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Year : 2023  |  Volume : 26  |  Issue : 3  |  Page : 265-270
A confocal laser scanning microscopic evaluation of nonthermal atmospheric plasma on the dentinal tubule penetration of bioceramic and epoxy resin-based root canal sealers

1 Department of Conservative Dentistry and Endodontics, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India
2 Clinical Practitioner, Suraksha Dental Clinic, Guntur, Andhra Pradesh, India

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Date of Submission09-Jan-2023
Date of Decision27-Jan-2023
Date of Acceptance30-Jan-2023
Date of Web Publication16-May-2023


Aim: Using confocal laser scanning microscopy (CLSM), the current study assessed the impact of nonthermal atmospheric plasma (NTAP) on the dentinal tubule penetration of bioceramic and epoxy resin-based root canal sealers (CLSM).
Materials and Methods: Forty human mandibular premolar teeth with a single root that had just undergone extraction were chosen and biomechanical preparation of root canals was done with ProTaper Gold rotary Nickel-titanium instruments. Samples were divided into four groups (n = 10). Group 1: Bioceramic sealer (BioRoot RCS); Group 2: Epoxy resin-based sealer (AH Plus) without application of NTAP; Group 3: Bioceramic sealer (BioRoot RCS); and Group 4: Epoxy resin-based sealer (AH Plus) with the application of NTAP for 30 s. In Groups 3 and 4, all of the samples underwent obturation with the appropriate sealers following NTAP application. For determination of the sealer's dentinal tubule penetration values, slices with a thickness of 2 mm were taken from the middle third of sample's root and examined using CLSM. The acquired data were statistically analyzed with one-way analysis of variance and the Post hoc Tukey's test. The cutoff for statistical significance was P < 0.05.
Results: In comparison to other groups, the maximum sealer penetration values into dentinal tubules were significantly higher in Group 3 for Bioceramic sealer with NTAP application and Group 4 for Epoxy resin-based sealer with NTAP application.
Conclusion: NTAP application increased the dentinal tubule penetration of bioceramic and epoxy resin-based sealers in comparison to groups without NTAP application.

Keywords: Bioceramic sealer; confocal laser scanning microscopy; dentinal tubule penetration; nonthermal atmospheric plasma

How to cite this article:
Garlapati R, Gali PK, Bolla N, Guptha Anila BS, Vemuri S, Naidu BP. A confocal laser scanning microscopic evaluation of nonthermal atmospheric plasma on the dentinal tubule penetration of bioceramic and epoxy resin-based root canal sealers. J Conserv Dent 2023;26:265-70

How to cite this URL:
Garlapati R, Gali PK, Bolla N, Guptha Anila BS, Vemuri S, Naidu BP. A confocal laser scanning microscopic evaluation of nonthermal atmospheric plasma on the dentinal tubule penetration of bioceramic and epoxy resin-based root canal sealers. J Conserv Dent [serial online] 2023 [cited 2023 Nov 28];26:265-70. Available from:

   Introduction Top

The success of endodontic therapy is influenced by a thorough root canal debridement, elimination of pathogenic microorganisms, using an inert root canal filler substance in the root canal that blocks the entry of microorganisms which in turn provides a fluid-tight seal.[1] The core obturation material and root canal sealers provide a seal that is fluid-tight inside the root canal during obturation. Root canal sealers fill the irregularities, dead spaces, and prevent the gap formed between the core materials and the dentinal walls through micromechanical retention or frictional resistance which is favorable in sustaining the unity of the sealer-dentin interface.[2]

Based on their chemical composition, many varieties of commercial root canal sealers are available. A sealer made with epoxy resin is AH plus Sealer (Dentsply Maillefer, Ballaigues, Switzerland). Due to its better physical properties, good adhesion to root dentin, less solubility, apical sealing ability, and antimicrobial action, it is broadly used for root canal filling. Due to its increased ability to penetrate the root canal's minute abnormalities and its extended setting time, AH Plus exhibits an increase in the mechanical interlocking of the sealer with root dentin. Balguerie et al. suggested that AH Plus sealer showed better adaptation to root canal wall, peritubular dentin, and deeper dentinal tubular penetration.[3] A more recent bioceramic-based sealer is Bioroot RCS (Septodont, France). It has good sealing ability as a repair material, showed improved quality of obturation of root canal, more biocompatible, less toxic, low solubility, and good radiopacity. Due to its self-adhesive properties, Bioroot RCS demonstrated a higher bond strength as compared to AH Plus, MTA Fillapex, and Sealapex, according to Dayanand Chole et al.[4]

The performance of root canal sealers is evaluated by their penetration into the root dentin. Sealer selection depends on two important aspects, i.e., penetration and adhesion. Previous research suggested that the type of root canal sealer and pretreatment of root canal walls affects the penetration and bond strength of sealer.[5],[6] Physical and chemical characteristics, including those of surface tension, viscosity, solubility, and particle size have an impact on the consistency and depth of a material's dentinal tubule penetration, according to Mamootil and Messer.[7]

There are various methods of improving the sealer penetration and their adhesion; nonthermal atmospheric plasma (NTAP) application is one of the methods Du et al. and Pan et al., in their analysis examined the feasibility of treating root canals contaminated biofilms of Enterococcus faecalis using NTAP and mentioned that NTAP was effective in disinfection of the root canals with E. faecalis biofilms. Thus, NTAP can be an effective and efficient adjunct to standard endodontic antimicrobial treatment.[8],[9] NTAP application reduces the contact angle, increases the surface wettability, and improves the surface energy of the materials thus increasing the bonding capability of the plasma-treated surfaces. NTAP application improves the hydrophilic property of dentinal tubules which helps in deeper root canal sealers penetration.[10]

Various microscopy techniques are available for the evaluation of the depth of root canal sealer penetration. Confocal laser scanning microscopy (CLSM) was superior in providing thorough details regarding the presence of sealer and its distribution across the dentinal tubules.[11] In order to acquire and process images, CLSM uses a system that combines physical-chemical principles, optical microscopy, and computing resources. The excitation of fluorophores, which can permeate through enamel, dentin, and biofilms, was encouraged by the use of the laser source. This allowed for the detection of their interior structures and the formation of multiple two-dimensional pictures.[12]

Fewer studies have examined the impact of NTAP on the penetration of bioceramic and epoxy resin-based sealers into dentinal tubules. Current confocal laser scanning microscope investigation therefore assessed and compared the impact of NTAP application on the penetration of bioceramic and epoxy resin-based sealers into root dentin.

   Materials and Methods Top

Sample preparation

Forty human-extracted single-rooted mandibular premolars (n = 40) were chosen. Crowns were decoronated with a low-speed handpiece below the cementoenamel junction, standardizing all root lengths to 15 mm. Using #10 K-file (Dentsply Maillefer, Switzerland), the root canal's patency was achieved and the working length was determined 1 mm short of the apex.

Biomechanical preparation was done using ProTaper Gold (Dentsply Sirona, USA) nickel–titanium rotary instruments. Each time a file is changed, to eliminate the smear layer left behind from instrumentation, root canals were irrigated with 2 ml of 5.25% sodium hypochlorite (Prime Dental Products, India) and with 2 ml of 17% ethylenediaminetetraacetic acid (MD-Cleanser, Korea). Finally, 5 ml of distilled water was used to flush the canals, and paper points were used to dry them. Forty samples (n = 40) were divided each contained ten samples (n = 10), as follows:

  • Group 1: Bioceramic sealer (BioRoot RCS) without application of NTAP
  • Group 2: Epoxy resin-based sealer (AH Plus) without application of NTAP
  • Group 3: Bioceramic sealer (BioRoot RCS) with application of NTAP
  • Group 4: Epoxy resin based-sealer (AH Plus) with application of NTAP.

Nonthermal atmospheric plasma application

After biomechanical preparation, samples in Groups 3 and 4 were given NTAP treatment. A glass reactor with a 30 cm length and 5 cm diameter was employed for the NTAP application. Using a mechanical pump, the glass reactor is evacuated to pressures under 2 Pa. The glass reactor was fueled with gas and inflated to a pressure of 10 Pa. Helium-argon gas was used to treat all samples at 60 watts for 30 s and the length of the plasma jet maintained was almost about 15 mm. The sample and the plasma jet's tip's distance during the procedure was about approximately 5 mm. NTAP is dispersed as a plasma jet at a greater flow rate of 5 L/min [Figure 1].[13],[14] NTAP is a partially ionized gas at room temperature. Even if NTAP is applied at the canal orifice, the depth of NTAP penetration into the root dentin may be due to the highly energetic particles bombarding plasma-treated surfaces, which cause the energy of these species to be transferred to the solid surfaces of the root dentin.
Figure 1: Nonthermal atmospheric plasma application

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By using Rhodamine B (Sigma-Aldrich, USA), each sealer was fluorescently labeled at about 0.1 w/w ratio for analysis in confocal laser scanning microscope. In accordance with the instructions provided by the manufacturer, both sealers were manipulated. Following NTAP application, all samples in the control and experimental groups were obturated using the cold lateral compaction technique with the respective sealer and gutta-percha points (Dentsply Maillefer, Switzerland). For 1 week, all of the samples were kept at 37°C with 100% humidity. The roots from each sample were mounted on self-curing acrylic resin cylinders and a hard tissue microtome (SRM Institute of Science and Technology, Kattakulathur) was used to produce 2 mm slices from the middle third portion of the root from each sample.

Measurement of dentinal tubule penetration values using confocal laser scanning microscopy

Images of slices were taken after each slice's coronal surface was polished using silicon carbide abrasive paper and viewed under CLSM at ×10. Zeiss Zen software was used to analyze the images. The maximum depth of root canal sealer penetration was measured in micrometers (μm) [Figure 2] from the root canal wall to the point of deepest sealer penetration.
Figure 2: Confocal laser scanning microscope images showing dentinal tubule penetration of root canal sealers in Group 1, 2, 3 and 4

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Statistical analysis

MS Excel was used to enter the data and Statistical Package for the Social Sciences version 21.0 (Statistical Package for Social sciences Inc., IBM Corporation, Chicago, Illinois, United States) was used to analyze it. Shapiro–Wilk test was used to find the normality. Mean and standard deviation was used to illustrate descriptive statistics. Ninety-five percent confidence intervals were calculated. One-way analysis of variance with a Post hoc Tukey's test was used to determine significance. Statistics were deemed significant at P < 0.05.

   Results Top

The results obtained have exhibited that NTAP-treated samples, i.e., Group 3 and Group 4 had a statistically significant increase in depth of sealer penetration compared to that of their respective control groups, i.e., Group 1 and Group 2 (P < 0.05). Group 3 showed higher mean penetration (1278.90 ± 47.23) followed by Group 4 (958.32 ± 74.99) which is statistically significant. In between-group comparison was done where Group 3 showed higher sealer penetration values than Group 1 and also Group 4 has shown higher sealer penetration values than Group 2 which were statistically significant [Table 1] and [Graph 1].
Table 1: Mean values and standard deviation of the sealer penetration (µm) into the dentinal tubules of four groups

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   Discussion Top

Sealers are used to fill the morphologic root canal system irregularities and penetrate dentinal tubules to provide a hermetic seal of the root canal system and to prevent microleakage.[15] The number and diameter of dentinal tubules, dimensions of the root canal as well as chemical and physical characteristics of the root canal sealers, among other variables, all affect the depth of penetration of the sealers. Removal of the smear layer, the type of obturating material, and the type of obturation technique may also improve the penetration depth of sealer.[11]

Dentinal tubules at the coronal third of the root canal have a greater diameter (4.32 μm) than the middle third (3.74 μm) and the apical third (1.73 μm). In root dentin, there is homogeneous distribution of intertubular dentin. Dentinal tubules at the coronal third and middle third of the root canal converge from cemento-dentinal junction to the root canal wall whereas in the apical third dentinal tubules were distorted, which were more convoluted, and had a smaller diameter than the middle third.[16] Apical dentin displays less tubule density with some areas completely devoid of tubules and also showed poor permeability due to age changes and sclerosis of dentinal tubules (Carrigan et al. 1984).[7],[17] The presence of numerous dentinal tubules with a larger diameter is identified in the coronal third of the root canal which may lead to the easy penetrability of the sealers into dentinal tubules. Sealers showed better adaptation and deeper penetration in the middle third of the root canals.[16] Therefore, in the current study, the middle third of the root portion was examined for sealer penetrability for standardization.

When evaluating the adaptability and penetration of root canal sealers, CLSM has advantages over scanning electron microscopy and other methods.[18] At lower magnifications, when viewing horizontal slices, presence of Rhodamine B fluorescence in dentinal tubules enables one to observe adaptation and penetration depth of sealers. At higher magnification, it is therefore simple to confirm a three-dimensional view of sealer adaptation into the root canal and dentinal tubules. Rhodamine B dye labeling is necessary to examine the degree of adaption of root canal sealer and its dentinal tubules penetration, as shown by past investigations employing CLSM. According to the American Dental Association requirements, the sealers labeled with 0.1% Rhodamine B dye variations in flow. Hence, CLSM was selected in the methodology of this study.[19]

In the current investigation, bioceramic sealer after NTAP application (Group 3) exhibited greater sealer penetration values into the dentinal tubules than epoxy resin-based sealer after NTAP application (Group 4). Yesildal Yeter et al. (2022) examined the impact of plasma on Endosequence BC sealer penetration into the dentinal tubules and reported that plasma improved the percentage of penetration values of Endosequence BC root canal sealer when used with a single cone technique.[20] In the course of setting, BioRoot RCS sealer creates a chemical bond with the root dentine by producing hydroxyapatite. In addition, this Bioceramic sealer can easily spread over the dentinal walls due to its low contact angle.[21] Changes on the root canal dentine surface after NTAP application such as wettability, chemical interactions, and grafting hydrophilic groups onto the surface could increase the penetration of Bioceramic sealer into the root dentin. After 30 s NTAP application, the contact angle values decrease and the low contact angle values define the hydrophilic surface properties.[22] However, carbonyl groups of dentin surface increase after NTAP application. These carbonyl groups enhance the hydrophilic property of the dentin surface which might have caused higher sealer penetration in Groups 3 and 4 compared to other groups.[23]

According to Prado et al. the wettability of epoxy resin-based sealer and surface-free energy of the dentin was improved by Argon Plasma which helped the sealer adhere to dentin surfaces.[24] The method of resin-based sealers adhering to dentin was improved by argon plasma. When the epoxide ring opens, the collagen's exposed amino groups react with AH Plus to establish covalent bonds between the collagen and resin.[21],[25] AH plus has greater penetrability into the micro-irregularities of dentinal tubules because of its good flow ability. The consistency and particle size of the sealer have an impact on how well it flows, which in turn affects tubular penetration. AH Plus sealer flow is greater because of the presence of increased concentration of epoxy resin. The long-term polymerization duration and creep capacity affect the mechanical interlocking between AH Plus sealer and the root canal dentin.[26] There are minimal studies which evaluated the application of NTAP on the penetration of root canal sealer.[6],[20],[27] Menezes et al. stated that NTAP applicated showed superior penetration of MTA-Fillapex when compared AH Plus sealer.[6] The penetration rates of AH Plus and Endosequence BC sealers have not been impacted by NTAP, according to Gunes et al. However, the following NTAP treatment, Endosequence BC sealer had maximal penetration than AH Plus sealer.[27]

Bioceramic sealer (Group 1) demonstrated higher sealer penetration values than epoxy resin-based sealer (Group 2) in the current investigation. These findings support a study by El Hachem et al. who found that BC sealer and novel tricalcium silicate sealer outperformed AH Plus sealer pertaining to dentinal tubule penetration.[28] Similar findings were observed in accordance with Cruz et al. and Uzunoglu-Özyürek et al. which showed that the penetrability of BioRoot RCS was higher up as compared with epoxy resin-based sealer.[29],[30] According to Pawar et al., leakage was exhibited due to insufficient adhesion between gutta-percha and the sealer. Owing to the resin it contains and its quicker setting time, AH Plus has a propensity to shrink and induce early debonding from the root canal.[31]

The application of NTAP resulted in dentin substrate surface alteration without topographical destructive effects. Negative results after 45 s are linked to a prolonged plasma exposure that may begin destroying the inorganic structure of the dentin by reducing the phosphate species in relation to the carboxylic groups.[32]

To the best of the author's knowledge, there is no enough published research on the use of NTAP on patients in endodontics. The complexity and availability of delivery systems have limited the usage of in vitro studies as per the current published data. On the other hand, delivery tips available at present are not to the scale of intraoral usage. Additional research should be conducted to learn more about the impact of NTAP on the root dentin analyze the various application times, and develop a standard protocol for NTAP treatment.

   Conclusion Top

The current confocal laser scanning microscopic study concluded that the application of NTAP improved the penetration of epoxy resin-based and bioceramic sealers into the dentinal tubules in comparison with other groups. Bioceramic sealer with the application of NTAP showed better sealer penetration values than other groups.

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Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Dr. Roopadevi Garlapati
Department of Conservative Dentistry and Endodontics, Sibar Institute of Dental Sciences, Takkellapadu, Guntur, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcd.jcd_22_23

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