Root canal contamination or exposure to lipopolysaccharide differentially modulate prostaglandin E 2 and leukotriene B 4 signaling in apical periodontitis

Abstract Purpose To evaluate the kinetics of apical periodontitis development in vivo , induced either by contamination of the root canals by microorganisms from the oral cavity or by inoculation of bacterial lipopolysaccharide (LPS) and the regulation of major enzymes and receptors involved in the arachidonic acid metabolism. Methodology Apical periodontitis was induced in C57BL6 mice (n=96), by root canal exposure to oral cavity (n=48 teeth) or inoculation of LPS (10 µL of a suspension of 0.1 µg/µL) from E. coli into the root canals (n= 48 teeth). Healthy teeth were used as control (n=48 teeth). After 7, 14, 21 and 28 days the animals were euthanized and tissues removed for histopathological and qRT-PCR analyses. Histological analysis data were analyzed using two-way ANOVA followed by Sidak's test, and qRT-PCR data using two-way ANOVA followed by Tukey's test (α=0.05). Results Contamination by microorganisms led to the development of apical periodontitis, characterized by the recruitment of inflammatory cells and bone tissue resorption, whereas inoculation of LPS induced inflammatory cells recruitment without bone resorption. Both stimuli induced mRNA expression for cyclooxygenase-2 and 5-lipoxygenase enzymes. Expression of prostaglandin E 2 and leukotriene B 4 cell surface receptors were more stimulated by LPS. Regarding nuclear peroxisome proliferator-activated receptors (PPAR), oral contamination induced the synthesis of mRNA for PPARδ, differently from inoculation of LPS, that induced PPARα and PPARγ expression. Conclusions Contamination of the root canals by microorganisms from oral cavity induced the development of apical periodontitis differently than by inoculation with LPS, characterized by less bone loss than the first model. Regardless of the model used, it was found a local increase in the synthesis of mRNA for the enzymes 5-lipoxygenase and cyclooxygenase-2 of the arachidonic acid metabolism, as well as in the surface and nuclear receptors for the lipid mediators prostaglandin E2 and leukotriene B4.

Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage

Abstract Objectives: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. Methodology: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). Results: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. Conclusions: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Murine Experimental Root Canal Infection: Cytokine Expression in Response to F. nucleatum and E. faecalis

Abstract The aim of this study was to evaluate the gene expression of proinflammatory (RANKL, TNF-a and IFN-g) and regulatory (TGF-b and IL-10) cytokines as reaction to experimental infection by mono or bi-association of Fusobacterium nucleatum (ATCC 10953) and Enterococcus faecalis (ATCC 19433). F. nucleatum and E. faecalis, either in mono- or bi-association were inoculated into the root canal system (RCS) of Balb/c mice. Animals were sacrificed at 10 and 20 days after infection and periapical tissues surrounding the root were collected. The mRNA expression of the cytokines RANKL, TNF-a, IFN- g, TGF-b and IL-10 was assessed using real-time PCR. The Kruskal-Wallis test was used for statistical analysis. F. nucleatum mono-infection induced high expression of RANKL and TNF-a, while its modulation was due to IL-10. High expression of IFN-g at day 20 was up-regulated by E. faecalis and RANKL; TNF-a was up-regulated by an independent mechanism via IL-10 and TGF-b. Bi-association (F. nucleatum and E. faecalis) stimulated high expression of RANKL, TNF-a and IFN-g, which seemed to be modulated by TGF-b 20 days later. The gene expression of proinflammatory cytokines was more prominent in the earlier periods of the experimental periapical infection, which concomitantly decreased in the later period. This expression may be regulated by IL-10 and TGF-b in an infection-specific condition

Resumo O objetivo deste trabalho foi avaliar a expressão gênica de citocinas pró-inflamatórias (RANKL, TNF-a e IFN-g) e regulatórias (TGF-b e IL-10) em resposta à infecção experimental por Fusobacterium nucleatum (ATCC 10953) e Enterococcus faecalis (ATCC 19433) como mono-infecção ou em bi-associação. F. nucleatum e E. faecalis foram inoculados no sistema de canais radiculares de camundongos Balb/c, tanto isoladas como em bi-associação. Os animais foram sacrificados em 10 e 20 dias após a infecção, e os tecidos periapicais foram coletados. As expressões do mRNA das citocinas RANKL, TNF-a, IFN-g, TGF-b e IL-10 foram analisadas por meio do real-time PCR. O teste de Kruskal-Wallis foi utilizado para análise estatística. A mono-infecção com F. nucleatum induziu alta expressão de RANKL e TNF-a, enquanto sua modulação ocorreu devido à IL-10. A alta expressão de IFN-g no dia 20 foi regulada positivamente por E. faecalis e RANKL; TNF-a foi regulada positivamente por um mecanismo independente via IL-10 e TGF-b. A bi-associação (F. nucleatum e E. faecalis) estimulou uma alta expressão de RANKL, TNF-a e IFN-g, que parece ser modulada por TGF-b após 20 dias. A expressão gênica de citocinas pró-inflamatórias foi mais proeminente nos estágios iniciais da infecção periapical experimental, com concomitante redução no período tardio. Este fenômeno pode ser regulado por IL-10 e TGF-b em uma condição de infecção específica.

The effect of calcium hydroxide placement on pH and calcium concentration in periapical environment: An in vitro study.

Aims: Comparing the calcium concentration and pH levels of Ca(OH) 2 medicament placing in pulp chamber and root canal. Materials and Methods: Ninety-nine extracted human mandibular second premolars were instrumented to size #40 k file. Nine teeth served as the control group and the remaining teeth were assigned into two groups. Group 1-Ca(OH) 2 was placed in the dried pulp chamber, while root canals remained wet with normal saline; group 2-Ca(OH) 2 was placed in dried root canals. In control group, canals remained wet without medication. Each group was divided into 3 sub-groups of 15 teeth in which pH and calcium concentration were measured in three intervals of 2 days, 1 week, and 2 weeks by pH meter and atomic absorption spectrometer system, respectively. Findings were assessed using Kruskal-Wallis and t-test. Results: At 1 and 2 weeks, the calcium concentration had increased without being significantly different from Ca(OH) 2 placed either in the root canal or in the pulp chamber. Ca(OH) 2 placed in the pulp chamber or root canal provided similar pH values (P=0.362). Conclusions: Placing Ca(OH) 2 in pulp chamber is as effective as placing it in the root canal.

Dentin permeability of the apical third in different groups of teeth

This ex vivo study evaluated dentin permeability of the root canal in the apical third of different human groups of teeth. Eighty teeth were used, 8 from each dental group: maxillary and mandibular central incisors, lateral incisors and canines, maxillary first premolars (buccal and palatal roots), mandibular first premolars, and maxillary and mandibular second premolars, totalizing 88 roots that were distributed in 11 groups. The root canals were instrumented, irrigated with 1 percent NaOCl and 15 percent EDTA. Roots were immersed in 10 percent copper sulfate for 30 min and then in 1 percent rubeanic acid alcohol solution for the same period; this chemical reaction reveals dentin permeability by the formation of copper rubeanate, which is a dark-colored compound. Semi-serial 100-µm-thick cross-sections were obtained from the apical third of the roots. Five sections of each apical third were washed, dehydrated, cleared and mounted on glass slides for examination under optical microscopy. The percentage of copper ion infiltration and the amount of tubular dentin were quantified by morphometric analysis. The penetration of copper ions in the apical third ranged from 4.60 to 16.66 percent. The mandibular central and lateral incisors presented the highest dentin permeability (16.66 percent), while the maxillary canines and mandibular second and first premolars presented the lowest dentin permeability (4.60 percent, 4.80 percent and 5.71 percent, respectively; p<0.001). The other teeth presented intermediate permeability. In conclusion, dye penetration into dentin tubules at the apical region is strongly dependent on the group of teeth evaluated.

Este estudo ex vivo avaliou a permeabilidade da dentina do canal radicular do terço apical de diferentes grupos de dentes humanos. Foram utilizados 80 dentes, sendo 8 de cada grupo dental superior e inferior: incisivos centrais, incisivos laterais, caninos, primeiros pré-molares superiores (raízes vestibulares e palatinas), primeiros pré-molares inferiores, segundos pré-molares superiores e inferiores, totalizando 88 raízes, as quais foram distribuídas em 11 grupos. Os canais foram instrumentados, irrigados com NaOCl a 1 por cento e EDTA a 15 por cento. As raízes foram imersas em sulfato de cobre a 10 por cento por 30 min e acido rubeânico a 1 por cento pelo mesmo período. Esta reação química revela a permeabilidade da dentina por meio da formação de um complexo escurecido denominado rubeanato de cobre. Hemi-secções de 100 µm de espessura foram obtidas do terço apical da raiz. Cinco secções do terço apical foram lavadas, desidratadas, diafanizadas e montadas em lâminas para análise em microscopia óptica. A porcentagem de infiltração de íons cobre e a quantidade de dentina tubular foram quantificadas por meio de análise morfométrica. A penetração de íons cobre no terço apical da raiz variou de 4,60 por cento a 16,66 por cento. Os incisivos centrais e laterais apresentaram a maior permeabilidade dentinária (16,66 por cento), e os caninos superiores e segundos e primeiros pré-molares inferiores as menores (4,60 por cento, 4,80 por cento e 5,71 por cento, respectivamente; p<0,001). Os outros dentes apresentaram permeabilidade intermediaria. Conclui-se que a penetração de corante nos túbulos dentinários da região apical é extremamente dependente do grupo de dentes avaliado.

Effect of endodontic smear layer and various solvents on the calcium ion diffusion through radicular dentin--an in vitro study.

External root resorption and ankylosis remains the major cause of failure of replanted teeth. This study was conducted to explore the different ways to increase the pH of periradicular area in order to overcome the problem of root resorption and ankylosis. 60 freshly extracted permanent anteriors were used after removing the crown at CEJ. After biomechanical preparation Ca (OH)2 was injected and assays were done using EDTA, Citric and tannic acid Assays were repeated. Calcium diffusion and pH in the root exterior was measured using spectrophotometer. Results showed that dentin is permeable to calcium & hydroxyl ions and placement of Ca (OH)2 in the canal resulted in its increased recovery and alkaline pH periradicularly. Smear layer removal did not result in significant increase in Ca++ recovery or alkaline pH however combination of EDTA & NaOCl was found best than the other two.

Frequência do desvio apical quando do emprego de técnica escalonada e do "canal finder system" / Apical deviation in root canal preparation using the step-back technique and the canal finder system

Os autores estudaram a presença de desvio apical quando da utilizaçäo de técnica escalonada e de aparelho automatizado, o "Canal Finder System", no preparo do canal radicular. Sessenta canais simulados curvos, confeccionados em blocos de resina, foram utilizados e agrupados em número de quinze, perfazendo quatro grupos. No Grupo I, empregou-se a técnica escalonada, tendo como instrumento memória o de número 30, e, para o Grupo II, o de número 35. Os Grupos III e IV foram preparados empregando-se o "Canal Finder System" com variaçäo do instrumento memória, conforme os grupos anteriores. A menor presença de desvio apical ocorreu no Grupo III, em que o aparato mecânico foi utilizado, tendo, como instrumento memória, o de número 30, seguido do Grupo IV, que teve como memória o de número 35. A maior presença de desvio apical ocorreu nos Grupos I e II, nos quais a técnica escalonada foi empregada, tendo como instrumento memória, respectivamente, o de 30 e o de 35, com frequência de 33,3 por cento e 53,3 por cento

Avaliaçäo da ocorrência de desvio apical, tendo como fonte de variaçäo o instrumento memória, quando do emprego da técnica escalonada / The evaluate the presenca of apical zip, having a means of variation the memory file, for the step-back technique

Este trabalho objetivou estudar a ocorrência do desvio apical, tendo como fonte de variaçäo o instrumento memória, quando do emprego da técnica escalonada. Empregou-se setenta e cinco canais curvos simulados, os quais foram divididos em 3 grupos, onde desenvolveu-se a técnica escalonada, variando-se o instrumento memória nos números 25, 30 e 35. Assim, após o preparo dos canais simulados, os resultados evidenciaram o desvio apical nos três grupos, numa porcentagem de 32 por cento, 56 por cento e 76 por cento, respectivamente