Araçatuba 2022 JÚLIA GUERRA DE ANDRADE Avaliação do efeito sinérgico da associação do hipoclorito de sódio com dióxido de carbono contra o biofilme de Enterococcus faecalis Araçatuba 2022 JÚLIA GUERRA DE ANDRADE Avaliação do efeito sinérgico da associação do hipoclorito de sódio com dióxido de carbono contra o biofilme de Enterococcus faecalis Dissertação apresentada à Faculdade de Odontologia da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Campus de Araçatuba, para obtenção de título de Mestre em Ciência Odontológica - Área de Concentração: Endodontia. Orientador: Prof. Assoc. Rogério de Castilho Jacinto Catalogação na Publicação (CIP) Diretoria Técnica de Biblioteca e Documentação – FOA / UNESP Andrade, Júlia Guerra de. A553a Avaliação do efeito sinérgico da pressurização do hipoclo- rito de sódio com dióxido de carbono contra o biofilme de Enterococcus faecalis / Júlia Guerra de Andrade. - Araçatuba, 2022 32 f. : il. ; tab. Dissertação (Mestrado) – Universidade Estadual Paulista, Faculdade de Odontologia de Araçatuba Orientador: Prof. Rogério de Castilho Jacinto 1. Irrigantes do canal radicular 2. Hipoclorito de sódio 3. Dióxido de carbono 4. Bactérias 5. Biofilmes I. T. Black D24 CDD 617.67 Claudio Hideo Matsumoto CRB-8/5550 DADOS CURRICULARES Júlia Guerra de Andrade Nascimento 27.07.1993 – Itabira- MG Filiação Léa Lúcia Guerra de Andrade Marcos Martins de Andrade 2002-2006 Curso de Graduação em Odontologia pela Pontifícia Universidade Católica de Minas Gerais- PUC/MG 2008-2010 Curso de Especialização em Endodontia pela Faculdade de Odontologia da Universidade de São Paulo – USP/SP 2007-2019 Curso de MBA Gestão Empresarial com Ênfase em Finanças pela Faculdade de Ciências Administrativas e contábeis de Itabira – FUNCESI/MG 2020-2022 Desenvolvimento do projeto de Mestrado em Ciências Odontológicas Área de Endodontia Faculdade de Odontologia de Araçatuba- UNESP Associações CROMG – Conselho Regional de Odontologia de Minas Gerais. CROSP - Conselho Regional de Odontologia de São Paulo. SBPqO - Sociedade Brasileira de Pesquisa Odontológica Dedico este trabalho, À minha filha, Teresa, que tem o olhar tão puro e um sorriso inocente que me servem de guia para querer ser sempre o melhor de mim. Obrigada por suportar minha ausência em tantos momentos que Deus permita que eu seja exemplo pra você! AGRADECIMENTOS À Deus, agradeço por ser tão presente em minha vida, por trilhar meu caminho e entregar meu destino nas mãos de pessoas tão especiais…. Meus pais Marcos e Léa, obrigada por me fazerem ser tanto! Em cada parte minha, cada conquista e cada reconhecimento, vocês estavam por trás. Na minha ausência, foram a presença que a Teresa precisava… vocês são meus melhores ouvidos, meu mais sólido chão. Graças a vocês estou concretizando este sonho! Obrigada, à minha filha, por me escolher sua mãe. Por me fazer querer ser melhor a cada dia….tenho certeza que a felicidade estará sempre com você! Agradeço ao meu querido irmão Hugo, minha cunhada Carol e meu afilhado Thiago, sempre presentes na minha vida! Que sorte eu tenho em poder contar com vocês…. Sofremos e brindamos juntos! Sempre! Agradeço também, à toda minha família, em especial, à tia Dô, que sempre acreditou em mim, que sabe falar a palavra certa na hora certa e que me dá forças para seguir em frente! Ao Rafael, por se tornar tão importante na minha vida. Obrigada por estar ao meu lado em todos os momentos! À Rosane, que sonhou junto comigo toda essa conquista, e à Pati, que se aventurou nas estradas Minas/São Paulo para que tudo isso fosse possível! Leio todos os dias a mensagem que você me enviou! Ao meu orientador, prof. Dr. Rogério, por ter acreditado e me ajudado a realizar um sonho, que faz parte do meu projeto de vida. Ter você como meu orientador foi um privilégio que a vida me ofertou. Quanta paciência em me conduzir até aqui! Meu sincero agradecimento pela orientação valiosa, confiança e amizade. À Carol Loureiro, por ter sido meu anjo guia durante todo esse tempo! Minha gratidão por cada vez que você me amparou, por todo tempo que você se dedicou em me ajudar. Sobra grandeza em seu coração! Admiro sua capacidade de descomplicar o complicado! Aos amigos da Pós-Graduação, Ana Paula Fernandes, Pedro Chaves, Nathalia Machado, Henrique Banci, Cristiane Cantiga, Carolina Barros, Flávio Faria e Ana Maria Vasques que me acolheram com tanto carinho quando cheguei à Araçatuba! Ver o entusiasmo e dedicação de vocês me motivou ainda mais a não desistir do meu mestrado. À Flávia Plazza, por me receber durante bons dias em sua morada. E aos queridos amigos que ingressaram comigo na Pós-Graduação, Marcelo Seron, Ana Cláudia Rodrigues, Gabriela Pacheco, Bharbara Moura, Lucas Chalub, Camila Arantes, Larissa Nunes, por serem tão especiais, cada um da sua maneira, e por se fazerem tão presentes, mesmo de longe! Agradeço imensamente a amizade de vocês! À Equipe de Docentes da Endodontia FOA/UNESP, professores Eloi Dezan Junior, João Eduardo Gomes Filho, Gustavo Sivieri de Araújo, Luciano Tavares Ângelo Cintra e mais uma vez, ao professor Rogério de Castilho Jacinto, aos quais tive a honra de poder compartilhar dos seus conhecimentos, seja durante os seminários, das disciplinas que cursei ou nas conversas informais que tivemos pelos corredores da faculdade. Por vocês meu respeito e gratidão. Ao professor Antônio Chaves-Neto por disponibilizar de seu tempo e conhecimento para enriquecer o nosso projeto. À Banca do Exame Geral de Qualificação, composta pelos professores Frederico Canato Martinho e Francisco Montagner, que tanto contribuíram para compor esse trabalho. Agradeço a toda equipe do Departamento de Odontologia Preventiva e Restauradora, da Faculdade de Odontologia de Araçatuba, em especial ao Jorge e Carlos por serem tão atenciosos. À Faculdade de Odontologia de Araçatuba, na pessoa dos professores: Prof. Titular Glauco Issamu Miyahara, digníssimo Diretor e Prof. Titular Alberto Botazzo Delbem, digníssimo Vice-Diretor. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - por financiar a minha bolsa de mestrado durante um ano para que esse trabalho pudesse ser realizado. “Mas se desejarmos fortemente o melhor e, principalmente, lutarmos pelo melhor... O melhor vai se instalar em nossa vida. Porque sou do tamanho daquilo que vejo, e não do tamanho da minha altura.” Carlos Drummond de Andrade ANDRADE, J.G. Avaliação do efeito sinérgico da associação do hipoclorito de sódio com dióxido de carbono contra o biofilme de Enterococcus faecalis. 2022. 46 f. Dissertação (Mestrado) em Ciência Odontológicas - área Endodontia, Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista, 2022. RESUMO Objetivo: Avaliar a eficácia da associação do dióxido de carbono (CO2) pressurizado ao hipoclorito de sódio (NaOCl) na inativação do biofilme de Enterococcus faecalis presentes no interior dos canais radiculares e nos túbulos dentinários por meio de cultura microbiológica. Material e métodos: 40 pré-molares inferiores humanos extraídos, previamente esterilizados, com único canal, foram contaminados com E. faecalis por um período de 10 dias. Os dentes, então, foram distribuídos aleatoriamente em quatro grupos experimentais (n=10) de acordo com o protocolo de irrigação, conforme segue: irrigação convencional com hipoclorito de sódio 2,5% (NaOCl); irrigação convencional com NaOCl 2,5% associado ao dióxido de carbono (CO2); solução salina estéril; solução salina estéril associada ao CO2. O pH e temperatura da solução de NaOCl 2,5% foram analisados puro e em combinação com a adição de CO2 em diferentes pressões (1 a 10) usando um pHmetro digital calibrado a 25°C. As medições foram registradas em 30 e 60 segundos após cada adição de CO2. A carga microbiana no interior dos canais radiculares, foi avaliada pela contagem de unidades formadoras de colônias nas placas comparando a coleta inicial (S1) com a coleta do canal após a irrigação (S2) e nos túbulos dentinários, avaliada por meio da contagem de unidades formadoras de colônia através da coleta de raspas de dentina dos túbulos dentinários após a irrigação nos 3 terços da raiz. O teste Two Way Repeated Measures ANOVA seguido de Post-Hoc de Student-Newman-Keuls foi usado para Comparação intergrupos e diferentes tempos de amostragem. Uma comparação intragrupo com terços radiculares também foi realizada por meio do teste Two Way Repeated Measures ANOVA seguido de Student-Newman-Keuls ao nível de significância estabelecido em 5% (p < 0,05). Resultados: Bactérias cultiváveis estavam presentes em todas as amostras S1 (p > 0,05). Todos os protocolos de irrigação foram eficazes na redução da carga bacteriana, independente da solução utilizada (p < 0,05). Houve diferença estatística nos grupos do NaOCl 2,5% e NaOCl 2,5% associado ao CO2 comparados à solução fisiológica, no interior dos canais radiculares. Em relação à análise dentinária, o grupo NaOCl 2,5% + CO2 apresentou diferença significativa em todos os terços, com maior redução de UFC/ml no terço cervical. Não foi observada diferença significativa ao comparar os terços nos grupos de solução salina estéril. Após 10 pressões, houve redução do valor do pH para 7,43 e aumento da temperatura da solução para 28°C. Conclusão: Embora a associação de CO2 com NaOCl 2,5% favoreça sua capacidade de reduzir bactérias no interior do canal radicular, não potencializou seu efeito bactericida no interior dos túbulos dentinários. Palavras-chave: irrigação endodôntica, hipoclorito de sódio, dióxido de carbono, bactérias, biofilme ANDRADE, J.G. Evaluation of the synergistic effect of association of sodium hypochlorite with carbon dioxide on Enterococcus faecalis biofilm. 2022. 46 f. Dissertação (Mestrado) em Ciência Odontológicas - área Endodontia, Faculdade de Odontologia de Araçatuba, Universidade Estadual Paulista, 2022. ABSTRACT Objective: to evaluate the effectiveness of the association of pressurized carbon dioxide (CO2) with sodium hypochlorite (NaOCl) in the inactivation of the Enterococcus faecalis biofilm present inside the root canals and in the dentinal tubules through microbiological culture. Material and methods: 40 extracted human lower premolars previously sterilized, with a single canal, were contaminated with E. faecalis for a period of 10 days. The teeth were randomly distributed into four experimental groups (n=10) according to the irrigation protocol, as follows: conventional irrigation with 2.5% sodium hypochlorite (NaOCl); conventional irrigation with 2.5% NaOCl associated with carbon dioxide (CO2); sterile saline solution; sterile saline solution associated with CO2. The pH and temperature of the 2.5% NaOCl solution were analyzed neat and in combination with the addition of CO2 at different pressures (1 to 10) using a digital pH meter calibrated at 25° C. Measurements were recorded at 30 and 60 seconds after each CO2 addition. The microbial load inside the root canals was evaluated by counting colony forming units in the plates comparing the initial collection (S1) with the collection of the canal after irrigation (S2) and in the dentinal tubules, evaluated by counting colony forming units by collecting dentin shavings from dentinal tubules after irrigation in the 3 thirds of the root. Data normality was verified by the Shapiro-Wilk test. The Two Way Repeated Measures ANOVA Student-Newman-Keuls Post-Hoc test was used for intergroup comparison and different sampling times. One Way Analysis of Variance on Ranks was used for % of CFU reduction. An intragroup comparison with root thirds was also performed using the Student- Newman-Keuls test. The Two Way Repeated Measures ANOVA Student-Newman-Keuls Post-Hoc test was used for intergroup comparison and different sampling times. An intragroup comparison with root thirds was also performed using the Student-Newman-Keuls test. The significance level was set at 5% (p < 0.05). Results: Cultivable bacteria were present in all S1 samples (p>0.05). All protocols and irrigation were effective in reducing bacterial load, regardless of the solution used (p < 0.05). There was a statistical difference in the 2.5% NaOCl and 2.5% NaOCl groups associated with CO2 compared to saline solution. Regarding the dentin analysis, the 2.5% NaOCl + CO2 group showed a significant difference in all thirds, with a greater reduction of CFU/ml in the cervical third. No significant difference was observed when comparing the thirds in the sterile saline groups. After 10 pressures, there was a reduction in the pH value to 7.43 and an increase in the temperature of the solution to 28°C. Conclusion: Although the association of CO2 with 2.5% NaOCl favors its ability to reduce bacteria inside the root canal, it did not potentiate its bactericidal effect on the inside of the dentinal tubules. Keywords: root canal irrigants, sodium hypochlorite, carbon dioxide, bactéria, biofilm LISTA DE FIGURAS Figure 1 - SEM image of Enterococcus faecalis biofilm formation in the root canal walls and within the dentinal tubules. 4 Figure 2 - Study design 7 Figure 3 - pH measurement and temperature of 2.5% NaOCl solution during the addition of CO2 by the carbonator machine. 10 LISTA DE TABELAS Table 1 - Mean (Standard Deviation) of CFU/ml counts (log10) before and after irrigation protocols (IP), and percentage of CFU reduction 8 Table 2 - Mean (Standard Deviation) of CFU/ml counts (log10) after irrigation protocols (IP) in the different thirds. 9 SUMÁRIO INTRODUCTION 1 MATERIAL AND METHODS 2 Sample selection and preparation 2 Cultivation of E. faecalis and specimen contamination 3 Experimental groups 4 Microbiological collection 6 Dentine samples 6 pH Measurement 7 Statistical analysis 7 RESULTS 8 DISCUSSION 10 CONCLUSION 12 REFERENCES 13 Anexo 1 – Parecer do Comitê de Ética em Pesquisa (CEP) 20 Anexo 2 – Guia para submissão do artigo na revista Brazilian Oral Research 24 1 Introduction The ability of carbon dioxide (CO2) to inactivate microorganisms has gained special attention. Pressurized carbon dioxide (PCD) has shown great potential to inhibit several pathogenic species in both aqueous and non-aqueous products1-3. Among the advantages of using PCD are its low cost, low viscosity, non-toxicity and zero surface tension, which allows its rapid penetration into complex structures and porous materials4. Previous studies have shown the effectiveness of PCD in inactivating microorganisms5-7. In addition, the effect of PCD resulted in better disinfection when associated with sodium hypochlorite (NaOCl) in the inactivation of Enterococcus spp. in seawater1. In fact, the bactericidal activity of chlorine depends on the proportion of hypochlorous acid (HOCl), which is the predominant form at low pH levels8. It is assumed that a reduction in pH caused by CO2 may help to increase the proportion of HOCl, thus increasing its bactericidal activity9. Therefore, pressurization of NaOCl with CO2 may be an alternative to potentiate the antimicrobial activity of NaOCl in the root canal disinfection. Root canal irrigation is critical to endodontic treatment success as it performs several functions including cleaning, lubrication, and disinfection of the root canal system10. Irrigation optimizes the disinfection in the regions untouched by the instruments such as apical delta, isthmus, and dentinal tubules11-13. NaOCl is the most used endodontic irrigant, due to its antimicrobial activity and capacity of dissolving organic tissues14,15. It has been tested in different concentrations for root canal disinfection, ranging from 0.5% to 6%16. Although the antimicrobial activity of NaOCl is directly proportional to its concentration, it should be considered that at high concentrations its cytotoxicity also increases in periapical tissues18-20. The literature also suggests that reducing the pH of NaOCl between 6 and 7.5 leads to better antimicrobial activity of the solution.21 2 The persistence of microorganisms and their virulence factors within root canals can also contribute to the endodontic treatment failure11,22. Persistent endodontic infections harbor a polymicrobial community with a predominance of Gram-positive facultative anaerobes, such as E. faecalis23-26. It has a biofilm-forming capacity even in areas of anatomical complexity, and also in dentinal tubules27-30. In addition to playing a crucial role in bacterial coaggregation, E. faecalis has other virulence factors that confer its resistance to endodontic therapy31,32. To date, there are no studies related to the antimicrobial activity of NaOCl pressurized with CO2 for root canal disinfection. The aim of the present in vitro study was to evaluate the effectiveness of the association of PCD with NaOCl in the inactivation of E. faecalis biofilm inside root canals and dentinal tubules through microbiological culture. The null hypothesis tested was that pressurizing NaOCl with CO2 does not promote a significant difference in the reduction of colony forming unit (CFU) compared with NaOCl in root canal disinfection. Materials and methods Sample selection and preparation This study was approved by the Research Ethics Committee of School of Dentistry, Araçatuba - UNESP (CAAE: 34692620.8.0000.5420). Forty human mandibular premolars, extracted for periodontal or orthodontic reasons, were selected for this study. Digital radiographs were taken in the mesiodistal and buccolingual directions to select only permanent teeth with a single root canal. Teeth with caries, root fractures, open apex, multi-rooted teeth, root curvatures > 10°, calcified canals, resorption defects, dental posts or prosthetic crowns, and previous endodontic treatment were excluded. All teeth were stored in 0.9% saline until use. Remnants of periodontal tissues were removed with periodontal curettes. The teeth 3 crowns were sectioned transversely using a water‐cooled diamond disk (American Burrs, Santa Catarina, Brazil) and the radicular samples were standardized in 16 mm lengths. The length was determined by inserting a size 10 K file (Dentsply Sirona, Ballaigues, Switzerland) in the root canal until the tip was visible in the region of the apical foramen under magnification33. All root canals were instrumented with a rotary file up to 40.04 (MK life, RS, Brazil) following the manufacturer's recommendations regarding speed and torque. During instrumentation, 1 ml of 2.5% NaOCl (Rioquímica, São José do Rio Preto, SP, Brazil) was administered after each file. The irrigation protocol to clean the entrance of dentin tubules was adapted of Ferraz et al34. All teeth were submitted to an ultrasonic bath for 10 min in 17% EDTA (Biodinâmica, Ibiporã, PR, Brazil), followed by 10 min in a 5.25% NaOCl (Apothimed, SP, Brazil) bath to remove smear layer. The samples were neutralized with 5 ml of 5% sodium thiosulfate (Merck KGaA, Darmstadt, Germany). Each sample was immersed in approximately 700 ml of brain and heart infusion broth (BHI, Kasvi, Paraná, Brazil), and ultrasonically activated for 1 min to allow penetration of the culture medium into the root canal irregularities. The teeth were sterilized in an autoclave for 30 min at 121°C and they were kept in an incubator at 37°C for 48 h to check the effectiveness of the sterilization treatment. Cultivation of E. faecalis and specimen contamination The contamination protocol of the specimen was adapted of Carvalho et al.35 Pure culture of E. faecalis (ATCC 29212) was cultivated in BHI broth at 37°C for 24 hours. The bacterial culture was transferred to another tube containing BHI and incubated, overnight at 37º C to achieve exponential growth. This culture was adjusted to McFarland standard #1 (3×108 CFU/ml) using a spectrophotometer (BioTek Instruments, Winooski, USA). The inoculum (800 μl) was inserted into the 2 ml Eppendorf tubes with the specimen and centrifuged (Eppendorf Centrifuge 5702-R, Eppendorf, Hamburg, Germany) in sequence at 1,400, 2,000, 3,600 and 4 5,600 g in double cycles of five minutes. The inoculum was renewed at each centrifugation cycle. After the eight centrifugation cycles, the sterile BHI broth was inserted into 2 mL tubes, agitated in vortex (Phoenix Luferco, Araraquara, Brazil), and incubated at 37 ° C for 24 hours. For 10 days, bacterial cultivation was carried out using sterile culture medium (BHI broth, Kasvi) with centrifugation (3,600 g for 5 minutes at 25° C) and alternate days to allow bacterial penetration into the root canal system. The sterile BHI broth (1 ml) was renewed every 48 hours, on the days of centrifugation. All procedures were performed under aseptic conditions in a laminar flow cabinet (Veco Bioseg 12 Ltda, Campinas, Brazil). On day 10, the samples were removed from the Eppendorf tubes as well as the excesses of the culture medium and the external root surfaces were cleaned with sterile gauze. Bacterial penetration into the dentinal tubules using this technique was confirmed by scanning electron microscopy (SEM) in a pilot study, as observed in figure 1. Figure 1. SEM image of Enterococcus faecalis biofilm formation in the root canal walls (a) and within the dentinal tubules (b). Experimental groups The sample size calculation was based on the data of a previous investigation35-37 which indicated 10 teeth per group with α-type error = 0.05 and power β = 0.80. Twelve teeth per group were included, considering the possibility of tooth loss throughout the study. (a) (b) 5 The samples were assigned into 4 experimental groups, according to the irrigation protocol used, as follows: • 2.5% NaOCl group: the root canal was irrigated and manually agitated with a #15 K file using 15 ml of 2.5% NaOCl, 5 ml 17% EDTA, and 5 ml of 2.5% NaOCl for 20 seconds each. Then, 1 ml of 5% sodium thiosulfate for 1 min to inactivate NaOCl. The total volume of irrigation with 2.5% NaOCl was 20 ml35,38. • 2.5% NaOCl + CO2 group: the irrigation procedures were essentially the same as those described for the 2.5% NaOCl group, except that the irrigating solution used was sodium hypochlorite associated with carbon dioxide introduced by the machine for aerating jet water (Sodastream Industries LTD, Kfar Saba, Israel). • Sterile saline group: with similar irrigation procedures described above, except to the irrigation solution. In this group the sterile saline solution was use. The total volume of irrigation was 20 ml. • Sterile saline + CO2 group: the irrigation procedures were similar other groups, except that the irrigating solution used was a sterile saline solution associated with carbon dioxide. After contamination, the samples were mounted on a sterile aluminum table. The apical foramen of each experimental sample was sealed with a fast-setting epoxy resin to prevent apical bacterial leakage and to create a closed canal. All irrigation procedures were performed with 30-G NaviTip needles (Ultradent Products Inc, Indaiatuba, Brazil) always placed 1 mm short from the working length. The addition of CO2 in 2.5% NaOCl + CO2 group and Sterile saline + CO2 group was carried out through the machine for aerating jet water (Sodastream Industries LTD, Kfar Saba, Israel). A gas cylinder (CO2) was threaded on the back of the machine, and then a bottle of the equipment was completed to the existing mark (800 ml) with the desired solution and attached to the machine. The button, located at the top, to add the gas, was activated 6 10 times to standardize the volume of CO2 in the solutions. Microbiological collection The method used for sample collection in the present study was previously reported by Yamamoto et al.39 Briefly, bacteriological samples were collected from all root canals before (S1) and after the irrigation protocols (S2) using three sterile paper points (Dentsply Maillefer). Each cone was kept for one minute in the root canal and transferred to 2 ml Eppendorf tubes containing 1 ml of Ringer's solution (Sigma-Aldrich, St Luis, MI, USA). Each tube was vortexed for 30 s. The samples were homogenized and diluted to 10-4 before and 10-1 after irrigation protocols. Then each dilution was plated on BHI agar and incubated at 37 ° C for 48 h. The colony-forming units (CFU/ml) grown were counted. Dentine samples Each tooth was sectioned into three thirds (cervical, medium and apical). Dentin debris were removed from the root canals to collect bacteria inside the dentinal tubules using increasing‐diameter sterile diamond‐tipped conical burs [4137 (ISO 025), KGSorensen, São Paulo, SP, Brazil] driven by a low-speed electric motor (Dentsply Sirona). The samples obtained from each third were individually stored in 2 ml Eppendorf tubes containing Ringer's solution 7 (Berber et al. 2006), plated on BHI agar, and incubated at 37 °C for 48 h to count colony-forming units (CFU). Figure 2. Study design. A- Extracted tooth used in experimental groups. B- Sample preparation: standardization of the root canal length (16 mm) and endodontic treatment with Rotatory Files #40/04. C- Root canal filled with BHI solution. D- Sample contamination with E. faecalis for 10 days. E- Experimental groups. F- Root canal sample collection after irrigation protocols. G- Dentine sample collection. pH Measurement The pH of the 2.5% NaOCl solution was analyzed pure and in combination with the addition of CO2 at different pressures (1 to 10) using a digital pH meter (PHS3BW model, Bel Engineering, Monza, Italy) calibrated at 25°C. Measurements were recorded at 30 and 60 seconds after each addition of CO2. Statistical analysis The data collected (CFUs) were statistically analyzed by using Sigma Plot 12.0 for Windows (Systat Software Inc., San Jose, USA). The Two Way Repeated Measures ANOVA 8 followed by Student-Newman-Keuls Post-Hoc test was used for inter-group comparison and different sampling times. One Way Analysis of Variance on Ranks was used for percentager of CFU reduction. An intra-group comparison with root thirds was also performe d using the Two Way Repeated Measures ANOVA Student-Newman-Keuls test. The significance level was set at 5% (p < 0.05). Results Table 1 shows inter-group E. faecalis counts before and after irrigation protocols, as the percentage of bacterial reduction. Cultivable bacteria were present in all samples S1 (40/40). All irrigation protocols were effective in reducing bacterial load (p < 0.05). Irrigation using 2.5% NaOCl as the irrigation solution was more effective than the sterile saline solution (p = 0.001), regardless of the presence of CO2. The association with CO2 improves the effectiveness of root canal decontamination when associated with 2.5% NaOCl. However, incorporate CO2 in sterile saline solution did not increase decontamination of the samples (p > 0.05). The inter- and intra-group analysis are presented in Table 2. 2.5% NaOCl + CO2 group showed a significant difference in all thirds, with greater reduction of CFU/ml in the cervical third. No significant difference was observed when comparing the thirds in sterile saline groups. Table 1. Mean (Standard Deviation) of CFU/ml counts (log10) before and after irrigation protocols (IP). Irrigation Protocol Before IP After IP 2.5% NaOCl 6.5 Aa (0.5) 1.2 Ba (1.2) 2.5% NaOCl + CO2 6.3 Aa (0.4) 0.4 Bb (0.7) 9 Sterile saline 6.7 Aa (0.4) 5.0 Bc (0.2) Sterile saline + CO2 6.9 Aa (0.3) 4.9 Bc (0.2) p < 0.05 Means followed by different letters indicate statistically significant differences. The horizontal uppercase letters show the comparison within the same group before and after irrigation. The lowercase vertical letters show the comparison within the different groups using different irrigation protocols at the same time. Table 2. Mean (Standard Deviation) of CFU/ml counts (log10) after irrigation protocols (IP) in the different thirds. Thirds Irrigation Protocol 2.5% NaOCl 2.5% NaOCl + CO2 Sterile saline Sterile saline + CO2 Cervical 0.8 Aa 0.2 Aa 3.9 Ba 4.1 Ba (1.3) (0.5) (0.6) (0.6) Medium 0.7 Aa 1.0 Ab 4.2 Ba 3.9 Ba (1.6) (1.4) (0.4) (0.9) Apical 2.7 Ab 3.5 Ac 4.5 Ba 4.5 Ba (1.8) (0.6) (0.3) (0.5) p <0.05 Means followed by different letters indicate statistically significant differences. The horizontal uppercase letters show the comparison within the different groups using different irrigation protocols. The lowercase vertical letters show the comparison between the thirds after irrigation. The pure 2.5% NaOCl solution has an alkaline pH value (13.39 - 13.51) at 25°C. With the increase in gas injections, the pH value of the solution decreased, reaching a pH value of 7.43 in a total of 10 injections (Figure 3). The temperature of the solution also changed, increasing 10 from 25.3 °C in the pure solution to 28 °C in the solution with 10 injections of CO2. Figure 3. pH measurement and temperature of 2.5% NaOCl solution during the addition of CO2 by the carbonator machine. Discussion Pressurized carbon dioxide (PCD) has been used as a sterilization technique both in the food preservation and water treatment industry, with a potential of being useful in many other applications1,5,40. Many studies have investigated the effects of PCD on pathogenic organisms, vegetative cells and spores, yeasts and molds, showing great potential to inhibit several pathogenic species5-7. However, to the best of our knowledge, this is the first study to investigate the effectiveness of the association of pressurized CO2 with NaOCl in human substrate and in biofilm inactivation. Although high concentration NaOCl solution has greater antimicrobial activity, its potential to irritate periapical tissues is more critical compared with the lower concentrations41. In this view, our aim was to test the 2.5% NaOCl associated with pressurized CO2 in order to improve its antibacterial activity and serve as an alternative to high concentration solutions. The presence of cultivable bacteria was revealed in 100% of the initial samples (S1), 14 25.3 °C 26 °C 13 26.6 °C 12 26.7 °C 11 10 27 °C 27 °C 9 27 °C 27.2 °C 27.4 °C 8 27.5 °C 28 °C 7 0 1 2 3 4 5 6 7 8 9 10 times of CO2 injections by the carbonator machine 30 s 60 s 11 validating the contamination protocol. E. faecalis is widely used in models of ex vivo bacteriological studies for several reasons: it survives isolated in root canals42, it is resistant to endodontic treatment43 and it is an easy bacterium to grow in the laboratory44. Biofilm sampling was performed through collection with sterilized paper tips, which despite limitations, such as collecting only cultivable bacteria present inside the root canals, is a well-established protocol14,45,46. For sampling the biofilm inside the dentinal tubules, dentin samples were obtained with drills right after the biomechanical procedures. This method has been shown to be effective in other work45. The microbiological culture technique was used in our study to assess the effectiveness of irrigants, which is the most used evaluation method in studies (87%)47. In the present study, PCD associated with 2.5% NaOCl was more effective against E. faecalis biofilm within the root canals (p < 0.05) when compared with 2.5% NaOCl. The significant synergistic benefit of this association has also being observed in previous publications1,48. The reduction in pH of 2.5% NaOCl caused by CO2 observed in the present study, may be the most effective factor in improving disinfection9, probably due to the increase in the proportion of HOCl in NaOCl, which has high bactericidal activity8. In our experiment, the gas injection by the carbonator machine was activated 10 times, to reach pH 7.4, as shown in the pH curve. The influence of pH on the antibacterial activity of NaOCl in ex vivo infected root canals can be explained by the fact that the acidification of NaOCl favors its ability to kill bacteria as the lowest values of cell viability were found in 2.5% NaOCl groups at acidic pH49. Likewise, the 4.2% NaOCl solution proved to be more effective in terms of its bactericidal action with the acidification of the solution to pH 6.550. Regarding the influence of temperature on the effectiveness NaOCl of the solution, it is not possible to infer that the increase in temperature observed by the addition of CO2, would have any influence as few articles have been published. Previous in vitro studies have suggested that increasing the temperature of a low-concentration NaOCl solution improved its tissue dissolution capacity51 12 and increased its antimicrobial action52. However, in a randomized controlled clinical study, no extra antibacterial efficacy was observed with NaOCl preheating54. Another in vivo study showed that pre-warmed (60°C) NaOCl begins to reduce to body temperature (35.7°C) within minutes as soon as the solution touches the root canal wall55. Regarding disinfection within the dentinal tubules, the results of this study suggest that the penetration capacity of NaOCl inside the dentinal tubules did not increase with the addition of pressurized CO2 (p > 0.05). This result was contrary to the expectation that the association of the physicochemical properties of the liquid (solvation), and of the gas (diffusivity), combined with the low surface tension of CO2 would increase penetration into dentin40. However, it is important to point out that the PCD technique in microbial inactivation in the food industry is associated with an agitation to promote the solubilization of CO2, thus improving its cellular penetration and increasing its antimicrobial activity. Therefore, for further research, it is suggested to evaluate the association of passive ultrasonic irrigation (PUI) that uses ultrasonic tips to stir solutions with PCD in the root canals. Studies on the antibacterial effect of PUI show significantly better results than conventional syringe irrigation13,56,57. Despite the constant search for a disinfection protocol that totally eliminates bacteria, the great anatomical variation and its complexity make this impossible. 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Journal of Endodontics. 2003 Sep;29(9):562–4. https://www.sciencedirect.com/science/article/pii/S0300571220301585 20 Anexo 1 – Parecer do Comitê de Ética em Pesquisa (CEP) 21 22 23 24 Anexo 2 – Guia para submissão do artigo na revista Brazilian Oral Research 25 26 27 28 29