Análise in vitro do extrato de citronela (Cymbopogon nardus) e de enxaguatórios bucais comerciais sobre propriedades físicas e microbiológicas de materiais utilizados na confecção de prótese tipo protocolo
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Data
2017-06-23
Autores
Cunha, Bruno Guandalini [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O uso de soluções enxaguatórias é uma ferramenta no controle de patologias relacionadas ao uso de próteses. A efetividade antimicrobiana e citotoxicidade de alguns enxaguatórios comercialmente disponíveis ainda são questionadas, tornando a fitoterapia uma alternativa a ser pesquisada. Sendo assim, este estudo teve como objetivos: 1) avaliar in vitro a eficácia de formulações à base do extrato de citronela (CN) em biofilmes monoespécie em formação e formado, sobre amostras de resina acrílica ativada termicamente (RAAT) e liga de níquel-cromo (LNC), contaminadas por S. aureus e C. albicans, simulando uma prótese protocolo em meio bucal, em comparação com dois enxaguatórios comerciais sem etanol (LT – Listerine Zero e CHX – Periogard sem etanol); 2) avaliar in vitro a citotoxicidade das soluções em células epiteliais HaCat, simulando o contato com os enxaguatórios por 1 min; 3) avaliar, in vitro, o efeito de enxaguatórios bucais comerciais e da solução teste à base do óleo de CN a 10,9%, na alteração de rugosidade e cor de materiais utilizados na confecção de prótese tipo protocolo, sendo eles: duas marcas de dentes artificiais (DA - Trilux e Vivodent), RAAT e LNC. A alteração de cor da LNC não foi avaliada. Para o primeiro objetivo, após a obtenção da concentração bactericida/fungicida mínima (CBM/CFM) da CN contra S. aureus e C. albicans, foram confeccionadas amostras de RAAT e LNC. A CBM/CFM da CN foi multiplicada por 5x e 10x para a formulação de dois enxaguatórios. As superfícies das amostras com o biofilme em formação (4 h de adesão) e formado (24 h) foram submetidas à ação das soluções à base de CN, Periogard sem etanol (CHX) e Listerine Zero (LT). A simulação dos bochechos foi realizada em dois momentos diferentes, sendo a primeira simulação logo após o tempo de adesão de 4 h (biofilme em formação) e 24 h (biofilme formado), e a segunda simulação, 6 h após a primeira simulação, para cada microrganismo. O ensaio de citotoxicidade das soluções foi realizado em células epiteliais HaCat, simulando o contato com os enxaguatórios por 1 min e quantificado pelo método de MTT. Para a análise das propriedades físicas, amostras dos três materiais foram distribuídas em 6 grupos (n=10), de acordo com a solução enxaguatória: GI - Saliva artificial; GII - Periogard sem etanol; GIII - Periogard com etanol; GIV - Listerine Zero; GV – Listerine Tartar Control; GVI - Solução teste à base do CN à 10,9%, sendo submetidas à imersão e agitação por 180 min, simulando bochechos diários por 6 meses. A alteração de superfície das amostras foi avaliada em rugosímetro, no qual um grupo sem imersão foi utilizado como controle (GC); e a alteração de cor, em espectrofotômetro. Como resultados da ação antibiofilme das soluções testadas, observou-se que independente da superfície, todas as soluções impediram o desenvolvimento dos biofilmes de S. aureus e C. albicans, quando aplicados no início de sua formação (4 h). Para os biofilmes formados (24 h), todas as soluções diferiram dos respectivos controles, em resina ou metal, mostrando atividade contra biofilmes de S. aureus e C. albicans. CN 5x CBM/CFM e CN 10x CBM/CFM tiveram os melhores efeitos antibiofilme, não diferindo estatisticamente entre si, independente da superfície da amostra. Ambas as concentrações de CN tiveram efeito superior à CHX e LT, independente da superfície. Para os ensaios de citotoxicidade, todas as soluções diluídas em 50 e 25% causaram citotoxicidade celular. A partir da diluição de 3,12%, todas as soluções permitiram mais de 70% de viabilidade celular. Como resultados da alteração de cor foi possível observar que todas as soluções promoveram um ΔE acima de 3,3 para RAAT, valor considerado clinicamente inaceitável. Para os DA não foi notada nenhuma diferença estatisticamente significante em relação ao grupo controle. Para a análise de superfície, a rugosidade da RAAT foi alterada somente no grupo GIV – Listerine Tartar Control, enquanto que não houve nenhuma alteração estatisticamente significante para os DA e LNC. Pode-se concluir que ambas as concentrações do enxaguatório à base de citronela tiveram os maiores efeitos antibiofilme, comparativamente às soluções comerciais sem etanol, contra S. aureus e C. albicans. Todas as soluções enxaguatórias foram citotóxicas nas suas concentrações iniciais, entretanto, após diluição seriada, considerando as concentrações dos princípios ativos dos enxaguatórios comerciais, CN teve o menor efeito citotóxico. A formulação teste à base de 10,9% de CN mostrou-se segura em seu uso como solução enxaguatória, no que se refere às propriedades físicas estudadas.
The use of mouthrinses is a tool in the control of pathologies related to the use of prostheses. The antimicrobial effectiveness and cytotoxicity of some commercially available mouthrinses are still questioned, making phytotherapy an alternative to be researched. Therefore, this study aimed: 1) to evaluate in vitro the efficacy of formulations based on the citronella extract (CN) in monospecific biofilms, in formation and formed, on samples of thermally activated acrylic resin (RAAT) and nickel-chromium (LNC), contamineted by S. aureus and C. albicans, simulating a protocol prosthesis in buccal medium, compared to two alcohol free commercial mouthrinses (LT - Listerine Zero and CHX - Periogard alcohol free); 2) to evaluate in vitro the cytotoxicity of solutions in HaCat epithelial cells, simulating contact with the mouthrinses for 1 min; 3) to evaluate in vitro the effect of commercial mouthrinses and the test solution based on the extract of CN at 10.9%, in the alteration of roughness and color of materials used in the manufacturing of protocol type prosthesis; i.e. two tooth marks (DA - Trilux and Vivodent), RAAT and LNC. The color change of the LNC was not evaluated. For the first objective, samples were prepared after obtaining the minimum bactericidal / fungicidal concentration (CBM / CFM) of CN against S. aureus and C. albicans, RAAT and LNC. The CN’s CBM / CFM was multiplied by 5x and 10x for the formulation of two mouthrinses. The surfaces of the samples with the biofilm in formation (4 h of adhesion) and formed (24 h) were subjected to the action of solutions based on CN, Periogard alcohol free (CHX) and Listerine Zero (LT). The simulation of mouthwashing was performed at two different moments; the first simulation after the time of adhesion of 4 h (biofilm in formation) and 24 h (formed biofilm), and the second simulation, 6 h after the first simulation, for each microorganism. The cytotoxicity assay of the solutions was performed on HaCat epithelial cells, simulating contact with the mouthrinse for 1 min and quantified by the MTT method. For the analysis of the physical properties, samples of three materials were distributed in 6 groups (n = 10) according to the mouthrinse: GI - Artificial saliva; GII - Periogard alcohol free; GIII - Periogard with alcohol; GIV - Listerine Zero; GV- Listerine Tartar Control; GVI - Test solution based on CN at 10.9%, and submitted to immersion and agitation for 180 min, simulating daily mouthwashing for 6 months. The surface change of the samples was evaluated in a rugosimeter and the color change in a spectrophotometer. A group without immersion was used as control (GC). As a result of the antibiofilm action of the tested solutions, it was observed that, regardless of the surface, all solutions prevented the development of the biofilms of S. aureus and C. albicans, when applied at the beginning of its formation (4 h). For the formed biofilm (24 h), all solutions differed from the respective resin or metal controls, showing activity against biofilms of S. aureus and C. albicans. CN 5x CBM / CFM and CN 10x CBM / CFM had the best antibiofilm effects, not statistically different from each other, regardless of the sample surface. Both CN concentrations presented a superior effect than CHX and LT, regardless of the surface. For cytotoxicity assays, all solutions diluted at 50 and 25% caused large cell cytotoxicity. From the 3.12% dilution, all solutions allowed more than 70% cell viability. From the results of the color change, it was possible to observe that all solutions promoted a ΔE above 3.3 for RAAT; a value considered clinically unacceptable. For DA, no significant statistical difference was observed in relation to the control group. For the surface analysis, RAAT roughness was altered only in the GIV - Listerine Tartar Control group, whereas there were no significant statistical alterations for the DA and LNC. It can be concluded that both concentrations of citronella-based mouthrinse had the highest antibiofilm effects compared to commercial alcohol free solutions against S. aureus and C. albicans. All commercial mouthrinses were cytotoxic at their initial concentrations, however, after serial dilution, considering the concentrations of active principles of commercial mouthrinses, CN had the lowest cytotoxic effect. The test formulation based on 10.9% CN was safe in its use as a mouthrinse solution in respect of the physical properties studied.
The use of mouthrinses is a tool in the control of pathologies related to the use of prostheses. The antimicrobial effectiveness and cytotoxicity of some commercially available mouthrinses are still questioned, making phytotherapy an alternative to be researched. Therefore, this study aimed: 1) to evaluate in vitro the efficacy of formulations based on the citronella extract (CN) in monospecific biofilms, in formation and formed, on samples of thermally activated acrylic resin (RAAT) and nickel-chromium (LNC), contamineted by S. aureus and C. albicans, simulating a protocol prosthesis in buccal medium, compared to two alcohol free commercial mouthrinses (LT - Listerine Zero and CHX - Periogard alcohol free); 2) to evaluate in vitro the cytotoxicity of solutions in HaCat epithelial cells, simulating contact with the mouthrinses for 1 min; 3) to evaluate in vitro the effect of commercial mouthrinses and the test solution based on the extract of CN at 10.9%, in the alteration of roughness and color of materials used in the manufacturing of protocol type prosthesis; i.e. two tooth marks (DA - Trilux and Vivodent), RAAT and LNC. The color change of the LNC was not evaluated. For the first objective, samples were prepared after obtaining the minimum bactericidal / fungicidal concentration (CBM / CFM) of CN against S. aureus and C. albicans, RAAT and LNC. The CN’s CBM / CFM was multiplied by 5x and 10x for the formulation of two mouthrinses. The surfaces of the samples with the biofilm in formation (4 h of adhesion) and formed (24 h) were subjected to the action of solutions based on CN, Periogard alcohol free (CHX) and Listerine Zero (LT). The simulation of mouthwashing was performed at two different moments; the first simulation after the time of adhesion of 4 h (biofilm in formation) and 24 h (formed biofilm), and the second simulation, 6 h after the first simulation, for each microorganism. The cytotoxicity assay of the solutions was performed on HaCat epithelial cells, simulating contact with the mouthrinse for 1 min and quantified by the MTT method. For the analysis of the physical properties, samples of three materials were distributed in 6 groups (n = 10) according to the mouthrinse: GI - Artificial saliva; GII - Periogard alcohol free; GIII - Periogard with alcohol; GIV - Listerine Zero; GV- Listerine Tartar Control; GVI - Test solution based on CN at 10.9%, and submitted to immersion and agitation for 180 min, simulating daily mouthwashing for 6 months. The surface change of the samples was evaluated in a rugosimeter and the color change in a spectrophotometer. A group without immersion was used as control (GC). As a result of the antibiofilm action of the tested solutions, it was observed that, regardless of the surface, all solutions prevented the development of the biofilms of S. aureus and C. albicans, when applied at the beginning of its formation (4 h). For the formed biofilm (24 h), all solutions differed from the respective resin or metal controls, showing activity against biofilms of S. aureus and C. albicans. CN 5x CBM / CFM and CN 10x CBM / CFM had the best antibiofilm effects, not statistically different from each other, regardless of the sample surface. Both CN concentrations presented a superior effect than CHX and LT, regardless of the surface. For cytotoxicity assays, all solutions diluted at 50 and 25% caused large cell cytotoxicity. From the 3.12% dilution, all solutions allowed more than 70% cell viability. From the results of the color change, it was possible to observe that all solutions promoted a ΔE above 3.3 for RAAT; a value considered clinically unacceptable. For DA, no significant statistical difference was observed in relation to the control group. For the surface analysis, RAAT roughness was altered only in the GIV - Listerine Tartar Control group, whereas there were no significant statistical alterations for the DA and LNC. It can be concluded that both concentrations of citronella-based mouthrinse had the highest antibiofilm effects compared to commercial alcohol free solutions against S. aureus and C. albicans. All commercial mouthrinses were cytotoxic at their initial concentrations, however, after serial dilution, considering the concentrations of active principles of commercial mouthrinses, CN had the lowest cytotoxic effect. The test formulation based on 10.9% CN was safe in its use as a mouthrinse solution in respect of the physical properties studied.
Descrição
Palavras-chave
Prótese total, Resinas acrílicas, Desinfecção, Cymbopogon, Teste de materiais, Propriedades físicas, Biofilmes, Staphylococcus aureus, Candida albicans, Denture, Acrylic resins, Disinfection, Cymbopogon, Material testing, Physical properties, Biofilms