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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 11  |  Issue : 3  |  Page : 350-357

Effect of Q-Mix® and apple vinegar on penetration of alizarin red dye through dentinal tubules: A comparative confocal microscopic study


1 Endodontic Department, Faculty of Dentistry, Alexandria University, Alexandria, Egypt
2 Department of Histology and Cell Biology, Faculty of Medicine, Alexandria University, Alexandria, Egypt

Date of Submission10-Aug-2020
Date of Acceptance28-Sep-2020
Date of Web Publication3-Sep-2021

Correspondence Address:
Dr, Samar Mahmoud Mohamed Saied
13 Champollion St., Azarita, Alexandria
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sej.sej_205_20

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  Abstract 

Introduction: Effective treatment of an endodontic therapy aims for removal of microorganisms from the root canal. Therefore, intraradicular removal of the smear layer is essential for successful treatment. The aim of the study was to analyze area and percentage of alizarin dye penetration into dentinal tubules to investigate the elimination of smear layer among Q-Mix®, apple cider vinegar (ACV), and ethylenediaminetetraacetic acid (EDTA) using confocal laser scanning microscopy (CLSM).
Materials and Methods: Thirty mandibular premolars were subjected to decoronation to 15-mm standardized length. Root canals were prepared and randomly allocated to three groups according to the final irrigation used: Group I: 10 mL of Q-Mix for 1 min, Group II: 10 mL of ACV for 1 min, and Group III: 10 mL 17% EDTA for 1 min. The samples were irrigated with 2.5% sodium hypochlorite labeled with 0.2% alizarin red dye and then horizontally sectioned to be examined under CLSM. Data were statistically analyzed. The level of statistical significance was set at P < 0.05.
Results: No statistically significant difference (P < 0.05) was present in the area and percentage of penetration of alizarin dye into the dentinal tubules among Q-Mix, ACV, and EDTA groups at the distinct levels of the canal walls.
Conclusions: Q-Mix, ACV, and EDTA revealed effective cleaning ability in the coronal and middle thirds when compared to the apical canal section.

Keywords: Apple cider vinegar, confocal laser scanning microscopy, ethylenediaminetetraacetic acid, Q-Mix®, smear layer


How to cite this article:
Saied SM, Moussa SM, Leheta NA, Mourad GM. Effect of Q-Mix® and apple vinegar on penetration of alizarin red dye through dentinal tubules: A comparative confocal microscopic study. Saudi Endod J 2021;11:350-7

How to cite this URL:
Saied SM, Moussa SM, Leheta NA, Mourad GM. Effect of Q-Mix® and apple vinegar on penetration of alizarin red dye through dentinal tubules: A comparative confocal microscopic study. Saudi Endod J [serial online] 2021 [cited 2021 Dec 4];11:350-7. Available from: https://www.saudiendodj.com/text.asp?2021/11/3/350/325396


  Introduction Top


Successful endodontic therapy mainly targets the reduction of microorganisms and prevents reinfection of the root canal system. It is a combination of instrumentation and irrigation of the root canal with a chemically active substance to clean infected and/or inflamed root canal systems.[1]

However, such mechanical preparation was inadvertently accompanied by an amorphous layer termed “smear layer” formed of organic and inorganic components that are loaded into dentinal tubules up to 40 μm in depth. This layer adversely affects the access of solutions, pastes, and medicaments that could be used into the infected dentinal tubules.[2] Thus, combination of different irrigants is needed to remove both components of the smear layer.[3]

Sodium hypochlorite (NaOCl) is still the universal irrigant applied through endodontic therapy on account of its antimicrobial properties in addition to the competence to dissolve organic tissue.[3] However, the inorganic contents of the smear layer cannot be eliminated, so NaOCl has been used with chelating agents such as ethylenediaminetetraacetic acid (EDTA) 17% for smear layer removal. This combination represents the current optimal irrigation protocol.[4],[5]

The Q-Mix® solution is an endodontic irrigant composed of four components which are a chelating agent of polyaminocarboxylic acid, an antimicrobial agent represented in a bisbiguanide, a surfactant, and deionized water.[6] It has the ability to eliminate the smear layer in addition to its antimicrobial efficiency.[7],[8] As it causes less dentin erosion than EDTA, it can be used as a final irrigant and substitute for EDTA.[8]

On the other hand, various natural products have been considered to be used as root canal irrigants due to their antimicrobial action with fewer side effects.[9]

Investigations of using apple cider vinegar (ACV) as a natural product in the chemo-mechanical preparation of root canals have also been accomplished for smear layer removal,[10],[11] but it needs further investigations. Furthermore, its bactericidal action against Staphylococcus aureus and Enterococcus faecalis, the most considerably characteristic microorganisms found in relation to endodontic infections, has been revealed.[12]

Confocal laser scanning microscopy (CLSM) provides the advantage of observations being made in near-normal conditions, as no special processing technique is required. CLSM has less chances of producing artifacts in comparison to scanning electron microscope (SEM).[13]

Thus, the aim of the current study was to analyze the area and percentage of alizarin red dye penetration into dentinal tubules to investigate the elimination of smear layer among Q-Mix, ACV, and EDTA using CLSM.


  Materials and Methods Top


This study was conducted on thirty human single-canaled mandibular premolar teeth extracted for periodontal reasons with fully developed root apices from the outpatient clinic of Oral Surgery Department, after the ethical approval obtained from the Research Ethics Committee, Faculty of Dentistry, Alexandria University, Egypt (institutional review board 00010556 – IORG 00008839).

Preparation of teeth

The teeth were examined visually and radiographically. Single-rooted canals without calcifications, cracks, root caries, attrition, and external resorption were selected. Teeth with severe canal curvatures, apical fractures, or those that were previously obturated were excluded. The teeth were disinfected in 2.5% NaOCl solution for 15 min and scaled to remove debris, calculus, and organic tissues using an ultrasonic scaler, and then stored in 0.9% saline. Decoronation was performed for root standardization at 15-mm length using a diamond disc (ökoDENT, Preußer oHG, Germany) with a straight handpiece under water cooling. A stainless steel 10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) was inserted in each root canal to check apical patency, and then a 1 mm was subtracted from the mentioned length to obtain a working length of 14 mm.

Chemo-mechanical preparation was performed by ProTaper Next® system (Dentsply Maillefer, Ballaigues, Switzerland) on a rotary motor up to X3 of the apical diameter 0.3 mm and taper 7%. Irrigation was proceeded with 2 mL of NaOCl (2.5%) following each instrument shift using a 30G side vented needle (PPH CERKAMED, Stalowa Wola, Poland) for 1 min.

The exterior part of each root was covered with wax apically to prevent the irrigant from dripping through the apical foramen.

Irrigation groups

The canals received irrigation with 5 mL of distilled water after instrumentation to get away any remnant of NaOCl solution. The roots were distributed randomly to three groups and final irrigation protocol was followed:

  • Group I (n = 10): 10 mL of Q-Mix (Dentsply Tulsa Dental Specialities, Tulsa, OK, USA) for 1 min
  • Group II (n = 10): 10 mL of 5% ACV (Andrea Milano, Napoli, Italy) for 1 min
  • Group III (n = 10): 10 mL of 17% EDTA (Dharma Research Inc., Miami, FL, USA) for 1 min (control group).


In an apical and coronal motion, the roots were irrigated with a side vented 30G needle within a 1-mm distance shorter than the working length. Manual dynamic agitation of the final irrigants was performed using master gutta percha points (Dentsply Maillefer, Ballaigues, Switzerland) at a value of 100 strokes in a time of approximately 1 min.[14]

The root canals were rinsed with 5 mL distilled water and then dried using sterile absorbent paper points (Dentsply Maillefer, Ballaigues, Switzerland).

Confocal laser scanning microscopy

For fluorescence inspection under CLSM, irrigation was performed by 2.5 mL of 2.5% NaOCl mixed with 2.5 mL of 0.2% fluorescent alizarin red (Sigma Aldrich Corporation, USA) to obtain a total 5 mL of labeled NaOCl for each canal.

A 30G side vented needle was used for a 1-min irrigation accompanied by manual dynamic agitation using the master gutta-percha points (Dentsply Maillefer, Ballaigues, Switzerland) at a value of 100 strokes for approximately 1 min.

Subsequently, the paper points (Dentsply Maillefer, Ballaigues, Switzerland) were used for drying all the root canals, and horizontal sectioning was performed using a water-cooled microtome saw on a slow speed in 1-mm-thick sections at 3, 5, and 8 mm from the anatomical apex. The sections were ground to approximately 500-μm thickness and polished, followed by mounting onto glass slides.

The slides underwent examination by Leica TCS-SPII CLSM (Leica, Mannheim, Germany) at a magnification of × 5 with an emission wavelength of 580 nm. Partial images were taken to produce a montage using Adobe Photoshop software (Adobe systems Inc., San Jose, California, USA) program when the whole canal could not be detected in one image at the same time.

The samples were preserved away from light for CLSM analysis of dentinal tubule penetration at the apical, middle, and coronal thirds of the canal [Figure 1].
Figure 1: Representative confocal laser scanning microscopy images from Q-Mix, apple cider vinegar, and ethylenediaminetetraacetic acid groups showing apical, middle, and coronal thirds with objective lens ×5

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The method defined by Gharib et al.[15] was followed for evaluation of CLSM images. ImageJ analysis program (ImageJ software, National Institutes of Health (NIH), USA) was applied on the imported digital images to obtain the measurement of total dentinal tubule penetration area and percentage of NaOCl labeled with alizarin dye into the canal walls.

Image acquisition and analysis

The whole area of the root and the area of the inner lumen of the canal walls were outlined for each image and then measured with the aid of ImageJ measuring tool. Subtraction of the value of the area of the inner lumen of the canal walls from the whole area of the root was done to obtain the result of the total cross-sectional area of the canal wall for each section.

The penetrated areas with the labeled irrigant along the canal walls were outlined for the presence of alizarin red fluorescent dye and then measured automatically using image analysis in ImageJ software [Figure 2].
Figure 2: Image sequence showing image routine analysis using ImageJ software to represent automatic outline and measurement of the labeled irrigant (a) CLSM image imported in imageJ program. (b,c) Color thresholding to trace the area of penetration to be measured. (d) Automatic selection of the area of penetration to be measured

Click here to view


The values of area of penetration were then converted into percentage of penetration by dividing the area of penetration by the total cross-sectional area of the canal wall.

The measurements were repeated twice to ensure reproducibility. Intra-reliability was tested using intraclass correlation (ICC).[16] For statistical analysis, values were calculated in micrometers and then transformed into square millimeters.

Statistical analysis

Sample size calculation was done using GPower version 3.1.9.2 (Heinrich Heine University Düsseldorf, Germany) software.[17] Data were analyzed using IBM SPSS statistical software (version 23), (SPSS Inc., Chicago, IL, USA) and statistical significance was set at P < 0.05. Normality was checked for all variables using descriptive statistics, plots (histogram, boxplots), and tests of normality.

One-way ANOVA with repeated measures was used for comparing variables among the three groups at different regions for quantitative normally distributed variables (percentage of penetration), whereas Kruskal–Wallis and Friedman's tests were used for nonnormally distributed variables (area of penetration).

These were followed by post hoc multiple comparisons using Bonferroni adjustment for multiple pair-wise comparisons with an adjusted significance level (P < 0.02).[18]


  Results Top


Medians, means, and standard deviations of dentinal tubule areas of penetration and percentages of penetration in the different groups are shown in [Table 1] and [Table 2]. The ICC coefficient ranged from 0.96 to 1.00.
Table 1: Area of penetration of irrigant solution (mm2) in the three experimental groups

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Table 2: Percentage of penetration of irrigant solution in the three experimental groups

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Evaluation of area of penetration of irrigating solution

On between-group comparison, no statistically significant difference (P < 0.05) was present in the area of penetration among the tested groups at the different levels of the canal walls. Median values showed that coronal and middle thirds demonstrated the highest area of penetration in Q-Mix, followed by ACV and EDTA. For the apical thirds, ACV showed the highest area of penetration followed by EDTA and finally Q-Mix [[Table 1] and [Figure 3]].
Figure 3: Bar chart showing median area of penetration of the labeled irrigant in the three experimental groups

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For intragroup comparison, there was a statistically significant difference (P > 0.02) within the Q-Mix and ACV groups between the apical and both the coronal and middle thirds. Better penetration and cleaning ability were noticed in the coronal and middle thirds compared to the apical third with no statistical difference mentioned between the coronal and middle thirds [Table 3], while the EDTA group showed no statistically significant difference (P = 0.15) among the coronal, middle, and apical sections.
Table 3: Post hoc multiple comparisons of area of penetration using Bonferroni adjustment

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Evaluation of percentage of penetration of irrigating solution

On between-group comparison, no statistically significant difference (P < 0.05) was present in the percentage of penetration among the tested groups at the different levels of the canal walls. The mean (standard deviation) values showed that the coronal and middle thirds demonstrated the highest percentage of penetration in Q-Mix, followed by ACV and EDTA. For the apical thirds, ACV showed the highest percentage of penetration followed by EDTA and Q-Mix [[Table 2] and [Figure 4]].
Figure 4: Bar chart showing the mean percentage of penetration of the labeled irrigant in the three experimental groups

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For intragroup comparison, [Table 4] shows a statistically significant difference (P > 0.02) within Q-Mix and ACV groups between the apical and both the coronal and middle thirds. The coronal and middle thirds presented better percentage of penetration compared to the apical third but no statistically significant difference (P = 1) was observed between the coronal and middle thirds. The EDTA group showed no statistically significant difference (P = 0.07) among the coronal, middle, and apical thirds.
Table 4: Post hoc multiple comparisons for percentage of penetration using Bonferroni adjustment

Click here to view



  Discussion Top


Mechanical instrumentation should be accompanied by irrigating solutions to guarantee complete cleanliness and disinfection of the root canal system.[1],[19] Consequently, the chosen irrigants should rely on its capability of removing the smear layer.

NaOCl has proved to be the most common irrigant used worldwide. However, NaOCl has shown reduction in the mechanical resistance of dentin and cannot bring out the inorganic component of the smear layer.[8],[10] In addition, reports pointed to the allergic reactions and accidents caused by NaOCl during root canal therapy.[20] Searching for an irrigating solution that obtains better biocompatibility than NaOCl and removes the components of smear layer is needed.

In the current study, single-canaled mandibular premolars were used because they are almost straight canals that may facilitate the flow and penetration of the irrigant in the apical third because the presence of curvature may limit to some extent the entrance in the depth of the irrigant. This came in accordance with Attur et al.,[21] Jagzap et al.,[22] and Akcay et al.[23]

Several precautions were implied within the study for standardization and balancing which include: initial apical diameter, total length, mechanical preparation, amount of irrigant, and gauge of the irrigating needle and its penetration.

All the canals underwent preparation till X3 ProTaper file so that sufficient amount of irrigant could reach the intracanal walls and achieve cleaning in balance with the preservation of radicular dentin thickness, as stated by Brunson et al.[24]

In the current study, a 30G needle tip was positioned shorter than the working length by 1 mm so that the needle could penetrate till the apical third of the canal. The delivery of the irrigant has been proved to be 1 mm away from the irrigating tip.[25]

The 17% EDTA solution is the gold standard irrigant in the effective elimination of smear layer. The ionized EDTA can chelate calcium ions within the dentin effectively. However, its disadvantages are represented in its cytotoxicity and inhibition of the macrophage function, resulting in alteration of the inflammatory response in periapical lesions.[22] Hence, searching for more biocompatible irrigants with less severe impact on periapical tissues was needed.

Q-Mix was chosen for this study as it is considered an antimicrobial agent. EDTA found in Q-Mix effectively removes the smear layer, while chlorhexidine (CHX) adsorbs onto the dentin surface and prevents the microbial colonization on dentinal walls.[7]

Furthermore, no interaction was observed between Q-Mix and remnant of NaOCl to form an orange-brown precipitate if used directly for the final rinse. The chemical design of Q-Mix prevents precipitate formation when EDTA and CHX are mixed together.[26],[27]

Apple vinegar, also known as apple cider vinegar, was selected due to biocompatibility and high content of organic acids. Maleic acid has been assumed as an essential component in ACV, with fungicidal and bactericidal properties.[12] A noticeable medicinal potency has also been determined for the numerous mineral components found in ACV such as potassium, phosphorus, fluoride, calcium, sulfur, silicon, and magnesium. Other elements such as pectin, beta-carotene, amino acids, and enzymes have also been found to affect the immune system through attacking the free radicals.[10],[12]

Data regarding ACV and its effect on smear layer removal are lacking. Therefore, more investigations were needed.

Manual dynamic agitation was also used for cleaning and elimination of microbial biofilm. It is simple, fast, and cost-effective. Moreover, it could disinfect the gutta-percha cone before canal obturation.[14]

In the present study, NaOCl (2.5%) labeled with alizarin red dye was applied as an indicator to investigate the ability of various irrigating solutions to remove the smear layer from distinct regions of the canal walls by tracing fluorescent alizarin red dye penetrated through the dentinal tubules under CLSM. Alizarin was used due to its availability, validity, and safety.[28]

In addition, intracanal rinsing with labeled NaOCl was applied without obturation of the canal to trace the penetration of the irrigant within the dentinal tubules with fewer steps required. This came in accordance with Küçük and Kermeoğlu,[13] Akcay et al.,[23] and Vadhana et al.[25]

However, other investigators traced the influence of final irrigation regimen by the penetration of sealer labeled with fluorescent dye under CLSM. It could be a good indicator for assessment of the degree of smear layer eliminated in the in vitro model.[27],[29]

Meanwhile, Akcay et al.[23] reported that the stage of obturating the root canal could affect the penetration of the final irrigating solution regarding the pressure exerted during the lateral compaction.

Confocal microscopic analysis was chosen in the current study. This method was in accordance to Akcay et al.,[23] Vadhana et al.,[25] and Chaudhry et al.,[27] who selected CLSM because it directs the depth of field and eradicates or lessens background information away from the focal plane. Moreover, it gathers sequential optical sections regardless of the specimen thickness. Significant improvement of the contrast and definition was noticed over wide-field procedures because of the decrease in background fluorescence and promoted signal-to-noise ratio. It also supports detection and inspection up to 10 μm below the specimen's surface.

The utilization of CLSM is a nondamaging approach during specimen handling, empowering the observations to be performed under close to normal environmental conditions, reducing technical artifacts.[30] However, this strategy could not inspect the large root section in one image. Hence, several images were picked up and gathered to produce one image.[23]

Other investigators used SEM to estimate the removal of intraradicular smear layer,[7],[10],[31] but the methodology was time consuming. The gold sputtering and vacuum stages in which the samples were exposed to through SEM examination could compromise the appropriate evaluation of the images and lower their contrast when compared to CLSM. This is due to artifact production.[13],[32]

For evaluation of dentinal tubule penetration under CLSM, the following two parameters have been monitored: area of penetration and the percentage of penetration of the dye inside the dentinal tubules as the direction of dentinal tubules could affect the results. The measurements of the total dentinal tubule penetration area have been carried out using the ImageJ program. This program exhibits its capability of calculating area and pixel value statistics of user-defined selections and intensity–threshold objects.[33]

The results have shown no difference in the area and percentage of dentinal tubule penetration and smear layer elimination using Q-Mix, ACV, or EDTA as a final rinse for the root canal. These results came in agreement with those of Stojicic et al.[7] and Dai et al.,[31] who demonstrated that Q-Mix was as effective as EDTA in smear layer removal regardless of the canal level. Furthermore, Ballal et al.[34] found that there was no significant difference between Q-Mix, EDTA, and maleic acid (which is considered the most important component of ACV) in eliminating the smear layer from the coronal and middle thirds. However, for the apical part, maleic acid performed significantly better than EDTA and Q-Mix. This could account on the acidic pH of 7% solution of maleic acid.

On the contrary, Eliot et al.[26] found that Q-Mix presented significantly higher cleaning effect than 17% EDTA. In their study, different formulas of Q-Mix were used with larger sample size and all showed better effect in the cleaning ability in the coronal and middle thirds of the canals compared to the apical third.

Based on the observation of the numerical values of the obtained data, Q-Mix gave better cleaning ability, followed by ACV, compared to EDTA in the middle and coronal sections of the canal. This observation came in line with that of Ballal et al.[34] who found that Q-Mix more effectively removed the smear layer compared to 17% EDTA. This is attributed to the presence of CHX and EDTA in Q-Mix that showed a synergistic effect. Moreover, a surface-active agent helped to reduce the surface tension of the solutions and increase their wettability and penetration of an irrigant in the canal.[7]

Regarding ACV, no significant difference was found between ACV and EDTA in cleaning efficacy of the canal walls. However, it was noticed from the obtained data that ACV showed more cleaning efficacy in the middle and coronal thirds compared to 17% EDTA solution, and this was in accordance with Ballal et al.[35] Moreover, Candeiro et al.[10] reported that ACV used in association with or without EDTA was found to be efficient to remove smear layer when used as an endodontic irrigant.

However, this came in disagreement with Spanó et al.[36] who stated that EDTA and citric acid were more effective in smear layer removal than ACV. Moreover, Kirchhoff et al.[11] found that 17% EDTA enabled greater removal of smear layer compared to ACV and other organic products with no statistical difference among the coronal, middle, and apical regions of the root canals. This could be attributed to the methodology applied, in which the solution used had free passage through the apical foramen and that was different from the present study, in which the apical foramen was sealed using wax.

Statistically significant difference was found in the current study between apical, coronal, and middle thirds when intragroup comparison was performed. Coronal and middle thirds showed better penetration, whereas apical third showed the least amount of penetration. This result came in accordance with that of Jagzap et al.,[22] Aydın et al.,[29] and Ballal et al.[35]

This result is attributed to the dentinal architecture. The chelating agent may appear that it is less effective in the apical area due to less calcification of intertubular dentin in comparison to intratubular dentin that may reveal its sclerosis with different degrees. Moreover, removal of smear layer is better in coronal and middle sections due to better accessibility to receive the irrigants due to which the volume and velocity of irrigant are highly effective in these regions in addition to larger diameter and number of dentinal tubules.[27] Dentinal tubule density diminishes apically, so does the permeability of dentin. Furthermore, the mineralization of radicular dentin is not uniform. Some areas of apical dentin show more mineralization while other areas completely destitute of tubules in their structure.[29],[37]

One of the limitations of the present study was tubular sclerosis of dentinal tubules and its relation to the age of the patient that were not taken into consideration. Furthermore, surface tension, temperature, and time application of the tested chelating agents should be investigated.


  Conclusions Top


Within the limitations of the study, it can be concluded that:

  1. Q-Mix, ACV, and EDTA promoted the cleaning ability and penetration of dentinal tubules
  2. The penetration of the irrigants in the coronal and middle sections is superior to that of the apical section
  3. ACV is a promising biocompatible natural substance that efficiently affects the penetration of the dentinal tubules and eliminates the smear layer in vitro.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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