The role of Candida albicans in root caries biofilms: an RNA-seq analysis.

Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.

Dual wavelength irradiation antimicrobial photodynamic therapy using indocyanine green and metformin doped with nano-curcumin as an efficient adjunctive endodontic treatment modality.

BACKGROUND: Indocyanine green (ICG) doped with nano-curcumin (N-CUR) can increase photosensitivity in antimicrobial photodynamic therapy (aPDT). Since metformin (Met) provides a synergistic advantage with photosensitivity, it was conjugated with N-CUR@ICG. Aim of the study was to evaluate the photosensitizing effect of N-CUR@ICG-Met used as a new photosensitizer in dual wavelengths irradiation (diode laser and light-emitting diode, LED) aPDT in root canals infected with Enterococcus faecalis biofilm. MATERIALS AND METHODS: Following synthesis and confirmation of N-CUR@ICG-Met by Scanning electrone microscope (SEM), Fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) and Zetasizer analysis, the mature microbial biofilm was formed. The quantitative and qualitative evaluations of E. faecalis biofilm were made using microbial viability and SEM analysis of the following groups of treatment modalities (n = 5): 1- N-CUR, 2- ICG, 3- Met, 4- N-CUR@ICG, 5- N-CUR@ICG-Met, 6- Diode laser, 7- LED, 8- aPDT/diode, 9- aPDT/LED, 10- aPDT/diode laser + LED, 11- aPDT/LED + diode laser, 12- 2.5 % sodium hypochlorite (NaOCl). In addition, E. faecalis not treated served as negative control. Data were statistically analyzed using One-way Analysis of Variance (ANOVA) and the post-Hoc Bonferroni tests with a level of significance set at P < 0.05. RESULTS: SEM, FT-IR, XRD, Zetasizer analysis confirmed successful doping of ICG-Met onto/into N-CUR. The treatment modality of N-CUR@ICG-Met mediated aPDT/diode laser, aPDT/LED, aPDT/diode laser + LED, and aPDT/LED + diode laser statistically decreased the cell viability of E. faecalis for 69.40 %, 75.52 %, 82.74 %, and 83.84 %, respectively compared with the negative control group (P < 0.05). The double wavelengths irradiation technique, which exposed the N-CUR@ICG-Met with diode laser irradiation and immediately after with LED (i.e. group No. 10), as well as, N-CUR@ICG-Met with LED irradiation and immediately after with diode laser (i.e. group No. 11) reduced the viable E. faecalis count in biofilm structures statistically more than the other treatment modalities (P < 0.05). CONCLUSIONS: N-CUR@ICG-Met as a new photosensitizer in dual wavelengths irradiation method may improve anti-biofilm activity of aPDT against E. faecalis.

The role of Candida albicans in root caries biofilms: an RNA-seq analysis

Abstract Objective This study sought to analyze the gene expression of Candida albicans in sound root surface and root caries lesions, exploring its role in root caries pathogenesis. Methodology The differential gene expression of C. albicans and the specific genes related to cariogenic traits were studied in association with samples of biofilm collected from exposed sound root surface (SRS, n=10) and from biofilm and carious dentin of active root carious lesions (RC, n=9). The total microbial RNA was extracted, and the cDNA libraries were prepared and sequenced on the Illumina Hi-Seq2500. Unique reads were mapped to 163 oral microbial reference genomes including two chromosomes of C. albicans SC5314 (14,217 genes). The putative presence of C. albicans was estimated (sum of reads/total number of genes≥1) in each sample. Count data were normalized (using the DESeq method package) to analyze differential gene expression (using the DESeq2R package) applying the Benjamini-Hochberg correction (FDR<0.05). Results Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) were up-regulated on SRS, and their functions are related to biofilm formation. Seven genes ( UTP20 , FDR=0.018; ITR1 , FDR=0.036; DHN6 , FDR=0.046; CaO19.7197 , FDR=0.046; CaO19.7838 , FDR=0.046; STT4 , FDR=0.046; GUT1 , FDR=0.046) were up-regulated on RC and their functions are related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation. The use of alternative carbon sources, including lactate, and the ability to form hypha may be a unique trait of C. albicans influencing biofilm virulence. Conclusions C. albicans is metabolically active in SRS and RC biofilm, with different roles in health and disease.

Using autofluorescence to detect bacterial contamination in root fractures.

OBJECTIVES: Conventional methods for detecting root fractures cannot assess their depth or bacterial contamination. This study was designed to measure the autofluorescence emitted from a root fracture, with the aim of determining whether this is a suitable method for quantifying the depth and bacterial invasion of a fracture. METHODS: This in vitro study investigated 33 mandibular second molars with periapical lesions that had been extracted after finding root fractures in endodontically treated teeth during intentional replantation or diagnostic surgery. The root fractures were scanned using a fluorescence technique, and the association between fluorescence parameters and fracture depth was analyzed. The significance of the association between the red fluorescence among autofluorescence parameters and bacterial contamination within the fracture was examined. RESULTS: When the depth of the root fractures was evaluated by micro computed tomography, the scattering of light in the fractures increased with depth, and there was a gradual increase in the quantitative fluorescence parameter indicating the deepest point (ΔFmax) in the fractures. In addition, we observed red fluorescence on the outer surface of deeper fractures. The tooth fractures exhibiting red fluorescence were evaluated for bacterial contamination associated with red-fluorescent porphyrin, which revealed bacterial invasion into these fractures. On the other hand, non-red-fluorescing fractures contained necrotic tissue, debris, and irritants. CONCLUSIONS: This viable fluorescent technique can potentially quantify the depth of root fractures and be used as a risk indicator for root fractures with periodontal inflammation. CLINICAL SIGNIFICANCE: The auto-fluorescence technique can be used to detect depth and bacterial contamination of root fractures. It is postulated that the auto-fluorescence can be used as a risk indicator of deep fractures and can replace conventional fracture detection methods.

Effect of Sweeteners on Root Dentine Demineralization Using a Microcosm Biofilm Model / Efecto de los Edulcorantes en la Desmineralización de la Dentina Radicular Utilizando un Modelo de Biofilm Microcosmo

ABSTRACT: The aim of the present study was to evaluate the effect of commercial sweeteners on root dentin demineralization using a microcosm biofilm model. Bovine dentin specimens with pre-determined surface hardness were randomized into six groups according to the studied sweeteners: sucralose, stevia, saccharin, aspartame. Sucrose was used as a positive control and an untreated group as a negative control. The specimens were submitted to biofilm development from one saliva donor and the cariogenic challenge occurred on subsequent five days, twice a day. At the end, the percentage of surface hardness loss (%SHL) and biomass was determined and submitted to ANOVA followed by Tukey's test. Sucrose presented the highest rate of demineralization, however, all sweeteners tested lead to a statistically higher root demineralization compared to the negative control (p <0.05). Sucrose caused greater demineralization in root dentin, however, the sweeteners were also able to induce it under this biofilm model.

RESUMEN: El objetivo del presente estudio fue evaluar el efecto de los edulcorantes comerciales en la desmineralización de la dentina radicular utilizando un modelo de biofilm microcosmo. Se asignaron al azar muestras de dentina bovina con una dureza de la superficie predeterminada de acuerdo con los edulcorantes estudiados: sucralosa, estevia, sacarina, aspartame. La sacarosa se utilizó como control positivo y un grupo no tratado como control negativo. Las muestras se enviaron al desarrollo de biopelículas de un donante de saliva y el desafío cariogénico se produjo en los siguientes cinco días, dos veces al día. Al final, se determinó el porcentaje de pérdida de dureza de la superficie (% PDS) y biomasa y se aplicó un estudio estadístico de ANOVA seguido de la prueba de Tukey. La sacarosa presentó la mayor tasa de desmineralización; sin embargo, todos los endulzantes probados condujeron a una desmineralización de la raíz estadísticamente mayor en comparación con el control negativo (p<0,05). La sacarosa causó una mayor desmineralización en la dentina de raíz, sin embargo, los edulcorantes también fueron capaces de inducirla bajo este modelo de biofilm.

In vitro evaluation by quantitative real-time PCR and culturing of the effectiveness of disinfection of multispecies biofilms in root canals by two irrigation systems.

OBJECTIVES: The purpose of this in vitro study was by using quantitative real-time PCR and culturing to determine the effectiveness of two irrigation and cleaning systems in removing multispecies oral biofilms from root canals. MATERIAL AND METHODS: Twenty extracted human molars were instrumented to size #15/.02 and then cleaned with the GentleWave (GW) System. The teeth were autoclaved to provide the same sterile baseline. The molars were filled with mixed plaque suspended in BHI and centrifuged to inoculate the biofilms. After 2 weeks of incubation, the teeth were randomly divided into two treatment groups. In GW group (26 canals), the teeth were further instrumented to size #15/04, and in PiezoFlow (PF) group (30 canals) to #35/.04. The teeth were then cleaned either with GW System or ProUltra PiezoFlow Active Ultrasonic System using 3% sodium hypochlorite NaOCl, 8% EDTA, and sterile water as irrigants. Samples (S1, S2, and S3) for bacterial cultures were taken from 13 canals before and after instrumentation and after final cleaning. Quantitative real-time PCR was performed from all 56 canals, and universal bacterial, one genus, and one species-specific primers were used to determine the presence of microorganisms in samples from root canals before and after instrumentation and after final cleaning. Statistical analyses were performed using the Mann-Whitney U test with the significance level set at P < 0.05. RESULTS: Bacterial culturing from the canal samples revealed strong reduction of bacteria from S1 to S2 in both groups after instrumentation and irrigation with water only. No growth was detected in any of the S3 samples after cleaning in either group. A highly significant reduction in bacterial DNA was recorded by qPCR for both groups (P < 0.001). GW System showed more constant and a significantly higher reduction of total microbial DNA (P = 0.007), Enterococcus faecalis DNA (P = 0.011) and Streptococcus spp. DNA (P = 0.029) than the Ultrasonic System. The amount of residual microbial DNA calculated as an average of residual DNA in each individual canal in PF group was 1.99% and in GW group 0.09%. CONCLUSIONS: While both systems demonstrated a highly effective reduction of intracanal bacterial DNA, the final total amount and variation in the number of residual bacterial DNA was significantly smaller in the GW group. CLINICAL RELEVANCE: Elimination of microbes from the infected root canal system is regarded as the key for long-term clinical success. While both GentleWave and Ultrasonic Systems used with NaOCl and EDTA demonstrated a highly effective reduction of intracanal bacterial DNA; GW produced higher reduction and better predictability.

Starch Combined with Sucrose Provokes Greater Root Dentine Demineralization than Sucrose Alone.

Since there is no consensus about whether starch increases the cariogenic potential of sucrose, we used a validated 3-species biofilm model to evaluate if starch combined with sucrose provokes higher root dentine demineralization than sucrose alone. Biofilms (n = 18) composed by Streptococcus mutans (the most cariogenic bacteria), Actinomces naeslundii (which has amylolytic activity), and Streptococcus gordonii (which binds salivary amylase) were formed on root dentine slabs under exposure 8 ×/day to one of the following treatments: 0.9% NaCl, 1% starch, 10% sucrose, or a combination of 1% starch and 10% sucrose. Before each treatment, biofilms were pretreated with human whole saliva for 1 min. The pH of the culture medium was measured daily as an indicator of biofilm acidogenicity. After 96 h of growth, the biofilms were collected, and the biomass, bacteria viability, and polysaccharides were analyzed. Dentine demineralization was assessed by surface hardness loss (% SHL). Biofilm bioarchitecture was analyzed using confocal laser scanning microscopy. Treatment with a starch and sucrose combination provoked higher (p = 0.01) dentine demineralization than sucrose alone (% SHL = 53.2 ± 7.0 vs. 43.2 ± 8.7). This was supported by lower pH values (p = 0.007) of the culture medium after daily exposure to the starch and sucrose combination compared with sucrose (4.89 ± 0.29 vs. 5.19 ± 0.32). Microbiological and biochemical findings did not differ between biofilms treated with the combination of starch and sucrose and sucrose alone (p > 0.05). Our findings give support to the hypothesis that a starch and sucrose combination is more cariogenic for root dentine than sucrose alone.

Development of the gingival sulcus at the time of tooth eruption and the influence of genetic factors.

Tooth eruption is characterized by a concert of mechanisms that result in the emergence of teeth in the oral cavity. Genetic variants seem to regulate this process and the formation of a gingival sulcus around the teeth. Interindividual variability in the response to microbial triggers in the sulcus plays an important role in the onset and progression of periodontal diseases. Host genetic variants can influence this variability, affecting the response of the host to the subgingival biofilm. Genetic factors affecting tooth eruption could potentially influence susceptibility to periodontal diseases and, specifically, susceptibility to localized aggressive periodontitis. This review aims to discuss the evidence available for the role of host genetic variants in tooth eruption and to and to give some directions for prospective research in this topic.

The periodontal pocket: pathogenesis, histopathology and consequences.

The conversion of junctional epithelium to pocket epithelium is regarded as a hallmark in the development of periodontitis. Knowledge of factors contributing to the initiation and progression of pocket formation is important and may result in the development of better preventive measures and improve healing outcomes after therapeutic interventions. The periodontal pocket is a pathologically deepened gingival sulcus. In healthy periodontal conditions, the defense mechanisms are generally sufficient to control the constant microbiological challenge through a normally functioning junctional epithelium and the concentrated powerful mass of inflammatory and immune cells and macromolecules transmigrating through this epithelium. In contrast, destruction of the structural integrity of the junctional epithelium, which includes disruption of cell-to-cell contacts and detachment from the tooth surface, consequently leading to pocket formation, disequilibrates this delicate defense system. Deepening of the pocket apically, and also horizontal expansion of the biofilm on the tooth root, puts this system to a grueling test. There is no more this powerful concentration of defense cells and macromolecules that are discharged at the sulcus bottom and that face a relatively small biofilm surface in the gingival sulcus. In a pocket situation, the defense cells and the macromolecules are directly discharged into the periodontal pocket and the majority of epithelial cells directly face the biofilm. The thinning of the epithelium and its ulceration increase the chance for invasion of microorganisms and their products into the soft connective tissue and this aggravates the situation. Depending on the severity and duration of disease, a vicious circle may develop in the pocket environment, which is difficult or impossible to break without therapeutic intervention.

A Comparison of Er:YAG Laser with Photon-Initiated Photoacoustic Streaming, Nd:YAG Laser, and Conventional Irrigation on the Eradication of Root Dentinal Tubule Infection by Enterococcus faecalis Biofilms: A Scanning Electron Microscopy Study.

This study evaluated the antimicrobial efficacy of Er:YAG laser activation with photon-initiated photoacoustic streaming (PIPS), Nd:YAG laser disinfection, and conventional irrigation on Enterococcus faecalis biofilms using scanning electron microscopy (SEM). Biofilms were grown on 110 root halves and divided into the following: Groups 1 and 2 (saline and 1% NaOCl with apical position of PIPS, resp.), Groups 3 and 4 (saline and 1% NaOCl with coronal position of PIPS, resp.), Groups 5 and 6 (Nd:YAG laser after saline and 1% NaOCl irrigation, resp.) and Groups 7, 8, and 9 (conventional irrigation with 1% NaOCl, 6% NaOCl, and saline, resp.). SEM images of the apical, middle, and coronal levels were examined using a scoring system. Score differences between Groups 1 and 2 were insignificant at all levels in the remaining biofilm. Group 4 had significantly greater bacterial elimination than Group 3 at all levels. Differences in Nd:YAG laser irradiation between Groups 5 and 6 were insignificant. Groups 7 and 8 were insignificantly different, except at the middle level. Saline group had a higher percentage of biofilms than the others. In this study, PIPS activation with NaOCl eliminates more E. faecalis biofilms in all root canals regardless of the position of the fiber tip.

Antifungal Effects of Synthetic Human Beta-defensin-3-C15 Peptide on Candida albicans-infected Root Dentin.

INTRODUCTION: The aim of this study was to assess the antifungal efficacy of a synthetic human beta-defensin-3-C15 peptide (HBD3-C15) in Candida albicans-infected human root dentin. METHODS: Standardized root dentin blocks were prepared (6-mm thick, 0.7-mm-wide canal) from single-rooted human permanent premolars and infected with C. albicans for 3 weeks. They were randomly divided into 4 groups (n = 8/group), and their canals were filled with calcium hydroxide (CH), HBD3-C15 peptide, or chlorhexidine digluconate (CHX, 2%) as disinfectants or saline as control. After 1 week of disinfection, dentinal debris were harvested at depths of 200 and 400 µm from the canal lumen, and incubated in Yeast broth for 72 hours at 37°C. Then, colony-forming units (CFU) were measured to assess the antifungal efficacy of each medicament and analyzed statistically. RESULTS: All medicaments showed significantly lower CFU than saline (P < .05), and their antifungal efficacies were similar at both 200- and 400-µm tubular depths (P > .05). HBD3-C15 had similar antifungal efficacy to that of CHX at both depths (P > .05), and both medicaments had significantly lower CFU than CH at both depths (P < .05). CONCLUSIONS: In this ex vivo model of C. albicans-infected human root dentin, the antifungal efficacy of synthetic HBD3-C15 was comparable with CHX.

Does the Removal of Cementum Facilitate Bacterial Penetration into Dentinal Tubules In Vitro?

INTRODUCTION: The importance of an intact layer of cementum on the root surface in preventing bacterial penetration into radicular dentin has not been sufficiently investigated. The aim of this in vitro study was to determine the effect of the absence of cementum from the root surface and the length of the infection period (2 or 4 weeks) on the maximum depth of bacterial penetration and the percentage of sectors lined with bacteria. METHODS: Sound, single-rooted extracted teeth with closed apices were randomly assigned to either a control group (cementum present [CP]) or an experimental group (cementum removed [CR]). Each group was further divided randomly into 2 subgroups: 2-week infection (CP2 and CR2) and 4-week infection (CP4 and CR4). Teeth were then artificially infected with Enterococcus faecalis and prepared for histology. RESULTS: A total of 107 teeth were available for histologic examination, 25 teeth in CP2, 31 teeth in CP4, 27 teeth in CR2, and 24 teeth in CR4. Pairwise comparisons revealed statistically significant differences in the maximum depth of bacterial penetration for the following combinations: CP2-CR2, CP2-CR4, CP4-CR2, and CP4-CR4 (P < .001). Pairwise comparisons also revealed a statistically significant difference in the percentage of sectors lined with bacteria for CP2-CR2, CP2-CP4, and CP2-CR4 (P < .001). CONCLUSIONS: The results support the hypothesis that the absence of cementum facilitates bacterial penetration into dentinal tubules. Results also suggest that the process of radicular dentin infection is time dependent and highlight the importance of early treatment of infected teeth, especially in situations in which cementum discontinuity is suspected.

pH Response and Tooth Surface Solubility at the Tooth/Bacteria Interface.

Evaluating the physiochemical processes at the tooth surface/bacteria interface is important for elucidating the etiology of dental caries. This study aimed to compare the mineral solubility and protein degradation of coronal enamel (CE) and root dentin (RD), and investigate the involvement of dissolved components in bacteria-induced pH changes using a model of tooth/bacteria interface. An experimental apparatus forming a well was made of polymethyl methacrylate, and a bovine tooth (CE or RD) specimen was fixed at the bottom of the well. A miniature pH electrode was placed on the tooth, and Streptococcus mutans NCTC 10449 cells, grown in 0.5% glucose-containing complex medium, were packed into the well. The pH at the tooth/S. mutans interface was monitored continuously for 120 min after the addition of 0.5% glucose at 37°C. S. mutans cells were recovered from the wells, and the amounts of lactate and calcium were measured using a portable lactate meter and a fluorescent dye, respectively. Proteolytic activity was also evaluated fluorometrically. The pH of the RD/S. mutans interface was significantly higher than that of the CE/S. mutans interface (30 min: 6.37 ± 0.12 vs. 6.18 ± 0.11, 60 min: 6.08 ± 0.14 vs. 5.66 ± 0.27, 90 min: 5.49 ± 0.24 vs. 5.14 ± 0.22, p < 0.05). Greater amounts of calcium were dissolved from RD (3.19 ± 0.74 µg/mL) than from CE (1.84 ± 0.68 µg/mL; p < 0.05), while similar amounts of lactate were produced. Proteolytic activity was not detected at any of the interfaces. These results indicate that RD is more soluble to bacteria-induced acidification than CE. This method can contribute to the evaluation and development of caries-preventive materials.

Persistent extra-radicular bacterial biofilm in endodontically treated human teeth: scanning electron microscopy analysis after apical surgery.

Biofilms are the main cause of endodontic failures. Even the best executed endodontic treatment can fail, when the infection is resistant to treatment or when it is located in inaccessible areas, such as the external surface of the root apex. The purpose of this study was to evaluate, by scanning electron microscopy, the presence of bacterial biofilm on endodontically treated teeth considered clinical failures and suitable for apical surgery. Root apices were collected from 20 teeth undergoing apical surgery and one negative control and analyzed under SEM. Digital photomicrographs of the root apices of 21 specimens at different magnifications were taken. Data were analyzed using descriptive statistics. Apical biofilms were observed in 100% of root canal treatments considered endodontic failure. Topographical analysis of the root apices revealed areas of resorption, microcracks, and apical foramina in 90%, 80%, and 50% of cases, respectively. Within the limits of this study, it can be concluded that endodontic failures present bacterial biofilm in areas inaccessible to conventional endodontic treatment, such as the external surfaces of the root apex.

Evaluation of Bacteriological Profile in the Apical Root Segment of the Patients with Primary Apical Periodontitis.

INTRODUCTION: Apical periodontitis usually results from bacterial accumulation and contamination occurring in the root-canal system, and extending beyond the apical foramen to involve the periapical tissues. Literature has a paucity of the studies that stress on the division and analysis of the pulp canal segments. The reason for this disparity might be the technique used for collecting the samples from the pulp canals. Hence, we carried out the present study to evaluate the microbial flora in the apical part of the roots with necrotic pulp canals. MATERIALS AND METHODS: The present study included the assessment of 40 freshly extracted teeth that had necrotized pulpal tissue along with the presence of periapical periodontal lesions. Removal of the soft tissue lesions attached to the root portion of the teeth along with apical periodontal lesions was done with the help of scalpel blade, after rinsing them with a sterile solution of saline. Thorough cleaning of the root surfaces was done with hydrogen peroxide followed by rapid disinfection with the help of sodium hypochlorite at varying concentrations. Sectioning of the root portion of all the specimens with the help of a disk was done perpendicular to the long axis of the teeth at a distance of roughly 5 to 6 mm from the teeth's apicalmost point. Cryotubes were used for transferring the specimens of apical portions containing 1 mL of buffer and were subjected to immediate frozen processing at a temperature of -20°C. A 10 K-type file was used for the initial collection of the samples followed by subsequent incubation of the files and paper pints in the incubation cabinet. Subsequent deoxyribonucleic acid (DNA) extraction from the samples was done following the procedure described by Siqueira et al. Paster et al's modification of the reverse-capture checkerboard assay was used in the present study. Semiquantitative data were used for overcoming the difficulties arising due to obtaining the counts of the polymerase chain reaction (PCR)-based analysis of specimens. RESULTS: A positive result for the 16S ribosomal ribonucleic acid (rRNA) gene primer was observed only in two examined specimens of all the samples of the apical portion of the root canals in the present study. Negative result was shown by all the control group specimens, which were sterile samples. Presence of bacteria was confirmed by PCR in 38 out of 40 examined specimens. Amount of bacterial taxa, out of these 24 samples, ranged up to 6. Pseudoramibacter alactolyticus, Porphyromonas endodontalis, Dialister oral species, Bacteroidetes species, Streptococcus species, Olsenella uli, Synergistes species, Fusobacterium nucleatum, Parvimonas micra, Treponema denticola, and Filifactor alocis were the specific species detected. Bacteroidetes species was the only species that were detected at levels at or above 105. Heavy bacterial infections were noticed in more than 45% of the cases at the periradicular part of the root canals. CONCLUSION: Microbial flora of the apical segment of the root with necrotized pulp tissue comprises a vast variety of pathogenic bacteria. CLINICAL SIGNIFICANCE: For better prognosis of the treatment of such cases, adequate knowledge of the microbial flora of the root, especially the apical portion is necessary.

Removal of simulated biofilm: an evaluation of the effect on root surfaces roughness after scaling.

BACKGROUND: Despite the development of less invasive devices, a debate exists about the benefits and risks of hand versus powered root surface instrumentation used in supportive periodontal therapy (SPT). The aim of the in vitro study was to differentially compare plaque removal efficacy and root surface roughening of newly developed sonic, ultrasonic scaler, and curettes in the hands of experienced versus less experienced operators. MATERIALS AND METHODS: Sonic (AIR), ultrasonic devices (TIG), and double-gracey curettes (GRA) were utilized by seven experienced (EO) and four less experienced operators (LO) for root surface instrumentation of standardized plastic teeth on manikins' heads in a randomized sequence. The proportion of residual simulated plaque (RSP area in %) was planimetrically assessed, and the average root surface roughness produced (Ra and ∆Ra in µm) was measured by a precision profilometer. RESULTS: The uninstrumented root surfaces showed a Ra of (median (Q25/Q75)) 1.00 µm (0.83/1.16). Following instrumentation, EO left significantly less RSP than LO regardless of the used instruments (20.00 % (10.00/34.00) vs. 26.00 % (12.00/44.00) p < 0.001), whereas the ∆Ra values (0.29 µm (-0.04/0.96) vs. 0.35 µm (-0.04/1.01), p = 0.237) failed to show significant differences. The surface roughness was higher with GRA followed by AIR then TIG regardless of operators' experience (p < 0.001). CONCLUSION: Within the limits of the present study, the sonic device was most efficient in plaque removal, while the ultrasonic device produced the least surface roughness. CLINICAL RELEVANCE: All three tested instruments seem effective in the mechanical root debridement during SPT, whereat the ultrasonic device show the smoothest root surface of all.

Efficacy of calcium oxide and calcium hydroxide nanoparticles on the elimination of Enterococcus faecalis in human root dentin.

OBJECTIVES: The objective of this study was to compare the antibacterial effect of calcium oxide nanoparticles (CONPs) and calcium hydroxide nanoparticles (CHNPs) against Enterococcus faecalis in a dentinal block model. MATERIALS AND METHODS: E. faecalis strain JCM 7783 was introduced into dentinal tubules of semicylindrical dentin specimens by centrifugation and incubated for 1 week. Fifty microliters of CONPs or CHNPs was placed on the root canal side of the infected dentin specimens. The specimens were then incubated in aerobic condition at 37 °C and 100 % relative humidity for 1 week. The treated dentin specimens were subjected to fluorescent staining and confocal laser scanning microscopy (CLSM) to analyze the proportions of non-vital and vital bacterial cells inside the dentinal tubules. Scanning electron microscopy (SEM) was used to confirm the effect of the medicaments on the bacteria in the dentinal tubules. Calcium oxide (CO) and calcium hydroxide (CH) were used as controls. RESULTS: Based on the CLSM and SEM analyses, CHNPs were more efficient than CONPs in the elimination of the bacteria in the dentinal tubules. CONPs significantly killed more E. faecalis than CO and CH (P < .05). Neither CO nor CH was able to kill the bacteria. CONCLUSIONS: CHNPs were more effective than CONPs in the elimination of E. faecalis in dentinal tubules. CLINICAL RELEVANCE: CHNPs are effective nanoparticles in killing endodontic bacteria present in dentinal tubules. They have potential as an intracanal medicament, which may be beneficial in root canal therapy.

2-Hydroxyisocaproic acid is bactericidal in human dental root canals ex vivo.

AIM: To compare the activity of 2-hydroxyisocaproic acid (HICA), calcium hydroxide (Ca(OH)2 ) and chlorhexidine digluconate (CHG) against Enterococcus faecalis T-75359 (root canal isolate) in the root canals of extracted human teeth. METHODOLOGY: Bacterial suspensions (108  cfu mL-1 ) were incubated in root canals with 0.9 mm diameter root blocks (n = 73) for 21 days. Bacterial penetration into dentine was analysed by the Brown and Brenn method (n = 5). Canals (n = 17/group) were medicated with 40% of HICA paste, 40% of Ca(OH)2 paste, 2% of CHG solution or 0.9% of saline solution for 7 days. Samples taken from the inner (first 0.1 mm) and deeper (second 0.1 mm) dentine, and residual roots were cultured in broth for 24 h. Bacterial growth was detected by spectrophotometry (optical density, OD) and confirmed by culture on agar. The OD data were analysed with Kruskal-Wallis and Friedman with Wilcoxon signed-rank test between and within groups, respectively, and agar culture data with Pearson chi-square with Mann-Whitney and Cochran with McNemar tests, respectively (P < 0.05). RESULTS: Bacterial invasion into dentine tubules was confirmed. In deeper dentine, HICA inhibited >90% of bacterial growth in comparison with saline. No bacterial growth was observed in 82-100% of inner and deeper dentine samples. CHG prevented the growth in 88%, Ca(OH)2 in 59-76% and saline in 65-71%, respectively. HICA was significantly more active than Ca(OH)2 (P = 0.008) in the residual roots. The viability testing on agar showed essentially the same result. CONCLUSION: HICA paste exerted superior activity against E. faecalis and could have potential for root canal medication.

Adjunctive Steps for Disinfection of the Mandibular Molar Root Canal System: A Correlative Bacteriologic, Micro-Computed Tomography, and Cryopulverization Approach.

INTRODUCTION: This study evaluated the disinfecting ability of chemomechanical preparation with rotary nickel-titanium instruments, followed by 2 distinct adjunctive procedures in the root canals of extracted mandibular molars by means of a correlative analytical approach. METHODS: Twenty-two extracted mandibular molars were selected and anatomically matched between groups on the basis of micro-computed tomographic analysis. In the first phase of the experiment, root canals were contaminated with Enterococcus faecalis and subjected to chemomechanical preparation with BT RaCe instruments and 2.5% NaOCl irrigation. Then either XP-Endo Finisher instrument or passive ultrasonic irrigation was used to supplement disinfection. Micro-computed tomography was used to show whether the percentage of unprepared areas correlated to bacterial counts. In the second phase, the same teeth were contaminated once again, and the adjunctive procedures were used. Samples from the isthmus area of mesial roots and the apical 5-mm fragment of distal roots were obtained by cryopulverization. Samples taken before and after treatment steps in both phases were evaluated by quantitative polymerase chain reaction and statistically analyzed. RESULTS: In phase 1, preparation in both groups resulted in substantial decrease of bacterial counts (P < .001). The adjunctive approaches led to a further small bacterial reduction, which was significant for XP-Endo Finisher (P < .05). No significant differences were observed between groups for persisting bacterial counts. Correlative analysis revealed no statistically significant relationship between bacterial reduction and the percentage of unprepared areas (P > .05). In phase 2, both methods had significant antibacterial effects in the main canal, but none of them could predictably disinfect the isthmus/recess areas. CONCLUSIONS: Both XP-Endo Finisher and passive ultrasonic irrigation exhibited antibacterial effectiveness, but only the former caused a significant reduction in the bacterial counts after chemomechanical preparation. None of them were effective in predictably disinfecting the isthmus/recess areas.

Ecological Hypothesis of Dentin and Root Caries.

Recent advances regarding the caries process indicate that ecological phenomena induced by bacterial acid production tilt the de- and remineralization balance of the dental hard tissues towards demineralization through bacterial acid-induced adaptation and selection within the microbiota - from the dynamic stability stage to the aciduric stage via the acidogenic stage [Takahashi and Nyvad, 2008]. Dentin and root caries can also be partly explained by this hypothesis; however, the fact that these tissues contain a considerable amount of organic material suggests that protein degradation is involved in caries formation. In this review, we compiled relevant histological, biochemical, and microbiological information about dentin/root caries and refined the hypothesis by adding degradation of the organic matrix (the proteolytic stage) to the abovementioned stages. Bacterial acidification not only induces demineralization and exposure of the organic matrix in dentin/root surfaces but also activation of dentin-embedded and salivary matrix metalloproteinases and cathepsins. These phenomena initiate degradation of the demineralized organic matrix in dentin/root surfaces. While a bacterial involvement has never been confirmed in the initial degradation of organic material, the detection of proteolytic/amino acid-degrading bacteria and bacterial metabolites in dentin and root caries suggests a bacterial digestion and metabolism of partly degraded matrix. Moreover, bacterial metabolites might induce pulpitis as an inflammatory/immunomodulatory factor. Root and dentin surfaces are always at risk of becoming demineralized in the oral cavity, and exposed organic materials can be degraded by host-derived proteases contained in saliva and dentin itself. New approaches to the prevention and treatment of root/dentin caries are required.