Efficacy of the GentleWave System in the removal of biofilm from the mesial roots of mandibular molars before and after minimal instrumentation: An ex vivo study.

AIM: To compare the efficacy of Enterococcus faecalis biofilm removal using the GentleWave System (GWS) (Sonendo Inc, CA) on non-instrumented versus minimally instrumented root canal systems. METHODOLOGY: Thirty-four mandibular molars were autoclaved and allocated to four groups: Negative control (n = 5); positive control (n = 5); Group 1: non-instrumentation + GWS (NI + GWS) (n = 12); and Group 2: minimal instrumentation + GWS (MI + GWS) (n = 12). Of 34 samples, 24 samples with Vertucci type 2 configuration within the mesial root of each sample were allocated to Groups 1 and 2 and then matched based on the working length and root canal configuration. After inoculation of samples with E. faecalis for 3 weeks, the GWS was used on Group 1 without any instrumentation and Group 2 after instrumentation of mesial canals until size 20/06v. CFU and SEM analysis were used. RESULTS: Log10 (CFU/mL) from the positive control, and Group 1 and 2 were 7.41 ± 0.53, 3.41 ± 1.54, and 3.21 ± 1.54, respectively. Both groups showed a statistically significant difference in the reduction of viable E. faecalis cells compared to the positive control (Group 1 [p = .0001] and Group 2 [p < .0001]), whilst showing no significant difference between the two tested groups (p < .05). CONCLUSION: The use of GWS on the non-instrumented root canal system could be an effective disinfection protocol in removing the biofilm without dentin debris formation.

Effect of supplementary sodium hypochlorite agitation techniques on an ex vivo oral multispecies biofilm during passive disinfection of simulated immature roots.

AIM: To compare the effect of different sodium hypochlorite (NaOCl) agitation techniques on an ex vivo oral multispecies biofilm during passive disinfection of simulated immature roots. METHODOLOGY: Extracted human teeth were prepared to simulate immature roots. They were infected with a dental plaque-derived multispecies biofilm and cultured for 14 days. The roots were randomly designated into four groups: (1) negative control (PBS), (2) 1.5% NaOCl (CNI), (3) CNI + Ultrasonic activation (UA), (4) CNI + EasyClean agitation (ECA), (5) CNI + XP-endo finisher agitation (XPF), and (6) positive control (6% NaOCl). Biofilm samples were collected from the root canals and used to determine the number of viable cells (colony-forming units), scanning electron microscopy, and 16S rRNA gene sequencing. The mean colony-forming units per mL (CFU/mL) were analysed using One-way anova. 16S rRNA sequencing data were analysed for alpha (observed OTUs, Shannon index, and Chao1) and beta diversity (Bray-Curtis dissimilarities). The LEfSe analysis was used to determine the effect of treatment procedures on the abundance of root canal microbiota. The significance was set at .05. RESULTS: PBS and CNI samples had significantly higher CFU/mL counts than UA, ECA, XPF, and 6% NaOCl samples (p < .05). The pre-treatment, PBS, and CNI groups had significantly greater alpha diversity than the UA, ECA, XPF, and 6% NaOCl groups (p < .05). NaOCl agitation groups and the 6% NaOCl group achieved a more pronounced reduction in bacteria from the genera Fusobacterium, Actinomyces, Porphyromonas, and Capnocytophaga. CONCLUSIONS: The effectiveness of passive disinfection protocols was enhanced by NaOCl agitation techniques, suggesting that this supplementary method can improve the outcome of revitalization procedures.

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.

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.

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.

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.

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.

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.

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.

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.

Collagen-like peptide sequences inhibit bacterial invasion of root dentine.

AIM: To investigate the effects of peptides derived from the sequence of collagen to inhibit penetration of human or bovine dentine by species of streptococci and enterococci. METHODOLOGY: Blocks of human or bovine root dentine were infected for 14 days with bacterial cultures, in the presence or absence of various collagen-like peptide sequences. Invasion of dentinal tubules was determined from microscopic images of histochemically stained dentine thin sections. Extent of invasion was expressed as tubule invasion index (TI), or tubule invasion factor (TIF) which, in addition to the density of invasion, took into account the depth of invasion. Data were analysed by two-way anova. RESULTS: Streptococcus gordonii, Streptococcus mutans and Enterococcus faecalis were associated with heavy invasion (TI >2.5, TIF >4) of human or bovine root dentinal tubules, with E. faecalis being the most penetrative. Incorporation of peptides Gly-Pro-Ala or Gly-Pro-Hyp into the in vitro model system significantly reduced (P < 0.05) dentine invasion by the three species of highly invasive organisms. Inhibition of bacterial invasion by the peptides was dose dependent, and the peptides did not inhibit bacterial growth in culture. CONCLUSION: Specific collagen-like peptide sequences inhibited the invasion of dentine in vitro by a range of oral bacteria. The peptides likely act as competitive inhibitors blocking bacterial collagen receptors and could potentially allow for target-specific control of dentine infections.

Endodontic drug delivery for root surface disinfection: a laboratory feasibility evaluation.

OBJECTIVES: This study aims to assess the potential of a mixture of three antibiotics (TreVitaMix, TVM) as an intracanal dressing to disinfect the outer root surface by applying a new in vitro model. MATERIALS AND METHODS: Fifty freshly extracted bovine roots were endodontically treated. Forty samples were then thoroughly scaled, mounted to petri dishes, gas sterilized, and randomly allocated to four groups (n = 10/group) according to their intracanal medication: sterile saline (NaCl; control, A); the TVM carrier material alone, i.e., propylene glycol (PG; B); TVM (C); and calcium hydroxide (D). In an additional group (E), the cementum was not removed and TVM was placed. Petri dishes were filled with Fastidious Anaerobe Agar, inoculated with Fusobacterium nucleatum suspension and then anaerobically incubated during 48-h intervals at 37 °C up to 192 h. Inhibition zones around the roots were then measured after each incubation period (mm(2)). RESULTS: Only teeth inoculated with the TVM dressing showed inhibition at all time points, whereas the other treatments showed no peri-radicular growing inhibition. Presence of cementum had no negative effect on disinfection (p = 0.9320). CONCLUSION: TVM was able to penetrate through the dentine and inhibit the bacterial growth of F. nucleatum up to 192 h. CLINICAL RELEVANCE: TVM might have the potential to sustainably disinfect the outer root surface in perio-endo lesions and serve as an adjunctive antimicrobial agent.

Cold plasma: a novel approach to treat infected dentin-a combined ex vivo and in vitro study.

OBJECTIVES: The aim of this study was to evaluate the antimicrobial effect of cold plasma (CP) on infected dentin surfaces in vitro and ex vivo. MATERIALS AND METHODS: To examine the effect of cold plasma on root surfaces, 24 root surfaces were infected with Streptococcus mitis. Specimens were randomly divided into three groups: Within the control group (C), root surfaces were rinsed with NaCl; root surfaces in the second group were additionally scaled and root planed (SRP), and in the third group, root surfaces were rinsed, scaled, root planed, and in addition, CP was applied (SRP + CP). To examine the effect of CP on root caries lesions (RCLs), 16 freshly extracted teeth with symmetrical carious lesions were equally divided into two groups. In the control group, carious lesions were treated with chlorhexidine (CHX), whereas CHX was applied in conjunction with CP in the test group (CHX + CP). For microbiological analysis, dentin samples were serially diluted and CFU counts were estimated after 24 h of incubation. RESULTS: Compared to C, mean CFU values for SRP and SRP + CP were significantly lower (p < 0.05). In addition, mean CFUs for SRP + CP were reduced to 0 and, therefore, significantly lower than SRP (2.98 log CFU/mL) alone (p = 0.000, Mann-Whitney U). Regarding RCLs, significantly lower mean CFU values were observed for CHX + CP when compared to CHX (4.45 vs. 2.67 log CFU/mL, p = 0.002, Mann-Whitney U test). CONCLUSIONS: For disinfection of exposed root surfaces, the adjunctive application of CP is promising. In addition, the combined application of CHX + CP has the potential to disinfect root dentin surfaces. CLINICAL RELEVANCE: It was shown that the combination of cold plasma with CHX is the best available option for the disinfection of root surfaces.

[Microbial Community Structure on the Root Surface of Patients with Periodontitis.]

OBJECTIVES: To study the microbial community structure on the root surface of patients with periodontitis. METHODS: Bacterial plaque and tissues from the root neck (RN group),root middle (RM group) and root tine (RT group) of six teeth with mobility 3 in one patient with periodontitis were sampled.The V3V4 region of 16S rRNA was sequenced on the Illumina MiSeq platform.The microbial community structure was analyzed by Mothur,Qiime and SPSS software. RESULTS: The principal component analysis (PCoA) results indicated that the RM samples had a similar microbial community structure as that of the RT samples,which was significant different from that of the RN samples.Thirteen phyla were detected in the three groups of samples,which included 7 dominant phyla.29 dominant genera were detected in 184 genera.The abundance of Bacteroidetes_[G-6] and Peptostre ptococcaceae_[XI][G-4] had a positive correlation with the depth of the collection site of samples (P<0.05),while the abundance of Prevotella,Selenomonas,Corynebacterium and Olsenella had a negative correlation with the depth of the collection site of samples (P<0.05). CONCLUSIONS: There is region-specificity of microbial community structure on the root surface of patients with periodontitis.

The collagen binding protein Cnm contributes to oral colonization and cariogenicity of Streptococcus mutans OMZ175.

Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm(+) native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm(+) isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity.

Use of a dissolved oxygen microsensor for assessing the viability and thickness of microbial biofilm on root surfaces.

AIM: To evaluate the use of a dissolved oxygen microsensor (DOMS) for assessing the viability and thickness of microbial biofilms on the apical external surface of contaminated human tooth roots. METHODOLOGY: Apical biofilm formation was evaluated in 15 roots contaminated in vitro with a polymicrobial mixture of Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans for 7, 21 and 60 days and in three freshly extracted roots with associated radiographically visible periapical lesions. In each root, the thickness and viability (measured by the amount of dissolved oxygen) of biofilm formed on the apical 2 mm were examined with the DOMS. Scanning electron microscopy (SEM) was used as an auxiliary analysis to confirm the existence of the biofilms detected by the DOMS. RESULTS: The DOMS detected dissolved oxygen on the biofilms formed on the three residual roots up to thickness of 375 µm, 480 µm and 1650 µm. In the 15 roots contaminated in vitro, the DOMS detected dissolved oxygen in six specimens up to thicknesses from 75 to 250 µm, and the intensity of the metabolic activity (biofilm thickness) was directly proportional to the contamination time. SEM confirmed the presence of biofilm in all roots. CONCLUSION: The dissolved oxygen microsensor allowed the measurement of the amount of dissolved oxygen in the biofilm, which is indicative of the intensity of the microbial metabolic activity (viability), correlating the results with biofilm thickness. The DOMS was effective in freshly extracted roots, but had limitations in roots contaminated in vitro after short periods (7 and 21 days) of contamination.

Protein and bacteria binding to exposed root surfaces and the adjacent enamel surfaces in vivo.

Exposure of root surfaces due to inflammatory tissue breakdown is a clinical characteristic of periodontitis. The gingival margin may further recede during treatment. Pellicles and early dental plaque on enamel surfaces of periodontitis patients have previously been described. The binding properties of exposed root surfaces, which may affect the incorporation of proteins from especially the GCF into the enamel pellicle and thereby early dental plaque formation are largely unknown. The aim of this study was to examine if exposed root surfaces could affect pellicle and initial dental plaque formation on the enamel surface by the analysis of proteins and early adhering bacteria binding to the exposed root surfaces and to the adjacent, gingival enamel surface. Supragingival pellicle and plaque samples were taken from exposed root surfaces and the adjacent enamel surfaces in eleven surgically treated periodontitis patients. For comparison, samples were taken from enamel surfaces of teeth not in need of treatment. Additionally, subgingival bacterial samples were taken. Pellicle proteins were analysed by SDS-PAGE, immunoblotting and image analysis, and bacterial samples by culturing. Significantly more plasma proteins and bacteria were found on the exposed root surfaces than on the enamel. The depth of the gingival recessions was negatively correlated to the amount of plasma proteins in the enamel pellicle. Actinomyces spp. were most frequently found on the exposed root surfaces. The total viable counts and streptococci (%TVC) were positively correlated between subgingival samples and samples from the root surface and enamel of surgically treated teeth. A positive correlation was also found for the findings of Gram-negative anaerobes in subgingival samples and samples from the enamel surface. Our findings suggest that an exposed root surface has binding properties different from an enamel surface and could affect early biofilm formation on the adjacent enamel surface.

Promotion of endodontic lesions in rats by a novel extraradicular biofilm model using obturation materials.

Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular biofilm development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an in vivo extraradicular biofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extraradicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular first molars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into the mesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks of pulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria were identified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites. Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, in the extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulp exposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experimental sites than in the control sites (P < 0.05 by Welch's t test). These results suggest that we developed an extraradicular biofilm model in rats and that extraradicular biofilms affect developing periapical lesions.