|Year : 2022 | Volume
| Issue : 3 | Page : 261-266
Atomic force microscopy study on the effect of different irrigation regimens on the surface roughness of human root canal dentin
I Tsenova-Ilieva1, S Simeonova2, E Karova1
1 Department of Conservative Dentistry, Faculty of Dental Medicine, Medical University of Sofia, Sofia, Bulgaria
2 Department of Physical Chemistry, Faculty of Chemistry and Pharmacy, Sofia University, Sofia, Bulgaria
|Date of Submission||01-Apr-2021|
|Date of Acceptance||13-Sep-2021|
|Date of Web Publication||16-Mar-2022|
Dr. I Tsenova-Ilieva
Department of Conservative Dentistry, Faculty of Dental Medicine, Medical University of Sofia, Sofia, Bulgaria. 1 blv. St. G. Sofiiski, Sofia - 1431
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Endodontic irrigants could alter the mechanical properties of root canal dentin, such as its roughness. Aim: To compare the effect of various irrigation protocols on root canal dentin roughness with respect to their application time, concentration, and additional ultrasonic activation. Materials and Methods: Forty single-rooted, non-endodontically treated upper incisors were decoronated and further sectioned longitudinally in a buccolingual direction. The surfaces of all eighty samples were polished and randomly segregated to eight equal groups (n = 10): Group A: 2% NaOCl/2 min followed by 17% EDTA/2 min; Group B: 2% NaOCl/5 min followed by 17% EDTA/5 min; Group C: 5.25% NaOCl/2 min followed by 17% EDTA/2 min; Group D: 5.25% NaOCl/5 min followed by 17% EDTA/5 min; Group E: 2% NaOCl solution, ultrasonically activated for 2 min; Group F: 5.25% NaOCl, ultrasonically activated for 2 min; Group G: 2% NaOCl/2 min followed by 17% EDTA/2 min, both ultrasonically activated; Group H: Control group (distilled water). Results: All irrigation regimens increased root dentin surface roughness in comparison with the control group. The analysis of the values revealed significant differences between the arithmetical average roughness values (Ra) in the groups (p < 0.001). A statistically significant increase in the Ra parameter was observed in groups E, F, G, and D. The irrigation protocol in Group A showed a significantly smoother dentin surface than those with the ultrasonic activation. Conclusion: All tested irrigants increased root canal dentin roughness and the effect was time and concentration-dependent. The ultrasonic activation of the disinfection solutions roughened the root dentin surface significantly.
Keywords: Atomic force microscopy, dentin roughness, EDTA, irrigation, sodium hypochlorite, ultrasonic activation
|How to cite this article:|
Tsenova-Ilieva I, Simeonova S, Karova E. Atomic force microscopy study on the effect of different irrigation regimens on the surface roughness of human root canal dentin. Niger J Clin Pract 2022;25:261-6
|How to cite this URL:|
Tsenova-Ilieva I, Simeonova S, Karova E. Atomic force microscopy study on the effect of different irrigation regimens on the surface roughness of human root canal dentin. Niger J Clin Pract [serial online] 2022 [cited 2022 Dec 10];25:261-6. Available from: https://www.njcponline.com/text.asp?2022/25/3/261/339705
| Introduction|| |
The success of endodontic treatment depends on the acquisition of proper endodontic access, thorough debridement, shaping, and three-dimensional filling of the root canal space., Adequate removal of smear layer can only be achieved by a combination of mechanical instrumentation and chemical disinfection through various irrigating solutions.,
Sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA) are among the most widely used and recommended endodontic irrigants. As an antibacterial agent with proteolytic action, NaOCl is able to dissolve vital and necrotic pulp tissue, affecting only the organic part of the smear layer., EDTA is a chelator that removes the mineralized portion of the smear layer by binding single calcium ions present in the hydroxyapatite crystals of dentin.,, Thus, successive rinses of NaOCl and EDTA are preferred for a more efficient reduction of bacterial load.
Despite their favorable qualities, endodontic irrigants are proven to cause alterations in the chemical composition of dentin that are time and concentration-dependent., The modification of the Ca/P ratio affects the original proportion of organic to inorganic components. This causes a significant decrease of dentin's microhardness and elasticity, and an increase of its permeability, solubility, and surface roughness.,,,,
From a clinical point of view, surface irregularities could benefit the adhesion of the root canal sealer to the dentinal wall. However, uneven surfaces might promote bacterial colonization and biofilm formation.,,,,
Various approaches have been proposed to measure the dentin surface roughness such as atomic force microscopy (AFM), computerized roughness testing, stylus profilometry.,, Almost since its invention by Binning et al. in 1986, AFM has evolved into one of the most widely used imaging tools in varied scientific fields., AFM enables the mapping of the sample topography by following an atomic-force field on a surface by non-destructive probes., As a result, a detailed topographical three-dimensional image of an object surface with vertical and lateral resolution within the nanometric scale is obtained.
The aim of the current study was to compare the effect of seven endodontic irrigation regimens on root canal dentin roughness. The null hypothesis tested was that there would be no significant difference between the impact of the disinfection protocols with respect to their concentration and additional ultrasonic activation at different time exposures.
| Materials and Methods|| |
Selection of teeth and sample preparation
The Research Ethics Committee of Medical University Sofia, Sofia, Bulgaria approved the experiment with a protocol №838/05.03.2020. The approval of the ethics committee was obtained with a protocol ? 838 from the 5th March 2020. Forty human, non-endodontically treated, upper central incisors of patients aged 40–55 years were obtained from a pool of people who recently extracted teeth for periodontal reasons in the Department of Oral and Maxillofacial Surgery, Faculty of Dental Medicine, Medical University Sofia, Bulgaria. Immediately after, extracted teeth were stored in an 0.1% water solution of thymol. The external surfaces of all samples were cleaned from calculus and plaque by using hand periodontal curettes. All incisors were radiographed to confirm the presence of a single canal. Teeth were selected on the basis of their relative dimensions and morphological similarities. The external surface of all samples was examined under a stereomicroscope Leica S6, (Leica Microsystems, Wetzlar, Germany) for detection of root defects, cracks, or fractures. Teeth with more than one root canal, apical curvature more than 5°, immature root apices, calcification, resorptions, fractured root apex, and any root or coronal defects, including cracks, were excluded from the study and replaced with new ones. All teeth were decoronated at the level of the cementoenamel junction by using a finishing high-speed diamond bur under copious water cooling. Each root was sectioned longitudinally into two parallel dentin slices by a diamond saw microtome Leica SP 1600 (Leica Microsystems, Wetzlar, Germany) under water cooling.
The total eighty dentin segments were horizontally embedded in an auto polymerizing acrylic resin Meliodent RR (Kulzer, Hanau, Germany), exposing the root canal dentin surfaces. Each specimen was ground polished with a series of increasingly finer polishing discs OptiDisc (Kerr Dental, Orange, CA, USA) in the following order: Extra-Coarse (80 μm), Coarse/Medium (40 μm), Fine (20 μm), Extra-Fine (10 μm), under copious water cooling by using a low-speed handpiece. Each disc was used for 1 min and changed in every three samples.
All 80 samples were randomly assigned into eight equal groups (n = 10), depending on the irrigation regimen to be used, as follows:
Group A The samples were successively immersed for 2 min in 2% NaOCl and 17% EDTA solution.
Group B: The samples were successively immersed for 5 min in 2% NaOCl and 17% EDTA solution.
Group C: The samples were successively immersed for 2 min in 5.25% NaOCl and 17% EDTA solution.
Group D: The samples were successively immersed for 5 min in 5.25% NaOCl and 17% EDTA solution.
Group E: The samples were immersed in 2% NaOCl solution and ultrasonically activated for 2 min.
Group F: The samples were immersed in 5.25% NaOCl and ultrasonically activated for 2 min.
Group G: The samples were successively immersed for 2 min in 2% NaOCl and 17% EDTA. Both solutions were ultrasonically activated.
Group H: Control group: The samples were immersed in 50 ml distilled water for 5 min.
Exposed root canal dentinal surfaces of each root half were immersed in glass plates containing 50 ml of the disinfection solutions at room temperature for the given time periods. The irrigants were renewed after each specimen. The additional agitation of the irrigants was achieved by an ultrasonic device, Digital Ultrasonic Cleaner CD-4820 (Shenzhen Codyson Electrical Co., Ltd, China), at 42,000 Hz frequency.
At the end of each treatment period, the samples were rinsed with 50 ml distilled water to eliminate the chemical interaction between the irrigants and the prolonged effect of the chelating agent, and dried with sterile blotting paper.
AFM surface roughness testing
The surface roughness of all specimens was determined by using an atomic force microscope MultiMode V (Veeco Instruments Inc.) and Controller NanoScope V (Bruker Ltd, Germany). The testing was performed in four areas of the mid-root region of each segment by soft tapping mode AFM probes (Tap 150Al-G, Budget Sensors, Innovative solutions Ltd., Bulgaria) with an aluminum reflective coating. A dynamic, tapping mode, in an ambient experimental environment, at a scanning speed of 0.5 Hz and scanning size of 10 μm × 10 μm was applied. All images were obtained in a topographic scanning mode, 512 × 512 pixels in JPG. format and further analyzed by NanoScope software.
The Ra parameter, describing the overall roughness of the dentin surface, was registered. It is defined as the arithmetical average value of all absolute distances of the roughness profile from the centreline within the measuring length. The arithmetical mean roughness value (Ra, nm) for each sample was estimated and averaged to produce the final roughness value of each specimen.
Data were tabulated for statistical analysis using the IBM SPSS Statistics 23.0 software (International Business Machines Corporation, New York, NY, USA). Mean and standard deviations (SD) were calculated for all the variables. Kolmogorov-Smirnov test was used to establish the normality of the data distribution. Statistical analysis for intergroup comparison of the values of Ra was carried out using the non-parametric Kruskal-Wallis H test. Post hoc Dunn-Bonferroni test was performed to find out the pair-wise mean roughness difference between the two groups. The confidence level was set at (p < 0.05) for (α = 0.05).
| Results|| |
Kolmogorov-Smirnov test showed that the data was not normally distributed. The analysis of the surface roughness values (Kruskal-Wallis H test) revealed significant differences between the Ra values in the experimental groups P < 0.001 [Table 1]. All irrigation regimens increased root dentin surface roughness in comparison with the control group (Group H) [Figure 1].
|Figure 1: Two- and three-dimensional reconstructions of the scanned areas of root canal dentin. (US – ultrasonic)|
Click here to view
The intergroup comparison after the post hoc test is listed in [Table 2]. Root dentin surfaces in groups where the disinfection solutions were activated ultrasonically (Groups E, F, G) and Group D, in which treatment was done with 5.25% NaOCl for a longer period of 5 min, were significantly rougher than those in the control group (Group H). The irrigation protocol, in which the irrigants were applied in the lowest concentration for the shortest time exposure (Group A), showed significantly smoother dentin surface than those with the ultrasonic activation.
| Discussion|| |
All of the tested irrigation regimens in the current study increased the root dentin surface roughness compared to the control group. Our results are in line with previous statements that disinfection solutions used during the endodontic treatment may cause alterations of the mechanical properties of radicular dentin, such as its roughness.,,,,,
Only a few authors utilize AFM for observation of surface topography of root dentin.,, This methodology was chosen on the basis of its essential advantages, one of them being the little or no pretreatment of the sample, which enables repeated scanning of one and the same specimen. Moreover, due to the high resolution of the images, the surface characteristics of the examined area can be determined quantitatively with high mathematical accuracy.,
In an attempt to ensure a reproducible and unbiased evaluation, all of the roots were sectioned longitudinally into buccal and lingual segments which are in agreement with previous methodologies.,,,,,, Our experimental design differs from earlier investigations where root canals were mechanically shaped prior to the action of the endodontic solutions.,, The intact root canal provides a plane for measurement as close as the original morphology of the dentinal surface and enables comparative investigation of the isolated effect of irrigation regimens.
Tartari et al. stated that despite the heterogeneous structure of the different root thirds, they performed equally in response to direct contact of irrigation agents with the root surface. The mid-root region was used for the current testing, approximately halfway between the central lumen and root cementum, where the size and distribution of dentinal tubules are relatively uniform. This was done to minimize the effect of structural variations of different teeth and to establish standardized experimental conditions for all groups.
There is a lack of consensus in the literature regarding the optimal application time and concentration of the endodontic irrigants necessary for thorough smear layer removal without altering the mechanical properties of root dentin. The concentration and time exposure of the irrigants in our study resembled the most widely used in clinical practice regimens for chemical disinfection.,,
An increase in the surface roughness of root dentin has been reported in several studies after the single-use of 2.5%, 5.25% NaOCl, and 17% EDTA solutions.,,, Nevertheless, there is insufficient evidence in the research data concerning the successive action of NaOCl and EDTA on dentin roughness. Keine et al. stated that the combined application of NaOCl and EDTA led to the greatest increase in radicular dentin roughness when compared to the saline control group. We observed that in the groups where these two solutions were applied without additional activation, the roughness was increased to a different extent, though the effect between them remained statistically insignificant. Radicular dentin surface was significantly more uneven in comparison to the control group only after the treatment in Group D (5.25% NaOCl/5 min + 17% EDTA/5 min), thus the null hypothesis was rejected.
Tartari et al. investigated the effect of separate use of irrigants and reported that the increase of root dentin surface roughness was only due to the effect of the chelating agents. They further concluded that the sole use of 2.5% NaOCl and its application before or after chelators (EDTA, citric acid) did not alter this characteristic of dentin. Akbulut et al. reported similar findings in their study about the effect of different kinds of fruit vinegar. We assume that these dissimilarities with our results are due to the different methods, devices, and irrigation regimens used.
It has been advocated that additional activation of various irrigation solutions could benefit their disinfecting abilities. However, this effect has not been studied in depth regarding the change of surface roughness of root dentin.
It might be speculated that additional activation of the disinfection irrigants could result in a greater increase of dentin roughness compared to that of the non-activated groups using the same or higher concentrations of NaOCl. The analysis revealed significant differences between each of the ultrasonically activated groups (Group E, F, G) and the one with samples treated with the lowest concentration of solutions for the shortest time period (Group A). The observed alteration of the dentinal surface could be attributed to the continuous movement of the irrigant and the effect of cavitation, which directly enhances the effectiveness of sodium hypochlorite and the demineralizing action of chelators.
A possible limitation of the present study is that the experimental conditions differ markedly from the clinical situation. The immersion treatment requires a greater amount of the irrigants compared to the volume used during the endodontic treatment. Additionally, the ultrasonic activation contrasts with the methods applied clinically. AFM scanning is an expensive, labor and time-consuming process that requires dedicated software and trained operator for the acquisition and interpretation of the results. Thus, we did not test the specimens at the beginning of the procedure. There is no data regarding the baseline roughness values of the root canal dentin surface, which might be considered as another limitation of the current investigation. Further experiments are needed to assess the effect of various irrigant regimens on radicular dentin roughness in a close-end root canal with or without ultrasonic agitation.
| Conclusions|| |
Within the limitations of the current in vitro study, it can be concluded that:
- All tested irrigants increased root canal dentin roughness and the effect was time and concentration-dependent.
- The ultrasonic activation of the disinfection solutions roughened the root dentin surface significantly.
This study was funded by the Scientific Council of Medical University – Sofia, Bulgaria, Grant Project № 8326/22.11.2018; Contract № 90/23.04.2019.
Financial support and sponsorship
Grant Project № 8326/22.11.2018; Contract № 90/23.04.2019.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]