Creation of well-balanced experimental groups for comparative endodontic laboratory studies: a new proposal based on micro-CT and in silico methods.

AIM: To introduce a new method to select anatomically matched teeth using micro-computed tomographic (micro-CT) technology. METHODOLOGY: Single-rooted mandibular incisors with a single root canal (n = 60) were selected and distributed into three experimental groups according to the method used for matching 10 pairs of teeth in each group. In group 1, the pairs of mandibular incisors were randomly selected from a pool of teeth. In group 2, teeth were paired based on the measurement of canal width 5 mm from the root apex using radiographs taken from buccolingual and mesiodistal directions. In group 3, teeth were scanned (pixel size of 14.25 µm) and pair-matched based on the anatomical aspects of the root canal, named aspect ratio (AR), volume and three-dimensional canal geometry. After allocating the specimens into groups 1 and 2, the teeth were scanned and the canal morphology evaluated as in group 3. A bivariate Pearson's regression analysis was performed correlating the individual AR values of each pair, and the correlation coefficient was used to estimate the strength of the pair-matching process. One-way anova post hoc Tukey's tests were applied for pairwise comparisons at a significance level of 5%. RESULTS: The micro-CT revealed that 100% of the samples had strong (80%) or very strong (20%) correlations with respect to AR values. Analysis of the radiographic method revealed strong correlation in two pairs (20%), but most of the samples had weak (30%) or negligible (30%) correlation coefficients. The randomization method resulted in three pairs (30%) with very strong correlations, whilst 50% had weak or negligible rates. A significant difference in correlation coefficients was observed in the micro-CT method compared to the other groups (P < 0.05), whilst no difference was detected between radiographic and randomized methods (P > 0.05). Eta-squared (η2 ) calculations demonstrated a very high effect size in the micro-CT group for selecting pairs (0.99) and lower effect sizes in the radiographic (0.67) and randomized (0.66) groups. CONCLUSIONS: Use of Micro-CT was able to provide better control of the confounding effect that anatomical variances in tooth morphology may have on the results in experiments with matched-pair design.

Anatomical danger zone reconsidered: a micro-CT study on dentine thickness in mandibular molars.

AIM: To investigate the smallest dentine thickness in mesial canals of mandibular molars along the cervical and middle thirds of the root by means of a micro-computed tomographic (micro-CT) technology and digital image analysis. METHODOLOGY: Fifty mesial roots of mandibular molars having two independent canals (mesiobuccal and mesiolingual), in the coronal and middle levels, were selected and scanned in a micro-CT device. After reconstruction procedures, approximately 468 slices per root covering the 7 mm below the furcation area of the mesial root were analysed to measure the smallest dentine thickness (danger zone [DZ]) in each slice from both distal and mesial regions of the mesial canals by an automatic segmentation process. RESULTS: The DZ values in the mesiobuccal canals varied from 0.67 to 1.93 mm, with an average of 1.13 ± 0.21 mm. For the mesiolingual canals, the DZ varied from 0.77 to 1.89 mm with an average of 1.10 ± 0.21 mm. There was no correspondence in the DZ between the mesiobuccal and mesiolingual canals at the same cross-sectional level in 71% of the specimens. Moreover, the smallest dentine thickness was towards the mesial region of the roots in 22% and 18% of the mesiolingual and mesiobuccal canals, respectively. (Figs 1 and 2 CONCLUSIONS: The smallest dentine thickness was on the mesial plane of the roots in about 40% of the canals. The vertical location of the DZ in relation to the furcation area was in the middle third of the root.

Optimization of digital image processing to determine quantum dots' height and density from atomic force microscopy.

An optimized method of digital image processing to interpret quantum dots' height measurements obtained by atomic force microscopy is presented. The method was developed by combining well-known digital image processing techniques and particle recognition algorithms. The properties of quantum dot structures strongly depend on dots' height, among other features. Determination of their height is sensitive to small variations in their digital image processing parameters, which can generate misleading results. Comparing the results obtained with two image processing techniques - a conventional method and the new method proposed herein - with the data obtained by determining the height of quantum dots one by one within a fixed area, showed that the optimized method leads to more accurate results. Moreover, the log-normal distribution, which is often used to represent natural processes, shows a better fit to the quantum dots' height histogram obtained with the proposed method. Finally, the quantum dots' height obtained were used to calculate the predicted photoluminescence peak energies which were compared with the experimental data. Again, a better match was observed when using the proposed method to evaluate the quantum dots' height.

Exploiting the potential of free software to evaluate root canal biomechanical preparation outcomes through micro-CT images.

AIM: To propose an automated image processing routine based on free software to quantify root canal preparation outcomes in pairs of sound and instrumented roots after micro-CT scanning procedures. METHODOLOGY: Seven mesial roots of human mandibular molars with different canal configuration systems were studied: (i) Vertucci's type 1, (ii) Vertucci's type 2, (iii) two individual canals, (iv) Vertucci's type 6, canals (v) with and (vi) without debris, and (vii) canal with visible pulp calcification. All teeth were instrumented with the BioRaCe system and scanned in a Skyscan 1173 micro-CT before and after canal preparation. After reconstruction, the instrumented stack of images (IS) was registered against the preoperative sound stack of images (SS). Image processing included contrast equalization and noise filtering. Sound canal volumes were obtained by a minimum threshold. For the IS, a fixed conservative threshold was chosen as the best compromise between instrumented canal and dentine whilst avoiding debris, resulting in instrumented canal plus empty spaces. Arithmetic and logical operations between sound and instrumented stacks were used to identify debris. Noninstrumented dentine was calculated using a minimum threshold in the IS and subtracting from the SS and total debris. Removed dentine volume was obtained by subtracting SS from IS. RESULTS: Quantitative data on total debris present in the root canal space after instrumentation, noninstrumented areas and removed dentine volume were obtained for each test case, as well as three-dimensional volume renderings. CONCLUSION: After standardization of acquisition, reconstruction and image processing micro-CT images, a quantitative approach for calculation of root canal biomechanical outcomes was achieved using free software.

Lack of correlation between sealer penetration into dentinal tubules and sealability in nonbonded root fillings.

AIM: To determine the correlation between leakage and sealer penetration into dentinal tubule. METHODOLOGY: The root canal of 60 mandibular central incisor teeth were prepared and filled under standardized conditions. Teeth were placed into a device to assess glucose penetration using 15 psi pressure application. After 1 h, glucose concentrations in the lower chamber were measured using an enzymatic reaction. Each specimen was then sectioned horizontally at 3, 6 and 8 mm from the apex, and a standard metallographic preparation was performed. The coronally facing surface of each slice was examined in a high-resolution stereomicroscope and under Confocal Laser Scanning Microscope. For each slice, the external outline of the root canal, the internal circumference of the root canal walls and the areas along the canal walls where the sealer had penetrated into the dentinal tubules (sealer tags) were outlined and measured. Afterwards, the percentage of the sealer penetration in each section was calculated. The Spearman correlation test was used to verify the correlation between the variables studied (glucose leakage and sealer penetration into dentinal tubules). RESULTS: The means and standard deviations of glucose leakage were 0.744 g L(-1) (±0.5), and the percentage of the dentinal tubule sealer penetration varied between teeth from 5.6% to 42.21%, with an average of 13.7 ± 17.2%. The Spearman correlation test revealed no significant correlation between the two factors analysed (P = 0.082). The r(2) value was equal to 0.12. CONCLUSIONS: There was no significant correlation between sealability sealer penetration into dentinal tubules. The lack of correlation reported is of relevance as sealer penetration into dentinal tubules has been used as an advantageous property during the launch of new root filling materials and techniques.

Gold nanoparticles on the surface of soda-lime glass: morphological, linear and nonlinear optical characterization.

Materials presenting high optical nonlinearity, such as materials containing metal nanoparticles (NPs), can be used in various applications in photonics. This motivated the research presented in this paper, where morphological, linear and nonlinear optical characteristics of gold NPs on the surface of bulk soda-lime glass substrates were investigated as a function of nanoparticle height. The NPs were obtained by annealing gold (Au) thin films previously deposited on the substrates. Pixel intensity histogram fitting on Atomic Force Microscopy (AFM) images was performed to obtain the thickness of the deposited film. Image analysis was employed to obtain the statistical distribution of the average height of the NPs. In addition, absorbance spectra of the samples before and after annealing were measured. Finally, the nonlinear refractive index (n2) and the nonlinear absorption index (α2) at 800 nm were obtained before and after annealing by using the thermally managed eclipse Z-scan (TM-EZ) technique with a Ti:Sapphire laser (150 fs pulses). Results show that both n2 and α2 at this wavelength change signs after the annealing and that the samples presented a high nonlinear refractive index.

Smear layer dissolution by peracetic acid of low concentration.

AIM: To test the effect of a noncaustic concentration of peracetic acid (PAA) in a standardized smear layer model. METHODOLOGY: The smear layer dissolution kinetics of 0.5% PAA on human dentine were compared to those of 2.25% PAA and 17% ethylenediaminetetraacetic acid (EDTA) solutions. Coronal dentine discs were prepared from six human maxillary molars. A standardized smear layer was produced on the pulpal side of each disc. The smear layer-covered surface was divided into three similar areas and then exposed to one of the three solutions tested. Co-site image sequences (around 40, 500 ×) of the specific areas were obtained after four cumulative demineralisation times (15, 30, 60 and 180 s). An image processing and analysis sequence measured sets of images, providing data of area fraction (AF, dentine-free area in % of total analysis area). A general linear model for repeated measures was used to verify the influence of time and solution type over the change in AF from baseline (ΔAF). RESULTS: Overall, EDTA and 2.25% PAA produced higher ΔAF values than the 0.5% PAA solution (P < 0.05). No significant difference was observed in ΔAF between 15 s and 30 s (P > 0.05). After 60 s of etching, all tested solutions produced similar ΔAF (P > 0.05), whereas at 180 s, ΔAF of both EDTA and 2.25% PAA continued to increase (P > 0.05). CONCLUSIONS: After 60 s of contact, the 0.5% PAA solution dissolved smear layer as well as 2.25% PAA and 17% EDTA.

Two- and three-dimensional profilometer assessments to determine titanium roughness.

In this study, a comparative topography analysis of titanium (Ti) surfaces was performed using two- (2D) and three-dimensional (3D) profilometers. Ti samples were either only sandblasted (SB) using Al(2)O(3) particles or were SB and received an additional chemical treatment using a solution of 4% HF (SLA). Samples with no treatment were used as a control group (C). Therefore, three different surfaces were evaluated: SB, SLA and C. The Ti surface topographies were evaluated by scanning electron microscopy. An Ra roughness measurement was performed on each Ti sample by 2D and 3D profilometers. Surface roughness was also characterized using amplitude, spatial and hybrid 3D parameters. 2D and 3D profilometer analyses produced very close results. Mean Ra values range from 0.19 to 0.25 (C, p<0.05), 0.84 to 0.99 (SLA, p<0.05) and 0.98 to 1 microm (SB, p>0.05). The statistically different Ra values depending on the surface studied may be explained by methodological and technical differences. Also, 3D profilometer seems to be the more appropriate analytical method for measuring the roughness of Ti surfaces because it also describes surface organization.

Dentine demineralization when subjected to EDTA with or without various wetting agents: a co-site digital optical microscopy study.

AIM: To analyse quantitatively the chelating ability of ethylenediaminetetraacetic acid (EDTA) and three common EDTA-based associations with wetting agents. METHODOLOGY: Twelve maxillary human molars were selected, from which 3 mm thick discs were obtained from the cervical third of the root. Following the creation of standardized smear layer co-site microscopy image sequences of the dentine surface submitted to EDTA, EDTA plus 0.1% cetavlon (Sigma Chemical Co., St Louis, MO, USA), EDTA plus 1.25% sodium lauryl ether sulphate and SmearClear (Sybron Endo, Orange, CA, USA) were obtained after several cumulative demineralization times. Sixteen images were obtained of each dentine sample for each experimental time, at 1000 x magnification. An image processing and analysis sequence was used to measure the area of open tubules for each experimental time. Thus, it was possible to follow the demineralization process and quantitatively analyse the effect of the various substances. The Student's t-test was used to assess differences between experimental groups. Results EDTA solution had the strongest effect at all experimental times whilst the association of EDTA with wetting agents showed a weaker chelating effect and this difference was statistically significant (P < 0.05). CONCLUSIONS: (i) The EDTA solution had the strongest effect at all experimental times (P < 0.05); (ii) the association of EDTA with wetting agents did not improve the chelating power of the solution; (iii) co-site optical microscopy represents a powerful approach to compare directly, longitudinally and quantitatively the ability of the chelating solutions.

High concentration of residual aluminum oxide on titanium surface inhibits extracellular matrix mineralization.

In the present study we characterized titanium (Ti) surfaces submitted to different treatments and evaluated the response of osteoblasts derived from human alveolar bone to these surfaces. Five different surfaces were evaluated: ground (G), ground and chemical etched (G1-HF for 60 s), sand blasted (SB-Al(2)O(3) particles 65 mum), sand blasted and chemical etched (SLA1-HF for 60 s and SLA2-HF for 13 s). Surface morphology was evaluated under SEM and roughness parameters by contact scanning instrument. The presence of Al(2)O(3) was detected by EDS and the amount calculated by digital analyses. Osteoblasts were cultured on these surfaces and it was evaluated: cell adhesion, proliferation, and viability, alkaline phosphatase activity, total protein content, and matrix mineralization formation. Physical and chemical treatments produced very different surface morphologies. Al(2)O(3) residues were detected on SB and SLA2 surfaces. Only matrix mineralization formation was affected by different surface treatments, being increased on rough surface (SLA1) and reduced on surface with high amount of Al(2)O(3) residues (SB). On the basis of these findings, it is possible to conclude that high concentration of residual Al(2)O(3) negatively interfere with the process of matrix mineralization formation in contact with Ti implant surfaces.

The effect of the canal-filled area on the bacterial leakage of oval-shaped canals.

AIM: To compare the sealing ability and the canal-filled area (gutta-percha + sealer) of three current filling techniques in oval-shaped canals. METHODOLOGY: A total of 50 oval-shaped root canals were prepared and root filled as follows: G1: lateral condensation (n = 10), G2: System B (n = 10) and G3: Thermafil system (n = 10). All teeth were mounted in a two-chamber apparatus and the coronal access was exposed to human saliva. The appearance of turbidity in the BHI broth over a 15-week period was observed. A cross section of each tooth was made 5 mm from the apex and the samples were prepared for microscopic analysis. Digital image measurements of the cross-sectional area and the area filled by gutta-percha and sealer were performed. Log-rank and Fisher's exact tests were used to analyse the leakage data. Student's t-test was used to analyse the filled-area data. RESULTS: Overall, 30% of the specimens of G1 and G2 and 20% of G3 demonstrated leakage after 15 weeks and no significant difference was found amongst the groups (P > 0.05). The percentage of canal-filled area was 68, 70 and 78%, respectively (P > 0.05). The correlation analysis revealed no significant relation between the pattern of bacterial leakage and canal-filled area (P = 0.128). CONCLUSIONS: No significant difference in apical sealing and canal-filled area in oval-shaped canals was seen between the three filling techniques. No significant correlation was found between the quality of the apical seal and the filled-area of the root canal space.

Comparison of the percentage of gutta-percha-filled area obtained by Thermafil and System B.

The aim of this study was to determine the percentage of gutta-percha-filled area (GPFA) obtained by Thermafil and System B techniques using light microscopy and digital image processing. Forty-five human mandibular first molars were prepared and obturated as follows: Group 1: lateral condensation (n = 15); Group 2: System B (n = 15); and Group 3: Thermafil system (n = 15). Horizontal sections were cut 4 and 6 mm from the apical foramen of each tooth. The samples were metallographically prepared and taken through photomicrographs. Using digital analysis, the cross-sectional area of the canal and the gutta-percha areas were measured. The GPFA data obtained for the three groups were analysed using the non-parametric Friedman and Wilcoxon Signed-Ranks tests. Significant differences were found between Group 3 and Group 2-Group 1. No significant difference was found between Group 2 and Group 1. The Thermafil system produced significantly higher GPFAs than lateral condensation and System B techniques (P < 0.01). This result suggests that the Thermafil system can reduce sealer and voids.

Co-site digital optical microscopy and image analysis: an approach to evaluate the process of dentine demineralization.

AIM: To introduce and explore the potential of digital optical co-site microscopy and image analysis for the observation of changes in dentine surfaces during demineralization. The effect of ethylenediamine tetraacetic acid (EDTA) was evaluated quantitatively and longitudinally. METHODOLOGY: Three maxillary human molars were sectioned transversely at the cemento-enamel junction, and the crowns discarded. Subsequently, discs approximately 3 mm thick were cut in the cervical third of the root and a standardized smear layer produced. Co-site image sequences of the dentine surface subjected to 17% EDTA were obtained over the experimental period (15, 30, 60, 180 and 300 s). Sixteen images were obtained in each dentine sample for each experimental time, thus, a total of 48 image fields were obtained. For each field, an image analysis routine automatically discriminated open dentine tubules and measured their number, area fraction and minimum diameter, thus allowing the quantification of the demineralization process. The Student t-test was used to analyse the data. RESULTS: The number of open tubules remained essentially constant during the demineralization process. The area fraction increased from 9% to 32%. Tubule minimum diameter increased from 1.5 to 3.0 microm. The changes over time for the area fraction and minimum diameter were significant for comparison between all experimental times (P<0.05). CONCLUSIONS: The methodology developed for longitudinal observation of dentinal surfaces was fast, robust and reproducible. It could be easily extended to other chelating substances, thus contributing to the understanding of the demineralization process and in establishing an optimal time-effect relationship in the clinical application.

In situ atomic force microscopy and image analysis of dentine submitted to acid etching.

The effect of acid etching on root dentine was analyzed with Atomic Force Microscopy using a liquid cell that allowed image acquisition while the sample was kept in contact with the etching solution. The time evolution of the etching process was evaluated both qualitatively and quantitatively. Image sequences against etching time were obtained showing a clear trend of surface cleaning and opening of dentine tubules. Image analysis methods were employed to quantify the changes on the dentine surface, through the detection and morphological measurement of dentine tubules over time. The measurements indicated an overall increase in the diameter of the tubules against time, with a saturation value between 3.5 and 5.5 microm reached after approximately 300 seconds.

Real-time atomic force microscopy of root dentine during demineralization when subjected to chelating agents.

AIM: To explore the potential of atomic force microscopy (AFM) for the examination of changes to dentine surfaces during demineralization and evaluate qualitatively the effect of EDTA, EDTAC and citric acid. METHODOLOGY: Nine canine teeth were sectioned transversely at the cemento-enamel junction, and the crowns discarded. Subsequently, each root was embedded in an epoxy cylinder and discs approximately 5 mm thick were cut. A standard metallographic procedure was then used to prepare the surfaces for observation. From the central portion of these samples, two specimens were symmetrically prepared per tooth so that a total number of 18 samples was produced. To allow the use of a liquid cell during AFM, the samples were embedded in silicone rubber and were then randomly divided into three groups, as follows: group 1: 17% EDTA (pH 7.7), group 2: 17% EDTAC (pH 7.7) and group 3: 10% citric acid (pH 1.4). Topographical images were acquired during the demineralization process, allowing real-time observation of the dentine surface. Two operators assigned scores to the AFM images using a double-blind method. anova analysis with random effects (P < 0.05) was used to compare the results. RESULTS: The average scores were 6.13 +/- 0.35 for EDTAC, 7.36 +/- 0.23 for EDTA and 14.55 +/- 1.21 for citric acid. Citric acid was statistically different from EDTA and EDTAC while EDTA and EDTAC were not statistically different. CONCLUSIONS: The most effective demineralizing substance was citric acid. The methodology developed for real-time observation of dentine surfaces is a valuable method to evaluate demineralization.

Evaluation of the effect of EDTA, EDTAC and citric acid on the microhardness of root dentine.

AIM: To evaluate the effect of citric acid, ethylenediaminetetraacetic acid (EDTA) and ethylenediaminetetraacetic acid plus Cetavlon (EDTAC) solutions on the microhardness of human root canal dentine. METHODOLOGY: Sixteen maxillary human canines were sectioned transversely at the cemento-enamel junction and the crowns were discarded. Subsequently, each root was embedded in an epoxy resin cylinder and their middle third sectioned horizontally into 4 mm thick slices. The samples were randomly divided into three groups according to the chelating agent employed, as follows (n = 6): group 1: EDTA 17%, group 2: EDTAC 17% and group 3: citric acid 10%. Dentine microhardness was then measured with a load of 50 g for 15 s. At the beginning of the experiment, reference microhardness values were obtained for samples without any etching (t = 0 min). The same samples were then exposed to 50 microL of the chelator solution for 1, 3 and 5 min. The Student's t-test (P < 0.05) was used to compare results for different times for each chelator and different chelators for each time. RESULTS: Microhardness decreased with increasing time of application of chelating solutions. There were no significant (P > 0.05) differences between initial microhardness for the three groups as well as after 1 min of application of the substances. After 3 min, EDTA produced a significantly greater reduction in microhardness. However, there was no significant difference between EDTA and EDTAC after 5 min. Citric acid caused significantly less reduction in microhardness. CONCLUSIONS: Overall, citric acid was least effective in reducing dentine hardness whilst EDTA had the strongest effect.

A low-cost non instrumental method for semiquantitative determination of mercury in fish.

A non instrumental method for the semiquantitative determination of mercury in fish is described. The sample is acid digested and the mercury vapor released after chemical reduction with SnCl2. The mercury vapor is then collected on a detecting paper covered with an emulsion of Cu2I2, 3% carboxymethylcellulose and MgCl2 as moistener agent. The colored Cu2[HgI4] complex is formed and the color intensity is proportional to the mercury concentration in the original sample. Parameters such as sample mass/digestion solution volume ratio, emulsion composition, paper covering technique, carrier gas flow were investigated. Comparative studies using CVAAS and digital color analysis after scanning the papers confirmed the applicability of the method to real samples.

A pattern recognition technique for the analysis of grain boundary structure by HREM.

A pattern recognition technique for the detection of structural units in high resolution images of interfaces is described. The technique uses cross-correlation functions as a means of locating atomic patterns characteristic for an interface and as a measure of similarity between related units. Application is not limited to periodic, or even to planar interfaces. Characteristic structural units can be extracted from an experimental image and some important parameters such as mirror or mirror glide symmetry, and rigid body displacements can be determined without knowledge of the imaging parameters. The technique allows an image of a structural unit with reduced specimen noise to be obtained by averaging over several similar units, even if a boundary is not periodic and not planar.