Comparison Of Fusobacterium Nucleatum And Porphyromonas Gingivalis Lipopolysaccharides Clinically Isolated From Root Canal Infection In The Induction Of Pro-inflammatory Cytokines Secretion

The aim of this study was to compare the biological activity of lipopolysaccharides (LPS) purified from Fusobacterium nucleatum and Porphyromonas gingivalis strains, both isolated from primary endodontic infection (PEI) in the levels of IL-1β and TNF-α released by macrophage cells. Moreover, LPS was purified from F. nucleatum and P. gingivalis American Type Collection (ATCC) and its biological activity was compared to respectively clinical isolates strains. F. nucleatum and P. gingivalis strains clinically isolated from PEI had their identification confirmed by sequencing the 16S rRNA gene. LPS from F. nucleatum and P. gingivalis and their respective ATCC strains were extracted by using Tri-reagent method. Macrophages (Raw 264.7) were stimulated with LPS at 100 ng/mL for 4, 8 and 12 h. Secretion of IL-1 β and TNF-α was also determined. Paired t-test, repeated measures ANOVA and one-way ANOVA were employed. All LPS induced significant production of IL-1β and TNF-α, with the former being secreted at higher levels than the latter in all time-points. F. nucleatum induced a higher expression of both cytokines compared to P. gingivalis (p<0.05). No differences were observed between clinical and ATCC strains, as both presented the same potential to induce pro-inflammatory response. It was concluded that F. nucleatum and P. gingivalis LPS presented different patterns of activation against macrophages as seen by the IL-1β and TNF-α production, which may contribute to the immunopathogenesis of apical periodontitis. Moreover, clinical and ATCC strains grown under the same in vitro environment conditions presented similar biological activity.27220220

Comparison of fusobacterium nucleatum and porphyromonas gingivalis lipopolysaccharides clinically isolated from root canal infection in the induction of pro-inflammatory cytokines secretion

The aim of this study was to compare the biological activity of lipopolysaccharides (LPS) purified from Fusobacterium nucleatum and Porphyromonas gingivalis strains, both isolated from primary endodontic infection (PEI) in the levels of IL-1β and TNF-α released by macrophage cells. Moreover, LPS was purified from F. nucleatum and P. gingivalis American Type Collection (ATCC) and its biological activity was compared to respectively clinical isolates strains. F. nucleatum and P. gingivalis strains clinically isolated from PEI had their identification confirmed by sequencing the 16S rRNA gene. LPS from F. nucleatum and P. gingivalis and their respective ATCC strains were extracted by using Tri-reagent method. Macrophages (Raw 264.7) were stimulated with LPS at 100 ng/mL for 4, 8 and 12 h. Secretion of IL-1 β and TNF-α was also determined. Paired t-test, repeated measures ANOVA and one-way ANOVA were employed. All LPS induced significant production of IL-1β and TNF-α, with the former being secreted at higher levels than the latter in all time-points. F. nucleatum induced a higher expression of both cytokines compared to P. gingivalis (p<0.05). No differences were observed between clinical and ATCC strains, as both presented the same potential to induce pro-inflammatory response. It was concluded that F. nucleatum and P. gingivalis LPS presented different patterns of activation against macrophages as seen by the IL-1β and TNF-α production, which may contribute to the immunopathogenesis of apical periodontitis. Moreover, clinical and ATCC strains grown under the same in vitro environment conditions presented similar biological activity272202207CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP302575/2009-0; 150557/2011-6; 308162/2014-510/19136-1; 10/17877- 4; 11/50051-5; 11/50510-0; 11/09047-4O objetivo deste estudo foi comparar a atividade biológica de lipopolissacarídeos (LPS) purificados a partir de linhagens de Fusobacterium nucleatum e Porphyromonas gingivalis, ambas isoladas de infecções endodônticas primárias (IEP) nos níveis de IL-1β e TNF-α produzidos por macrófagos. Adicionalmente, LPS foi purificado de F. nucleatum e P. gingivalis "American Type Collection" (ATCC) e sua atividade comparada às respectivas linhagens clinicamente isoladas. Linhagens de F. nucleatum e P. gingivalis isoladas clinicamente de IEP tiveram sua identificação confirmada por sequenciamento do gene 16S rRNA. LPS de F. nucleatum e P. gingivalis e das respectivas linhagens foram extraídos com o uso do método "Tri-reagent". Macrófagos (Raw 264.7) foram estimulados com LPS a 100 ng/mL por 4, 8 e 12 h. A secreção de IL-1β e de TNF-α foi determinada. Foram usados os testes t-pareado, ANOVA de medidas repetidas e ANOVA de um fator. Todos os LPS induziram a produção significante de IL-1β e TNF-α, sendo o primeiro secretado em mais altas concentrações que o último em todos os tempos avaliados. F. nucleatum induziu uma maior expressão de ambas as citocinas comparativamente ao P. gingivalis (p<0,05). Não foram observadas diferenças entre as linhagens clínica e ATCC, uma vez que ambas apresentaram o mesmo potencial de indução da resposta pró-inflamatória. Conclui-se que F. nucleatum e P. gingivalis possuem diferentes padrões de ativação dos macrófagos, como visto pela produção de IL-1β e TNF-α, o que pode contribuir para a imunopatogênese da periodontite apical. Ainda, linhagens clínica e ATCC mantidas no mesmo ambiente in vitro apresentaram ativação biológica semelhant

Comparison Of Endotoxin Levels Found In Primary And Secondary Endodontic Infections.

This clinical study was conducted to compare the levels of endotoxins (lipopolysaccharides [LPSs]) found in primary and secondary endodontic infections with apical periodontitis by correlating LPS contents with clinical/radiographic findings. In addition, the presence of target gram-negative anaerobic bacteria was also investigated. Samples were taken from 15 root canals with primary infections and 15 with secondary infections by using paper points. The limulus amebocyte lysate assay was used to quantify endotoxins, and the polymerase chain reaction technique (16S rDNA) was used for bacterial investigation. Endotoxins were detected in 100% of the root canal samples collected from primary (15/15) and secondary (15/15) infections with median values of 7.49 EU/mL and 3.96 EU/mL, respectively (P 3 mm) (P < .05). Prevotella nigrescens (10/15, 4/15), Fusobacterium nucleatum (5/15, 1/15), Treponema denticola (3/15, 1/15), and Treponema socranskii (5/15, 1/15) were detected in teeth with primary and secondary infections, respectively. P. endodontalis was present only in teeth with primary infections (5/15). Teeth with primary endodontic infections had higher contents of endotoxins and a more complex gram-negative bacterial community than teeth with secondary infections. Moreover, the levels of endotoxins were related to the severity of bone destruction in periapical tissues as well as the development of clinical features in teeth with primary infections.381082-

Comparison Of 2.5% Sodium Hypochlorite And 2% Chlorhexidine Gel On Oral Bacterial Lipopolysaccharide Reduction From Primarily Infected Root Canals.

This clinical study was conducted to compare the efficacy of chemomechanical preparation with 2.5% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) gel on eliminating oral bacterial lipopolysaccharide (LPS) in teeth with pulp necrosis and apical periodontitis. Fifty-four root canals were selected. Samples were collect before (s1) and after chemomechanical preparation (s2). Teeth were randomly divided into groups: GI, 2.5% NaOCl (n = 27) and GII, CHX gel (n = 27). Limulus amebocyte lysate assay was used to quantify endotoxins. Endotoxin was present in 100% of the samples investigated, with a median value of 272 endotoxin units (EU)/mL (GI) and of 152.46 EU/mL (GII). As a result of chemomechanical preparation, LPS content was reduced to a median value of 86 EU/mL (GI) and 85 EU/mL (GII). Higher percentage value of endotoxin reduction was found in GI (P < .05). The 2.5% NaOCl and 2% CHX gel were not effective in eliminating endotoxin from the primarily infected root canals.351350-

Proinflammatory Activity Of Primarily Infected Endodontic Content Against Macrophages After Different Phases Of The Root Canal Therapy.

This study investigated the presence of target bacterial species and the levels of endotoxins in teeth with apical periodontitis. Levels of inflammatory mediators (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) were determined after macrophage stimulation with endodontic content after different phases of endodontic therapy using different irrigants. Thirty primarily infected root canals were randomly assigned into 3 groups according to the irrigant used for root canal preparation (n = 10 per group): GI: 2.5% sodium hypochlorite, GII: 2% chlorhexidine gel, and GIII (control group): saline solution. Root canal samples were taken by using paper points before (s1) and after root canal instrumentation (s2), subsequently to 17% EDTA (s3), after 30 days of intracanal medication (Ca[OH]2 + saline solution) (s4), and before root canal obturation (s5). Polymerase chain reaction (16S recombinant DNA) and limulus amebocyte lysate assay were used for bacterial and endotoxin detection, respectively. Macrophages were stimulated with the root canal contents for IL-1β/TNF-α measurement using enzyme-linked immunosorbent assay. Porphyromonas gingivalis (17/30), Porphyromonas endodontalis (15/30), and Prevotella nigrescens (11/30) were the most prevalent bacterial species. At s1, endotoxins were detected in 100% of the root canals (median = 32.43 EU/mL). In parallel, substantial amounts of IL-1β and TNF-α were produced by endodontic content-stimulated macrophages. At s2, a significant reduction in endotoxin levels was observed in all groups, with GI presenting the greatest reduction (P < .05). After a root canal rinse with EDTA (s3), intracanal medication (s4), and before root canal obturation (s5), endotoxin levels reduced without differences between groups (P < .05). IL-1β and TNF-α release decreased proportionally to the levels of residual endotoxin (P < .05). Regardless of the use of sodium hypochlorite or CHX, the greatest endotoxin reduction occurs after chemomechanical preparation. Increasing steps of root canal therapy associated with intracanal medication enhances endotoxin reduction, leading to a progressively lower activation of proinflammatory cells such as macrophages

Macrophage Cell Activation with Acute Apical Abscess Contents Determined by Interleukin-1 Beta and Tumor Necrosis Factor Alpha Production

Introduction: This clinical study has investigated the antigenic activity of bacterial contents from exudates of acute apical abscesses (AAAs) and their paired root canal contents regarding the stimulation capacity by levels of interleukin (IL)-1 beta and tumor necrosis factor alpha (TNF-alpha) throughout the root canal treatment against macrophage cells. Methods: Paired samples of infected root canals and exudates of AAAs were collected from 10 subjects. Endodontic contents were sampled before (root canal sample [RCS] 1) and after chemomechanical preparation (RCS2) and after 30 days of intracanal medication with calcium hydroxide + chlorhexidine gel (Ca[OH](2) + CHX gel) (RCS3). Polymerase chain reaction (16S rDNA) was used for detection of the target bacteria, whereas limulus amebocyte lysate was used to measure endotoxin levels. Raw 264.7 macrophages were stimulated with AAA exudates from endodontic contents sampled in different moments of root canal treatment. Enzyme-linked immunosorbent assays were used to measure the levels of TNF-alpha and 11-1 beta. Results: Parvimonas micra, Porphyromonas endodontalis, Dialister pneumosintes, and Prevotella nigrescens were the most frequently detected species. Higher levels of endotoxins were found in samples from periapical exudates at RCS1 (P < .005). In fact, samples collected from periapical exudates showed a higher stimulation capacity at RCS1 (P < .05). A positive correlation was found between endotoxins from exudates with IL-1 beta (r = 0.97) and TNF-alpha (r = 0.88) production (P < .01). The significant reduction of endotoxins and bacterial species achieved by chemomechanical procedures (RCS2) resulted in a lower capacity of root canal contents to stimulate the cells compared with that at RCS1 (P < .05). The use of Ca(OH)(2) + CHX gel as an intracanal medication (RCS3) improved the removal of endotoxins and bacteria from infected root canals (P < .05) whose contents induced a lower stimulation capacity against macrophages cells at RCS1, RCS2, and RCS3 (P < .05). Conclusions: AAA exudates showed higher levels of endotoxins and showed a greater capacity of macrophage stimulation than the paired root canal samples. Moreover, the use of intracanal medication improved the removal of bacteria and endotoxins from infected root canals, which may have resulted in the reduction of the inflammatory potential of the root canal content.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Quantification of Endotoxins in Infected Root Canals and Acute Apical Abscess Exudates: Monitoring the Effectiveness of Root Canal Procedures in the Reduction of Endotoxins

Introduction: This clinical study was conducted to measure the endotoxin levels in infected root canals (RCs) and exudates related to acute apical abscesses (AAAs). In addition, the effectiveness of RC procedures in reducing the endotoxin levels in RCs was monitored. Methods: Paired samples of infected RCs and exudates from AAAs were collected from 10 subjects by using paper points. RCs samples were collected before (RCS1) and after chemomechanical preparation (CMP) (RCS2), after 17% EDTA (RCS3), and after 30 days of intracanal medication (Ca[OH](2) + chlorhexidine) (RCS4). A turbidimetric kinetic limulus amebocyte lysate assay was used for the measurement of endotoxins. Results: Endotoxins were detected in 100% of the baseline samples of AAAs and RCs (RCS1) with median values of 175 EU/mL and 41.5 EU/mL, respectively (P < .05). After CMP (RCS2), endotoxins were reduced to a median value of 0.54 EU/mL (P < .05). Subsequent irrigation with EDTA (RCS3) failed to present a significant effectiveness in reducing the endotoxin levels (median = 0.37 EU/mL) (P = .07). However, intracanal medication for 30 days (RCS4) reduced endotoxins to median values of 0.03 EU/mL (P < .01). Conclusions: The present study revealed a strong association between the high levels of endotoxins found in AAAs and RCs collected from the same tooth. Moreover, the effectiveness of CMP in reducing the endotoxin levels from RCs in acute endodontic infection was improved by the use of RC medication.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Advancing Photodynamic Therapy for Endodontic Disinfection with Nanoparticles: Present Evidence and Upcoming Approaches

The persistence of microorganisms in the root canal system is one of the leading causes of root canal treatment failure. Root canal anatomy is complex, and it is often a challenge to obtain optimal disinfection. Biofilms of putative pathogens hidden inside dentin tubules and other root canal ramifications may limit current disinfection protocols. The search for additional disinfection of the root canal has been intensely carried out over the last twenty years. Antimicrobial photodynamic therapy (aPDT) is an adjunctive, conservative, non-selective bacterial kill approach. aPDT has been used to improve root canals disinfection without inducing bacterial resistance. This review focuses on the up-to-date aPDT performance and upcoming promising strategies for disinfection of the root canal system. First, we summarized the barriers encountered by photosensitizer (PS) and light delivery applied to root canal disinfection. Second, we compile the most updated clinical literature. A systematic search for scientific articles was conducted in PubMed, MEDLINE, SCOPUS, and EMBASE to screen the related in vivo studies about this theme. Third, we summarized and critically analyzed the current developments to overcome the aPDT limitations, and we revealed upcoming perspectives in this scoping literature review. We present a timely and opportune review article focusing on the significant potential of aPDT in endodontic disinfection. aPDT offers multiple capabilities that may be considered toward the root canal system’s disinfection with future outlooks in nanosized-platforms’ design and performance