Detection of bacteria in dental samples using the Periodic acid-Schiff (PAS) histological stain.

The bacterial cell wall mainly consists of glycoproteins and polysaccharides, which could be detected in dental tissue with specific stain protocols. The present study aimed to investigate bacteria stainability in dental histological samples of human teeth by a histochemical method. Eight extracted teeth, because severely decayed, were decalcified, dehydrated, paraffin-embedded, and serially sectioned at 4 µm thickness each. The serial sections were then stained with Periodic acid-Schiff (PAS). Furthermore, SEM analysis was performed on the same slide of one previously histologically investigated tooth to acquire more details on the structures stained by the PAS method obtained from the histological procedures. Afterward, some American Type Culture Collection (ATCC) strains, smeared on glass slides, were stained following the staining method used in histological samples. Stained rod and cocci forms by PAS stain, observed under light microscopy, were predominantly detected inside dentinal tubules and root canal space of histologically examined specimens, suggesting their bacterial origin. Additional SEM analysis on the identical histological stained slide showed the precise nature of these forms (bacteria) and supplemental information regarding their vitality status. In addition, ATCC smeared strain samples showed variable PAS stainability of microorganisms investigated. Due to its properties, the PAS histochemical stain could be a valid and helpful aid for non- or weakly stainable microorganisms in infected tissues to be associated with other methods of investigation.

Post-Operative Quality of Life after Single-Visit Root Canal Treatment Employing Three Different Instrumentation Techniques-An Institutional Randomized Clinical Trial.

Root canal treatment (RCT) eliminates damaged pulpal tissue and protects the tooth from recurrent microbial invasion. Post-endodontic pain (PEP) is a frequently encountered complication of root canal therapy. It can have an impact on patients' quality of life (QoL) and their subjective perception of treatment options. Thus, a self-assessment questionnaire was used to evaluate and compare the influence of manual, rotary, and reciprocating file shaping procedures on immediate post-operative quality of life (POQoL) involving single-visit root canal therapy. It was a double-blinded, randomized, controlled clinical trial. A total of 120 participants were randomly assigned sequentially to three groups comprising 40 patients in each group: Group A: Hand K file (positive control); Group B: ProTaper Next file system; and Group C: WaveOne Gold. Post-operative pain was evaluated using a 4-point visual analog scale (VAS) after 12 h, 24 h, 48 h, 72 h, and 1 week. The highest post-operative pain was with manual instrumentation using hand K files, and the lowest was with reciprocating and rotating instrumentations. No significant difference was noted between the parameters of quality of life assessed, suggesting the filing system or technique had a similar effect.

Arduino Automated Microwave Oven for Tissue Decalcification.

Decalcification of hard tissues such as bone and teeth is a complex process that requires using chemicals such as acids and chelating agents. Acids act faster than chelating agents, but they have a greater risk of damaging biological samples. Increasing the reaction speed of the chelating agent may solve this issue. There are several strategies to speed up this process, and using microwaves seems to be one of the most effective. However, lab-dedicated microwave ovens are expensive, and their purchase may seem unjustified. Therefore, a low-cost modification of a commercial microwave oven, consisting of an Arduino automation device, has been developed. The setup has proven reliable for continuous work, thanks to implementing an electronic safety circuit. In addition, it may reduce the decalcification time using a chelating agent, achieving optimal results regarding tissue preservation and quality of histological sections.

A simplified method for detecting Gram-positive and Gram-negative bacteria in dental histological samples: A preliminary and comparative study.

OBJECTIVE: Bacteria identification inside the dental tissue is a complex procedure requiring specific protocols. This study aimed to compare two classical Gram staining methods with a new staining method proposed by the authors to detect Gram-positive and Gram-negative bacteria in dental histological samples of human dentin. METHODS: Ten human teeth, extracted because of various pathologies, were decalcified, dehydrated, and paraffin-embedded. Then, approximately 100 serial sections of 4 µm thickness were made per sample. The serial sections were placed on glass slides and were stained according to Brown-Brenn, Brown-Hopps, and a proposed modification of Brown-Brenn staining. Both ATCC strains, smeared on glass slides, were stained following each method's instructions used in histological samples. RESULTS: From a qualitative evaluation, the Brown-Brenn method resulted in better staining of Gram-positive bacteria, while the authors' proposed staining technique was more oriented towards Gram-negative bacteria. On the other hand, the Brown-Hopps showed quite a balance in detecting Gram-positive and Gram-negative bacteria. Unlike the Brown-Brenn stain, the other two protocols showed better stainability of Gram-negative microorganisms in bacterial-smeared samples. CONCLUSION: All staining techniques evaluated in this article can identify bacteria, but the outcome can change according to the staining procedure used.

Can the Concentration of Citric Acid Affect Its Cytotoxicity and Antimicrobial Activity?

BACKGROUND: There has been no unanimity concerning the ideal concentration of citric acid for safe use in clinical practice. This study evaluated the cytotoxicity and the antibacterial activity in infected dentinal tubules of 10% and 1% citric acid (CA) solutions. METHODS: The cytotoxicity of CA solutions in DMEM (diluted 1/10, 1/100) was assessed in L-929 fibroblasts. A broth macrodilution method (MIC and MBC) was used to assess CA antibacterial concentration. The antimicrobial activity of CA solutions was also evaluated after their final rinse inside root canals in previously Enterococcus faecalis-contaminated dentinal tubules. Ten infected dentine samples were rinsed for 5 min with 5% NaOCl and subsequently with 1% citric acid for 3 min. Another 10 were rinsed with 5% NaOCl and 10% citric acid for 3 min; the remaining four specimens were utilized as positive controls. Two uncontaminated specimens were used as negative controls. After LIVE/DEAD BacLight staining, the samples were assessed using CLSM to analyze the percentage of residual live and dead cells. RESULTS: Both undiluted and diluted CA solutions showed severe toxicity; no changes from normal morphology were displayed when diluted 1/100. The MIC and MBC of CA were 6.25 mg/mL and 12.50 mg/mL, respectively. CA solutions demonstrated significantly low levels of bacterial counts than the positive control group, reporting a value of 9.3% for the 10% solution versus the 1% solution (35.2%). CONCLUSIONS: Despite its valuable antimicrobial properties, the cytotoxic effects of citric acid should be considered during endodontic treatment.

The impact of irrigation protocols on epoxy sealer penetration depth in dentinal tubules. Study involving laser confocal microscopy.

The aim was to assess the impact of irrigation protocols ended with ethanol or chlorhexidine on AH Plus penetration into dentinal tubules. 45 root canals were prepared to ISO 40/04, divided into three groups and irrigated with three protocols: Group 1 (control): 5.25% NaOCl; Group 2: 40% CA (citric acid), 5.25% NaOCl, 40% CA, water, ethanol; Group 3: 40% CA, 5.25% NaOCl, 40% CA, water, 2% chlorhexidine. Canals were filled using vertical condensation technique with gutta-percha and fluorescein-stained AH Plus sealer. After 72 h, 1 mm thick cross-sections were cut at 2, 5, 8 mm from the apex. Confocal laser microscope was used to measure the sealer penetration into dentinal tubules. Mean depth of sealer penetration (in micrometres) was 107, 131, 170 (Group 1); 146, 233, 317 (Group 2); 185, 301, 542 (Group 3); in apical, middle and coronal parts, respectively. Irrigation protocol ended with chlorhexidine resulted in the deeper sealer penetration compared with alcohol.

Comparative Evaluation of the Penetration Depth into Dentinal Tubules of Three Endodontic Irrigants.

This study aimed to examine the penetration depth into dentinal tubules of some chelating agents. The 17% EDTA and two preparations containing surfactants (Smear Clear, Bioakt Endo) were tested. Surface tension and liquid viscosity were measured using a Dynamic Contact Angle Analyzer and a Haake rotational rheometer. To measure the penetration depth inside dentinal tubules, thirty maxillary central incisors were selected from a pool of extracted human permanent teeth and allocated to three experimental groups (10 samples each), as well as were mechanically shaped and cleansed with 5.25% NaOCl, followed by each of the chelators being labeled with 0.1 wt % Rhodamine B according to final irrigation protocol established. The samples were embedded in an epoxy resin, after which 200 µm thick transverse sections were obtained at 2, 5, and 8 mm from the apex with a saw microtome. The specimens were then observed using a confocal laser microscope (CLSM) and the penetration of the labeled solution was measured in every third of each sample. Statistical analysis was performed using ANOVA or Kruskal-Wallis tests according to the distribution of data, evaluated with the Shapiro-Wilk normality test. Viscosity and surface tension tests have shown that BioAKT Endo has the lowest values compared to EDTA and Smear Clear. The medium penetration depth did not significantly differ among the three irrigants, while it increased considerably from the apical to the coronal level in all groups. Additionally, the maximum penetration depth increased significantly from the apical to coronal level, while among groups, BioAKT Endo showed the highest values at the apical and middle level compared to the other irrigants. No significant differences were observed among the three groups in medium and maximum penetration depths when the entire root was considered. New irrigants containing surfactants show reduced surface tension and, in one case (BioAKT Endo), viscosity. The lowering of the surface tension allows for better penetration of liquids into dentinal tubules than EDTA alone, thus improving the cleaning of the root canal system.

Real-time co-site optical microscopy study on the morphological changes of the dentine's surface after citric acid and sodium hypochlorite: a single-tooth model.

BACKGROUND: To this day, the effects of sodium hypochlorite and chelating agents on the smear layer and on the dentine's surface, remain not fully examined. The study is aimed to analyze the dentine's surface treated with 40% citric acid and 5.25% sodium hypochlorite according to two irrigation protocols. MATERIALS AND METHODS: The study employed a computer-controlled Nikon Eclipse LV100 optical microscope. Ten roots split longitudinally with canals prepared mechanically using the MTwo system to a size of 40/04 were observed. The root halves were divided into two study groups, one half in each of the groups. According to two irrigation protocols, the dentine's surface was irrigated with 40% citric acid and 5.25% sodium hypochlorite, separated with water. Dentine surface was observed in a fixed place and photographed in 500 × magnification after each irrigation stage. The obtained images were then analyzed using computer software (NIS-Elements AR, GIMP-2.6, ImageJ 1.45s). RESULTS: Various speed of removal of the smear layer and varied morphological changes of the dentine's surface were observed in both examined groups. Double irrigation of the dentine with citric acid for 30 s resulted in complete removal of the smear layer, and double irrigation for 60 s resulted in increased mean diameter of the dentine tubules (degradation of the dentine's surface). CONCLUSIONS: Double alternating irrigation with citric acid and sodium hypochlorite for 30 s yielded satisfactory results, while double irrigation for 60 s resulted in increased mean diameter of the dentine tubules. A real time assessment of the dentine's surface changes after both tested solutions can improve understanding of the phenomena that occur during the irrigation and as a result it might help to improve clinical outcomes in root canal treatment.

Evaluation of Cytotoxicity and Antibacterial Activity of a New Class of Silver Citrate-Based Compounds as Endodontic Irrigants.

In the present study, the cytotoxicity and the antimicrobial activity of two silver citrate-based irrigant solutions were investigated. Cytotoxicity of various concentrations (0.25%, 0.5%, 1%, 2.5%, 5%) of both solutions (BioAKT and BioAKT Endo) was assessed on L-929 mouse fibroblasts using the MTT assay. For the quantitative analysis of components, an infrared (I.R.) spectroscopy was performed. The minimum inhibitory and minimal bactericidal concentrations (M.I.C. and M.B.C., respectively) were ascertained on Enterococcus faecalis strain ATCC 4083. For biofilm susceptibility after treatment with the irrigating agent, a minimum biofilm eradication concentration (M.B.E.C.) and confocal laser scanning microscope (C.L.S.M.) assays were performed. Quantification of E. faecalis cell biomass and percentage of live and dead cells in the biomass was appraised. Normality of data was analyzed using the D'Agostino & Pearson's test and the Shapiro-Wilk test. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's test. Both silver citrate solutions showed mouse fibroblasts viability >70% when diluted to 0.25% and 0.5%. Conversely, at higher concentrations, they were extremely cytotoxic. F.T.-IR spectroscopy measurements of both liquids showed the same spectra, indicating similar chemical characteristics. No substantial contrast in antimicrobial activity was observed among the two silver citrate solutions by using broth microdilution methods, biofilm susceptibility (MBEC-HTP device), and biomass screening using confocal laser scanning microscopy (C.L.S.M.) technique. Both solutions, used as root canal irrigants, exhibited significant antimicrobial activity and low cytocompatibility at dilutions greater than 0.5%.

Antimicrobial effectiveness of etidronate powder (Dual Rinse® HEDP) and two EDTA preparations against Enterococcus faecalis: a preliminary laboratory study.

This study aimed to evaluate the antimicrobial and antibiofilm activity of two chelating agents: ethylenediaminetetraacetic acid (EDTA) combined or not with detergents, and etidronic acid combined with sterile saline. The bacterial inhibitory and bactericidal concentrations (MIC and MBC, respectively) were determined on Enterococcus faecalis ATCC 4083 strain. Antimicrobial tests were performed on a biofilm model after treatment with the chelating agents at different times (1, 3, and 5 min) using a biofilm eradication concentration (MBEC) and confocal laser scanning microscope (CLSM) assays. Quantification of cell biomass and percentage of live and dead cells in the biomass was assessed for each group. The normality of the distributions for each variable was assessed using the D'Agostino and Pearson's omnibus normality test. The comparison of bacterial viability among groups and between any two groups was performed using the non-parametric Kruskal-Wallis one-way analysis of variance and the Dunn's test, respectively. No significant between-group difference was observed regarding biomass reduction. On the other hand, EDTA combined with detergents displayed a substantial increase of the dead bacteria ranging between 35 and 43%; whereas, the number of cells killed in the control group and in the other treated groups always ranged between 1 and 6%, at all experimental times. The addition of detergents to EDTA can improve its anti-biofilm activity by reducing EPS production and enhancing the killing of sessile bacterial cells. Clinical relevance EDTA presents a relevant antimicrobial activity when combined with surface-active agents.

Dual Rinse® HEDP increases the surface tension of NaOCl but may increase its dentin disinfection efficacy.

The aim of this study was to evaluate the surface tension and the antimicrobial activity in infected dentin of a NaOCl solution combined with an etidronate powder (Dual Rinse® HEDP), compared to pure NaOCl and the classic NaOCl + EDTA irrigating sequence, respectively. The surface tension of three irrigants was measured by Wilhelmy technique. To evaluate the antimicrobial activity of the solutions, 26 human teeth were contaminated for 5 days with E. faecalis. After bacterial contamination, ten samples were irrigated with NaOCl followed by EDTA, another ten with NaOCl/Dual Rinse® HEDP, and four were used as positive controls. Two specimens not contaminated were used as negative controls. After live/dead BacLight staining, samples were examined by CLSM for analyzing % of residual live and dead cells. Comparison of bacterial viability between and within groups was performed using the Mann-Whitney test for independent samples and the Wilcoxon signed-rank test, respectively. The mean surface tension of EDTA was significantly lower than that of the other irrigants tested (p < 0.001). Conversely, the surface tension of NaOCl/Dual Rinse® HEDP solution was significantly higher than that of all the other solutions (p < 0.001). Residual bacterial viability in the NaOCl/Dual Rinse® HEDP (1.71%) was significantly lower (p = 0.019) than in the NaOCl + EDTA group (3.77%). All of the experimental groups showed significantly lower proportion of viable bacterial cells than the positive control group (p < 0.01). Clinical relevance adding etidronate to NaOCl increases its antimicrobial effect in dentinal tubules even though increases its surface tension.

Smear Layer Removing Ability of Root Canal Irrigation Solutions: A Review.

AIM: The purpose of this review is to address the smear layer removing the ability of root different canal irrigants including ethylenediaminetetraacetic acid (EDTA), a mixture of tetracycline, acid and detergent (MTAD), tetraclean, citric acid, Q-Mix, maleic acid, and smear clear. BACKGROUND: Smear layer is a layer which is produced during instrumentation. It contains both organic and inorganic materials. It may also contain bacteria and their byproduct considering the root canal situation. This layer may prevent the penetration of the healing material introduced to the canal to the interior of the dentinal tubules. In addition, it can affect the adaptation of sealing materials to the root canal walls. REVIEW RESULTS: The smear layer removal ability of MTAD is superior and faster than EDTA. Tetraclean is similarly composed of an acid, an antibiotic, and a detergent. The presence of doxycycline is believed to help the smear layer removal ability of these irrigants. Antibiotics such as tetracycline had similar smear layer removal ability as even citric acid. EDTA is an amino acid with a chelating ability that sequestrates metal ions. Some believed EDTA smear layer removing ability is better than MTAD, tetraclean, SmearClear, and 20% citric acid which is controversial in case of comparison between MTAD and EDTA. Phosphoric acid is efficient enough to be comparable to EDTA in removing the smear layer. Maleic acid is an organic compound with acid etching smear layer removal. Its ability seems to be similar or even better than EDTA. Citric acid as another organic acid is believed to be used as smear removing agent. HEBP is another chelating agent that can be used in combination with NaOCl; however, HEBP is a weak decalcifying agent compared to EDTA and hence cannot be applied as a mere final rinse. QMiX is a combination of CHX, EDTA, and detergent and should be used at final rinse. It is believed that QMiX is as efficient as EDTA. Smear clear is a 17% EDTA solution including an anionic and cationic (cetrimide) surfactant. The ability of QMiX is similar to EDTA. CONCLUSION: There are different canal irrigation solutions with various smear layer removal ability that some should be used as a mere final rinse and some should not. CLINICAL SIGNIFICANCE: The usage of canal irrigation solutions depends on the clinical situation and preference of the dentists. This study provides a good guide for clinician of the field.

Clinical and Histological Findings of Post-Treatment Infection in the Presence of Vertical Root Fracture and Apical Periodontitis: Case Reports.

Apical periodontitis and vertical root fracture (VRF) might coexist in the same root, thus representing a di-agnostic challenge in endodontics. Diagnosis should be based on detailed clinical examination and precise radiographic interpretation in addition to histological examination, if needed. The histological findings of the case presented showed the possible coexistence of a VRF in the coronal third of the root and signs of apical periodontitis caused by intracanal infection in the apical third of the same root. The presentation of this case underlines the importance of histological examination as a valid complementary diagnostic tool with clinical and radiographic examinations in reaching an accurate diagnosis once the root has been extracted.

Sodium hypochlorite penetration into dentinal tubules after manual dynamic agitation and ultrasonic activation: a histochemical evaluation.

The aim of this study was to compare the effects of Manual Dynamic Agitation and Passive Ultrasonic Irrigation on sodium hypochlorite (NaOCl) penetration into dentinal tubules using its bleaching ability. Thirty-four single-rooted teeth with round-shaped root canals were distributed in two homogeneous groups and one control group, characterized by different NaOCl activation systems: Manual Dynamic Agitation and Passive Ultrasonic Irrigation. After instrumentation, all root canals were stained with 10% copper sulphate solution followed by 1% rubeanic acid alcohol solution under vacuum. Final irrigation was performed with 5 mL of 5.25% NaOCl solution for 1 min and activated with Manual Dynamic Agitation or Passive Ultrasonic Irrigation for another 1 min depending on the treatment group. The teeth were transversely sectioned at the middle portion of the apical, middle, and coronal thirds and observed under light microscope. NaOCl solution penetration was evaluated by measuring the percentage of bleached circumference of the root canal relative to the stained circumference, bleached areas, mean, and maximum penetration depth. No differences in the evaluated parameters were observed between groups (p > 0.05). Within groups, an increase of values was recorded from apical to coronal direction as for percentage of staining, percentage of bleaching and bleached area. NaOCl penetration into dentinal tubules did not significantly vary among the three levels. No significant differences in penetration of sodium hypochlorite into dentinal tubules when activated by means of Manual Dynamic Agitation or Passive Ultrasonic Irrigation were observed in the apical, middle, and coronal thirds of teeth with single straight round root canals.

Lasers in Apicoectomy: A Brief Review.

Since the invention of laser, various applications for lasers in endodontics have been proposed, such as disinfection of the root canal system, canal shaping, pulp diagnosis, and apico-ectomy. One of the major applications of laser in endodontics is apicoectomy. The aim of this article is to review the benefits and drawbacks of laser applications in apicoectomy, including effect on apical seal, effect on dentin permeability, effect on postsurgery pain, effect on crack formation, effect on root-end morphology, effect on treatment outcome, and connective tissue response to laser-treated dentin.

Effect of Surfactants on the Efficacy of Root Canal Irrigants: A Review.

The mechanical debridement of the root canal system fails to completely remove debris from the root canal walls. So, one or more irrigants must be used for complete disinfection of the root canal system (RCS). One possible way to improve the bactericidal efficacy of the disinfecting solutions is to incorporate different detergents as surface active agents to help reduce the surface tension and increase the wettability of the solutions. Surfactants have been added to various root canal irrigation solutions. The purpose of this paper is to review the effects of adding surfactants to established root canal irrigants and new surfactant-containing root canal irrigants.

Antimicrobial Effect and Surface Tension of Some Chelating Solutions with Added Surfactants.

This study assessed the antimicrobial efficacy and surface tension of established irrigating solutions with a new experimental chelating solution in infected dentin tubes. Twenty-five specimens were randomly assigned to each of the irrigating solutions. Twenty specimens were used as negative and positive controls. After 21 days of contamination with E. faecalis, the irrigating solutions MTAD, QMiX and Tetraclean NA were delivered into each infected root canal. The solutions were removed and dentin samples were withdrawn from the root canals with sterile low-speed round burs with increasing ISO diameters. The dentin powder samples obtained with each bur were immediately collected in separate test tubes containing 3 mL of BHI broth. After that, 100 µL from each test tube was cultured on blood agar. The grown colonies were counted and recorded as colony-forming units (CFU). The surface tension of the irrigants was measured using a Cahn DCA-322 Dynamic Contact Angle Analyzer. A Kruskal Wallis nonparametric ANOVA and a Friedman test were used (p<0.05). Tetraclean NA showed lower surface tension and CFU values than MTAD and QMiX. Better antibacterial action and low surface tension were observed for Tetraclean NA, probably due to the improved penetration into the root canal and dentinal tubes.

Antimicrobial Effect and Surface Tension of Some Chelating Solutions with Added Surfactants

Abstract This study assessed the antimicrobial efficacy and surface tension of established irrigating solutions with a new experimental chelating solution in infected dentin tubes. Twenty-five specimens were randomly assigned to each of the irrigating solutions. Twenty specimens were used as negative and positive controls. After 21 days of contamination with E. faecalis, the irrigating solutions MTAD, QMiX and Tetraclean NA were delivered into each infected root canal. The solutions were removed and dentin samples were withdrawn from the root canals with sterile low-speed round burs with increasing ISO diameters. The dentin powder samples obtained with each bur were immediately collected in separate test tubes containing 3 mL of BHI broth. After that, 100 μL from each test tube was cultured on blood agar. The grown colonies were counted and recorded as colony-forming units (CFU). The surface tension of the irrigants was measured using a Cahn DCA-322 Dynamic Contact Angle Analyzer. A Kruskal Wallis nonparametric ANOVA and a Friedman test were used (p<0.05). Tetraclean NA showed lower surface tension and CFU values than MTAD and QMiX. Better antibacterial action and low surface tension were observed for Tetraclean NA, probably due to the improved penetration into the root canal and dentinal tubes

Resumo Este estudo avaliou a eficácia antimicrobiana e tensão superficial de soluções irrigadoras e uma nova solução quelante em tubos de dentina infectada. Vinte e cinco espécimes foram aleatoriamente distribuídos conforme as soluções irrigantes. Decorrifdos 21 dias de contaminação com E. faecalis, a soluções de irrigação MTAD, QMiX e Tetraclean NA foram distribuídas em cada canal radicular infectado. As soluções foram removidas e as amostras de dentina foram retiradas dos canais radiculares com brocas esféricas de baixa velocidade com diâmetros ISO sucessivamente maiores. As amostras do pó de dentina obtidas com cada broca foram imediatamente colocadas em tubos de ensaio separados contendo 3 mL de caldo BHI. A seguir, 100 μL de cada amostra do tubo de teste foi cultivada em agar de sangue. As colônias crescidas foram contadas e registadas como unidades formadoras de colônias (UFC). A tensão superficial das soluções irrigantes foi medida utilizando o método de Wilhelmy. A análise não paramétrica de Kruskal-Wallis e o teste de Friedman foram utilizados (p<0,05). Tetraclean NA apresentou menor tensão de superfície e menores valores de UFC do que MTAD e QMiX. A melhor ação antibacteriana e baixa tensão superficial foram observadas para Tetraclean NA, provavelmente devido à melhor penetração no canal radicular e túbulos dentinários.