Outcomes of apexification in immature traumatised necrotic teeth and risk factors for premature tooth loss: A 20-year longitudinal study.

BACKGROUND/AIM: To evaluate the long-term survival of immature traumatized incisors with pulp necrosis and apical periodontitis after endodontic treatment with two apexification techniques (calcium hydroxide apexification and MTA-apical plug) and to identify major factors affecting the survival of these teeth. MATERIALS AND METHODS: Records of 2400 children and adolescents were screened for presence of traumatic dental injuries to immature incisors where endodontic treatment with the two apexification techniques was performed during January 2003 and December 2022, compared to a control group of mature teeth treated with conventional endodontic techniques. The studied variables were age; sex; apexification technique, presence of luxation and hard tissue injuries; preoperative root development stage (RDS), preoperative and postoperative periapical index (PAI), the time-point for tooth loss, and overall survival time in years. Kaplan-Meier estimates were used to graphically present the survival functions and Cox proportional hazard model to calculate hazard ratios (HR, 95% CI). RESULTS: The median survival time was 10 years for calcium hydroxide apexification, 16.1 for MTA-apexification, for luxation injuries other than intrusions and avulsions 15.5 years, for intrusions 12.5 years and for avulsions 6.8 years. The variables with significant negative impact on tooth survival were calcium hydroxide apexification, avulsion and postoperative PAI 3-5. No significant relationships were found for the variables MTA apexification, concussion; subluxation; lateral luxation; extrusion, intrusion, hard tissue injuries, preoperative RDS and PAI scores and postoperative PAI 1-2. After adjustment, the risk for premature tooth loss was 13.5 times higher in calcium hydroxide apexification, approximately 2 to 4 times higher in PAI 3-5, and 5.6 times higher in avulsions. CONCLUSIONS: Calcium hydroxide apexification, avulsion, and postoperative PAI 3-5 were identified as prognostic variables with significant negative impact on the risk for premature tooth loss.

Endodontic operative field asepsis: a comparison between general dentists and specialists.

OBJECTIVE: The aim was to evaluate the establishment of an aseptic endodontic operative field in general dentistry by assessing general dentists' ability to reduce the amount of contamination to a non-cultivable level, and to compare the operative field asepsis at a general dentistry clinic with that at an endodontic specialist clinic. MATERIALS AND METHODS: A total of 353 teeth were included in the study (153 in general dentistry, 200 at the specialist clinic). After isolation, control samples were taken, the operative fields disinfected with 30% hydrogen peroxide (1 min) followed by 5% iodine tincture or .5% chlorhexidine solution. Samples were collected from the access cavity area and buccal area, placed in a fluid thioglycolate medium, incubated (37°, 7 d), evaluated for growth/non-growth. RESULTS: Significantly more contamination was observed at the general dentistry clinic (31.6%, 95/301), than at the endodontic specialist clinic (7.0%, 27/386) (p <.001). In general dentistry, significantly more positive samples were collected in the buccal area than in the occlusal area. Significantly more positive samples were collected when the chlorhexidine protocol had been used, both in general dentistry (p <.001) and at the specialist clinic (p =.028). CONCLUSIONS: The result from this study shows insufficient endodontic aseptic control in general dentistry. At the specialist clinic, both disinfection protocols were able to reduce the amount of microorganisms to a non-cultivable level. The observed difference between the protocols may not reflect a true difference in the effectiveness of the antimicrobial solutions, as confounding factors may have contributed to the result.

Successful rehabilitation after multiple severe complications following orthognathic surgery: a case report.

BACKGROUND: Complications of orthognathic surgery are quite rare, but they cause suffering in affected individuals. The range of complications is broad and includes both hard and soft tissue. CASE PRESENTATION: We here present a case of a fully healthy woman without signs of impaired healing capacity. The patient underwent bimaxillary orthognathic surgery and experienced multiple complications both peri- and post-operatively. During the post operative period, the patient also suffered from soft tissue complications after an orthopaedic injury. Therefore, we referred the patient to her general practitioner for further medical investigation. We also present the result after restorative surgery and endodontic and prosthodontic treatment resulting in a successful rehabilitation. CONCLUSION: This case report clearly shows the need for a good collaboration between different odontological and medical fields to achieve a good and predictable result. In situations where normal healing processes do not occur, in-depth analysis must be carried out. HIGHLIGHTS: Orthognathic surgery affects soft and hard tissue which can result in adverse healing and complications. It is of great importance to follow up performed surgery to see late complications. Be restrictive with early re-operations when there are signs of necrosis. Always use a multidisciplinary approach when handling complications after surgery.

Endodontic pulp revitalization in traumatized necrotic immature permanent incisors: Early failures and long-term outcomes-A longitudinal cohort study.

AIM: This prospective cohort study evaluates clinical and radiographical outcomes of endodontic pulp revitalization (PR) of traumatized necrotic incisors. METHODOLOGY: Pulp revitalization was performed in 75 traumatized necrotic immature incisors from 71 patients. The radiographic outcome measures were continued root formation (width and length), root resorption, apex closure, periapical index, and root development stage. The clinical outcome measures were percussion pain, palpation pain, pathological tooth mobility, swelling, sinus tract, ankylosis, crown discolouration, response to pulp sensitivity test, and subjective pain. Treatment outcomes were categorized as a success based on the absence of clinical symptoms and when radiographic evidence was present for apical healing and continued root development. The performed statistical tests were repeated measures anova, pairwise comparisons of interactions (t-test), McNemar's test, and linear regression model. RESULTS: In 45 of 75 teeth (60%), PR was successful with the resolution of clinical and radiographic signs and continued root development. PR failed due to the absence of bleeding (n = 19) and persistent infection (n = 11). PR showed statistically significant increases in root length (11%), and dentinal wall thickness (30%), root maturation (pre-operative 3.38 [CI 1.88; 4.88]; post-operative 4.04, [CI 2.56; 5.52]) apical closure (71.4%), healing of pre-operative apical periodontitis (100%), and healing of pre-operative inflammatory root resorptions (100%). Three predictive variables for continued root maturation were identified - root development stage at entry (p = .0001, ß 0.649), [CI 0.431; 0.867], trauma to the soft tissues (p = .026, ß -0.012), [CI -0.0225; -0.015], and pre-operative dentinal wall thickness (p = .009, ß -0.001); [CI -0.001; 0.0001]. CONCLUSIONS: Our findings indicate that PR provides satisfactory clinical and radiographical outcomes in traumatized necrotic incisors. The failed cases were related to lack of bleeding and persistent infections, indicating that new techniques are needed to improve the predictability of PR.

Transcriptome Analysis Reveals Modulation of Human Stem Cells from the Apical Papilla by Species Associated with Dental Root Canal Infection.

Interaction of oral bacteria with stem cells from the apical papilla (SCAP) can negatively affect the success of regenerative endodontic treatment (RET). Through RNA-seq transcriptomic analysis, we studied the effect of the oral bacteria Fusobacterium nucleatum and Enterococcus faecalis, as well as their supernatants enriched by bacterial metabolites, on the osteo- and dentinogenic potential of SCAPs in vitro. We performed bulk RNA-seq, on the basis of which differential expression analysis (DEG) and gene ontology enrichment analysis (GO) were performed. DEG analysis showed that E. faecalis supernatant had the greatest effect on SCAPs, whereas F. nucleatum supernatant had the least effect (Tanimoto coefficient = 0.05). GO term enrichment analysis indicated that F. nucleatum upregulates the immune and inflammatory response of SCAPs, and E. faecalis suppresses cell proliferation and cell division processes. SCAP transcriptome profiles showed that under the influence of E. faecalis the upregulation of VEGFA, Runx2, and TBX3 genes occurred, which may negatively affect the SCAP's osteo- and odontogenic differentiation. F. nucleatum downregulates the expression of WDR5 and TBX2 and upregulates the expression of TBX3 and NFIL3 in SCAPs, the upregulation of which may be detrimental for SCAPs' differentiation potential. In conclusion, the present study shows that in vitro, F. nucleatum, E. faecalis, and their metabolites are capable of up- or downregulating the expression of genes that are necessary for dentinogenic and osteogenic processes to varying degrees, which eventually may result in unsuccessful RET outcomes. Transposition to the clinical context merits some reservations, which should be approached with caution.

Actinomyces radicidentis and Actinomyces haliotis, coccoid Actinomyces species isolated from the human oral cavity.

There are few reports on the bacterial species Actinomyces radicidentis in the literature. In this study, putative A. radicidentis isolates were collected from 16 root canal samples from 601 examined patients. The isolates were examined by biochemical tests, 16S rRNA gene sequencing, Arbitrarily-primed (AP-) PCR, antibiotic susceptibility testing, and MALDI-TOF analyses. In parallel, two A. radicidentis reference strains and two putative A. radicidentis isolates from United Kingdom were tested. Sixteen of the 18 isolates were confirmed as A. radicidentis. The remaining two isolates, both of which were isolated from root canals (one from Sweden and the other from the UK), but were identified as Actinomyces haliotis by sequencing âˆ¼ 1300 base pairs of the 16S rRNA-gene. This isolates had a divergent, but between them similar, AP-PCR pattern, and a common distribution of sequence signatures in the 16S rRNA gene, but were not identified by MALDI-TOF. A. haliotis is a close relative to A. radicidentis, hitherto only been described from a sea-snail. The identity of A. haliotis was confirmed by a phylogenetic tree based on 16S rRNA gene sequences with species specific sequences included, and by additional biochemical tests. The examined bacteria exhibited similar antibiotic susceptibility patterns when tested for 10 separate antibiotic classes with E-tests (bioMérieux). The MIC90 for ß-lactams (benzylpenicillin and cefuroxime) and vancomycin was 0.5 mg/L, for colistin and ciprofloxacin 8 mg/mL and for the other antibiotic classes ≤ 25 mg/mL The isolation of A. haliotis from infected dental root canals cast doubt on the accepted opinion that all Actinomyces infections have an endogenous source.

Microbiological assessment of success and failure in pulp revitalization: a randomized clinical trial using calcium hydroxide and chlorhexidine gluconate in traumatized immature necrotic teeth.

Aim: To compare differences in the disinfection efficacy of calcium hydroxide (CH) and chlorhexidine gluconate (CHD) dressings in pulp revitalization (PR) of traumatized immature necrotic teeth; to investigate the microflora in successful/failed PR and whether bacterial persistence influences the outcomes of PR. Methods: Microbiological assessment of the average bacterial load (CFU/sample) and bacterial diversity (taxa/sample) was performed on 41 teeth at three timepoints (S2-before, S3-after debridement and S5- after root canal dressing). Results: The primary microflora was more diverse in successful cases than in failed. Decreases in CFU/sample and taxa/sample occurred S2 - S3, though new increases occurred at S5 in the CHD subgroup (successful and failed) and CFU/sample in the CH subgroup (failed). At S5, the successful cases showed more bacterial decreases. No specific species was associated with the outcomes with no statistical differences between the disinfection efficacy. Conclusions: There were no statistical differences in CH and CHD efficacy. At S5, microflora persisted in both successful and failed outcomes, but the abundance and diversity increased significantly only in the failed cases. The successful outcomes presented higher diversity and higher decreases of the primary microflora at S5 than the failed outcomes. The abundance and diversity increased significantly at S5 only in failed cases.

Gram-negative quorum sensing signalling enhances biofilm formation and virulence traits in gram-positive pathogen Enterococcus faecalis.

Acyl-homoserine lactones (AHLs) are typical quorum-sensing molecules of gram-negative bacteria. Recent evidence suggests that AHLs may also affect gram-positives, although knowledge of these interactions remains scarce. Here, we assessed the effect of AHLs on biofilm formation and transcriptional regulations in the gram-positive Enterococcus faecalis. Five E. faecalis strains were investigated herein. Crystal violet was employed to quantify the biomass formed, and confocal microscopy in combination with SYTO9/PI allowed the visualisation of biofilms' structure. The differential expression of 10 genes involved in quorum-sensing, biofilm formation and stress responses was evaluated using reverse-transcription-qPCR. The AHL exposure significantly increased biofilm production in strain ATCC 29212 and two isolates from infected dental roots, UmID4 and UmID5. In strains ATCC 29212 and UmID7, AHLs up-regulated the quorum-sensing genes (fsrC, cylA), the adhesins ace, efaA and asa1, together with the glycosyltransferase epaQ. In strain UmID7, AHL exposure additionally up-regulated two membrane-stress response genes (σV, groEL) associated with increased stress-tolerance and virulence. Altogether, our results demonstrate that AHLs promote biofilm formation and up-regulate a transcriptional network involved in virulence and stress tolerance in several E. faecalis strains. These data provide yet-unreported insights into E. faecalis biofilm responses to AHLs, a family of molecules long-considered the monopole of gram-negative signalling.

Additional Treatment Indicative of an Unfavorable Endodontic Outcome in a Swedish County-A 10-year Observational Study.

INTRODUCTION: This study's aim was to calculate the incidence of first additional endodontic treatment or extraction as the result of an unfavorable endodontic outcome following orthograde root canal treatment (RCT) performed by general dental practitioners during a 10-year period and to identify possible predictors for outcomes. METHODS: A randomized cohort of 280 individuals (and as many teeth) with an orthograde RCT was followed for over 10 years. Dental records were reviewed, and individuals were recalled when data were missing. The following terminal events indicative of unfavorable endodontic treatment outcome were orthograde retreatment, surgical endodontics, and tooth extractions exclusively due to endodontic reasons. Selected variables related to individuals and treatment (pre-, intra-, and postoperative) were harvested to analyze possible associations with the terminal events. Unadjusted survival analysis and Cox regression analysis were performed and P < .05 was considered statistically significant. RESULTS: Terminal events were registered for 22 teeth/individuals and 17 of these were orthograde retreatments. The cumulative 10-year survival of RCTs was 92.7% (standard error 1.7%), with a higher yearly incidence during the first 2 years. The univariate analysis identified 5 factors associated with the outcome. There were too few events to perform a multivariate analysis. CONCLUSIONS: The mean incidence of additional treatment indicative of unfavorable endodontic outcome was 0.7% per year during the first 10 years, but the mean incidence was greater during the first 2 years. Five factors were associated with an unfavorable outcome; however, confounders cannot be excluded from the associations.

Profiling Antibiotic Susceptibility among Distinct Enterococcus faecalis Isolates from Dental Root Canals.

Enterococcus faecalis, a leading multi-resistant nosocomial pathogen, is also the most frequently retrieved species from persistently infected dental root canals, suggesting that the oral cavity is a possible reservoir for resistant strains. However, antimicrobial susceptibility testing (AST) for oral enterococci remains scarce. Here, we examined the AST profiles of 37 E. faecalis strains, including thirty-four endodontic isolates, two vanA-type vancomycin-resistant isolates, and the reference strain ATCC-29212. Using Etest gradient strips and established EUCAST standards, we determined minimum inhibitory concentrations (MICs) for amoxicillin, vancomycin, clindamycin, tigecycline, linezolid, and daptomycin. Results revealed that most endodontic isolates were susceptible to amoxicillin and vancomycin, with varying levels of intrinsic resistance to clindamycin. Isolates exceeding the clindamycin MIC of the ATCC-29212 strain were further tested against last-resort antibiotics, with 7/27 exhibiting MICs matching the susceptibility breakpoint for tigecycline, and 1/27 reaching that of linezolid. Both vanA isolates confirmed vancomycin resistance and demonstrated resistance to tigecycline. In conclusion, while most endodontic isolates remained susceptible to first-line antibiotics, several displayed marked intrinsic clindamycin resistance, and MICs matched tigecycline's breakpoint. The discovery of tigecycline resistance in vanA isolates highlights the propensity of clinical clone clusters to acquire multidrug resistance. Our results emphasize the importance of implementing AST strategies in dental practices for continued resistance surveillance.

Exploring the impact of oral bacteria remnants on stem cells from the Apical papilla: mineralization potential and inflammatory response.

Introduction: Bacterial persistence is considered one of the main causal factors for regenerative endodontic treatment (RET) failure in immature permanent teeth. This interference is claimed to be caused by the interaction of bacteria that reside in the root canal with the stem cells that are one of the essentials for RET. The aim of the study was to investigate whether prolonged exposure of stem cells from the apical papilla (SCAP) to bacterial remnants of Fusobacterium nucleatum, Actinomyces gerensceriae, Slackia exigua, Enterococcus faecalis, Peptostreptococcaceae yurii, commonly found in infected traumatized root canals, and the probiotic bacteria Lactobacillus gasseri and Limosilactobacillus reuteri, can alter SCAP's inflammatory response and mineralization potential. Methods: To assess the effect of bacterial remnants on SCAP, we used UV-C-inactivated bacteria (as cell wall-associated virulence factors) and bacterial DNA. Histochemical staining using Osteoimage Mineralization Assay and Alizarin Red analysis was performed to study SCAP mineralization, while inflammatory and osteo/odontogenic-related responses of SCAPs were assessed with Multiplex ELISA. Results: We showed that mineralization promotion was greater with UV C-inactivated bacteria compared to bacterial DNA. Immunofluorescence analysis detected that the early mineralization marker alkaline phosphatase (ALP) was increased by the level of E. coli lipopolysaccharide (LPS) positive control in the case of UV-C-inactivated bacteria; meanwhile, DNA treatment decreased the level of ALP compared to the positive control. SCAP's secretome assessed with Multiplex ELISA showed the upregulation of pro-inflammatory factors IL-6, IL-8, GM-CSF, IL-1b, neurotrophic factor BDNF, and angiogenic factor VEGF, induced by UV-C-killed bacteria. Discussion: The results suggest that long term stimulation (for 21 days) of SCAP with UV-C-inactivated bacteria stimulate their mineralization and inflammatory response, while DNA influence has no such effect, which opens up new ideas about the nature of RET failure.

Photo-oxidative stress response and virulence traits are co-regulated in E. faecalis after antimicrobial photodynamic therapy.

Knowledge of photo-oxidative stress responses in bacteria that survive antimicrobial photodynamic therapy (aPDT) is scarce. Whereas aPDT is attracting growing clinical interest, subsequent stress responses are crucial to evaluate as they may lead to the up-regulation of pathogenic traits. Here, we aimed to assess transcriptional responses to sublethal aPDT-stress and identify potential connections with virulence-related genes. Six Enterococcus faecalis strains were investigated; ATCC 29212, three dental root-canal isolates labelled UmID1, UmID2 and UmID3 and two vancomycin-resistant isolates labelled A1 and A2. TMPyP was employed as a photosensitiser. A viability dose-response curve to increasing concentrations of TMPyP was determined by culture plating. Differential expression of genes involved in oxidative stress responses (dps and hypR), general stress responses (dnaK, sigma-factorV and relA), virulence-related genes (ace, fsrC and gelE) and vancomycin-resistance (vanA) was assessed by reverse-transcription qPCR. TMPyP-mediated aPDT inactivated all strains with comparable efficiencies. TMPyP at 0.015 µM was selected to induce sublethal photo-oxidative stress. Despite heterogeneities in gene expression between strains, transcriptional profiles revealed up-regulations of transcripts dps, hypR as well as dnaK and sigma factorV after exposure to TMPyP alone and to light-irradiated TMPyP. Specifically, the alternative sigma factorV reached up to 39 ± 113-fold (median ± IQR) (p = 0.0369) in strain A2. Up-regulation of the quorum sensing operon, fsr, and its downstream virulence-related gelatinase gelE were also observed in strains ATCC-29212, A1, A2 and UmID3. Finally, photo-oxidative stress induced vanA-type vancomycin-resistance gene in both carrier isolates, reaching up to 3.3 ± 17-fold in strain A2 (p = 0.015). These findings indicate that, while aPDT successfully inactivates vancomycin-resistant and naïve strains of E. faecalis, subpopulations of surviving cells respond by co-ordinately up-regulating a network of genes involved in stress survival and virulence. This includes the induction of vancomycin-resistance genes in carrier isolates. These data may provide the mechanistic basis to circumvent bacterial responses and improve future clinical protocols.

The Effect of Extra Educational Elements on the Confidence of Undergraduate Dental Students Learning to Administer Local Anaesthesia.

Local anaesthesia is taught early in the practical part of dental programs. However, dental students express uncertainty and concern before their practical training in local anaesthesia. The aim of this study was to evaluate how extra educational elements in the teaching of local anaesthesia affect students' confidence using local anaesthesia. The students were divided into three groups (A, B and C). Group A received the same education that was used the previous year (i.e., four hours of theoretical lectures followed by four hours of practical exercises performed on a fellow student). Group B did their practical training on fellow students in groups of three, with each student taking turns performing, receiving and observing the procedure. Group C received training using an anatomically correct model before their practical training on a fellow student. After each training step, the students completed a questionnaire about their confidence administering local anaesthesia. The students experienced a significant increase in confidence after each educational step. Combining theory and practical instruction, including the use of anatomically correct models and peer instruction, improved students' confidence in administering local anaesthesia. The greatest increase in confidence was in the students placed in groups of three where each student performed, received and observed the procedure.

CBCT influences endodontic therapeutic decision-making in immature traumatized teeth with suspected pulp necrosis: a before-after study.

OBJECTIVE: To evaluate the impact of cone-beam computed tomography (CBCT) in endodontic therapeutic decision-making of immature traumatized teeth with suspected pulp necrosis. METHODS: Over two years, consecutive patients with a dental trauma in their front teeth (apex >0.5 mm) and with suspected pulp necrosis based on clinical and radiographic findings were referred to a specialist clinic in Sweden. Fifteen patients aged 6-13 (18 teeth) were included and clinically examined by an endodontist. Intraoral radiographs and CBCT examinations were obtained. Five practitioners, three endodontists and two residents in endodontics, used these examinations to determine the most appropriate treatment for the 18 cases (all central incisors) on two occasions scheduled 19 weeks apart. On the first occasion, the practitioners had access to clinical information and the intraoral radiographs ('before' CBCT); on the second occasion, the practitioners had also access to a radiologist report and the CBCT images ('after' CBCT). Their treatment plans - no treatment, watchful waiting, endodontic orthograde treatment, or extraction - were made anonymously and independently. Results were analysed using descriptive statistics and Wilcoxon signed-rank test. RESULTS: 'After' CBCT, practitioners changed treatment plans in 30% of the 90 assessments, 74% of which were more aggressive (p = 0.028). In 49% of the assessments, practitioners who chose the watchful and waiting treatment plan 'before' CBCT changed to a more aggressive therapy such as endodontic orthograde treatment and extraction 'after' CBCT (p = 0.005). CONCLUSION: This study provides evidence that CBCT influences endodontic therapeutic decision-making regarding immature traumatised teeth with suspected pulp necrosis, chiefly when expectant management (i.e., watchful and waiting) was selected before access to CBCT.

New Insights into the Microbial Profiles of Infected Root Canals in Traumatized Teeth.

Traumatic dental injuries in young individuals are often exposed to the invasion of oral microorganisms that leads to pulp necrosis. Infective necrosis in permanent teeth not-fully-developed causes aberrant root formation. Regeneration endodontic treatments (RETs) have shown promising results by promoting continued root development by stem cells. Critical to the success of RET is the thorough disinfection of the pulpal space. To establish effective antimicrobial protocols for root canal disinfection, the invading microorganisms need to be identified. In the present study, we use a combination of culture-based and high-throughput molecular sequencing techniques to investigate the microbial profiles from traumatized teeth (30 cases) and controls, i.e., teeth with pulp infections not caused by trauma (32 cases). Overall, a high microbial diversity in traumatized necrotic teeth was observed. Eubacterium yurii subsps. yurii and margaretiae, as well as key 'bridging oral species' F. nucleatum sp., Polymorphum and Corynebacterium matruchotti, were highly associated with traumatized teeth. The microbial compositions of traumatized teeth differed considerably from those of infected teeth not caused by trauma. Age and tooth position also influence microbial compositions. In conclusion, we show that the root canal microflora of traumatized teeth is highly diverse, and it differs from root canal infections not caused by trauma.

Phylogenetic Analysis of Filifactor alocis Strains Isolated from Several Oral Infections Identified a Novel RTX Toxin, FtxA.

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod of the phylum Firmicutes, and is considered an emerging pathogen in various oral infections, including periodontitis. We here aimed to perform phylogenetic analysis of a genome-sequenced F. alocis type strain (ATCC 35896; CCUG 47790), as well as nine clinical oral strains that we have independently isolated and sequenced, for identification and deeper characterization of novel genomic elements of virulence in this species. We identified that 60% of the strains carried a gene encoding a hitherto unrecognized member of the large repeats-in-toxins (RTX) family, which we have designated as FtxA. The clinical infection origin of the ftxA-positive isolates largely varied. However, according to MLST, a clear monophylogeny was reveled for all ftxA-positive strains, along with a high co-occurrence of lactate dehydrogenase (ldh)-positivity. Cloning and expression of ftxA in E. coli, and purification of soluble FtxA yielded a protein of the predicted molecular size of approximately 250 kDa. Additional functional and proteomics analyses using both the recombinant protein and the ftxA-positive, and -negative isolates may reveal a possible role and mechanism(s) of FtxA in the virulence properties of F.alocis, and whether the gene might be a candidate diagnostic marker for more virulent strains.

Contamination Controls for Analysis of Root Canal Samples by Molecular Methods: An Overlooked and Unsolved Problem.

INTRODUCTION: It has been almost 20 years since molecular methods were first described for the analysis of root canal microbial flora. Contamination control samples are essential to establish DNA decontamination before taking root canal samples, and this review assessed those studies. METHODS: Using PubMed, a search was conducted for studies using molecular microbial analysis for the investigation of endodontic samples. Studies were grouped according to the cleaning protocol, acquisition methods, and processing of control samples taken to check for contamination. RESULTS: Of 136 studies applying molecular analysis to root canal samples, 21 studies performed surface cleaning and checking nucleotide decontamination with contamination control samples processed by polymerase chain reaction. Only 1 study described disinfection, sampling from the access cavity, and processing by polymerase chain reaction and reported the result; that study reported that all samples contained contaminating bacterial DNA. CONCLUSIONS: Cleaning, disinfection, and checking for contamination are basic scientific prerequisites for this type of investigation; yet, this review identifies it as an overlooked issue. On the basis of this review, we call for improved scientific practice in this field.

Preservation of bacterial DNA by human dentin.

INTRODUCTION: The capacity of dentin and collagen to bind DNA and protect against spontaneous and nuclease-induced degradation was evaluated individually and by the incubation of DNA with nuclease-producing bacteria in a mixed culture. METHODS: Extracted Fusobacterium nucleatum DNA was incubated with dentin shavings or collagen for 90 minutes. The DNA-bound substrates were incubated in different media (water, sera, and DNase I) for up to 3 months. Amplifiable DNA was released from dentin using EDTA,or from collagen using proteinase K, and evaluated by polymerase chain reaction (PCR). The stability of dentin-bound DNA was also assessed in a mixed culture (Parvimonas micra and Pseudoramibacter alactolyticus) containing a DNase-producing species, Prevotella intermedia. Samples were analyzed for amplifiable DNA. RESULTS: In water, dentin-bound DNA was recoverable by PCR at 3 months compared with no detectable DNA after 4 weeks in controls (no dentin). DNA bound to collagen was detectable by PCR after 3 months of incubation in water. In 10% human sera, amplifiable DNA was detectable at 3 months when dentin bound and in controls (no dentin). In mixed bacterial culture, dentin-bound DNA was recoverable throughout the experimental period (3 months), compared with no recoverable F. nucleatum DNA within 24 hours in controls (no dentin). CONCLUSIONS: There is a strong binding affinity between DNA and dentin, and between DNA and serum proteins or collagen. These substrates preserve DNA against natural decomposition and protect DNA from nuclease activity, factors that may confound molecular analysis of the endodontic microbiota yet favor paleomicrobiological studies of ancient DNA.

DNA binding to hydroxyapatite: a potential mechanism for preservation of microbial DNA.

INTRODUCTION: Molecular methods are increasingly being deployed for analysis of the microbial flora in the root canal. Such methods are based on the assumption that recovered DNA is associated with the active endodontic infection, yet paleomicrobiology research is based on the recovery of ancient DNA from centuries-old tooth and bone samples, which points to considerable longevity of the DNA molecule in these tissues. The main component of dentin and bone is the mineral hydroxyapatite. This study assessed DNA binding to hydroxyapatite and whether this binding affinity stabilizes the DNA molecule in various media. METHODS: DNA was extracted from Fusobacterium nucleatum and added to ceramic hydroxyapatite for 90 minutes. The DNA-bound hydroxyapatite was incubated in different media (ie, water, sera, and DNase I) for up to 3 months. At predetermined intervals, the recovery of detectable DNA was assessed by releasing the DNA from the hydroxyapatite using EDTA and evaluating the presence of DNA by gel electrophoresis and polymerase chain reaction (PCR) amplification. RESULTS: When incubated with hydroxyapatite, nonamplified DNA was detectable after 3 months in water, sera, and DNase I. In contrast, DNA incubated in the same media (without hydroxyapatite) decomposed to levels below the detection level of PCR within 3 weeks, with the exception of DNA in sera in which PCR revealed a weak positive amplification product. CONCLUSIONS: These results confirm a specific binding affinity of hydroxyapatite for DNA. Hydroxyapatite-bound DNA is more resistant to decay and less susceptible to degradation by serum and nucleases, which may account for the long-term persistence of DNA in bone and tooth.

Persistence of dead-cell bacterial DNA in ex vivo root canals and influence of nucleases on DNA decay in vitro.

OBJECTIVE: The fate of DNA from bacteria that do not survive in the root canal is uncertain, yet DNA longevity may confound recovery of authentic etiologic participants in the disease process. This study assessed the recovery of PCR-detectable DNA in ex vivo human root canals and some environmental factors on the decay of microbial DNA. STUDY DESIGN: Heat-killed Enterococcus faecalis cells were inoculated into instrumented human root canals ex vivo, and samples were taken at intervals over 2 years and analyzed by polymerase chain reaction. In an in vitro assay, heat-killed E. faecalis cells and extracted E. faecalis DNA were inoculated into various media, DNase, and culture of a DNase-producing species, Prevotella intermedia. Recovery of DNA was assessed by gel electrophoresis. RESULTS: In ex vivo human teeth, amplifiable DNA was recovered after 1 and 2 years (in 14/15 and 21/25 teeth, respectively). In vitro experiments showed that extracted DNA incubated in different media (water, 10%-50% sera, and DNase) progressively decomposed to levels below the detection limit. In corresponding assays, cell-bound DNA was more resistant to decay. CONCLUSION: Amplifiable DNA is preserved after cell death, but the critical determinant is the form of DNA. Free DNA undergoes spontaneous and enzymatic decomposition, whereas cell-bound E. faecalis DNA persists for long periods.