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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.

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.

Les pommiers sont la cible de nombreux ravageurs et maladies fongiques, à l’origine de pertes économiques importantes. Les agriculteurs sont dans l’obligation de traiter les micro-organismes responsables des maladies fongiques, notamment à l’aide de pesticides. Les produits phytosanitaires sont aujourd’hui très controversés. Ainsi, les pouvoirs publics, français et européens, contrôlent leur emploi par de nombreuses règlementations. Par conséquent, il apparaît nécessaire de proposer une alternative peu rémanente permettant de contrôler le développement fongique. L’objectif de cette thèse est d’évaluer l’efficacité d’une alternative innovante basée sur l’emploi d’eau chargée en ozone contre quatre espèces fongiques à l’origine des principales maladies du pommier : Venturia inaequalis, Botrytis cinerea, Neofabraea alba et Phytophthora syringae. Dans un premier temps, ce travail de thèse a permis de vérifier l’action anti-fongique de l’ozone sur les suspensions de spores des quatre espèces fongiques ainsi que sur différents stades de développement des spores de V.inaequalis. Il a été mis en évidence une différence de sensibilité selon l’espèce et le stade de développement considérés. Par ailleurs, l’altération de la membrane sous l’action de l’ozone a été observée par plusieurs expérimentations : quantification du degré de peroxydation des phospholipides membranaires, observations des spores au Microscope Electronique à Balayage et au Microscope Optique après colorations. Dans un second temps, l’effet de l’eau chargée en ozone a été évalué sur des jeunes plants de pommiers préalablement contaminés par les spores de V.inaequalis. Cette étude a confirmé l’action anti-fongique de l’eau chargée en ozone. Il a également été observé l’importance des quantités d’eau et d’ozone apportées qui doivent être maîtrisées pour optimiser l’efficacité du traitement. Ces résultats permettent de valider la candidature de l’ozone comme fongicide alternatif à l’emploi de produits phytosanitaires. Les modalités d’application devront faire l’objet de recherches supplémentaires en vue du transfert de la technologie du laboratoire au verger. / Apple trees are the target of a lot of pests and fungal diseases, responsible for many economic losses. Farmers treat these micro-organisms mainly with pesticides. Plant-protection products are today very controversial. French and European authorities control their use by means of numerous regulations. Therefore, an alternative solution with a low remanence must be found in order to limit fungal diseases. The aim of this thesis is to evaluate the innovative process efficiency based on the use of ozonated water against four fungal species: Venturia inaequalis, Botrytis cinerea, Neofabraea alba and Phytophthora syringae. Firstly, this work of thesis verified the ozone antifungal action on spores suspensions of the four fungal species and on different stages of development of V.inaequalis spores. According to the species and stages of development considered, a difference of sensibility was detected. Moreover, alteration of the membrane, due to ozone action, was observed thanks to several experimentations: quantification of phospholipids peroxidation, spore observations with Scanning Electron Microscopy and with Optical Microscopy after staining. Secondly, the ozonated water effects on seedlings previously contaminated by V.inaequalis spores were evaluated. This study confirmed the anti-fungal action of water containing ozone. It was also observed the importance of the water and of ozone quantities supplied. These quantities must be controlled to optimize the antifungal action of the treatment. These results validated the ozone application as a fungicide alternative of use of pesticides. Additional researches will be made concerning the treatment modalities: the aim is to transfer the technology from the laboratory to the orchard.

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.

The objectives of this project were to evaluate the efficacies of electrolyzed oxidizing (EO) and ozonated waters as antimicrobial agents for enhancing the microbiological safety of fresh strawberries (Fragaria x ananassa). The influence of sodium chloride (NaCl) concentrations used for preparing EO water was evaluated on their bactericidal activities against naturally occurring aerobic mesophiles on strawberries with a contact time of 5, 10, or 15 min. EO water and ozonated water containing about 1.90 ppm ozone were evaluated and compared with sodium hypochlorite (NaOCl) solution on their capabilities to inactivate and control the growth of Listeria monocytogenes and Escherichia coli O157:H7 inoculated onto strawberries stored at 4 ± 1 °C for up to 15 d, respectively. Post-treatment neutralization of fruit surfaces by washing was also investigated. More than 2 log₁₀ CFU/g reduction of mesophilic aerobic bacteria was achieved in samples washed for 10 or 15 min in EO water prepared from 0.10% (w/v) NaCl solution. Bactericidal activity of treatment solutions against L. monocytogenes and E. coli O157:H7 was not affected by post-treatment neutralization, and their effectiveness against both pathogens in whole fruit tissues did not significantly increase with increasing exposure time. The EO water had an equivalent antibacterial effect as compare with NaOCl in eliminating L. monocytogenes and E. coli O157:H7 on whole strawberry tissues. Fruit surfaces washing with distilled water resulted in 1.90 and 1.27 log₁₀ CFU/ml of rinse fluid reduction of L. monocytogenes and E. coli O157:H7, respectively, whereas ≥ 2.60 log₁₀ CFU/ml of rinse fluid reduction of L. monocytogenes and up to 2.35 and 3.12 log reduction of E. coli O157:H7 were observed on fruit surfaces washed with EO water and NaOCl solution, respectively. However, EO water and NaOCl solution treatments did not exhibit a higher microbicidal activity than water treatment during refrigeration storage. The ozone treatment on inoculated strawberries was not remarkably effective in removing and eliminating pathogens on the whole fruit tissues, but the populations of L. monocytogenes and E. coli O157:H7 were significantly decreased after ozone treatment regardless of the exposure time. The number of L. monocytogenes and E. coli O157:H7 on fruit surfaces was decreased by 2.17 and 2.02 log₁₀ CFU/ml of rinse fluid, respectively, after washing with ozonated water for 10 min. / Graduation date: 2008

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: One of the main objectives of endodontic therapy is to reduce microbes and remove inflamed pulpal tissue within the root canal system (RCS). This is accomplished through chemomechanical debridement of the RCS using hand and rotary instrumentation along with an antimicrobial irrigant. Today, the most commonly used irrigant is sodium hypochlorite (NaOCl), often at concentrations toxic to human cells. The use of ozone as an endodontic irrigant is a novel technique that has been proven to be antimicrobial against several microorganisms. However, independent research is lacking on ozone’s efficacy against an established endodontic biofilm. If ozone’s efficacy against biofilms is confirmed, the use of toxic and potentially dangerous sodium hypochlorite could be replaced in some clinical situations (i.e., regeneration, immature teeth, resorption) with a safer and effective alternative. Objective: The aim of the current study was to evaluate the anti-biofilm activity of different concentrations of ozonated water compared to various concentrations of NaOCl against an established endodontic biofilm of Enterococcus faecalis in root canal treated teeth in vitro. Materials and Methods: The crowns of similarly sized, maxillary anterior teeth were removed, and the roots cut to a standard length (12 mm). All root canals were instrumented to a standard size. Specimens were sterilized and then inoculated with E. faecalis, which were allowed to grow for two weeks to form an established biofilm. There were six treatment groups: 1) 6% NaOCl; 2) 1.5% NaOCl; 3) 16µg/mL ozonated water; 4) 25µg/mL ozonated water; 5) 50µg/mL ozonated water, and 6) saline. Following treatment, samples were collected, plated, and incubated for two days. The number of CFU/mL were determined, and samples visualized using confocal imaging. The effect of treatment group on bacterial counts was made using one-way ANOVA followed by pair-wise comparisons. Null Hypothesis: Endodontically treated teeth irrigated with ozonated water will not demonstrate a statistically significant decrease in the E. faecalis biofilm compared to those treated with sodium hypochlorite Results: CFUs were converted to log10 and compared using Fisher’s Exact tests or one-way ANOVA followed by pair-wise tests. In all observations utilizing NaOCl irrigation, no colonies formed following treatment. The two NaOCl groups, with 0 CFU/mL, were significantly different than the other four groups (p=0.009). Saline showed a trend towards higher CFU/mL than 50 µg/ml O3 (p=0.068). None of the other comparisons approached statistical significance (p=0.453 25 µg/ml O3, p=0.606 16 µg/ml O3, p=0.999 25 µg/ml O3 vs 50 µg/ml O3, p=0.990 16 µg/ml O3 vs 50 µg/ml O3, p=1.000 16 µg/ml O3 vs 25 µg/ml O3). Confocal imaging helped illustrate effects of irrigation and confirm CFU findings. Conclusion: The results of this study failed to reject the null hypothesis. There was a statistically significant difference in the E. faecalis biofilm remaining in the groups treated with ozonated water compared to those treated with NaOCl. However, there was a trend towards higher CFU/mL in the saline group compared to the 50µg/mL ozonated water group. According to this finding, future studies should evaluate the effects of higher concentrations of ozonated water against an established E. faecalis biofilm. In addition, other follow-up studies might include ozonated water’s effect on human cells, such as the stem cells of the apical papilla that are so critical to the success of regenerative endodontic procedures. Due to university and laboratory closures caused by the COVID-19 pandemic, this project was stopped short and an insufficient sample size did not allow for proper statistical power. Additional occasions should be run upon the university’s re-opening to allow for proper statistical power.

L’esca est une maladie du bois de la vigne complexe et mal connue, contre laquelle aucun moyen de lutte efficace n’existe à ce jour. Ce travail s’est concentré sur les interactions précoces entre Vitis vinifera L. et les champignons associés au « young esca » P. chlamydospora et P. aleophilum dans deux types de tissus lignifiés : l’entre-nœud et le nœud (modèle plaie de taille). La colonisation 6 et 12 semaines après traitement des souches transformées P. aleophilum::gfp7 et P. chlamydospora::gfp1 a été observée. Les deux espèces coloniseraient différents tissus dans les premières semaines suivant l’infection. Les fibres du xylème constitueraient un tissu essentiel lors de l’interaction précoce entre P. aleophilum et la vigne, tandis que P. chlamydospora::gfp1 a seulement colonisé les vaisseaux du xylème après 12 semaines. Le bois de la vigne présenterait des réponses spécifiques à la présence de P. aleophilum 6 semaines après traitement, puis générales à la blessure 12 semaines après traitement, en microscopie. L’hypothèse de la spécificité de la réponse induite dans le bois de la vigne par ces deux espèces a été confirmée en étudiant l’expression de 11 gènes associés à la défense 10 h, 24 h, 48 h et 120 hpi. La réponse précoce du bois de la vigne serait spécifique suivant l’identité des pathogènes. Les tissus de l’entre-nœud ont été induits différemment par la blessure par rapport aux tissus dans la région nodale. Un modèle pour le criblage d’agents de biocontrôle ou d’éliciteurs contre l’esca en quelques mois en conditions de laboratoire a permis le développement d’un moyen de lutte durable et novateur, l’eau ozonée. L’eau ozonée présente des propriétés sporicides remarquables contre P. aleophilum in vitro. In planta l’application d’eau ozonée sur une blessure infectée en modèle plaie de taille a réduit de moitié la quantité de mycélium capable de se développer dans le bois 9 semaines après inoculation. / Esca is a complex pathosystem, poorly understood, affecting grapevine’s trunk. Currently there is not efficient tools to control esca disease. This study investigated early events between grapevine and fungi associated to young esca P. aleophilum and P. chlamydospora in two different woody tissues: at the internode and at the nodal (pruning wound model) levels. The colonization of transformed strains P. aleophilum::gfp7 and P. chlamydospora::gfp1 was observed 6 and 12 weeks post inoculation. Fungal species colonized different tissues during the first weeks of infection. Xylem fibres could be the key site of P. aleophilum-grapevine early interactions, whereas P. chlamydospora::gfp1 only colonized xylem vessels 12 weeks post inoculation. Grapevine wood may respond specifically to P. aleophilum after 6 weeks. This response seemed to be related to healing in microscopy, and thus aspecific 12 weeks post inoculation. The specificity of induced response in the wood of Vitis vinifera L. was confirmed by studying gene expressions of 11 defense-related genes 10 h, 24 h, 48 h and 120 hpi. The early response of woody tissues could be specific to pathogens identity in grapevine. Wounding damages induced defense-related genes differently according the tissues (internode vs node). A model for screening biological control agents or elicitors in laboratory conditions, within months, has been used to develop a sustainable and original control tool: ozonated water. Ozonated water presented remarkable sporicidal properties on P. aleophilum in vitro. Infected pruning wound treated with ozonated water reduced the P. aleophilum inoculum able to develop in the injured wood by a factor two 9 weeks post inoculation.

Deionised-ozonated water, so-called DIO3 appears to be an ideal alternative for usage in semiconductor industry. The utilisation of DIO3 for removal of photoresist from silicon wafers is faster, cheaper, and more environmental-friendly compared to classical technology based on mixture of sulphuric acid with hydrogen peroxide, so-called SPM. The diploma thesis deals firstly with research into ozone and ozonated water and their possible applications. Next sections describe two prototypes of generators for DIO3, that were assembled in CSVG a.s. Testing of parameters for generators on dissolved-ozone concentration is also a part of this thesis. Moreover, thesis involves tests, that were carrier out in ON Semiconductor in Rožnov pod Radhoštěm. These tests compare efficiency of cleaning by classical technology based on SPM and DIO3 approach.