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%. 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

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 (

Parameters of the antimicrobial activity and cytotoxicity of ozone for use in Endodontics

Este estudo analisou a ação do ozônio como coadjuvante à terapia endodôntica. Assim, (I) avaliou da eficiência da água ozonizada na redução microbiana realizada sobre suspensão bacteriana de Enterococcus faecalis, Pseudomonas aeruginosa e Staphylococcus aureus estudo in vitro; (II) testou a eficiência da água ozonizada e o gás ozônio na redução microbiana realizada em canais radiculares contaminados estudo ex vivo; e, (III) avaliou a reação biológica de cultura de fibroblastos sob aplicação do ozônio pelo método MTT. Inicialmente a água ozonizada nas concentrações de 5, 20 e 40 g/mL foi aplicada diretamente sobre as suspensões bacterianas separadamente por um minuto. Em seguida, foram feitas diluições seriadas e o plaqueamento para posterior contagem das UFC/mL. Na segunda parte, 180 dentes foram inoculados com 10 L das suspensões bacterianas separadamente e incubados por 7 dias. Em seguida foram submetidos aos grupos experimentais: Grupo I: controle de contaminação; Grupo II: instrumentação rotatória associando o hipoclorito de sódio 1% ao Endo-PTC gel e irrigação final com EDTA-T; Grupo III: protocolo semelhante ao Grupo II com a aplicação final do gás ozônio na concentração de 40 g/mL; e, Grupo IV: protocolo semelhante ao Grupo II com a irrigação final com a água ozonizada na concentração de 40 g/mL. Na sequencia de cada grupo foi feita coleta microbiológica, diluição seriada e plaqueamento por 24 horas e contagem das UFC/mL. Na terceira parte, a análise da viabilidade celular dos fibroblastos. O PBS foi ozonizado nas concentrações de 10, 20 e 40 g/mL e aplicado sobre as células. Os dados foram tabulados e submetidos à análise estatística pelo método de Kruskal-Wallis, complementado pelo método de Dunn (p<0.05). A primeira parte do estudo mostrou que o E. faecalis apresentou crescimento nas concentrações de 5 e 20 g/mL. Na análise da P. aeruginosa e o S. aureus não foi possível detectar UFC/mL. Já no estudo ex vivo, o Grupo IV foi o mais efetivo contra os microrganismos testados, com diferença estatística para os demais grupos, quando dos dentes contaminados com a P. aeruginosa. O estudo da viabilidade mostrou que o ozônio, em 0 horas, ocasionou uma queda na viabilidade celular que foi revertida nos demais tempos experimentais. A concentração de 40 g/mL foi a que proporcionou maior estímulo ao final da avaliação, com diferença estatística significante ao Grupo Controle. Diante das metodologias aplicadas, pode-se concluir que (I) a concentração de 40 g/mL foi a mais efetiva para reduzir o número dos microrganismos testados; (II) o protocolo instituído para a fase de preparo químico cirúrgico que associou a terapia tradicional com a água ozonizada na concentração de 40 g/mL foi o mais efetivo; (III) o ozônio em contato com os fibroblastos de gengiva, inicialmente causou diminuição na viabilidade celular, que foi revertida nos tempos experimentais subsequentes. A concentração de 40 g/mL foi a que causou maior redução no número de células viáveis, porém foi a que proporcionou maior estímulo ao final do experimento, mostrando-se biocompatível com a linhagem celular testada; e, (IV) diante do conjunto dos dados experimentais, a água ozonizada na concentração de 40 g/mL, comparada com as demais concentrações testadas, é a que apresenta melhor desempenho quanto ao seu poder antimicrobiano e biocompatibilidade, justificando sua inserção no arsenal clínico na terapia endodôntica.This study analyzed the action of ozone as an adjunct to endodontic therapy. So (I) evaluated the effectiveness of ozonated water in microbial reducing performed on bacterial suspension of Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus - in vitro study (II) tested the efficiency of ozone gas and ozonated water in reducing microbial held in contaminated root canals - ex vivo study, and (III) evaluated the biological response of fibroblasts culture in the application of ozone, according to MTT method. In the first part of the experiment the ozonated water at concentrations of 5, 20 and 40 g/mL was placed in direct contact with the bacterial suspensions separately for one minute. Then, serial dilutions were made for plating and subsequent counting of CFU/mL. In the second part, 180 teeth were inoculated with 10 L of bacterial suspensions and incubated separately for 7 days. They were then subjected to experimental groups: Group I: contamination control, Group II: rotary instrumentation combining 1%sodium hypochlorite and gel Endo-PTC, final irrigation with EDTA-T, Group III: similar protocol to Group II with final application of ozone gas in concentration of 40 g/mL, and Group IV: protocol similar to Group II with a final irrigation with ozonated water at concentration of 40 g/mL. Following each group was done microbiological collecting and serial dilution and plating for 24 hours and subsequent counting of CFU/mL. In the third part, the analysis of cell viability of gingival fibroblasts was performed by MTT method. The PBS has been ozonized at concentrations of 10, 20 and 40 g/mL and applied on the cells. Data were tabulated and analyzed statistically by Kruskal-Wallis method, complemented by Dunn\'s method (p <0.05). The first part of the study showed that E.faecalis grew in concentrations of 5 and 20 g/mL. The P. aeruginosa and S. aureus were susceptible to all three concentrations of ozonated water, it was not possible to detect CFU/mL. In the second part of the study, the Group IV was the most effective against the tested microorganisms, with statistical difference for the other groups, especially when the teeth were infected with P. aeruginosa. Finally, the third part of the study showed that ozone, at 0 hours, caused a decrease in cell viability that was reversed in the other experimental times. The concentration of 40 g/mL provided the greatest stimulus on the final evaluation period, with statistically significant difference to the control group. Given the methodologies applied, it can be concluded that (I) the concentration of 40 g/mL was most effective at reducing the number of microorganisms, (II) the protocol established for the standard chemical preparation stage associated with ozonated water at a concentration of 40 g/mL was the most effective, (III) the ozone in contact with the gingival fibroblasts, initially caused a decrease in cell viability, which was reversed in subsequent experimental times. The concentration of 40 g/mL was the one that caused a greater reduction in the number of viable cells, however, was that provided the greatest stimulus to the end of the experiment and proved to be biocompatible with the cell line tested, and (IV) on the set of experimental data, the ozonated water at a concentration of 40 g/mL , compared with the other concentrations tested, it has the best performance in terms of its biocompatibility and antimicrobial power, justifying their inclusion in the clinical arsenal in endodontic therapy

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.Molecular studies highlight the limitations of the traditional endodontic protocol in eliminating bacteria from the root canal. Although chemo-mechanical procedures (CMP) provide a drastic bacterial reduction, many canals remain infected after this treatment step. Thus, studies point out to the need for technical complementation to enhance root canal disinfection after CMP. Thus, the aim of this clinical study was to evaluate, by molecular methods based on DNA and RNA, the effect of supplementary preparation methods on root canal disinfection. Microbiological samples from the root canals of 20 single rooted teeth with apical periodontitis were performed at different steps of endodontic treatment: prior to preparation (S1); after chemo-mechanical procedures with Reciproc system associated with 2.5% NaOCl (S2) irrigation; after passive ultrasonic irrigation, or PUI (S3); and after calcium hydroxide intracanal medication (S4). The samples were submitted to DNA and RNA extraction. The RNA was submitted to reverse transcription reaction (RT-PCR) to make the complementary DNA double strand (cDNA). DNA and cDNA were submitted to qPCR reactions with universal primers for the 16S rRNA region of the Bacteria domain. The metabolic activity of bacteria was verified by the relation of rRNA and rDNA levels as determined by qPCR assays. Data were analyzed by Wilcoxon test for paired samples (p <0.05). S1 samples from the 20 cases had high levels of bacterial rDNA (median: 1.25 x 105, range 1.83 x 104 - 9.2 x 106) and rRNA (median: 5.47 x 105, range 7.8 x 104 - 5.95 x 107). Seventeen canals (85%) showed rDNA positive qPCR reactions in post-preparation samples (S2). The reduction in rDNA after preparation was statistically significant (p = 0.0003), with a median of 2.5 x 104 (range 2.26 x 103 - 9.52 x 104) copies of rDNA in S2. In turn, rRNA levels (median: 7.84 x 104, range 2.91 x 103 - 1.09 x 106) were higher than rDNA levels (p = 0.01), suggesting that bacteria were metabolically active in S2. After PUI, the number of rDNA-positive samples dropped to 12, representing a significant reduction compared to S2 samples (p = 0.008). In addition, PUI significantly reduced rDNA levels (median 2.94 x 103, range 2.70 x 103 - 1.09 x 105) compared to S2 samples (p = 0.01). In rRNA-based analysis, S3 levels (median: 03 x 104, 1.82 x 103 - 1.39 x 105 range) showed no significant difference compared to rDNA levels (p = 0.07), suggesting that there was a reduction in bacterial metabolism after PUI. In S4, the number of rDNA positive cases (n = 13) and rDNA levels (median: 3.73 x 104, range 1.98 x 103 - 3.21 x 105) were slightly higher when compared to S3 samples, but without significant differences. However, rRNA levels (median: 1.08 x 105, range 3.41 x 103 - 1.60 x 106) were higher than rDNA levels (p = 0.02) in S4 samples, suggesting that bacteria restored their metabolic activity despite the intracanal medicament use. Therefore, it was concluded that passive ultrasonic irrigation contributed to root canal disinfection, promoting a reduction in bacterial number and metabolism. On the other hand, bacteria remained active in the root canals after calcium hydroxide use as intracanal medication in teeth with apical periodontitis

Comparison of the antimicrobial activity of three different concentrations of aqueous ozone on Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis – in vitro study

The root canal treatment has been dramatically changed since the emergence of ozone therapy.Due toits potent antimicrobial activity and biocompatibility, ozone can be applied as an agent to improve decontamination procedures and possibly increase success rates. The aim of this study was to compare the antimicrobial activity of 3different concentrations of aqueous ozone on Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis.This study used a standardized suspension of 3 bacteria. These suspensions were cultivated and spectrophotometrically adjusted to a final concentration of 4.46X108 CFU/mL.Then they were submitted to groups: Group I – aqueous ozone 2 µg/mL; Group II – aqueous ozone 5 µg/mL; Group III – aqueous ozone 8 µg/mL, and Group Control – bidistilled cold sterile water with no ozone. Bidistilled cold sterile water was ozonated for 5 minutes. Then 10 mL of each suspension were added to 90 mL of each group in a glass flask. After 1 minute of contact, 1 mL of each flask was added to 9 mL of 0.1% sodium tiossulfate to neutralize the ozone, and then serial dilution was performed. After 24 hours of incubation, the CFU counting was performed. In parallel, 1 mL of each glass flask was added to 9 mL of TSB broth and incubated for 7 days for visual analyses of turbidity broth. The results showed Group I presented Enterococcus faecalis growth. Group II and III presented no CFU counting, but aqueous ozone at 5 µg/mL presented 2 bleary tubes of Enterococcus faecalis, indicating bacterium growth. Group III (aqueous ozone 8 µg/mL) showed no CFU counting or blurry broth. According to the applied methodology, it was possible to conclude that the aqueous ozone in concentration of 8 µg/mL was the most efficient to eliminate the three evaluated bacteria

Ozone therapy in Dentistry - Where we are and where we are going to?

There has been very little research to support clinical practices in dental ozone therapies. The protocols used in dental ozone therapy should fulfill the general guidelines and requirements commonly recognized by healthcare professionals and authorities as evidence-based medicine. To meet these criteria, both ozone dental research and the clinical practice of dentistry should converge. The positive results and advantages of dental ozone therapy should define standard parameters that the dental ozone equipment manufacturers need to follow to develop ozone systems that meet the requirements of both dentists and researchers. The aim of this paper is to review the available published research and to compare it to what the majority of practicing dentists are applying in their practice. Four databases (PubMed; Ovid Medline; Cochrane; ISCO3) were used to search for articles covering the use of ozone in dentistry. Using the key words “ozone in dentistry” on PubMed (last accessed November 2017) retrieved 295 articles. Articles not related to dental ozone and general reviews were excluded (70). The resulting sample size of 225 articles, as well as the retrieved analysis results, are highly indicative to be able to draw conclusions and to formulate future recommendations. To our knowledge, this is the first attempt to perform such an analysis. It is not the aim of this paper to critique the published research or the clinical practice of dental ozone therapy. The aim is to elicit the required modifications to research protocols and to evaluate whether dental ozone manufacturers provide the required equipment. This review study used different groupings to evaluate the results. In the “ozone gas only group”, a clear deviation between research and clinical practice was noted. In all the other groups, the results were, in general, more congruent. Most of the current dental ozone research has focused on the antimicrobial effects of ozone, using either just ozone gas only, ozonated water only or ozonated oils only. It is highly recommended that dental ozone research change its path. Clinicians have expectations that research will support their clinical uses of ozone. We need to meet the expectations of clinicians through adopting new and different studies. We need to create research that goes past the conventional and well-studied antimicrobial potential of ozone and by using ozone both in gas and water, not separately, plus ozonated oils when applicable...  

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

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%. 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

Ozone therapy: adjuvant to endodontic treatment in a subluxation case – case report

INTRODUCTION: An injury to the tooth-supporting structures results in increased mobility, but without displacement of the tooth. Bleeding in gingival sulcus is a pathognomonic signal. The tooth is positive to a percussion test. Follow-up is required to test dental vitality. When pulp necrosis occurs, root canal treatment is indicated. Ozone therapy comes as a new possibility in root canal treatment to promote high disinfection and increase apical healing. This manuscript reports a subluxation case followed by pulp necrosis with an extended apical radiolucent image. CASE REPORT: During conventional root canal treatment, 100 mL of 16 µg/mL ozonated water and 100 mL of 40 µg/mL ozone gas were applied into the root canal, and calcium hydroxide dressing was used. Apical radiolucent image decreased into 40 days. According to the literature and clinical data, ozone therapy has a great potential to be incorporated into endodontic therapy. Its biostimulator effects and antimicrobial potential are evident and corroborated by the literature. CONCLUSION: According to the literature and the case report, ozone therapy is suitable to be used as an adjuvant to root canal treatment