Investigation of LPS pathobiology in endodotic infections and its susceptibility to different therapeutic modalities

Orientador: Brenda Paula Figueiredo de Almeida GomesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Estratégias clínicas devem voltar-se para a máxima desagregação/redução da carga microbiana, assim como para eliminação e/ou inativação de lipopolissacarídeos (LPS) e demais produtos tóxicos e assim, reduzir o potencial inflamatório do conteúdo endodôntico aos tecidos periapicais. Os objetivos do presente estudo foram: 1) Avaliar a efetividade do sistema Reciproc (R25) em comparação com diferentes sistemas rotatórios na remoção de bactérias e endotoxinas de canais radiculares contaminados (artigo 1); Investigar a influência da interação entre substâncias antimicrobianas utilizadas na terapia endodôntica e o substrato Limulus Amebocyte Lysate (LAL) para quantificação de endotoxinas, com o objetivo de identificar substâncias interferentes quando da leitura dos níveis de endotoxinas em amostras clínicas (artigo 2); Monitorar "in vivo" a efetividade do preparo químico-mecânico (PQM) com NaOCl 2,5% e Clorexidina (CHX) gel 2% (tendo como controle o soro fisiológico [SS]) após o uso do EDTA 17% e medicação intracanal na redução de LPS, elucidando a contribuição de cada etapa da terapia endodôntica na redução de endotoxinas (artigo 3); Estudar o perfil da microbiota e níveis de endotoxinas nas infecções endodônticas primárias com lesão periapical, determinando o potencial inflamatório do conteúdo endodôntico contra macrófagos através da produção de IL1- ß e TNF-?. Além disso, comparar "in vivo" a efetividade do preparo químico-mecânico (PQM) com NaOCl 2,5% e Clorexidina (CHX) gel 2% e após o uso do EDTA 17% e medicação intracanal na eliminação de LPS, tendo como controle, soro fisiológico (SS) (artigo 4); Investigar a influência de diferentes substâncias químicas auxiliares utilizadas no tratamento endodôntico e da medicação à base de hidróxido de cálcio por diferentes períodos na estrutura e bioatividade do lipopolissacarídeo bacteriano, visando elucidar a ocorrência de possíveis implicações na detecção do lipideo A/ LPS e posteriormente, seu potencial imunoestimulador em receptores TLR-4 (artigo 5). Métodos: Amostras foram coletadas de canais radiculares utilizando cones de papel estéreis/despirogenizados. PCR (16s rDNA), contagem de unidades formadoras de colônias e método LAL foram utilizados. Macrófagos Raw 264.7 foram estimulados com conteúdo endodôntico infeccioso e determinação dos níveis de IL1- ß e TNF-? foi realizada por meio de ELISA. Espectrometria de massa (MALDI-TOF) e silver staining determinaram a presença de picos de lipídeo A e bandas correspondentes à presença de LPS após tratamentos, respectivamente. Em paralelo, ativação de TLR-4 foi realizada para determinação do potencial imunoestimulador do LPS. Resultados: Artigo 1- Não houve diferença entre o sistema Reciproc R25 e os diferentes sistemas rotatórios na remoção de bactérias e endotoxinas de canais radiculares contaminados, respectivamente: Reciproc (99.34% e 91.69%); GII - Mtwo (99.86% e 83.11%); GIII- ProTaper (99.93% e 78.56%); and GIV- FKG RaceTM (99.99% e 82.52%). Artigo 2- Água oxigenada 30%, Otosporin® e pasta tripla antibiótica causaram inibição/superestimação dos níveis de endotoxina em todas as diluições testadas. Artigo 3- PQM foi capaz de reduzir significativamente os níveis de LPS: NaOCl 2.5% (99.65%) (GI), CHX 2% (94.27%) (GII), e SS (96.79%) (GIII) (p 0.01). Article 2- Hydrogen peroxide 30%, Otosporin® and triple antibiotic paste caused inhibition/ enhancement of endotoxin levels in all dilutions. Article 3- Irrespective of the irrigant, CMP was able to significantly reduce LPS levels: NaOCl 2.5% (99.65%) (GI), CHX 2% (94.27%) (GII), and SS (96.79%) (GIII) (p .05). IL-1? and TNF-? were produced by macrophages stimulated with initial endodontic content. A proportional decrease of cytokine levels was found after each root canal procedure (p <0.05). Article 5- 5.25% NaOCl and Ca(OH)2 induced both loss of peak of lipid A and LPS detection, while no structural alteration was observed after LPS treatment with other auxiliary chemical substances. In parallel, LPSs treated with 5.25% NaOCl and Ca(OH)2 were significantly less potent in activating TLR4 when compared to other substances (p <0.0001). It was concluded that: Reciproc (R25) was as effective as the rotary multifile systems for bacteria and endotoxin removal from contaminated root canals (Article 1); The performance characteristics - accuracy and reproducibility- of the turbidimetric kinetic assay for quantification of endotoxins are influenced by the interaction of LAL substrate with Hydrogen peroxide 30%, Otosporin® and triple antibiotic paste (Article 2); High endotoxin reduction occurs after chemical-mechanical preparation and after the use of calcium hydroxide paste (Article 3). Additional endodontic procedures enhance endotoxin reduction (Article 3). Consequently, it was found a progressively lower activation of macrophages for IL-1? and TNF-? production (Article 4); 5.25% NaOCl and calcium hydroxide paste were capable of inducing loss of lipid A peaks and LPS detection, rendering a lower immunostimulatory potential against TLR-4 (Article 5)DoutoradoEndodontiaDoutora em Clínica Odontológica2013/02402-9FAPES

Influence of the apical enlargement size on the endotoxin level reduction of dental root canals

Gram-negative bacteria play an essential role in endodontic infections because they have virulence factors such as endotoxin. Due to its potential cytotoxic activity, special attention has been given to the removal/neutralization of this endotoxin in the root canal system. OBJECTIVE: The aim of this study was to evaluate the influence of the apical enlargement size (AES) by using rotary instruments on the endotoxin level reduction of dental root canals. MATERIAL AND METHODS: Forty root canals of the mandibular premolar teeth were used. Escherichia coli endotoxin (055: B55) was inoculated into thirty root canals. Ten teeth served as the negative control group. After the incubation period, the first endotoxin samples were collected from the root canals with a sterile/apyrogenic paper point for the analysis of the endotoxin units (EU/mL) present before instrumentation (S1). Specimen instrumentation was performed with the Mtwo® rotary system in the sequence 10/.04, 15/.05, 20/.06, 25/.06, 30/.05, 35/.04 and 40/.04. To monitor the effectiveness of increasing apical enlargement on endotoxin removal, the second endotoxin samples were collected from all the root canals after instrumentation with the following instruments: #25/.06- (S2); #30/.05- (S3); # 35/.04- (S4); and #40/.04- (S5). Limulus amebocyte lysate (LAL) was used to quantify the levels of endotoxin. The results were statistically compared by using repeated measures of ANOVA with post hoc Tukey testing. RESULTS: Increasing levels of endotoxin removal was achieved by large sized apical enlargement: S2 (AES #25/.06)- 89.2%, S3 (AES #30/.05)- 95.9%, S4 (AES #35/.04)- 97.8% and S5 (AES #40/.04)- 98.2%. Substantial reduction of endotoxin content was obtained in S4 and S5 compared to S2 (

Monitoring the effectiveness of root canal procedures on endotoxin levels found in teeth with chronic apical periodontitis

Objective: The aim of this study was to monitor the effectiveness of root canal procedures by using different irrigants and intracanal medication on endotoxin levels found in root canals of teeth with chronic apical periodontitis. Material and Methods: Thirty root canals of teeth with pulpal necrosis associated with periapical lesions were selected and randomly divided into groups according to the irrigants used: GI - 2.5% NaOCl, GII - 2% chlorhexidine (CHX) gel, and GIII - saline solution (SS) (all, n=10). Samples were collected with sterile/apyrogenic paper points before (S1) and after root canal instrumentation (S2), after use of 17% ethylenediaminetetraacetic acid (EDTA) (S3), and after 30 days of intracanal medication (Ca(OH)2+SS) (S4). A turbidimetric kinetic Limulus Amebocyte Lysate assay was used for endotoxin measurement. Results: Endotoxins were detected in 100% of the root canals investigated (30/30), with a median value of 18.70 EU/mL. After S2, significant median percentage reduction was observed in all groups, irrespective of the irrigant tested: 2.5% NaOCl (99.65%) (GI), 2% CHX (94.27%) (GII), and SS (96.79%) (GIII) (all p;0.05). Intracanal medication for 30 days was able to significantly reduce residual endotoxins: 2.5% NaOCl (90%) (GI), 2% CHX (88.8%) (GII), and SS (85.7%) (GIII,

Monitoring the effectiveness of root canal procedures on endotoxin levels found in teeth with chronic apical periodontitis

Objective: The aim of this study was to monitor the effectiveness of root canal procedures by using different irrigants and intracanal medication on endotoxin levels found in root canals of teeth with chronic apical periodontitis. Material and methods: Thirty root canals of teeth with pulpal necrosis associated with periapical lesions were selected and randomly divided into groups according to the irrigants used: GI - 2.5% NaOCl, GII - 2% chlorhexidine (CHX) gel, and GIII - saline solution (SS) (all, n=10). Samples were collected with sterile/apyrogenic paper points before (S1) and after root canal instrumentation (S2), after use of 17% ethylenediaminetetraacetic acid (EDTA) (S3), and after 30 days of intracanal medication (Ca(OH)2+SS) (S4). A turbidimetric kinetic Limulus Amebocyte Lysate assay was used for endotoxin measurement. Results: Endotoxins were detected in 100% of the root canals investigated (30/30), with a median value of 18.70 EU/mL. After S2, significant median percentage reduction was observed in all groups, irrespective of the irrigant tested: 2.5% NaOCl (99.65%) (GI), 2% CHX (94.27%) (GII), and SS (96.79%) (GIII) (all p0.05). Intracanal medication for 30 days was able to significantly reduce residual endotoxins: 2.5% NaOCl (90%) (GI), 2% CHX (88.8%) (GII), and SS (85.7%) (GIII, p<0.05). No differences were found in the endotoxin reduction when comparing S2 and S4 treatment groups. Conclusion: Our findings demonstrated the effectiveness of the mechanical action of the instruments along with the flow and backflow of irrigant enduring root canal instrumentation for the endotoxin removal from root canals of teeth with chronic apical periodontitis. Moreover, the use of intracanal medication for 30 days contributed for an improvement of endotoxin reduction226490495CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP302575/2009-0sem informação13/02402- 9; 10/13743-3; 10/19136-1; 10/17877-4; 11/09047-

Effect of chemo-mechanical preparation in the levels of endotoxin in primary endodontic infections and inflammatory potential of the infectious content regarding the production of pro-inflammatory cytokines

Orientador: Brenda Paula Figueiredo de Almeida GomesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Lipopolisacarídeos (LPS) - endotoxinas são capazes de estimular células a produzirem citocinas pró-inflamatórias envolvidas na destruição tecidual periapical. Os objetivos do presente estudo foram: 1) Verificar e quantificar endotoxinas em canais radiculares infectados de dentes com periodontite apical crônica e sua relação com sinais e sintomas clínicos de origem endodôntica; 2) Avaliar a efetividade do preparo químico-mecânico (PQM) com hipoclorito de sódio (NaOCl) 2,5%; clorexidina gel (CLX) 2% e soro fisiológico (SS-controle) na eliminação de endotoxinas; 3) Avaliar o potencial inflamatório do conteúdo endodôntico, antes (C1) e após a instrumentação do canal radicular (C2) com NaOCl 2,5%, CLX 2% ou soro fisiológico e após uso de EDTA 17% (C3) em cultura de células de macrófagos quanto à produção de citocinas pró-inflamatórias - IL-1?, TNF-?. Amostras foram coletadas de 30 canais radiculares com necrose pulpar e presença de lesão periapical em C1, C2 e C3 utilizando cones de papel estéreis/apirogênicos. Endotoxina foi detectada em 100% dos canais radiculares estudados, representada pela mediana de 18,70 EU/mL. Dentes com presença de dor à percussão e exsudação intracanal foram relacionados com níveis elevados de endotoxina (pC2>C3). Foi possível concluir que 1) Endotoxinas estavam presentes em todos os casos investigados, apresentando níveis mais elevados nos dentes com dor à percussão e exsudato intracanal. 2) O preparo químico-mecânico foi eficaz na redução do conteúdo de endotoxinas, independente da substância química auxiliar testada; 3) O potencial inflamatório do conteúdo endodôntico foi demonstrado pela produção de IL1- ? e TNF-?, exercendo maior atividade inflamatória contra macrófagos nas amostras iniciais, quando comparado com as obtidas após o preparo químico-mecânicoAbstract: Lipopolysaccharide (LPS)-endotoxin are able to stimulate cells to produce proinflammatory cytokines involved in the periapical tissue destruction. The aims of this study were: 1) To verify and quantify endotoxin in infected root canals of teeth with apical periodontitis and correlate the levels with clinical signs and symptoms of the primary endodontic infections; 2) To assess the effectiveness of chemo-mechanical preparation (CMP) with 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX) and saline solution (SS), as negative control, for the elimination of endotoxins; 3) To evaluate the inflammatory potential of endodontic content before (C1) and after the CMP (C2) with 2.5% NaOCl, 2% CHX or SS and after final rinse with 17% EDTA (C3) in macrophage cell culture regarding the production of proinflammatory cytokines - IL-1 ? and TNF-?- via the enzyme-linked immunosorbent assay (ELISA). Samples were collected from 30 canals with pulpal necrosis and apical periodontitis in C1, C2 and C3 using sterile non-pyrogenic/paper points. Endotoxin was detected in 100% of root canals studied, represented by the median of 18.70 EU/mL. Teeth with tenderness to percussion and intracanal exudate were related to high levels of endotoxin (pC2>C3). It was possible to conclude that 1) Endotoxin were present in all cases investigated, showing higher levels in the teeth with tenderness to percussion and intracanal exudate; 2) CMP was effective in reducing the endotoxic content, regardless of the auxiliary chemical substance; 3) The inflammatory potential of endodontic content was demonstrated by the production of IL1-? and TNF-? in all cases. The infectious content present in the root canal in the initial samples exerted greater inflammatory activity against macrophages compared to the residual content after CMPMestradoEndodontiaMestra em Clínica Odontológic