed on the use of the device for biofilm formation (48 hours). Microbiological step consisted in removal of enamel blocks of the device, application of the treatments (C+L+ and C-L-) and quantification of viable cells in specific culture media for Sm, Lc, Candida spp and unspecific media for MOs general growth. The data were processed and inferential analysis (MANOVA followed by ANOVA by a factor) showed that the factor "treatment group" was highly significant (p <0.001) on Sm and general MOs viability. Thus, in conclusion, although the efficacy of PDI mediated by CUR in order to reduce biofilms formed in vitro has been established, the efficacy is directly related to the type of vehicle used for solubilization of the photosensitizer. Furthermore, the protocol "CUR at 80 µM carried by hydrogel oleic acid associated with fluence of 18J/cm2 with pre-irradiation time of 5 minutes" was effective for the quantitative reduction of Sm and MOs in general of the biofilms which grown in situ and showed no deleterious effect on color stability, hardness and surface roughness of tooth enamel. However, for some resins, negative effects on these properties were observed. Thus, the use of this protocol appears to be safe on surfaces of enamel, while for resins, a careful evaluation prior to use should be made. Thus, more investigations are necessary in order to better understand the findings.
Descrição
Palavras-chave
Cárie Dentária, Curcumina, Fotoquimioterapia, Placa Dentária, Materiais Dentários, Curcumin, Dental Caries, Dental Materials, Dental Plaque, Photochemotherapy