Multiparameter image analysis to evaluate dentinal tubules patency after using different irrigation protocols

Mohamed Ahmed Elsayed

doi:10.4103/SDJ.SDJ_109_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 6 | Issue : 1 | Page : 36-41

Multiparameter image analysis to evaluate dentinal tubules patency after using different irrigation protocols

Mohamed Ahmed Elsayed
Department of Endodontics, RAK College of Dental Sciences, RAK Medical and Health Sciences University, Ras al Khaimah, UAE; Department of Endodontics, Faculty of Dentistry, Assuit University, Asyut, Egypt

Date of Submission	11-Oct-2021
Date of Decision	25-Nov-2021
Date of Acceptance	14-Dec-2021
Date of Web Publication	21-Feb-2022

Correspondence Address:
Mohamed Ahmed Elsayed
Department of Endodontics, Rak College of Dental Science, PO Box 12973, Ras al Khaimah, UAE

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/SDJ.SDJ_109_21

Abstract

Background: Scoring systems have been used to evaluate dentinal tubule patency in several papers; however, these conventional systems are time-consuming and rely primarily on evaluator estimation with a wide range of subjective variations. Objectives: A multiparameter, simple, and objective method is introduced to evaluate the ability of different irrigation protocols for smear layer removal and dentinal tubule patency enhancement. To show the applicability of this method, the effect of different irrigation regimens was evaluated. Methods: Ninety extracted single-rooted teeth were decapitated and shaped with ProTaper to size F4. According to the final irrigation protocol, specimens were divided into three groups: Group 1 (final irrigation of SmearOFF), Group 2 (17% EDTA), and Group 3 (negative control). Groups 1 and 2 were subdivided into two subgroups: Subgroup (A) was activated by an ultrasonic file, whereas samples in Subgroup (B) did not receive any activation. Samples were examined by scanning electron microscopy. Images were analyzed using ImageJ software, and the multiparameter image analysis method (relative total surface area, average size, and the number of opened dentinal tubules) was applied. One-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, was applied to reveal any significant differences (P < 0.05) among groups, activation techniques, and root thirds. Results: Passive ultrasonic activation led to a significant increase in the patency of dentinal tubules especially at the apical third, which was measured and shown by the triple parameter method. Conclusion: Digital image analysis shows potential and advantages for the objective evaluation of dentinal tubules and should replace the traditional subjective scoring system method.

Keywords: Dentinal tubules, digital image analysis, EDTA, ImageJ, passive ultrasonic activation, SmearOFF

How to cite this article:
Elsayed MA. Multiparameter image analysis to evaluate dentinal tubules patency after using different irrigation protocols. Sci Dent J 2022;6:36-41

How to cite this URL:
Elsayed MA. Multiparameter image analysis to evaluate dentinal tubules patency after using different irrigation protocols. Sci Dent J [serial online] 2022 [cited 2023 Mar 20];6:36-41. Available from: https://www.scidentj.com/text.asp?2022/6/1/36/338002

Background

Successful cleaning of the root canal space should achieve significant patency of dentinal tubules with minimal debris as well as a smear layer, possibly affecting the sealer penetration and sealing ability of obturation materials.^[1] Canal cleanliness is considerably dependent on the use of an effective irrigant and a proper irrigation technique.^[2],[3] Hence, an accurate assessment of dentinal tubule patency would clearly reflect the effectiveness of the studied regimens.^[4]

For decades, traditional scoring systems^[3],[5] have been used to assess the cleanliness of root canals in terms of the smear layer removal and patency of dentinal tubules.^{[6],[7],[8],[9]} However, these conventional scoring systems are primarily dependent on operators, with a wide range of subjective variations.^{[8],[10],[11]}

In this study, a user-friendly objective image analysis method is described in detail, including the application of several software features and plug-ins to achieve the most accurate results. This method is used to evaluate the effectiveness of two irrigant solutions in conjunction with or without passive ultrasonic activation. EDTA is known to exert an effect on the mineral matrix, and it can promote the removal of the smear layer and permit increased penetration of sealers into dentinal tubules.^[12] On the contrary, SmearOFF (Vista Dental Products, Racine, Wisconsin) is an irrigant solution containing tetrasodium EDTA (18% wt.) and CHX gluconate (<1% weight, wt).^[13],[14] Passive ultrasonic activation involves the activation of files without direct contact with dentin walls to produce acoustic streaming. It is believed to be highly effective in cleaning root canal dentin.^[15]

Materials and Methods

Selection and preparation of specimens

Ninety single-rooted teeth with straight roots and closed apices extracted for orthodontic reasons were collected from the clinics affiliated with the academic institution. The use of extracted teeth and study protocol were approved by the institutional research ethics board number AU-Rec 2018-05. Samples were randomly distributed into three groups according to the final irrigant solution: Group 1: final irrigation with SmearOFF (Vista Dental Products, Racine, Wisconsin) (n = 40); Group 2: final irrigation with 17% EDTA (Vista Dental Products), (n = 40), and Group 3: no final irrigant was applied (n = 10). Subsequently, Groups 1 and 2 were subdivided into two subgroups according to the irrigation method. In Subgroup A, an ultrasonic file was applied for activation of the irrigant solution, (n = 20), and in Subgroup B, no ultrasonic activation was applied, (n = 20). All root canals were mechanically prepared by the crown-down technique using Protaper rotary files (ProTaper NiTi, DENTSPLY Tulsa Dental Systems, Johnson City, Tennessee) up to the master apical file size F4; then, crowns were removed, and roots were adjusted to a standardized length of 16 mm. A small amount of wax was placed on the tip of each root to prevent the passage of irrigant solutions through the apical foramen.

Specimens in all groups were irrigated with 3 mL of sodium hypochlorite (NaOCl, 5.25%) after utilizing each file. All samples, except those in the control group, received 5 mL of the final irrigant (SmearOFF or 17% EDTA) for 1 min, which was delivered using a 27-gauge double-side-vent needle to reach the apical third.

Passive ultrasonic irrigation was performed using a magnetostrictive ultrasonic unit with a size 10 K ultrasonic file attached to the ultrasonic unit (Suprasson P5 Booster, Satelec, Merignac, France) using medium power. The ultrasonic file was placed into the canal 3 mm short of the working length without touching the canal walls to permit free vibration, and it was activated for six intervals.

Image acquisition by scanning electron microscopy

The samples were examined by scanning electron microscopy (SEM, JEOL JSM-5400, Tokyo, Japan). To prepare samples for SEM examination, two longitudinal grooves were made on the buccal and lingual surfaces of each root. The roots were grooved to three levels at 4, 8, and 12 mm from the root apices to define the coronal, middle, and apical thirds. Next, roots were split into two halves, and specimens were sputter-coated for SEM examination. Serial micrographs of the canal walls were recorded at 750x magnification.

Image analysis method

SEM images were analyzed using ImageJ software, which is a free open-source image processing program developed by the National Institute of Health (http://imagej.nih.gov/ij). Various original features in the program were adapted with additional plug-ins to match the study objectives. First, for standardization, all images were cropped to remove black edges to obtain accurate measurements by the analysis of only true dental tissue areas; consequently, the total surface area of all images after cropping is fixed to 260416 pixels [Figure 1]. In addition, the set scale function was applied to present the outcome measurements in micrometers. Eventually, the total surface area of the image was equal to 17996.28 μm².

Figure 1: Total surface area of every image that was measured by ImageJ after cropping and was found to be 260416 pixels

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Open dentinal tubules were identified by the determination of pixel intensity from the image histogram to identify a distinct outline of the open tubules. Then, the Adjust/Threshold function was used to measure the area of open dentinal tubules [Figure 2]. To count the dentinal tubules and calculate their average size, the same adjust/threshold function successively followed by the Process/Binary/Watershed function. The Watershed function was beneficial to outline some closely packed dentinal tubules that could be wrongly counted as one tubule [Figure 3].

Figure 2: Adjust/threshold function was used to identify the area to be measured. Blue areas correspond to the open dentinal tubules

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Figure 3: Watershed function to identify some closely packed dentinal tubules (yellow arrows) that could appear to be connected and might be falsely counted as one tubule. Closely packed dentinal tubules after outline separation (red arrows)

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Then, the Analyze Particles function was applied to configure the particle analyzer. Particles of a size less than that specified in the Size field (1 μm) were ignored. Accordingly, the minute branches of dentinal tubules of less than 1 μm were excluded from the counting process to obtain more precise measurements [Figure 4].

Figure 4: Tiny branches of dentinal tubules, which were excluded from the counting process to obtain more accurate results

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Finally, the Show/Outlines and Summarize functions were used to obtain automated numbering of the measured dentinal tubules as well as the final count and their average size, respectively [Figure 5].

Figure 5: Counting of the dentinal tubules: A number was assigned to each tubule

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Statistical analysis

Data were analyzed using Statistical Analysis System (SAS Institute Inc., North Carolina). One-way analysis of variance (ANOVA) was used to compare the study groups, followed by Tukey’s post hoc test for pair-wise comparison between the means when the ANOVA test was significant. The level of significance was set at P < 0.05.

Results

[Table 1] shows the mean value and standard deviation of the three outcome measures, including the number of open dentinal tubules, the percentage of the total surface area, and the average size of dentinal tubules in the coronal, middle, and apical thirds. A statistically significant difference between the studied groups in the three evaluated parameters was observed (P < 0.001). The mean value of the three outcome measures for the control group was significantly less than those of the experimental groups in all root sections.

Table 1: Mean value of the three parameters (number of open dentinal tubules, percentage of the total surface area, and average size of dentinal tubules) in the coronal, middle, and apical thirds

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Passive ultrasonic activation in combination with SmearOFF or 17% EDTA increased the number of open dentinal tubules in all thirds, but it was significantly different only in the apical third (472.5, 401.6). Similarly, ultrasonic activation in conjunction with SmearOFF or EDTA was associated with the increase in the percentage of the total surface area of open dentinal tubules in coronal and middle thirds, but with a significant difference in the apical third only (9.7% and 8.71%, respectively). Ultrasonic activation led to the increase in the average size of open dentinal tubules in all thirds, but the difference was not statistically significant.

Discussion

The most commonly used research methods to evaluate the removal of the smear layer, patency of dentinal tubules, and effectiveness of desensitizing agents on dentinal tubule occlusion^{[11],[16],[17]} are subjective because the methods directly depend on the perspective of evaluators.^[18] Digital evaluation of dentinal tubules using software is imperative to provide quantitative analysis and overcome bias and sources of error with panels of evaluators, such as fatigue and consistency during long periods.^[4],[9] The objective nature of digital analysis makes it more beneficial to assess large numbers of images at low costs due to less time required for training evaluators.^[4] However, the possible bias observed during area selection for capturing SEM images and in determining the pixel intensity for the threshold adjustment step should be considered.^[14]

In this study, an objective was introduced, and a simple semi-automatic method was used to evaluate the ability of different irrigation protocols to increase the patency of dentinal tubules. A previous study has confirmed an agreement between the traditional Hulsmann scoring system^[5] and digital image analysis;^[4] in this study, the accuracy of the digital method was first validated on a subset of samples of varying smear layer amounts, which was well correlated to observer interpretation (data not shown).

The accuracy and reproducibility of computerized image analysis were supported by previous studies.^[19],[20] However, most of these methods depend on only one parameter as the percentage of the total area corresponding to the lumen of dentin tubules^{[4],[14],[21]} or two parameters of the total surface area and the total number of opened tubules, respectively.^[22]

Ciocca et al.^[22] have reported that an automated analysis technique provides reliable identification and dentinal tubule numbering. In addition, George et al.^[4] and Ahmed et al.^[23] have used image processing software to determine the percentage of the total area of dentin tubules by counting the image pixels representing the tubules. They showed that their image analysis technique is well correlated with the conventional scoring approach, but it did offer a promising scope for the analysis of dentinal tubule characteristics, whereas Qian et al.^[24] have utilized image analysis software to evaluate the level of dentinal tubule erosion caused by different irrigant solutions.

To show the applicability of this method, the effects of SmearOFF and 17% EDTA with and without passive ultrasonic activation were compared. Images from three anatomical sections of the specimens were analyzed. Three parameters were utilized in this study to evaluate the patency of dentinal tubules (the percentage of the total area occupied by the lumen of dentin tubules, total number, and the average size of dentin tubules). The first two parameters have been reported previously to be indicators of tubular patency and cleaning efficacy of various irrigation protocols.^[4],[22]

This simple and rapid method permitted the simultaneous measurement of the number and size of hundreds of tubules without operator subjectivity and interference and did not require any specialized or sophisticated device or mathematical training. In contrast, a fully automated method requires complicated and expensive software, which has been described previously.^[22]

ImageJ software was set to overcome some inaccuracies that are typically observed in automated image analysis. For example, closely packed dentinal tubules could appear and be measured as one tubule; hence, the Watershed function is used to precisely distinguish and count these tubules as independent ones.

In addition, the software might count any openings as dentinal tubules, including small tubule branches or small artifacts. Therefore, the software is set to ignore all areas less than the value specified in the size field. The diameter of the dentinal tubules has been reported to range between 1 and 3 μm.^[25],[26] Accordingly, open areas less than 1 μm size were ignored.

Results revealed that ultrasonic activation renders a statistically significant increase in the number and surface area of open dentinal tubules in the apical third irrespective of the irrigant solution used. This result is supported by several studies and explained by the ability of passive ultrasonic activation to create cavitation and acoustic microstreaming.^{[27],[28],[29]} Fewer studies such as those reported by Ciucchi et al.^[30] and Abbott et al.^[31] did not exhibit such superior efficacy by the use of ultrasonic activation on the removal of the smear layer. The disagreement could be attributed to the difficult standardization of the activated instrument positioning in the root canal and displacement amplitude.^[15]

The effect of SmearOFF has been evaluated in previous studies.^[21],[32] This endodontic irrigant has been presented by the manufacturer as being specifically designed for smear layer removal and root canal cleaning. A nonsignificant difference between SmearOFF and EDTA in the three evaluated parameters at the coronal, middle, and apical third of the root was observed. These results have been confirmed by successive studies.^[21],[33] In the control group, images of specimens exhibited a heavy smear layer and debris covering the root canal surface.

Conclusion

Current root canal instrumentation typically leads to smear layer formation and dentinal tubule closure. Digital image analysis using simple methods such as ImageJ software shows the potential for objective evaluation of microscopic images of dentinal tubules and should eventually replace the traditional subjective scoring systems used in endodontic studies. A triple parameter outcome method (i.e., number, total surface area, and the average size of dentinal tubules, respectively) was applied to compare the effect of various irrigation protocols. The use of passive ultrasonic activation improved smear layer removal, especially in the apical third, which was shown by obtaining significantly more patent dentinal tubules.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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