Clinical evaluation of culturable bacteria, endotoxins and lipoteichoic acid in teeth with vital normal pulp tissues.

The objective of this study was to investigate the presence of culturable bacteria, endotoxins (LPS) and lipoteichoic acid (LTA) levels in teeth with normal vital pulp (NVP) with intact crowns (IC) and those with coronal restoration (CR) limited to the enamel level. A total of 20 teeth indicated for endodontic treatment due to prosthetic reasons were selected. Samples were collected from the root canals. The levels of cultivable bacteria, LPS and LTA were assessed. Statistical analyses were performed at significance level set at 5%. None of the teeth presented microbial growth. In the IC group, the LPS levels were limited to the lowest concentration of LPS. On the contrary, higher LPS and LTA levels were detected in teeth with CR. It was concluded that teeth with NVP and IC were negative for bacteria, LPS and LTA; while teeth with CR were positive for bacterial virulence factors.

Microbiological profile of root canals indicated for endodontic retreatment due to secondary endodontic infections or for prosthetic reasons.

OBJECTIVE: The objective of this study was to evaluate the microbiological profile of root canals indicated for endodontic retreatment due to secondary endodontic infections evidenced by the presence of chronic apical periodontitis (G1) or for prosthetic reasons, without clinical and radiographic signs of endodontic reinfection (G2). METHODS: Microbiological samples were collected from thirty teeth (N=30) out of which 15 were indicated for retreatment due to the presence of chronic apical periodontitis (G1) and 15 were indicated exclusively for prosthetic reasons (G2). Samples were collected from root canals before (S1), after chemomechanical preparation (S2), and after 30 days of intracanal medication composed of calcium hydroxide and 2% chlorhexidine gel (S3). The molecular analysis was performed using Nested-PCR for the detection of 17 bacterial species. The efficacy of each stage of the retreatment in reducing the microbial load was verified by counting colony-forming units (CFU). The statistical analysis considered a significance level of 5%. RESULTS: The results showed that bacteria were detected in 100% of the cases in S1, in both groups, by Nested PCR. The most frequently found species in S1 in both groups were Enterococcus faecalis, Fusobacterium nucleatum, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Parvimonas micra. The microbial load of G1 was higher than G2 in the initial samples (S1). Endodontic retreatment was effective in reducing the microbial load in G1 and G2. Statistically significant associations were found between specific bacteria and clinical signs and symptoms. CONCLUSION: It was concluded that the microbial community present in the root canal of teeth indicated for endodontic retreatment is mixed and heterogeneous. G1 and G2 differ in the number of species per case and microbial load. CLINICAL RELEVANCE: Endodontic retreatment was effective in reducing the microbial load. Statistically significant associations were found between specific bacteria and clinical signs and symptoms.

Efficacy of 6% Sodium Hypochlorite on Infectious Content of Teeth with Symptomatic Irreversible Pulpitis.

INTRODUCTION: The objective of this study was to monitor the effects of chemomechanical preparation (CMP) performed with 6% sodium hypochlorite and calcium hydroxide-based intracanal medication (ICM) on the levels and diversity of bacteria, endotoxins (lipopolysaccharides [LPS]), and lipoteichoic acid (LTA) in root canals of teeth with symptomatic irreversible pulpitis. METHODS: Samples were collected from 10 teeth with symptomatic irreversible pulpitis before CMP (S1), after CMP (S2), and after ICM (S3). The levels of bacteria, LPS, and LTA were assessed by using checkerboard DNA-DNA hybridization, LAL Pyrogent 5000, and enzyme-linked immunosorbent assay, respectively. Wilcoxon test, repeated-measures analysis of variance, and Tukey post hoc test were used for statistical analysis at a significance level of 5%. RESULTS: Forty species were detected at S1. Two species were eliminated after CMP and 5 after ICM. Resistant and pain-related species were detected in the root canals. Higher levels of culturable bacteria were detected at S1. However, CMP and ICM effectively reduced the microbial load in the root canals. Higher levels of LPS and LTA were detected at S1. CMP was effective in reducing both LPS and LTA (P < .05). ICM produced additional reduction in the levels of LPS (P > .05) and LTA (P < .05). CONCLUSIONS: Chemomechanical preparation using 6% sodium hypochlorite and calcium hydroxide-based intracanal medication were effective in reducing the levels of bacteria, LPS, and LTA in teeth with vital pulp and irreversibly inflamed pulp.

Antimicrobial Susceptibility and Characterization of Virulence Genes of Enterococcus faecalis Isolates from Teeth with Failure of the Endodontic Treatment.

INTRODUCTION: The aim of this study was to investigate the prevalence of virulence factors and the antimicrobial resistance of Enterococcus faecalis isolates of teeth with failure of the endodontic treatment. METHODS: Twenty root canal samples were collected from teeth with apical periodontitis. E. faecalis was firstly identified based on phenotypic features and then by 16S ribosomal RNA gene sequencing. The antimicrobial susceptibility was determined by the minimum inhibitory concentration (MIC) of amoxicillin, amoxicillin + clavulanate, azithromycin, benzylpenicillin, ciprofloxacin, clindamycin, chloramphenicol, doxycycline, erythromycin, gentamicin, metronidazole, moxifloxacin, rifampicin, tetracycline, and vancomycin using the E test method. Virulence factors (ace, asa, asa373, cylA, efaA, esp, and gelE) were detected by polymerase chain reaction assay. RESULTS: Amoxicillin + clavulanate was effective against all strains. Intermediate and total resistance was found against the majority of the tested antimicrobials. The susceptibility of some microorganisms to some antimicrobial agents changed according to the evaluation time. MIC50 and MIC90 also varied according to the evaluation time. In relation to the virulence factors of the E faecalis isolates, ace was detected in 100% of the strains, asa (60%), asa373 (15%), efaA (95%), esp (70%), and gelE (75%), whereas cylA was not detected. CONCLUSIONS: It was concluded that E. faecalis isolates from persistent endodontic infections showed varied degrees of intermediate/total resistance to several antimicrobial agents, with amoxicillin + clavulanate being the most effective agent. Moreover, the strains showed different patterns for virulence gene detection.

Quantification of Lipoteichoic Acid Contents and Cultivable Bacteria at the Different Phases of the Endodontic Retreatment.

INTRODUCTION: The infectious content of root canals, including bacteria and lipoteichoic acid (LTA), cause injuries to the periapical tissues. The purpose of this clinical study was to quantify the levels of both LTA and cultivable bacteria at the different phases of endodontic retreatment (ER) of teeth with post-treatment apical periodontitis. It also aimed to investigate the presence of gram-positive microorganisms before and after chemomechanical preparation (CMP) and intracanal medication (ICM). METHODS: Twenty infected root canals of single-rooted teeth were randomly assigned into 2 groups according to the chemical substance used for CMP (n = 10 per group): chlorhexidine (CHX) group, 2% CHX gel, and the sodium hypochlorite (NaOCl) group, 6% NaOCl. Root canal samples were taken using paper points before (S1) and after CMP (S2) and after 30 days of ICM with calcium hydroxide + 2% CHX gel (S3). Microorganisms were identified by the culture technique using biochemical tests. Cultivable bacteria were determined by counting the colony-forming unit. LTA levels were measured using the enzyme-linked immunosorbent assay (pg/mL). RESULTS: A total of 70 gram-positive species, out of 102 species isolated, were found in the root canals (54 in S1, 4 in S2, and 12 in S3). Enterococcus faecalis was the most frequent isolated taxon in all phases of the ER. LTA (574.0 ± 94.7) and cultivable bacteria (101.2 ± 79.2) were present in all S1 samples. CMP decreased the overall levels of cultivable bacteria by 99.4% and LTA by 24.8% (P < .05), whereas the total overall reduction level of ICM on viable bacteria was 99.5% and on LTA it was 38.6% (P < .05). CMP with 2% CHX gel (CHX group, 99.3%) was more effective (P < .05) than 6% NaOCl (NaOCl group, 92.1%) on bacterial reduction. Likewise, ICM showed a 100% reduction in the CHX group and 98.5% in the NaOCl group. Regarding the reduction of LTA, CMP with 2% CHX gel (CHX group, 26.9%) was more effective (P < .05) than 6% NaOCl (NaOCl group, 22.6%). In addition, ICM showed a 43.2% reduction in the CHX group and 36.2% in the NaOCl group (P > .05). CONCLUSIONS: The reduction rates of bacteria were higher than the LTA. Moreover, gram-positive microorganisms were present in all phases of the endodontic retreatment.

Efficacy of ethylene-diamine-tetra-acetic acid associated with chlorhexidine on intracanal medication removal: a scanning electron microscopy study.

The aim of this study was to evaluate the effectiveness of 17% ethylene-diamine-tetra-acetic acid (EDTA) used alone or associated with 2% chlorhexidine gel (CHX) on intracanal medications (ICM) removal. Sixty single-rooted human teeth with fully formed apex were selected. The cervical and middle thirds of each canal were prepared with Gates Glidden drills and rotary files. The apical third was shaped with hand files. The specimens were randomly divided into two groups depending on the ICM used after instrumentation: calcium hydroxide Ca(OH)(2) +CHX or Ca(OH)(2) +sterile saline (SS). After seven days, each group was divided into subgroups according to the protocol used for ICM removal: instrumentation and irrigation either with EDTA, CHX+EDTA, or SS (control groups). All specimens were sectioned and processed for observation of the apical thirds by using scanning electron microscopy. Two calibrated evaluators attributed scores to each specimen. The differences between the protocols for ICM removal were analyzed with Kruskal-Wallis and Mann-Whitney U tests. Friedman and Wilcoxon signed rank tests were used for comparison between the score of debris obtained in each root canal third. Remains of Ca(OH)(2) were found in all specimens independently of the protocol and ICM used (P > 0.05). Seventeen percent EDTA showed the best results in removing ICM when used alone (P < 0.05), particularly in those associated with CHX. It was concluded that the chelating agent 17% EDTA significantly improved the removal of ICM when used alone. Furthermore, the type of the vehicle associated with Ca(OH)(2) also plays a role in the ICM removal.

Microbial evaluation of traumatized teeth treated with triple antibiotic paste or calcium hydroxide with 2% chlorhexidine gel in pulp revascularization.

INTRODUCTION: Revascularization outcome depends on microbial elimination because apical repair will not happen in the presence of infected tissues. This study evaluated the microbial composition of traumatized immature teeth and assessed their reduction during different stages of the revascularization procedures performed with 2 intracanal medicaments. METHODS: Fifteen patients (7-17 years old) with immature teeth were submitted to the revascularization procedures; they were divided into 2 groups according to the intracanal medicament used: TAP group (n = 7), medicated with a triple antibiotic paste, and CHP group (n = 8), dressed with calcium hydroxide + 2% chlorhexidine gel. Samples were taken before any treatment (S1), after irrigation with 6% NaOCl (S2), after irrigation with 2% chlorhexidine (S3), after intracanal dressing (S4), and after 17% EDTA irrigation (S5). Cultivable bacteria recovered from the 5 stages were counted and identified by means of polymerase chain reaction assay (16S rRNA). RESULTS: Both groups had colony-forming unit counts significantly reduced after S2 (P < .05); however, no significant difference was found between the irrigants (S2 and S3, P = .99). No difference in bacteria counts was found between the intracanal medicaments used (P = .95). The most prevalent bacteria detected were Actinomyces naeslundii (66.67%), followed by Porphyromonas endodontalis, Parvimonas micra, and Fusobacterium nucleatum, which were detected in 33.34% of the root canals. An average of 2.13 species per canal was found, and no statistical correlation was observed between bacterial species and clinical/radiographic features. CONCLUSIONS: The microbial profile of infected immature teeth is similar to that of primarily infected permanent teeth. The greatest bacterial reduction was promoted by the irrigation solutions. The revascularization protocols that used the tested intracanal medicaments were efficient in reducing viable bacteria in necrotic immature teeth.

Evaluation of the colour change in enamel and dentine promoted by the interaction between 2% chlorhexidine and auxiliary chemical solutions.

To evaluate the colour change in enamel and dentine, promoted by interaction of 2% chlorhexidine gluconate (CHX) with 5.25% sodium hypochlorite (NaOCl) and 17% ethylenediaminetetraacetic acid (EDTA). Fragments containing enamel and dentine were obtained from the crowns of extracted bovine incisors. Before and after immersion of the samples in the substances, they were evaluated with reference to the colour of the enamel and dentine. The values obtained in numerical scores were subjected to statistical analysis using Wilcoxon test. A colour change in the enamel and dentine in groups treated with CHX gel + NaOCl and CHX gel + NaOCl + EDTA, and a change in colour only in the dentine in groups treated with CHX solution + NaOCl and CHX solution + NaOCl + EDTA. When used prior to NaOCl, CHX has the ability to induce a colour change in dental structures.

Oxidized linoleic acid metabolite-cytochrome P450 system (OLAM-CYP) is active in biopsy samples from patients with inflammatory dental pain.

Endogenous TRPV1 agonists such as oxidized linoleic acid metabolites (OLAMs) and the enzymes releasing them [eg, cytochrome P450 (CYP)] are up-regulated after inflammation in the rat. However, it is not known whether such agonists are elevated in human inflammatory pain conditions. Because TRPV1 is expressed in human dental pulp nociceptors, we hypothesized that OLAM-CYP machinery is active in this tissue type and is increased under painful inflammatory conditions such as irreversible pulpitis (IP). The aim of this study was to compare CYP expression and linoleic acid (LA) metabolism in normal vs inflamed human dental pulp. Our data showed that exogenous LA metabolism was significantly increased in IP tissues compared to normal tissues and that pretreatment with a CYP inhibitor, ketoconazole, significantly inhibited LA metabolism. Additionally, extracts obtained from LA-treated inflamed tissues evoked significant inward currents in trigeminal ganglia neurons and were blocked by pretreatment with the TRPV1 antagonist IRTX. Moreover, extracts obtained from ketoconazole-pretreated inflamed tissues significantly reduced inward currents in trigeminal ganglia neurons. These data suggest that LA metabolites produced in human inflamed tissues act as TRPV1 agonists and that the metabolite production can be targeted by CYP inhibition. In addition, immunohistochemical analysis of 2 CYP isoforms, CYP2J and CYP3A1, were shown to be predominately expressed in immune cells infiltrating the inflamed dental pulp, emphasizing the paracrine role of CYP enzymes in OLAM regulation. Collectively, our data indicate that the machinery responsible for OLAM production is up-regulated during inflammation and can be targeted to develop potential analgesics for inflammatory-induced dental pain.

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.

Barodontalgia: relato de dois casos clínicos / Barodontalgia: two case reports

O objetivo desse trabalho foi relatar dois casos clínicos sobre barodontalgia, que é um evento que envolve o aumento ou a diminuição da pressão atmosférica na prática de mergulho ou em viagens aéreas, causando sintomatologia. Os casos clínicos reportam barodontalgia na presença de vitalidade e de necrose pulpar. Tratamento endodõntico foi realizado no elemento envolvido, seguido da restauração coronária. Concluiu-se que o Cirurgião-Dentista deve ter conhecimento sobre a barodontalgia para considerá-Ia no diagnóstico diferencial da dor dentária comum.

The aim of this study was to report two cases of barodontalgia, which is an event that involves an increase or decrease of the atmospheric pressure in diving or air travei, causing symptomatology. The clinical cases reported barodontalgia in the presence of vital and necrotic pulp tissues. Endodontic treatment was performed in the tooth involved, followed by coronal restoration. It was concluded that the dentist must have the knowledge about barodontalgia in order to consider it in the differential diagnosis of common dental pain.

Deproteinization technique stabilizes the adhesion of the fiberglass post relined with resin composite to root canal.

To evaluate the effects of pretreatment of root dentin by 5.25% sodium hypochlorite (NaOCl) alone, associated with 2% chlorhexidine in gel base (CHX) and/or ethanol (EtOH), and the air-drying technique (Air) on the bond strength and adhesive durability of fiberglass post relined with resin composite to root dentin. A total of 100 bovine incisor roots were divided into 10 groups: G1 (control), irrigation with physiologic solution; G2, Air; G3, NaOCl; G4, NaOCl + Air; G5, NaOCl + EtOH; G6, NaOCl + EtOH + Air; G7, NaOCl + CHX; G8, NaOCl + CHX + Air; G9, NaOCl + CHX + EtOH; G10, NaOCl + CHX + EtOH + Air. Fiberglass post relined with resin composite was cemented and each group was randomly divided into two subgroups: 24 h of water storage and 12 months of water storage. The push-out test was performed and bond strength values were analyzed by ANOVA and Tukey's test. The use of NaOCl alone or associated with CHX had the highest values of bond strength with or without Air in the immediate and stored groups, being statistically similar to the immediate control group (p > 0.05). The groups using EtOH or Air alone had lower bond strength in the immediate and stored groups (p < 0.05). A significant decrease with the time of the bond strength in the control group was observed after 12 months of storage (p > 0.05). The use of NaOCl or NaOCl associated with CHX preserved the bond strength immediate and for 12 months. The air-drying technique and the other associations decreased the immediate bond strength values.

Comparison of endotoxin levels in previous studies on primary endodontic infections.

BACKGROUND: This study was performed to determine which of the quantitative methods, namely, chromogenic endpoint, chromogenic kinetic, and turbidimetric kinetic ones, best fit for the analysis of primary endodontic infections. METHODS: Twenty-one root canals with apical periodontitis were sampled with paper points. The same sample was analyzed by means of the endpoint chromogenic Limulus amebocyte lysate (LAL) assay (QCL), quantitative kinetic chromogenic LAL assay (KQCL), and kinetic turbidimetric LAL assay (Turbidimetric). RESULTS: All three LAL methods were effective in the recovery of endotoxin from root canal infection. Regardless of the method tested, endotoxin was detected in 100% of the root canals (21/21). The KQCL assay yielded a median value of endotoxin of 7.49 EU/mL, close to and not significantly different from those for the turbidimetric test (9.19 EU/mL) (both kinetic methods) (p > 0.05). In contrast, the endpoint QCL showed a median value of 34.20 EU/mL (p < 0.05). The comparison of the three methods revealed that both turbidimetric and KQCL methods were more precise, with best reproducibility (the coefficient variation between analysis of the root canal and its duplicate was lower than 10%). The inhibition/enhancement assay indicated a good interaction between the root canal samples with the turbidimetric method. CONCLUSION: This study has revealed that quantitative kinetic-turbidimetric and kinetic-chromogenic LAL methods are best fitted for the analysis of endotoxins in root canal infection, both being more precise and allowing better reproducibility compared with the endpoint-QCL assay.

Efeito antimicrobiano de diferentes substâncias químicas associadas ao preparo mecânico e da medicação intracanal em dentes de cães portadores de lesões periapicais induzidas / Efficacy of chemo-mechanical preparation with different substances and the use of a root canal medication in dog’s teeth with induced periapical lesion

Objetivo: avaliar o efeito antimicrobiano de diferentes substâncias químicas auxiliares durante o preparo mecânico de canais radiculares e de uma medicação intracanal.Métodos: foram utilizadas 55 raízes de dentes de cães portadores de lesões periapicais, divididas em cinco grupos experimentais de acordo com a substância química empregada durante o preparo mecânico: GI – solução salina; GII– natrosol gel; GIII – NaOCl 2,5%; GIV – CHX 2% gel; GV– CHX 2% solução. Foram realizadas coletas microbiológicas antes (s1) e após (s2) o preparo químico-mecânico e após o emprego de uma medicação à base de hidróxido de cálcio por 14 dias (s3). Após cada coleta, as amostras foram processadas e realizadas as contagens das unidades formadoras de colônias (UFC). Resultados: em s1, a contagem de UFCs variou de 5,5 x 105 a 1,5 x 106. Esses valores diminuíram significativamente (p<0,05) em s2. Não foi encontrada diferença significativa entre a coleta s2 e s3 (p>0,05).Conclusões: dentre as substâncias testadas, NaOCl 2,5%e CHX gel 2% demonstraram maior potencial antimicrobiano contra patógenos endodônticos in vivo

Objectives: to evaluate the effect of instrumentation, irrigationwith different substances and the use of calcium hydroxideon bacterial load and microbiota profile in dog’s teethwith pulp necrosis and periapical lesion. Methods: Fifty fiveroot canals were divided into groups: I) Saline (SSL) (n=11);II) natrosol gel (n=11); III) 2.5% NaOCl (n=11); IV) 2%CHX-gel (n=11); V) 2% CHX-solution (n=11). Endodonticsamples were cultured, microorganisms counted and themicrobiota analyzed at different sampling times — s1, s2and s3. Results: At s1, the mean CFU counts ranged from5.5 x105 to 1.5 x 106. These values dropped significantly ats2 (p<0.05). No statistical significant difference was foundbetween s2 and s3. Changes in root canal microbiota werefound at s2 and s3. Conclusion: Regardless the use of calciumhydroxide as a root canal medication, 2.5% NaOCl and2% CHX-gel demonstrated a potent antimicrobial activityagainst endododontic pathogens in vivo.

Clinical investigation of the efficacy of chemomechanical preparation with rotary nickel-titanium files for removal of endotoxin from primarily infected root canals.

INTRODUCTION: This clinical study was conducted to investigate the ability of chemomechanical preparation with 2.5% NaOCl + 17% ethylenediaminetetraacetic acid (EDTA) and rotary nickel-titanium (NiTi) system in removing endotoxin from primary root canal infection with apical periodontitis. METHODS: Twenty-one root canals with necrotic pulps were selected. Samples were collected before (s1) and after chemomechanical preparation (s2). The limulus amebocyte lysate (LAL) assay was used to quantify endotoxins. RESULTS: The LAL assay indicated that endotoxins were present in 100% of the root canals investigated (19/19) before (s1) and after chemomechanical preparation (s2). Analyses of the quantitative data revealed that the endotoxin content was significantly reduced at s2 (98.06%) compared with that at s1 (P < .05). CONCLUSIONS: Our findings indicate that chemomechanical preparation with 2.5% NaOCl + 17% EDTA and rotary NiTi files was effective in reducing endotoxin load in the root canal infection from primarily infected teeth with apical periodontitis.