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 (

Effect in periapical bone healing of Ozone therapy as adjuvant to endodontic treatment clinical-radiographic essay

A reparação dos tecidos periapicais testemunha o êxito da terapia endodôntica. Por intervir de forma favorável na reparação tecidual, a ozonioterapia é reconhecida como um importante coadjuvante ao protocolo terapêutico, sendo oportuno verificar sua eficácia no caso da terapia endodôntica. Para isso, foi avaliada, in vivo a influência do gás ozônio, da água ozonizada e óleo ozonizado na reparação tecidual em situações de necrose pulpar com alterações ósseas periapicais, diagnosticado e caracterizado como quadros de infecção primária. Foram feitas radiografias previamente e ao final do tratamento e novamente no terceiro e no sexto mês após a conclusão da terapia endodôntica. Respeitando-se os critérios de inclusão, 36 pacientes da Clínica Odontológica da Faculdade de Odontologia da Universidade de São Paulo foram divididos aleatoriamente em 3 grupos: Grupo I (12 dentes) Controle: tratamento endodôntico segundo protocolo da Disciplina de Endodontia da FOUSP; Grupo II (12 dentes) protocolo terapêutico empregado pelo Grupo I e associado ao final com irrigação de 10 mL de água bidestilada ozonizada na concentração de 8 g/mL; e, Grupo III (12 dentes) protocolo terapêutico empregado pelo Grupo I e associado ao final do preparo do canal da aplicação de 10 mL do gás ozônio na concentração de 40 g/mL. Foi utilizado como medicação intracanal o hidróxido de cálcio veiculado em PRP no Grupo I e o óleo de girassol ozonizado (Philozon, Santa Catarina, SC, Brasil) nos Grupos II e III. As medicações permaneceram por um período de 15 dias e em seguida foi feita obturação com cimento AH-Plus. A avaliação da reparação óssea periapical foi realizada analisando as radiografias digitalizadas no programa ImageLab. Após a delimitação e quantificação de pixels das lesões periapicais, as radiografias finais e controles de 3 e 6 meses foram comparadas, indicando o quanto de redução da radiolucência periapical. Paralelamente, foi feita a análise de sensibilidade pós-operatória utilizando uma Escala Visual Analógica. Os pacientes foram questionados com relação à sensibilidade previamente ao início do tratamento, 72 horas após consulta inicial e nos controles de 3 e 6 meses, devendo associá-la com uma nota de 0 a 10 de acordo com a intensidade. Os dados foram submetidos à análise de variância, complementado pelo teste de Tukey, com significância de 5%. Os três protocolos terapêuticos instituídos promoveram redução da imagem radiolúcida periapical sem diferença estatística significante. Na análise da sensibilidade, o Grupo III apresentou os melhores resultados, com diferença estatística significante em relação aos demais grupos. Com base nos dados obtidos e na análise estatística executada, foi possível concluir que a ozonioterapia, por meio do emprego do gás ozônio e água ozonizada na fase do preparo químico cirúrgico e do óleo ozonizado como medicação intracanal, interviu de maneira favorável na reparação, em situações de necrose pulpar com alterações ósseas periapicais, sendo o gás mais efetivo que a água ozonizada, com diferenças estatísticas significantes. Os 2 grupos experimentais de ozonioterapia comparado ao grupo controle (protocolo FOUSP) apresentaram padrão de reparação óssea semelhante. A utilização da água ozonizada como irrigante e do óleo ozonizado como medicação intracanal mostrou a melhor eficácia do ponto de vista sintomatológico.The success of the endodontic treatment is related to the repair of the periapical tissues. The ozone therapy is known for its therapeutic usefulness, favorable tissue healing, thus it seems suitable for evaluating its efficiency in the endodontic therapy. The aim of this study was to evaluate in vivo the influence of ozone gas, ozonated water and oil in bone repair in cases of pulpar necrosis with radiographic evidence of periapical bone loss associated with primary endodontic infection. Radiographs (Xrays) were taken prior and after the completion of the endodontic treatment, three and six months later. According to the enclosure criteria, 36 patients of the Dental Clinic of the School of Dentistry, University of Sao Paulo, were selected and randomly divided in three groups: Group I (12 teeth) Control: endodontic treatment protocol according to the discipline of Endodontics FOUSP, Group II (12 teeth): endodontic protocol associated with final irrigation with 10 mL of bidestilated ozonated water at concentration of 8 g/mL and Group III (12 teeth): endodontic protocol associated with application of 10 mL of ozone gas at concentration of 40 g/mL. Calcium Hydroxide was used as intracanal medication for 15 days. In Group I it was associated with anesthetic solution and in the Groups II and III associated with ozonated oil (Philozon, Santa Catarina, SC, Brazil). After this period, all teeth were obturated with AH-Plus root canal sealer. Evaluation of periapical bone repair was performed with Image Lab software after image digitalization. After delimitation and pixel quantification of the periapical lesion area of the images immediately after the endodontic treatment, 3 and 6-months follow up, the obtained data were compared indicating the quantum reduction of the periapical radiolucency. In parallel, a postoperative sensitivity analysis was performed using a Visual Analog Scale. Patients were asked to score the sensitivity from 0 to 10 according to its intensity prior to first appointment, 72 hours later the first visit, 3 and 6 months later. Data were submitted to analysis of variance (ANOVA), complemented by the Tukey test at 5% of significance. All the three groups promoted a reduction of the apical radiolucent image without statistical difference. In the sensitivity analysis, Group III showed the best results with statistically significant difference compared to the other groups. Based on the statistical data analysis we concluded that ozone therapy through the use of ozone gas and ozonated water during the root canal instrumentation and ozonated oil as intracanal medication, positively intervene in the repair of periapical bone loss. The ozone gas was more effective than the ozonated water with significant statistical difference. Group II and III (experimental groups) compared to Group I (endodontic protocol FOUSP) showed a similar pattern of bone repair. The use of ozonated water as irrigant and ozonated oil as intracanal dressing showed better efficacy in terms of symptomatology

Citotoxicity of antibiotics purposed as intracanal medicaments according to concentration and experimental period

Diante da presença de microrganismos resistentes à terapêutica endodôntica, novas formulações e alternativas medicamentosas têm sido investigadas. Desse modo, esse estudo teve como objetivo avaliar a citotoxicidade de diferentes medicações indicadas como medicação intracanal. Culturas de células de fibroblastos foram estimuladas de acordo com os seguintes grupos experimentais: Grupo I: controle; Grupo II: cloridrato de ciprofloxacina; Grupo III: cloridrato de clindamicina e Grupo IV: metronidazol. Para tal, as concentrações utilizadas para cada antibiótico foram de 5, 50, 150 e 300 mg/L, nos tempos experimentais de 24, 48, 72 e 96 horas. A citotoxicidade dessas substâncias foi avaliada usando a técnica de análise do MTT e leitura em espectrofotômetro de ELISA. Os resultados obtidos foram analisados pelo software BioEstat 4.0, por meio do teste de Kruskal-Wallis, complementado pelo teste de Dunn, com nível de significância de 5%. A viabilidade celular foi analisada diante da relação entre as diferentes concentrações e uma mesma concentração nos diferentes tempos experimentais. Diante dos resultados obtidos, foi possível concluir que: todos os antibióticos testados apresentaram citotoxicidade dosedependente. As concentrações de 5 e 50 mg/L, de todos os antibióticos, permitiram a viabilidade dos fibroblastos em todos os tempos avaliados. Independente da medicação avaliada, a viabilidade celular em 24 horas foi mais elevada em comparação aos demais tempos experimentais. A comparação entre os antibióticos nas mesmas concentrações, em diferentes tempos experimentais demonstrou que o gel de metronidazol apresentou menor citotoxicidade em 72 e 96 horas no confronto com os dois outros antibióticos, enquanto que o cloridrato de clindamicina apresentou maior viabilidade celular em relação ao cloridrato de ciprofloxacina em 72 horas.New medicaments have been assessed due to the presence of resistantmicroorganism to therapeutic procedures. Thus, this study aimed to evaluate the citotoxicity of differents drugs indicated as intracanal medicament. The human gingival fibroblasts were estimulated according to the following experimental groups: Group I: control; Group II: Ciprofloxacin Hydrochloride; Group III: Clindamicin Hydrochloride and Group IV: Metronidazole. The concentration used for each drugs were 5, 50, 150, and 300 mg/L, in the experimental time of 24, 48, 72, and 96 hours. The citotoxicicty were evaluated with MTT analysis and ELISA spectrophotometer reading. The results were evaluated by BioEstat 4.0 software, according to Kruskal- Wallis test supplemented by Dunn test with significance level of 5%. Cell viability analysis has been assessed in different concentrations in each experimental period. According to the results, it is possible to conclude that: all the tested drugs showed dose-dependent citotoxicity. The concentrations of 5 and 50mg/L of all groups allowed fibroblast viability in all evaluated periods. The cell viability in 24 hours was higher than the others experimental periods. The comparing between the antibiotics at the same concentrations, in different times showed the gel metronidazole obtained lowest citotoxicity at 72 and 96 hours when compared with the others antibiotics, meanwhile, the Clindamicin Hydrochloride showed the highest cell viability compared to Ciprofloxacin Hydrochloride, at 72 hours

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: 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

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

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