The antimicrobial effect of different ozone protocols applied in severe curved canals contaminated with Enterococcus faecalis: ex vivo study.

The aim of this study was to evaluate 3 different irrigation protocols using ozone on monoculture biofilm of Enterococcus faecalis. Fifty disto-buccal roots of maxillary first molars were instrumented up to the size 25.08, sterilized and then incubated with Enterococcus faecalis for 21 days for biofilm maturation in the root canal walls. After contamination, the initial collection (S1) was performed and the 50 samples were divided in 3 experimental groups (n = 15) and 1 positive control group (n = 5) as follows: Group1: Ozonated water (40 µg/ml); Group2: gaseous ozone (40 µg/ml); Group3: Ozonated water (40 µg/ml) under continuous ultrasonic irrigation (CUI) and, Group 4: 2.5% sodium hypochlorite (positive control). The final collection (S2) was performed using hedstroem files # 25 by scraping the root canal wall and absorbed paper cone. The collected samples were analyzed by CFU/ml count and qPCR. To compare the effect of irrigation methods and to investigate E. faecalis count data, Kruskal-Wallis and Wilcoxon nonparametric tests were used at p < 05. The values of the differences between the S1 values and the S2 values revealed that the groups did not differ significantly for either E. faecalis CFU count data (p = 0.713) or q-PCR (p = 0.185), however, it is observed that for all groups, the CFU count of E. faecalis decreased over 99%. For the q-PCR assay, the reduction was on the order of 74-95%. Only the NaOCl positive control group presented total elimination of CFU/mL bacterial counting. The ozone protocols assessed were similar in regard to Enterococcus faecalis reduction.

Ozone therapy in medicine and dentistry.

AIM: The purpose of this review is to present the potential for the incorporation of ozone therapy into the practice of dentistry. BACKGROUND: Ozone gas has a high oxidation potential and is 1.5 times greater than chloride when used as an antimicrobial agent against bacteria, viruses, fungi, and protozoa. It also has the capacity to stimulate blood circulation and the immune response. Such features justify the current interest in its application in medicine and dentistry and have been indicated for the treatment of 260 different pathologies. It can be used for the treatment of alveolitis as a replacement for antibiotic therapy, as a mouthwash for reducing the oral microflora, as well as the adherence of microorganisms to tooth surfaces. Ozone has been shown to stimulate remineralization of recent caries-affected teeth after a period of about six to eight weeks. CONCLUSION: The future of ozone therapy must focus on the establishment of safe and well-defined parameters in accordance with randomized, controlled trials to determine the precise indications and guidelines in order to treat various medical and dental pathologies. Scientific support, as suggested by demonstrated studies, for ozone therapy presents a potential for an atraumatic, biologically-based treatment for conditions encountered in dental practice.

Ozone therapy as an adjuvant for endondontic protocols: microbiological - ex vivo study and citotoxicity analyses.

OBJECTIVES: This study evaluated the antimicrobial efficacy of ozone therapy in teeth contaminated with Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus using a mono-species biofilm model. Parallel to this, the study aimed to evaluate the cytotoxicity of ozone for human gingival fibroblasts. Material and Methods: One hundred and eighty single-root teeth were contaminated with a mono-species biofilm of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus. Groups were formed: Group I - control; Group II - standard protocol; Group III - standard protocol + ozone gas at 40 µg/mL; and Group IV - standard protocol + aqueous ozone at 8 µg/mL. In parallel, human gingival fibroblasts were submitted to the MTT test. Cells were plated, then ozone was applied as follows: Group I (control) - broth medium; Group II - aqueous ozone at 2 µg/mL; Group III - aqueous ozone at 5 µg/mL; and Group IV - aqueous ozone at 8 µg/mL. Data were submitted to the Kruskal Wallis test and Bonferroni post hoc analyses to assess microbiology and cytotoxicity, respectively (p<0.05%). RESULTS: The results revealed antimicrobial efficacy by Group IV with no CFU count. The cytotoxicity assay showed Groups III and IV to be the most aggressive, providing a decrease in cell viability at hour 0 from 100% to 77.3% and 68.6%, respectively. Such a decrease in cell viability was reverted, and after 72 hours Groups III and IV provided the greatest increase in cell viability, being statistically different from Groups I and II. CONCLUSION: According to the applied methodology and the limitations of this study, it was possible to conclude that ozone therapy improved the decontamination of the root canal ex vivo. Ozone was toxic to the cells on first contact, but cell viability was recovered. Thus, these findings suggest that ozone might be useful to improve root canal results.

Ozone therapy as an adjuvant for endondontic protocols: microbiological – ex vivo study and citotoxicity analyses

ABSTRACT Objectives This study evaluated the antimicrobial efficacy of ozone therapy in teeth contaminated with Pseudomonas aeruginosa, Enterococcus faecalis, and Staphylococcus aureus using a mono-species biofilm model. Parallel to this, the study aimed to evaluate the cytotoxicity of ozone for human gingival fibroblasts. Material and Methods: One hundred and eighty single-root teeth were contaminated with a mono-species biofilm of Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus. Groups were formed: Group I – control; Group II – standard protocol; Group III – standard protocol + ozone gas at 40 µg/mL; and Group IV – standard protocol + aqueous ozone at 8 µg/mL. In parallel, human gingival fibroblasts were submitted to the MTT test. Cells were plated, then ozone was applied as follows: Group I (control) – broth medium; Group II – aqueous ozone at 2 µg/mL; Group III – aqueous ozone at 5 µg/mL; and Group IV – aqueous ozone at 8 µg/mL. Data were submitted to the Kruskal Wallis test and Bonferroni post hoc analyses to assess microbiology and cytotoxicity, respectively (p<0.05%). Results The results revealed antimicrobial efficacy by Group IV with no CFU count. The cytotoxicity assay showed Groups III and IV to be the most aggressive, providing a decrease in cell viability at hour 0 from 100% to 77.3% and 68.6%, respectively. Such a decrease in cell viability was reverted, and after 72 hours Groups III and IV provided the greatest increase in cell viability, being statistically different from Groups I and II. Conclusion According to the applied methodology and the limitations of this study, it was possible to conclude that ozone therapy improved the decontamination of the root canal ex vivo. Ozone was toxic to the cells on first contact, but cell viability was recovered. Thus, these findings suggest that ozone might be useful to improve root canal results.

Efeito da irrigação ultrassônica e da medicação intracanal com hidróxido de cálcio na quantidade e no metabolismo de bactérias que persistiram após o preparo dos canais radiculares de dentes com periodontite apical / Effect of ultrasonic irrigation and calcium hydroxide intracanal medication on the levels and metabolism of bacteria that persisted after root canal preparation of teeth with apical periodontitis

Estudos moleculares ressaltam as limitações do protocolo endodôntico tradicional em eliminar bactérias dos canais radiculares. Apesar do preparo químico-cirúrgico (PQC) promover uma drástica redução bacteriana, muitos canais continuam infectados após essa etapa do tratamento. Dessa forma, estudos apontam para a necessidade de complementação técnica para potencializar a desinfecção dos canais radiculares após o PQC. Assim, o objetivo deste estudo clínico foi avaliar, por métodos moleculares baseados em DNA e RNA, o efeito dos métodos complementares ao preparo na desinfecção dos canais radiculares. Coletas microbiológicas dos canais de 20 dentes unirradiculares com periodontite apical foram feitas em diferentes etapas do tratamento endodôntico: previamente ao preparo (S1); após o PQC realizado com sistema Reciproc associado à irrigação com NaOCl 2,5% (S2); após a irrigação ultrassônica passiva, denominada PUI (S3); e após a medicação intracanal à base de hidróxido de cálcio (S4). As amostras foram submetidas à extração de DNA e RNA. O RNA foi submetido à reação de transcrição reversa (RT-PCR) para confecção da fita dupla de DNA complementar (cDNA). DNA e cDNA foram submetidos a reações de qPCR, com iniciadores universais para a região 16S rRNA do domínio Bacteria. A atividade metabólica das bactérias foi verificada através da relação entre os níveis de rRNA e rDNA determinados pelos ensaios de qPCR. Os dados foram analisados pelo teste de Wilcoxon para amostras pareadas (p < 0,05). As amostras S1 dos 20 casos apresentaram altos níveis de rDNA (mediana: 1,25 x 105, intervalo 1,83 x 104 - 9,2 x 106) e rRNA bacteriano (mediana: 5,47 x 105, intervalo 7,8 x 104 - 5,95 x 107). Dezessete canais (85%) apresentaram reações qPCR positivas para rDNA nas amostras pós-preparo (S2). A redução de rDNA após o preparo foi estatisticamente significativa (p = 0,0003), com mediana de 2,5 x 104 (intervalo 2,26 x 103 - 9,52 x 104) cópias de rDNA em S2. Por sua vez, os níveis de rRNA (mediana: 7,84 x 104, intervalo 2,91 x 103 - 1,09 x 106) foram maiores que os níveis de rDNA (p = 0,01), sugerindo que essas bactérias estavam metabolicamente ativas em S2. Após a PUI, o número de amostras S3 com resultados positivos para rDNA caiu para 12, representando uma redução significativa em relação às amostras S2 (p = 0,008). Além disso, a PUI promoveu uma redução significativa dos níveis de rDNA (mediana 2,94 x 103, intervalo 2,70 x 103 - 1,09 x 105) em relação à amostras S2 (p = 0,01). Na análise baseada em rRNA, os níveis em S3 (mediana: 03 x 104, intervalo 1,82 x 103 - 1,39 x 105) não apresentaram diferença significativa em comparação aos níveis de rDNA (p = 0,07), sugerindo que houve uma redução do metabolismo bacteriano após a PUI. Em S4, o número de casos positivos para rDNA bacteriano (n = 13) e os níveis de rDNA (mediana: 3,73 x 104, intervalo 1,98 x 103 - 3,21 x 105) foram ligeiramente maiores quando comparados aos valores das amostras S3, porém sem diferenças significativas. Entretanto, os níveis de rRNA (mediana: 1,08 x 105, intervalo 3,41 x 103 - 1,60 x 106) foram maiores que os de rDNA (p = 0,02) nas amostras S4, sugerindo que as bactérias retomaram sua atividade metabólica apesar do uso da medicação intracanal. Portanto, foi possível concluir que a irrigação ultrassônica passiva contribuiu para a desinfecção dos canais radiculares, promovendo uma redução do número e do metabolismo de bactérias. Por outro lado, as bactérias persistiram ativas nos canais radiculares após o uso do hidróxido de cálcio como medicação intracanal em dentes com periodontite apical.

Time- and concentration-dependent cytotoxicity of antibiotics used in endodontic therapy.

OBJECTIVE: New drugs have to be assessed in endodontic therapy due to the presence of microorganisms resistant to therapeutic procedures. Thus, this study evaluated the time- and concentration-dependent cytotoxicity of different antibiotics used in endodontic therapy. MATERIAL AND METHODS: Human gingival fibroblasts were treated and divided into the following experimental groups: Group I - control; Group II - ciprofloxacin hydrochloride; Group III - clyndamicin hydrochloride; and Group IV - metronidazole. Each drug was used at concentrations of 5, 50, 150, and 300 mg/L for 24, 48, 72, and 96 h. Cytotoxicity was evaluated by the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and spectrophotometric reading of ELISA plates. The results were analyzed by BioEstat 4.0 software using Kruskal-Wallis and Dunn's tests at a significance level of 5%. Cell viability was assessed for the different concentrations and times. RESULTS: All drugs presented dose-dependent cytotoxicity. Concentrations of 5 and 50 mg/L produced viable fibroblasts at all experimental times in all groups. CONCLUSIONS: Cell viability at 24 h was greater than in the other experimental times. Comparison between the same concentrations of antibiotics at different times showed that metronidazole presented the highest cell viability at 72 and 96 h compared to the other antibiotics, whereas clyndamicin hydrochloride showed higher cell viability at 72 h than ciprofloxacin hydrochloride.

Efeito da irrigação ultrassônica e da medicação intracanal com hidróxido de cálcio na quantidade e no metabolismo de bactérias que persistiram após o preparo dos canais radiculares de dentes com periodontite apical / Effect of ultrasonic irrigation and calcium hydroxide intracanal medication on the levels and metabolism of bacteria that persisted after root canal preparation of teeth with apical periodontitis

Estudos moleculares ressaltam as limitações do protocolo endodôntico tradicional em eliminar bactérias dos canais radiculares. Apesar do preparo químico-cirúrgico (PQC) promover uma drástica redução bacteriana, muitos canais continuam infectados após essa etapa do tratamento. Dessa forma, estudos apontam para a necessidade de complementação técnica para potencializar a desinfecção dos canais radiculares após o PQC. Assim, o objetivo deste estudo clínico foi avaliar, por métodos moleculares baseados em DNA e RNA, o efeito dos métodos complementares ao preparo na desinfecção dos canais radiculares. Coletas microbiológicas dos canais de 20 dentes unirradiculares com periodontite apical foram feitas em diferentes etapas do tratamento endodôntico: previamente ao preparo (S1); após o PQC realizado com sistema Reciproc associado à irrigação com NaOCl 2,5% (S2); após a irrigação ultrassônica passiva, denominada PUI (S3); e após a medicação intracanal à base de hidróxido de cálcio (S4). As amostras foram submetidas à extração de DNA e RNA. O RNA foi submetido à reação de transcrição reversa (RT-PCR) para confecção da fita dupla de DNA complementar (cDNA). DNA e cDNA foram submetidos a reações de qPCR, com iniciadores universais para a região 16S rRNA do domínio Bacteria. A atividade metabólica das bactérias foi verificada através da relação entre os níveis de rRNA e rDNA determinados pelos ensaios de qPCR. Os dados foram analisados pelo teste de Wilcoxon para amostras pareadas (p < 0,05). As amostras S1 dos 20 casos apresentaram altos níveis de rDNA (mediana: 1,25 x 105, intervalo 1,83 x 104 - 9,2 x 106) e rRNA bacteriano (mediana: 5,47 x 105, intervalo 7,8 x 104 - 5,95 x 107). Dezessete canais (85%) apresentaram reações qPCR positivas para rDNA nas amostras pós-preparo (S2). A redução de rDNA após o preparo foi estatisticamente significativa (p = 0,0003), com mediana de 2,5 x 104 (intervalo 2,26 x 103 - 9,52 x 104) cópias de rDNA em S2. Por sua vez, os níveis de rRNA (mediana: 7,84 x 104, intervalo 2,91 x 103 - 1,09 x 106) foram maiores que os níveis de rDNA (p = 0,01), sugerindo que essas bactérias estavam metabolicamente ativas em S2. Após a PUI, o número de amostras S3 com resultados positivos para rDNA caiu para 12, representando uma redução significativa em relação às amostras S2 (p = 0,008). Além disso, a PUI promoveu uma redução significativa dos níveis de rDNA (mediana 2,94 x 103, intervalo 2,70 x 103 - 1,09 x 105) em relação à amostras S2 (p = 0,01). Na análise baseada em rRNA, os níveis em S3 (mediana: 03 x 104, intervalo 1,82 x 103 - 1,39 x 105) não apresentaram diferença significativa em comparação aos níveis de rDNA (p = 0,07), sugerindo que houve uma redução do metabolismo bacteriano após a PUI. Em S4, o número de casos positivos para rDNA bacteriano (n = 13) e os níveis de rDNA (mediana: 3,73 x 104, intervalo 1,98 x 103 - 3,21 x 105) foram ligeiramente maiores quando comparados aos valores das amostras S3, porém sem diferenças significativas. Entretanto, os níveis de rRNA (mediana: 1,08 x 105, intervalo 3,41 x 103 - 1,60 x 106) foram maiores que os de rDNA (p = 0,02) nas amostras S4, sugerindo que as bactérias retomaram sua atividade metabólica apesar do uso da medicação intracanal. Portanto, foi possível concluir que a irrigação ultrassônica passiva contribuiu para a desinfecção dos canais radiculares, promovendo uma redução do número e do metabolismo de bactérias. Por outro lado, as bactérias persistiram ativas nos canais radiculares após o uso do hidróxido de cálcio como medicação intracanal em dentes com periodontite apical.

Time- and concentration-dependent cytotoxicity of antibiotics used in endodontic therapy

OBJECTIVE: New drugs have to be assessed in endodontic therapy due to the presence of microorganisms resistant to therapeutic procedures. Thus, this study evaluated the time- and concentration-dependent cytotoxicity of different antibiotics used in endodontic therapy. MATERIAL AND METHODS: Human gingival fibroblasts were treated and divided into the following experimental groups: Group I - control; Group II - ciprofoxacin hydrochloride; Group III - clyndamicin hydrochloride; and Group IV - metronidazole. Each drug was used at concentrations of 5, 50, 150, and 300 mg/L for 24, 48, 72, and 96 h. Cytotoxicity was evaluated by the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and spectrophotometric reading of ELISA plates. The results were analyzed by BioEstat 4.0 software using Kruskal-Wallis and Dunn's tests at a signifcance level of 5 percent. Cell viability was assessed for the different concentrations and times. RESULTS: All drugs presented dose-dependent cytotoxicity. Concentrations of 5 and 50 mgjL produced viable fibroblasts at all experimental times in all groups. CONCLUSIONS: Cell viability at 24 h was greater than in the other experimental times. Comparison between the same concentrations of antibiotics at different times showed that metronidazole presented the highest cell viability at 72 and 96 h compared to the other antibiotics, whereas clyndamicin hydrochloride showed higher cell viability at 72 h than ciprofoxacin hydrochloride.