Potencial da terapia fotodinâmica na modulação da resposta inflamatória e nos fatores de virulência microbianos: avaliação em modelo de co-culturas tridimensionais de fibroblastos gengivais, Candida albicans e Staphylococcus aureus
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2024-03-13
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Universidade Estadual Paulista (Unesp)
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Este trabalho está dividido em três publicações cujos objetivos foram: a) realizar uma revisão sistemática e metanálise em ensaios clínicos para avaliar a eficácia da terapia fotodinâmica em lesões de mucosite oral (Publicação 1); b) avaliar o potencial in vitro de quatro diferentes fotossensibilizadores, sendo dois de origem sintética (Azul de Metileno e Fotoditazina) e dois de origem natural (Curcumina e extrato bruto de Senna alata), para terapia fotodinâmica antimicrobiana (TFDa) contra Candida albicans e Staphylococcus aureus cultivadas em suspensão e biofilme e c) avaliar a efetividade da TFDa mediada pelo extrato bruto de S. alata em modelo de cocultura celular tridimensional (3D) contendo fibroblastos gengivais, queratinócitos orais e colágeno tipo I. Publicação 1: A questão de pesquisa e as palavras-chave foram definidas seguindo a estratégia do PICO. A pesquisa do artigo foi realizada em diversas bases de dados, incluindo Embase, Scielo, Scopus, Cochrane Library, Web of Science, Science Direct e Pubmed. Foram selecionados ensaios clínicos randomizados para a revisão. O estudo foi conduzido conforme as normativas do PRISMA para revisão sistemática. Publicação 2: Foram utilizadas as seguintes cepas de referência em suspensão e biofilme: S. aureus ATCC 25923 e C. albicans ATCC 90028. Foram testados quatro agentes fotossensibilizadores (FS) em duas concentrações diferentes (Photodithazine (PDZ): 0.175 e 0.2 mg/mL; azul de metileno (AM): 0,2 e 2 mg/mL; Curcumina (CUR): 0.014 e 0.029 mg/mL; Extrato vegetal de S. alata: 0.05 mg/mL e 0.5 mg/mL. Dois protocolos de TFDa foram utilizados sobre biofilmes cultivados por 24 h e 48 hs: a) em único tratamento e b) em três aplicações sucessivas. As células microbianas em fase planctônica receberam apenas um único tratamento. A eficácia da terapia foi avaliada através da contagem de células viáveis após o tratamento (UFC/mL) e ensaio Live/Dead. O ensaio MTT foi utilizado para avaliar a citotoxicidade. O metabolismo de cada biofilme foi avaliado pelo ensaio XTT. Publicação 3: O modelo de cocultura 3D foi preparado e posteriormente infectado com S. aureus ATCC 25923 e C. albicans ATCC 90028 por 16 h, seguido pela aplicação da TFDa mediada pelo extrato bruto de S. alata nas concentrações de 0,05 e 0,5 mg/mL. Após 24 h do tratamento, foi avaliada a viabilidade microbiana (UFC/mL) e o dano celular pelo ensaio da lactato desidrogenase (LDH). AM nas concentrações de 0,2 e 2 mg/mL foram utilizados para fins de comparação. Os dados das publicações 2 e 3 foram analisados no programa IBM SPSS versão 20.0, com nível de significância de 5%. Dos 727 artigos analisados na publicação 1, foram selecionados 5 artigos para análise qualitativa e 2 para a meta-análise. Os resultados da meta-análise, incluindo nível de evidência e avaliação de risco de viés, demonstraram a eficácia da TFD na redução do tempo de cicatrização em combinação com a terapia a laser de baixa intensidade (TLBI), em comparação com a aplicação isolada da TLBI (p = 0,0005/I2 = 0%).Reduções totais na viabilidade microbiana foram encontradas na publicação 2 para TFDa mediada por curcumina (0.014 e 0.029 mg/mL), TFDa mediada por PDZ (0.175 e 0.2 mg/mL) e a TFDa mediada por S. alata (0.05 e 0.5 mg/mL) para ambos os micro-organismos cultivados em suspensão. Biofilmes de C. albicans cultivados por 24 horas mostraram redução total na viabilidade microbiana (UFC/mL), para TFDa mediada por AM (0,2 e 2,0 mg/mL) e TFDa mediada por S. alata (0.5 mg/mL) em ambos os protocolos utilizados (aplicação única e aplicações sucessivas). Biofilmes de C. albicans cultivados por 48 h apresentaram redução total na viabilidade microbiana para TFDa mediada por AM (2,0 mg/mL). Para S. aureus, houve redução significativa na viabilidade microbiana apenas para três aplicações de TFDa (S. alata: 0.05 e 0.5 mg/mL; AM: 0,2 e 2,0 mg/mL) em biofilmes cultivados por 24 h e em biofilmes cultivado por 48 h (S. alata: 5,0 mg/mL; MB: 0,2 e 2,0 mg/mL). As imagens CLSM para ensaio Live/Dead confirmaram os resultados encontrados para viabilidade microbiana. Todos os fotossensibilizadores apresentaram valores significativamente diminuídos para a atividade metabólica do biofilme de C. albicans e S. aureus cultivados por 24 horas tratados com uma única aplicação de TFDa. Além disso, as concentrações dos fotossensibilizadores e os parâmetros de irradiação utilizados no presente estudo apresentaram valores aceitáveis (≤70%) de citotoxicidade para ambas as linhagens celulares. Os resultados da publicação 1 destacam que a TFD é eficaz no tratamento da mucosite oral, contribuindo para a cicatrização de feridas, principalmente no tempo necessário para o reparo. Já os resultados da publicação 2 mostraram que a terapia fotodinâmica antimicrobiana (TFDa) mediada por diferentes FSs revelou-se seguro e eficiente para o controle de C. albicans e S. aureus. Adicionalmente, para a publicação 3, os melhores reduções de S. aureus e C. albicans foram encontradas para 0,5 mg/mL de S. alata e 2 mg/mL de AM, sendo que a TFDa mediada pelo extrato bruto de S. alata causam menos danos celulares em comparação com AM (p>0,05) e clorexidina 0,12%. Os resultados da publicação 1 destacam que a TFD apresenta resultados promissores para o tratamento da mucosite oral e pode ser uma opção terapêutica eficaz, contribuindo para a cicatrização dos tecidos lesionados principalmente no tempo necessário para o reparo. Já os resultados das publicações 2 e 3 mostraram que a terapia fotodinâmica antimicrobiana (TFDa) mediada pelo extrato bruto de S. alata revelou-se seguro e eficiente para o controle de C. albicans e S. aureus.
This study is divided into three publications whose objectives were: a) to conduct a systematic literature review, followed by a meta-analysis of clinical trials to evaluate the efficacy of photodynamic therapy in oral mucositis lesions (Publication 1); b) to evaluate the in vitro potential of four different photosensitizers, two of synthetic origin (Methylene Blue and Photodithazine) and two of natural origin (Curcumin and crude extract of Senna alata), to be used in antimicrobial photodynamic therapy (aPDT) against Candida albicans and Staphylococcus aureus cultured in suspension and biofilm; and c) to evaluate the effectiveness of aPDT mediated by the crude extract of S. alata in a three-dimensional (3D) cell co-culture model containing gingival fibroblasts, oral keratinocytes, and type I collagen. Publication 1: The research question and keywords were defined following the PICO methodology. The article search was conducted on various databases, including Embase, Scielo, Scopus, Cochrane Library, Web of Science, Science Direct, and PubMed. Randomized clinical trials were selected for the review. The study was conducted following PRISMA guidelines for systematic review. Publication 2: The following reference strains in suspension and biofilm were used: S. aureus ATCC 25923 and C. albicans ATCC 90028. Four photosensitizing agents (PS) were tested at two different concentrations (Photodithazine (PDZ): 0.175 and 0.2 mg/mL; Methylene Blue (MB): 0.2 and 2 mg/mL; Curcumin (CUR): 0.014 and 0.029 mg/mL; S. alata plant extract: 0.05 mg/mL and 0.5 mg/mL). Two aPDT protocols were used on biofilms cultured for 24 h and 48 h: a) single treatment and b) three successive applications. Planktonic microbial cells received only a single treatment. The therapy's efficacy was evaluated through viable cell counting after treatment (CFU/mL) and Live/Dead assay. The MTT assay was used to assess cytotoxicity. Biofilm metabolism was evaluated by the XTT assay. Publication 3: The 3D co-culture model was prepared and subsequently infected with S. aureus ATCC 25923 and C. albicans ATCC 90028 for 16 h, followed by application of aPDT mediated by the crude extract of S. alata at concentrations of 0.05 and 0.5 mg/mL. After 24 h of treatment, microbial viability (CFU/mL) and cell damage were assessed using the lactate dehydrogenase (LDH) assay. MB at concentrations of 0.2 and 2 mg/mL were used for comparison purposes. Data from publications 2 and 3 were analyzed using IBM SPSS version 20.0, with a significance level of 5%. Out of 727 articles analyzed in publication 1, 5 articles were selected for qualitative analysis and 2 for meta-analysis. The meta-analysis results, including the level of evidence and assessment of bias risk, demonstrated the efficacy of aPDT in reducing healing time in combination with low-level laser therapy (LLLT), compared to isolated LLLT application (p = 0.0005/I2 = 0%). Total reductions in microbial viability were found in publication 2 for aPDT mediated by curcumin (0.014 and 0.029 mg/mL), aPDT mediated by PDZ (0.175 and 0.2 mg/mL), and aPDT mediated by S. alata (0.05 and 0.5 mg/mL) for both microorganisms cultured in suspension. C. albicans biofilms cultured for 24 hours showed total reduction in microbial viability (CFU/mL) for aPDT mediated by MB (0.2 and 2.0 mg/mL) and aPDT mediated by S. alata (0.5 mg/mL) in both protocols used (single application and successive applications). C. albicans biofilms cultured for 48 h showed total reduction in microbial viability for aPDT mediated by MB (2.0 mg/mL). For S. aureus, significant reduction in microbial viability was observed only for three applications of aPDT (S. alata: 0.05 and 0.5 mg/mL; MB: 0.2 and 2.0 mg/mL) in biofilms cultured for 24 h and in biofilms cultured for 48 h (S. alata: 5.0 mg/mL; MB: 0.2 and 2.0 mg/mL). Live/Dead CLSM images confirmed the results found for microbial viability. All photosensitizers showed significantly reduced values for metabolic activity of C. albicans, and S. aureus biofilms cultured for 24 hours treated with a single application of aPDT. Additionally, the concentrations of photosensitizers and irradiation parameters used in this study showed acceptable levels (≤70%) of cytotoxicity for both cell lines. The results of publication 1 highlight that aPDT is for oral mucositis, contributing to tissue healing mainly in terms of repair time. On the other hand, the results of publications 2 and 3 showed that antimicrobial photodynamic therapy (aPDT) mediated by the crude extract of S. alata proved to be safe and efficient for C. albicans and S. aureus control.
This study is divided into three publications whose objectives were: a) to conduct a systematic literature review, followed by a meta-analysis of clinical trials to evaluate the efficacy of photodynamic therapy in oral mucositis lesions (Publication 1); b) to evaluate the in vitro potential of four different photosensitizers, two of synthetic origin (Methylene Blue and Photodithazine) and two of natural origin (Curcumin and crude extract of Senna alata), to be used in antimicrobial photodynamic therapy (aPDT) against Candida albicans and Staphylococcus aureus cultured in suspension and biofilm; and c) to evaluate the effectiveness of aPDT mediated by the crude extract of S. alata in a three-dimensional (3D) cell co-culture model containing gingival fibroblasts, oral keratinocytes, and type I collagen. Publication 1: The research question and keywords were defined following the PICO methodology. The article search was conducted on various databases, including Embase, Scielo, Scopus, Cochrane Library, Web of Science, Science Direct, and PubMed. Randomized clinical trials were selected for the review. The study was conducted following PRISMA guidelines for systematic review. Publication 2: The following reference strains in suspension and biofilm were used: S. aureus ATCC 25923 and C. albicans ATCC 90028. Four photosensitizing agents (PS) were tested at two different concentrations (Photodithazine (PDZ): 0.175 and 0.2 mg/mL; Methylene Blue (MB): 0.2 and 2 mg/mL; Curcumin (CUR): 0.014 and 0.029 mg/mL; S. alata plant extract: 0.05 mg/mL and 0.5 mg/mL). Two aPDT protocols were used on biofilms cultured for 24 h and 48 h: a) single treatment and b) three successive applications. Planktonic microbial cells received only a single treatment. The therapy's efficacy was evaluated through viable cell counting after treatment (CFU/mL) and Live/Dead assay. The MTT assay was used to assess cytotoxicity. Biofilm metabolism was evaluated by the XTT assay. Publication 3: The 3D co-culture model was prepared and subsequently infected with S. aureus ATCC 25923 and C. albicans ATCC 90028 for 16 h, followed by application of aPDT mediated by the crude extract of S. alata at concentrations of 0.05 and 0.5 mg/mL. After 24 h of treatment, microbial viability (CFU/mL) and cell damage were assessed using the lactate dehydrogenase (LDH) assay. MB at concentrations of 0.2 and 2 mg/mL were used for comparison purposes. Data from publications 2 and 3 were analyzed using IBM SPSS version 20.0, with a significance level of 5%. Out of 727 articles analyzed in publication 1, 5 articles were selected for qualitative analysis and 2 for meta-analysis. The meta-analysis results, including the level of evidence and assessment of bias risk, demonstrated the efficacy of aPDT in reducing healing time in combination with low-level laser therapy (LLLT), compared to isolated LLLT application (p = 0.0005/I2 = 0%). Total reductions in microbial viability were found in publication 2 for aPDT mediated by curcumin (0.014 and 0.029 mg/mL), aPDT mediated by PDZ (0.175 and 0.2 mg/mL), and aPDT mediated by S. alata (0.05 and 0.5 mg/mL) for both microorganisms cultured in suspension. C. albicans biofilms cultured for 24 hours showed total reduction in microbial viability (CFU/mL) for aPDT mediated by MB (0.2 and 2.0 mg/mL) and aPDT mediated by S. alata (0.5 mg/mL) in both protocols used (single application and successive applications). C. albicans biofilms cultured for 48 h showed total reduction in microbial viability for aPDT mediated by MB (2.0 mg/mL). For S. aureus, significant reduction in microbial viability was observed only for three applications of aPDT (S. alata: 0.05 and 0.5 mg/mL; MB: 0.2 and 2.0 mg/mL) in biofilms cultured for 24 h and in biofilms cultured for 48 h (S. alata: 5.0 mg/mL; MB: 0.2 and 2.0 mg/mL). Live/Dead CLSM images confirmed the results found for microbial viability. All photosensitizers showed significantly reduced values for metabolic activity of C. albicans, and S. aureus biofilms cultured for 24 hours treated with a single application of aPDT. Additionally, the concentrations of photosensitizers and irradiation parameters used in this study showed acceptable levels (≤70%) of cytotoxicity for both cell lines. The results of publication 1 highlight that aPDT is for oral mucositis, contributing to tissue healing mainly in terms of repair time. On the other hand, the results of publications 2 and 3 showed that antimicrobial photodynamic therapy (aPDT) mediated by the crude extract of S. alata proved to be safe and efficient for C. albicans and S. aureus control.
Descrição
Palavras-chave
Fotoquimioterapia, Infecções bacterianas e micoses, Revisão sistemática, Photochemotherapy, Bacterial infections and mycoses, Systematic review
Como citar
Oliveira AB. Potencial da terapia fotodinâmica na modulação da resposta inflamatória e nos fatores de virulência microbianos: avaliação em modelo de co-culturas tridimensionais de fibroblastos gengivais, Candida albicans e
Staphylococcus aureus [tese de doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2024.