Ultrastructural analysis of the root canal walls after simultaneous irrigation of different sodium hypochlorite concentration and 0.2% chlorhexidine gluconate

To determine whether sodium hypochlorite (NaOCl) with 0.2% chlorhexidine gluconate (CHX) leads to colour change and precipitate formation, and to ultrastructurally analyse the dentine surface after simultaneous irrigation with 0.5% NaOCl and 0.2% CHX. Four tubes in which 5ml of different NaOCl concentrations and 5ml 0,2% CHX were placed, were observed every 15 minutes in the first two hours and after 7 days. Mixture solutions were centrifuged at 800 rpm/4 min. A precipitate is observed under light-microscopy. Thirty-five single-rooted teeth were instrumented using crown-down technique with irrigation: Positive control: distilled water, Negative control: 0,5% NaOCl+15% EDTA, Experimental group: identical to the negative control, then canals were treated with 0.5% NaOCl+0.2% CHX. The longitudinal root sections were observed under scanning-electron-microscopy. The amount of debris was assessed with 5/score-system, and the results were analysed using the Mann-Whitney U test (p < 0.001). Change in colour was noticed immediately after the merger and it did not change with time. Mixture solutions showed considerable turbidity, but precipitate was observed only after centrifugation. Statistical analysis showed a significant difference in the dentinal wall purity when comparing the cervical and middle root canal thirds between the experimental and negative control group. The difference was also observed between the cervical and middle thirds compared to the apical third of the root canal within these groups. Interaction between NaOCl and CHX, as well as the creation of precipitates, depends not only on the concentration of NaOCl, but also on the concentration of CHX.     

Evaluation of different irrigation protocols concerning the formation of chemical smear layer

The aim of this work was to evaluate, by means of scanning electron microscopy (SEM) and electron dispersive spectroscopy (EDS), the different irrigation protocols concerning the formation of chemical smear layer (CSL). Fifty‐five teeth were instrumented by using distilled water (DW) as irrigant. Next, the teeth were submitted to a protocol for removal of the mechanical smear layer produced. The teeth were divided into 11 groups, according to irrigation regimen. Then, the teeth were prepared and analyzed with SEM and EDS. Only in Group 1 [1 mL sodium hypochlorite (NaOCl) + 10 mL DW + 1 mL chlorhexidine (CHX) solution], Group 2 [1 mL NaOCl + 10 mL DW + 1 mL ethylenediaminetetraacetic acid (EDTA) + 10 mL DW + 1 mL CHX solution], and Group 3 [1 mL NaOCl + 10 mL DW + 1 mL citric acid (CA) + 10 mL DW + 1 mL CHX solution], the formation of CSL was observed. In the groups where CHX gel was used alone or in association with EDTA, CA, phosphoric acid (PA), and CHX solution, no formation of CSL was observed. According to the results found in the cases in which one wants to associate NaOCl with CHX, the protocol using PA (1 mL NaOCl + 10 mL DW + 1 mL PA + 10 mL DW + 1 mL CHX solution) did not induce formation of CSL. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Effects of irrigation solutions on the surface of protaper instruments: A microscopy study

The aim of this study was to evaluate the effects of sodium hypochlorite (NaOCl), chlorhexidine digluconate (CHX), and mixture of tetracycline, citric acid, and detergent (MTAD) solutions on the surfaces of nickel titanium (NiTi) rotary files with scanning electron microscopy (SEM) and atomic force microscopy (AFM). Irrigation solutions including 2.5% NaOCl, 5% NaOCl, 2% CHX, and MTAD were used. Four new ProTaper rotary NiTi files (F3) were immersed in each four irrigation solutions for 10 min, separately. One untreated NiTi file was used as a control specimen. The surface analysis was performed with SEM and AFM. The surface analysis in AFM was performed on 12 different regions located between first and second millimeters from the tip. SEM images were taken with different magnifications. No surface alteration was recorded under SEM evaluation. AFM revealed that the root mean square (RMS) values of all treated specimens were statistically higher than the control sample (P < 0.05). The sample which was treated with CHX showed lowest RMS values in test groups. All tested solutions caused surface alterations. CHX demonstrated limited surface alterations when compared to the other tested solutions. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

Cleaning capacity of octenidine as root canal irrigant: A scanning electron microscopy study

The aim of this study was to assess the cleaning capacity of the octenidine hydrochloride (OCT) used as root canal irrigant by scanning electron microscopy (SEM) analysis. Sixty human unirradicular extracted teeth were randomly distributed in 6 groups (n = 10) according to irrigant solutions which were used during root canal preparation: G1, 0.1% OCT; G2, 2% chlorhexidine (CHX); G3, 2.5% sodium hypochlorite (NaOCl); G4, OCT + 17% ethylenediaminetetraacetic acid (EDTA); G5, 2.5% NaOCl + 17% EDTA and G6, distilled water. All specimens were instrumented with ProTaper system up to F4. Teeth were sectioned and prepared for SEM. The smear layer was evaluated using a 5‐score system and the data were analyzed by Kruskal–Wallis and Dunn (α = 0.05). In all root canal thirds there was no significant difference between OCT, CHX, NaOCl, and water groups (p > .05), and these groups showed higher smear layer values than NaOCl + EDTA and OCT + EDTA groups (p < .05). There was no significant difference between NaOCl + EDTA and OCT + EDTA groups (p > .05). It was concluded that OCT used as a single root canal irrigant presented poor cleaning capacity and could be used in association with a final irrigation with EDTA to obtain smear layer removal.     

Evaluation of the interaction between sodium hypochlorite and several formulations containing chlorhexidine and its effect on the radicular dentin—SEM and push‐out bond strength analysis

The aim of the current study was to evaluate the presence of debris and smear layer after endodontic irrigation with different formulations of 2% chlorhexidine gluconate (CHX) and its effects on the push‐out bond strength of an epoxy‐based sealer on the radicular dentin. One hundred extracted human canines were prepared to F5 instrument and irrigated with 2.5% sodium hypochlorite and 17% ethylenediaminetetraacetic acid. Fifty teeth were divided into five groups (n = 10), according to the final irrigation protocol with different 2% CHX formulations: G1 (control, no final rinse irrigation), G2 (CHX solution), G3 (CHX gel), G4 (Concepsis), and G5 (CHX Plus). In sequence, the specimens were submitted to scanning electron microscopy (SEM) analysis, in the cervical‐medium and medium‐apical segments, to evaluate the presence of debris and smear layer. The other 50 teeth were treated equally to a SEM study, but with the root canals filled with an epoxy‐based endodontic sealer and submitted to a push‐out bond strength test, in the cervical, middle, and apical thirds. G2, G3, G4, and G5 provided higher precipitation of the debris and smear layer than G1 (P < 0.05), but these groups were similar to each other (P > 0.05), in both segments. The values obtained in the push out test did not differ between groups, independent of the radicular third (P > 0.05). The CHXs formulations caused precipitation of the debris and smear layer on the radicular dentin, but these residues did not interfere in the push‐out bond strength of the epoxy‐based sealer. Microsc. Res. Tech. 77:17–22, 2014. © 2013 Wiley Periodicals, Inc.     

A procedure to prepare cultured cells in suspension for electron probe X-ray microanalysis: application to scanning and transmission electron microscopy

We describe a simple procedure to prepare cultured cells in suspension to analyse elemental content at the cellular level by electron probe X-ray microanalysis. Cells cultured in suspension were deposited onto polycarbonate tissue, culture plate well inserts, centrifuged at low g , washed to remove the extracellular medium, cryofixed and freeze-dried, and analysed in the scanning mode of a scanning electron microscope. We tested the effect of different washing solutions (150 m m ammonium acetate, 300 m m sucrose, and distilled water) on the elemental content of cultured cells in suspension. The results demonstrated that distilled water was the best washing solution to prepare cultured cells. In addition, the low Na content, high K content and high K/Na ratio of the cells indicated that this procedure, based on the centrifugation at low g followed by cryopreparation, constitutes a satisfactory method to prepare cultured cells in suspension. We also investigated the effects of different accelerating voltages on X-ray signal collection. The results showed that moderate accelerating voltages, i.e. 10–11 kV, should be used to analyse whole cells in the scanning mode of the scanning electron microscope. We show that this method of preparation makes it possible to prepare cryosections of the cultured cells, thus permitting analysis of the elemental content at the subcellular level, i.e. nucleus, cytoplasm and mitochondria, using a scanning transmission electron microscope.     

Improved quantitative electron probe X-ray microanalysis of biological cryosections using an EDS germanium detector and a super-atmosphere thin window (SuperATW)

A new Link energy-dispersive GEM detector with SuperATW window was tested for quantitative electron probe microanalysis of low calcium and sodium concentrations ([Ca], [Na]) in intracellular compartments of cardiac myocytes. We compare Ca profiles with high count statistics and similar peak area collected under the same conditions with either a Be-windowed Si and a Ge SuperATW detector. The height of the Ca peak was increased by 7%, the full width at half-maximum height was reduced by 9% with the Ge detector. The counts statistics of the Ca Kα peak improved by 9% and the partial overlap with the K Kβ peak was better deconvoluted. We calculate [Ca] and errors of the single measurement in mitochondria of guinea-pig cardiac myocytes from spectra acquired with a Si or Ge detector. For identical analysis conditions, the [Ca] were identical; however, with the Ge SuperATW detector, the calculated error of the single measurement was only 1/2.7 of that calculated from measurements with the Si detector. We compare the peak area of identical [Na] in spectra collected with the Be-windowed Si detector and Ge SuperATW detector. The peak area was significantly higher with the SuperATW Ge than with the Si detector and Be window, whereas the continuum in the range 4–10 keV was comparable, demonstrating the improved sensitivity for low atomic elements such as Na of the Ge SuperATW detector. [Na] and errors of the single measurement in mitochondria of quiescent guinea-pig cardiac myocytes were calculated from spectra acquired with the Si or the Ge detector. The use of the Ge SuperATW detector improved the detectability limit for sodium by more than 80% and reduced the error of the single measurement by a factor of 7–8.     

A cryoclamp for the rapid cryofixation of the isolated blood-perfused rabbit cardiac papillary muscle preparation at predefined times during the contraction cycle

There is increasing evidence that the distribution of monovalent cations in cardiac cells may be non-uniform, particularly in the region immediately beneath the sarcolemma, and we have proposed that a build-up of sodium in this region could be an important factor in the development of ischaemia-reperfusion injury. Electron probe X-ray microanalysis is ideal for the study of such changes in distribution but the application of the technique to this problem imposes severe requirements on the specimen and on the method for cryofixation. The specimen must be perfused through its vasculature so that it can be made truly ischaemic and be successfully reperfused. It is necessary to be able to cryofix the specimen without disturbance of its blood supply, electrical stimulation or temperature. It is also important to know the time in the contraction cycle when cryofixation occurs. Here we describe the design of an automated cryofixation device which can be used to cryofix a blood perfused papillary muscle preparation at predetermined time points in the contraction cycle. Preliminary data obtained from the analysis of rabbit papillary muscles subjected to varying periods of ischaemia are included as an example of the use of the cryoclamp.     

Evaluation of surface alterations in different retreatment nickel‐titanium files: AFM and SEM study

The aim of this study was to evaluate the surface changes of nickel titanium (Ni‐Ti) rotary retreatment files after three and five uses. Furthermore, the effects of 2% sodium hypochlorite and chloroform solutions and sterilization procedures on the NiTi rotary retreatment surfaces were investigated. ProTaper Retreatment files, R‐endo files, and Mtwo retreatment files were used for this study. The palatinal roots of maxillary molar teeth were obturated with gutta percha and Ah26. Retreatment procedures were performed with these retreatment file systems. The surface changes of untreated NiTi rotary files that were used three and five times, immersed in NaOCl and chloroform and subjected to sterilization procedures were investigated with atomic force microscopy (AFM) and scanning electron microscopy (SEM). The RMS values, three dimensional images and SEM images at various magnifications were obtained. RMS values showed that all three NiTi rotary retreatment file systems showed significant deteriorations after three and five uses. Cracks, damages and spiral construction deteriorations were detected in the SEM images after three and five uses. Furthermore, the Mtwo 15 file was broken off after five uses. AFM data indicated that 2% NaOCl caused significant surface deteriorations on NiTi rotary files and both AFM and SEM evaluation showed that chloroform solution and sterilization procedures did not cause significant surface deteriorations. In conclusion, ProTaper retreatment, R‐endo, and Mtwo retreatment files showed surface damages depending on retreatment procedures. Clinicians have to consider that retreatment files always have a tendency to break off after the third time they have been used. Microsc. Res. Tech. 78:356–362, 2015. © 2015 Wiley Periodicals, Inc.     

Effect of disinfectant solutions on gutta-percha and resilon cones

The aim of this study was to evaluate the effects of the 5.25% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), and MTAD solutions on the surface of gutta‐percha and Resilon cones by using atomic force microscopy (AFM). Accessory cones were washed and dried. The cones were randomly divided into six groups: gutta‐percha immersed in NaOCl, CHX, and MTAD, and Resilon immersed in NaOCl, CHX, and MTAD. AFM images of the same area were made in different periods of time. JPK? Image Processing Software was used to evaluate the images. The parameters used to evaluate the changes were RMS and line profiles. No statistically significant change was observed in the RMS values. The line profiles detected changes only for gutta‐percha surfaces after immersion in NaOCl and MTAD solutions. In conclusion, 5.25% NaOCl and MTAD are associated with local changes in surface roughness of gutta‐percha cones. No change was observed when 2% CHX was used. The use of all tested solutions did not produce any changes on Resilon surface. Microsc. Res. Tech. 75:791–795, 2012. © 2012 Wiley Periodicals, Inc.     

Evaluation of fibroblast attachment in root conditioning with Er,Cr:YSGG laser versus EDTA: A SEM study

The regeneration of periodontal support is a main concern in periodontal therapy. This study aims to investigate the efficacy of Er, Cr:YSGG laser and EDTA based conditioning in attachment of fibroblast on root surfaces. This in vitro study was conducted on 81 root plates (6 mm × 4 mm × 1 mm) prepared from 27 single‐rooted human mature teeth. The samples were divided into three groups: (1) Er, Cr: YSGG laser conditioning with a G6 tip (2.78 µm, 0.75 W, pulse duration of 140 µs, repetition rate of 20 Hz) for 5–7 s; (2) EDTA conditioning (17%, pH: 8) for 1 min; and (3) the control group which were exposed neither to EDTA nor laser. The viability and proliferation rates assessments were performed using MTT assay on days 3 and 5. In addition, the level of cell attachment was studied using scanning electron microscopy. The data indicated Er, Cr:YSGG conditioning increased cell viability by lapse of time (from days 3–5), with significantly better cell attachment compared to the other groups on days 3 and 5 (P < 0.05). In addition, increasing cell attachment in the EDTA conditioning group compared with the control group was statistically significant on day 5 but not on day 3 (P < 0.05). In conclusion, Er, Cr:YSGG laser conditioning can promote enhance fibroblast attachment on dentinal root surfaces more than EDTA. Microsc. Res. Tech. 78:317–322, 2015. © 2015 Wiley Periodicals, Inc.     

Influence of passive ultrasonic irrigation on the efficiency of various irrigation solutions in removing smear layer: a scanning electron microscope study

To evaluate the influence of passive ultrasonic irrigation (PUI) to remove the smear layer with different irrigation solutions. Seventy‐eight single rooted mandibular premolars were used. The working lengths were measured and the root canals were prepared by using nickel titanium rotary files. The specimens were subjected to six different irrigation regimens: sodium hypochloride (NaOCl) with classic irrigation (CI); NaOCl with PUI; EDTA with CI; EDTA with PUI; QMix with CI; and QMix with PUI. The specimens were split longitudinally. Scanning electron microscopy images were taken to evaluate the amount of residual smear layer and scored. Statistical analysis was performed with the Kruskal–Wallis test. The Dunn's test was used for post hoc testing after the Kruskal–Wallis test. The smear layer scores of apical thirds were significantly higher than coronal and middle thirds except for NaOCl + CI group (p < 0.001). The irrigation with PUI improved the efficacy of all irrigation solutions on removal of smear layer in both coronal and middle thirds (p < 0.001). The PUI activation increased the activity of NaOCl, EDTA, and QMix, respectively without any significance. All irrigation regimens were significantly more effective in coronal and middle thirds compared to apical third. PUI significantly increased the efficacy of smear layer removal regardless of irrigation solution.     

Residues of calcium hydroxide-based intracanal medication associated with different vehicles: a scanning electron microscopy evaluation

This study evaluated the presence of residues after removal of calcium hydroxide [Ca(OH)(2) ] associated with different vehicles. Thirty single-rooted teeth were instrumented to a master apical file #25 using 2.5% NaOCl as main irrigant and 17% trisodium EDTA (ethylenediaminetetraacetic acid) as final agent irrigant. Then, the root canals were dressed with Ca(OH)(2) associated with silicone oil (Group 1), 2% chlorhexidine gluconate (Group 2), or propylene glycol (Group 3). After coronal sealing, all teeth were kept in a moist environment at room temperature. After 7 days, the teeth were reopened and medicaments were removed using 5 mL of saline solution and instrumentation with master apical file followed by new irrigation with 5 mL of 2.5% NaOCl. Subsequently, teeth were split longitudinally and assessed by scanning electron microscopy. The wall cleanliness of the cervical and apical thirds of the roots were evaluated and scored by three blinded examiners. Statistical analysis was performed using Kruskal-Wallis and Wilcoxon tests at 5% level of significance. All roots had residues of Ca(OH)(2) on the canal walls. All experimental groups had similar results (P > 0.05) regardless of the third evaluated. There was significant difference between the apical and cervical thirds only in Group 3 (P < 0.05). Association of different vehicles to Ca(OH)(2) does not influence the persistence of residues on the root canal walls.