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-1ß 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 ß 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-1ß 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-1ß 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.

Comparison of Fusobacterium nucleatum and Porphyromonas gingivalis Lipopolysaccharides Clinically Isolated from Root Canal Infection in the Induction of Pro-Inflammatory Cytokines Secretion

Abstract 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.

Resumo O 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 semelhante.

Clinical comparison of the effectiveness of single-file reciprocating systems and rotary systems for removal of endotoxins and cultivable bacteria from primarily infected root canals.

INTRODUCTION: This clinical study was conducted to compare the effectiveness of single-file reciprocating systems and rotary systems in removing endotoxins and cultivable bacteria from primarily infected root canals. METHODS: Forty-eight primarily infected root canals were selected and randomly divided into 4 groups: WaveOne (Dentsply Maillefer, Ballaigues, Switzerland) (n = 12); Reciproc (VDW, Munich, Germany) (n = 12), ProTaper (Dentsply Maillefer) (n = 12), and Mtwo (VDW) (n = 12). Samples were collected before and after chemomechanical preparation. The irrigation was performed by using 2.5% sodium hypochlorite. A chromogenic limulus amebocyte lysate assay test was used to quantify endotoxins. Culture techniques were used to determine bacterial colony-forming unit counts. RESULTS: In the baseline samples (ie, samples collected before chemomechanical preparation), endotoxins and cultivable bacteria were recovered from 100% of the root canal samples. No differences were found in the median percentage values of endotoxin reduction achieved with reciprocating systems (ie, WaveOne [95.15%] and Reciproc [96.21%]) and with rotary systems (ie, ProTaper [97.98%] and Mtwo [96.34%]) (P < .05). Both single-file reciprocating systems (ie, WaveOne [99.45%] and Reciproc [99.93%]) and rotary systems (ProTaper [99.85%] and Mtwo [99.41%]) were effective in reducing the cultivable bacteria (all P < .05). Moreover, the culture analysis revealed no differences in bacterial load reduction (P > .05). CONCLUSIONS: Both single-file reciprocating systems (ie, WaveOne and Reciproc instruments) and rotary systems (ie, ProTaper and Mtwo instruments) showed similar effectiveness in reducing endotoxins and cultivable bacteria from primarily infected root canals, but they were not able to eliminate them from all root canals analyzed.

Quantitative and qualitative analysis of microorganisms in root-filled teeth with persistent infection: Monitoring of the endodontic retreatment.

OBJECTIVE: The aim of this study was to investigate in vivo microorganisms detected in root-filled teeth with post-treatment apical periodontitis and quantify colony-forming units (CFU) during endodontic retreatment. MATERIALS AND METHODS: Fifteen root-filled teeth had their previous gutta-percha removed and were randomly instrumented before being divided into three groups and medicated with either [Ca(OH)2 + 2% CHX gel], [Ca(OH)2 + 0.9% NaCl] or 2% CHX gel. Samples were taken after removal of gutta-percha (S1), after chemomechanical preparation using 2% CHX gel (S2), and after inter-appointment dressing (S3) for 7 or 14 days later. Cultivable bacteria recovered from infected root canals at the three stages were counted and identified by means of culture and PCR assay (16S rDNA). Quantitative data were statistically analyzed by using Mann-Whitney test in which pairs of groups were compared (P < 0.05). RESULTS: CFU counts decreased significantly from S1 to S2 (P < 0.05). No significant difference was found between S2 and S3 (P = 0.3093) for all three experimental groups. Chemomechanical preparation and intra-canal dressing promoted significant median reductions of 99.61% and 99.57%, respectively, in the number of bacteria compared to S1 samples. A total of 110 cultivable isolates were recovered by culture technique from 32 different species and 7 different genera. Out of the 13 target species-specific primer of bacteria analyzed, 11 were detected during endodontic retreatment. CONCLUSION: The great majority of taxa found in post-treatment samples were Gram-positive bacteria, although Gram-negative bacteria were found by molecular methods. Moreover, our results showed that gutta-percha removal and chemomechanical preparation are effective for root canal disinfection, whereas additional intra-canal dressing did not improve disinfection.

Comparison of endotoxin levels found in primary and secondary endodontic infections.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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 < .05). The median value of endotoxins found in the presence of clinical symptoms was significantly higher than in asymptomatic teeth with primary infections (P < .05). A positive correlation was found between endotoxin contents and a larger size of the radiolucent area (>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). CONCLUSIONS: 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.

Persistent extraradicular infection in root-filled asymptomatic human tooth: scanning electron microscopic analysis and microbial investigation after apical microsurgery.

INTRODUCTION: Procedural accidents have a negative effect on healing and might contribute to the persistence of infections in inaccessible apical areas, requiring surgical intervention. This report describes a case of persistent apical periodontitis of a lower left first molar associated with the sinus tract and a periapical lesion that required nonsurgical endodontic retreatment and apical surgery for resolution. METHODS: The tooth had received endodontic treatment 3 years ago and had to be retreated using the crown-down technique with chemical auxiliary substance (2% chlorhexidine gel), foramen patency, and enlargement and was filled in a single appointment. The occlusal access cavity was immediately restored with composite resin. After 1 month, it could be observed that the sinus tract persisted and, radiographically, the lesion remained unaltered. Therefore, endodontic microsurgery was indicated. Apical microsurgery was performed under magnification with the use of a dental operating microscope including apicectomy, root end with ultrasound, and sealing with mineral trioxide aggregate. A microbiological sample was collected from the apical lesion. The resected distal root apex was observed by scanning electron microscopy. RESULTS: The following species were detected: Actinomyces naeslundii and Actinomyces meyeri, Propionibacterium propionicum, Clostridium botullinum, Parvimonas micra, and Bacteroides ureolyticus; scanning electron microscopic analysis revealed bacterial biofilm surrounding the apical foramen and external radicular surface. Gutta-percha overfilling at the apex because of a zip caused during initial endodontic treatment could be observed. A 6-month follow-up showed apparent radiographic periapical healing, which progressed after 24 months. CONCLUSION: Gram-positive anaerobic bacteria and extraradicular biofilm seem to participate in the maintenance of persistent periapical pathology, and endodontic retreatment followed by periapical microsurgery proved to be a successful alternative in the resolution of persistent extraradicular infections.