Root canal treatment (RCT) is a widely accepted and effective modality for the management of pulpally involved teeth. Its success depends primarily on the thorough removal of necrotic and infected tissues, microorganisms, and debris from the root canal system, followed by a three-dimensional obturation that prevents reinfection [1]. Gutta-percha remains the most commonly used obturating core material due to its biocompatibility and dimensional stability; however, it lacks adhesion to root dentin and therefore requires a sealer to establish a hermetic seal [2]. An ideal sealer should adhere to canal walls, exhibit minimal shrinkage, be biocompatible, and provide long-term dimensional stability [3]. Root canal sealers can be categorized based on their composition into zinc oxide–eugenol, calcium hydroxide, resin, glass ionomer, silicone, and calcium silicate-based types [4]. Among these, resin-based sealers such as AH Plus (DentsplyDeTrey, Konstanz, Germany) have long been considered the “gold standard” due to their excellent physical properties, low solubility, and strong adhesion to dentin [5]. AH Plus is an epoxy resin–based sealer characterized by high radiopacity, good dimensional stability, and long working time, which enhances its penetration into microirregularities of the root canal system. However, its cytotoxic potential and difficulty in removal during retreatment remain drawbacks [6]. The development of calcium silicate–based sealers has introduced a new class of bioceramic sealers with bioactive properties, capable of forming hydroxyapatite and chemically bonding to dentin [7]. MTA Fillapex (Angelus, Londrina, Brazil) is a mineral trioxide aggregate–based sealer containing salicylate resin, natural resin, and bismuth oxide. It demonstrates good flow, alkaline pH, and the ability to induce mineralized tissue formation, although some studies have reported high solubility and variable sealing ability [8]. Another recent calcium silicate–based material, BioRoot RCS (Septodont, France), consists of tricalcium silicate, zirconium dioxide, and calcium chloride solution. It is bioactive, biocompatible, and capable of releasing calcium ions to promote biomineralization and form a chemical bond with dentin [9]. Despite these advancements, the complex anatomy of the root canal system often leaves uninstrumented areas containing residual pulp tissue, debris, or microorganisms that can lead to reinfection [10]. Microleakage due to incomplete obturation or poor sealer adaptation remains one of the major causes of endodontic failure. In such cases, endodontic retreatment becomes necessary to remove the previous filling materials and disinfect the canal system. The ease of removal of a sealer depends on its adhesion, solubility, and penetration into dentinal tubules. Deep sealer penetration, while beneficial for initial sealing, may complicate retreatability. Given these considerations, the present study was designed to evaluate and compare the dentinal tubule penetration and retreatability of three different root canal sealers — AH Plus, MTA Fillapex, and BioRoot RCS — using confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM). The study specifically assessed sealer penetration depth and residual material distribution within the coronal, middle, and apical thirds of the root canal system. The null hypothesis tested was that there would be no significant difference among the three sealers with respect to their dentinal tubule penetration and residual sealer remnants after retreatment. Aim of the study: To evaluate and compare the dentinal tubule penetration and retreatability of AH Plus, MTA Fillapex, and BioRoot RCS at different root canal levels using confocal and electron microscopic analyses.

Study Design and Study Setting This in-vitro experimental study was conducted in the Department of Conservative Dentistry and Endodontics, Regional Dental College and Hospital, Guwahati, Assam, during the academic year 2023–2024. All confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM) analyses were performed at the Indian Institute of Technology, Guwahati, Assam. Institutional ethical clearance was obtained prior to the commencement of the study. Sample Selection A total of 60 extracted human single-rooted permanent teeth with fully formed apices and straight canals (<10° curvature) were selected for the study. All teeth were cleaned of debris and soft tissue remnants and stored in 0.1% thymol solution until use. Inclusion Criteria • Single-rooted permanent teeth. • Teeth with a single canal and fully formed apices, verified radiographically in both buccolingual and mesiodistal angulations. Exclusion Criteria • Teeth with root caries, anatomical deformities, or visible cracks and root fractures. • Immature teeth with incompletely formed apices. • Teeth with multiple or curved canals verified radiographically. • Teeth with previous endodontic treatment. Specimen Preparation All teeth were decoronated at the cemento-enamel junction using a diamond disc under water coolant to obtain a standardized root length of 16 mm. The working length was determined by inserting a #10 K-file until visible at the apex and subtracting 1 mm. Root canals were prepared using the ProTaper Universal rotary file system (DentsplyMaillefer, Ballaigues, Switzerland) up to size F3 with an X-Smart Plus motor (DentsplyMaillefer) following the crown-down technique. During instrumentation, canals were irrigated with 5.25% sodium hypochlorite (NaOCl) after each file, followed by 17% ethylenediaminetetraacetic acid (EDTA) to remove the smear layer. Final irrigation was performed with distilled water, and canals were dried with ProTaper paper points. Grouping of Samples The 60 specimens were randomly divided into two main groups (n = 30 each): • Group 1 — Sealer penetration analysis (n = 30) • Group 2 — Retreatment analysis (n = 30) Each main group was further subdivided into three subgroups (n = 10 each) based on the sealer used: Subgroup Sealer Used Manufacturer Subgroup P₁ / R₁ AH Plus DentsplyDeTrey, Germany P₁ / R₁ P₂ / R₂ MTA Fillapex Angelus, Brazil P₂ / R₂ P₃ / R₃ BioRoot RCS Septodont, France P₃ / R₃ Obturation Procedure Each sealer was prepared according to the manufacturer’s instructions. For CLSM evaluation, sealers were fluorescently labelled with 0.1% Rhodamine B dye (Sigma-Aldrich, USA). Sealers were applied using a lentulo spiral, and obturation was performed with the lateral condensation technique using an F4 gutta-percha master cone (DentsplyMaillefer). The coronal access was sealed temporarily, and all specimens were stored in 100% humidity at 37°C for 14 days to ensure complete setting. Retreatment Procedure Retreatment was performed using the ProTaper Universal Retreatment System (DentsplyMaillefer). D1 (30/0.09), D2 (25/0.08), and D3 (20/0.07) files were used sequentially at the manufacturer’s recommended torque and speed to remove obturation material. Re-instrumentation was completed with ProTaper F4 files to the working length. Irrigation was performed using 5.25% NaOCl between instruments, followed by a final rinse with distilled water. No solvent was used during retreatment to prevent alteration of sealer residue morphology. Sectioning of Specimens After obturation or retreatment, each root was horizontally sectioned using a low-speed diamond disc under water irrigation to obtain 0.5 mm thick slices at three standardized levels: • Coronal third: 12–14 mm from the apex • Middle third: 7–9 mm from the apex • Apical third: 2–4 mm from the apex Each slice was mounted on a glass slide for CLSM analysis. Confocal Laser Scanning Microscopy (CLSM) Analysis All fluorescently labelled specimens were examined under a Confocal Laser Scanning Microscope (LSM 880, Carl Zeiss, Germany) at ×10 magnification. The depth of sealer penetration into dentinal tubules was measured in micrometres (µm) using ZEN imaging software (Zeiss) equipped with a digital measurement tool. Four measurements were taken per section (at 0°, 90°, 180°, and 270° orientations), and the mean penetration depth was recorded for each third. Field Emission Scanning Electron Microscopy (FESEM) Analysis Selected samples from the retreatment group were dehydrated in ascending ethanol concentrations and sputter-coated with gold before examination under FESEM (Gemini 300, Zeiss, Germany). Qualitative evaluation focused on the presence, morphology, and distribution of residual sealer tags and remnants within dentinal tubules across all root thirds. Statistical Analysis All quantitative data were analyzed using IBM SPSS Statistics (Version 25.0, IBM Corp., Armonk, NY, USA).Data normality was checked using the Shapiro–Wilk test. As data were non-parametric, • Intra-group comparisons (between coronal, middle, and apical thirds within each sealer) were analyzed using the Wilcoxon matched-pairs test. • Inter-group comparisons (among sealers at each root level) were assessed using the Kruskal–Wallis test followed by post-hoc pairwise analysis. A p-value < 0.05 was considered statistically significant

The present in-vitro study evaluated the dentinal tubule penetration and retreatability (residual sealer remnants) of three different endodontic sealers — AH Plus, MTA Fillapex, and BioRoot RCS — using confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM). All specimens were analyzed in the coronal, middle, and apical thirds of the root canal. The quantitative outcomes were expressed as mean ± standard deviation (SD) in micrometres (µm). Within each sealer group, comparisons among the three root canal thirds were performed using the Wilcoxon matched-pairs test, while comparisons among sealers within each third were analyzed using the Kruskal–Wallis test followed by post-hoc pairwise comparisons. Statistical significance was set at p < 0.05. Dentinal tubule penetration depth (CLSM) The mean depth of sealer penetration into dentinal tubules differed significantly among the coronal, middle, and apical thirds of the root canal for all tested sealers (p< 0.001). Within each sealer, penetration was highest in the coronal third, followed by the middle and apical thirds (Wilcoxon matched-pairs, p< 0.001). Among the three sealers, BioRoot RCS demonstrated the greatest penetration in the coronal (2235.06 ± 56.02 µm) and apical thirds (1364.03 ± 20.46 µm), while MTA Fillapex exhibited the highest penetration in the middle third (1757.49 ± 12.59 µm), which was not significantly different from BioRoot RCS (p = 0.422). AH Plus consistently showed the lowest penetration at all levels (p< 0.001). Overall, penetration depth decreased in the order coronal > middle > apical for each sealer (Table 1, Figure 1, Figure 4) Table 1. Mean ± SD dentinal tubule penetration depth (µm) and statistical comparisons Sealer Coronal (Mean ± SD) Middle (Mean ± SD) Apical (Mean ± SD) AH Plus 1424.48 ± 16.82 1384.83 ± 15.87 776.92 ± 15.42 MTA Fillapex 1999.34 ± 16.60 1757.49 ± 12.59 547.22 ± 13.91 BioRoot RCS 2235.06 ± 56.02 1742.55 ± 13.54 1364.03 ± 20.46 Residual sealer after retreatment (CLSM) The mean residual sealer values varied significantly among the coronal, middle, and apical thirds (p< 0.001). For all sealers, the coronal third retained the greatest amount of residual material, followed by the middle and apical thirds. BioRoot RCS showed the highest residuals in the coronal (1518.67 ± 15.26 µm) and middle thirds (893.34 ± 19.07 µm), whereas AH Plus exhibited the highest residuals in the apical third (441.80 ± 19.78 µm). The difference between BioRoot RCS and MTA Fillapex in the middle third was not statistically significant (p> 0.05). Overall, residual sealer quantity decreased in the order coronal > middle > apical for each sealer (Table 2, Figure 2, Figure 5) Table 2. Mean ± SD residual sealer remnant (µm) and intergroup comparisons (Kruskal–Wallis & post-hoc) Sealer Coronal (Mean ± SD) Middle (Mean ± SD) Apical (Mean ± SD) AH Plus 765.08 ± 19.67 470.18 ± 16.23 441.80 ± 19.78 MTA Fillapex 1010.35 ± 15.35 884.16 ± 61.09 250.22 ± 13.41 BioRoot RCS 1518.67 ± 15.26 893.34 ± 19.07 342.83 ± 22.51 Qualitative FESEM observations Field emission scanning electron microscopy (FESEM) images corroborated the quantitative CLSM findings. BioRoot RCS specimens exhibited dense, continuous sealer tags and adherent remnants, particularly in the coronal third of the canal walls. MTA Fillapex and AH Plus samples showed comparatively cleaner canal surfaces following retreatment, with fewer and discontinuous remnants. Across all sealers, the apical third revealed sparse and thinner residual films compared with the coronal and middle thirds. Representative FESEM micrographs are presented in Figure 3. Statistical summary All inter-third comparisons within each sealer were statistically significant (Wilcoxon matched-pairs test, p< 0.001), except for AH Plus between the middle and apical thirds (p = 0.013). All inter-sealer comparisons (Kruskal–Wallis test) were significant (p< 0.001) for the coronal and apical levels, indicating distinct differences among the sealers in these regions. At the middle third, no significant difference was observed between BioRoot RCS and MTA Fillapex (p> 0.05), while both differed significantly from AH Plus (p< 0.001). A summary of major statistical outcomes is provided in Table 3. Table 3. Summary of intergroup and intragroup statistical comparisons (p-values) Comparison Statistical Test Root Third(s) Significance (p-value) Interpretation Coronal vs Middle (within each sealer) Wilcoxon matched-pairs All sealers < 0.001 Significant difference Coronal vs Apical (within each sealer) Wilcoxon matched-pairs All sealers < 0.001 Significant difference Middle vs Apical (within each sealer) Wilcoxon matched-pairs AH Plus only 0.013 Significant (lower) Inter-sealer comparison (Coronal third) Kruskal–Wallis + Post-hoc Coronal < 0.001 BioRoot> MTA > AH Plus Inter-sealer comparison (Middle third) Kruskal–Wallis + Post-hoc Middle < 0.001 (overall), ns (BioRootvs MTA) BioRoot ≈ MTA > AH Plus Inter-sealer comparison (Apical third) Kruskal–Wallis + Post-hoc Apical < 0.001 BioRoot> AH Plus > MTA Overall, BioRoot RCS demonstrated the deepest dentinal tubule penetration and the highest residual sealer remnants after retreatment, followed in order by MTA Fillapex and AH Plus. Across all sealers, the coronal third consistently exhibited greater sealer penetration and more residual material than the middle and apical thirds, reflecting the progressive reduction of sealer adaptation and retrievability toward the apical region. These findings collectively indicate that the material composition and flow characteristics of the sealer significantly influence both its penetration potential and its retreatability within the root canal

The present in-vitro study evaluated the dentinal tubule penetration and retreatability of three root canal sealers—AH Plus, MTA Fillapex, and BioRoot RCS—using confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FESEM). Effective sealing and obturation are essential for the long-term success of endodontic therapy [11,12], and the sealer’s ability to penetrate into dentinal tubules enhances adaptation, mechanical retention, and microbial entombment [13,14]. Dentinal tubule penetration In the current study, all sealers showed the greatest dentinal tubule penetration in the coronal third, followed by the middle and apical thirds, corroborating earlier findings by Ravi et al. [20] and Dasari et al. [21]. The reduced penetration in the apical third can be attributed to smaller and fewer dentinal tubules, increased sclerosis, and limited irrigant exchange apically [22]. Among the tested materials, BioRoot RCS demonstrated the deepest sealer penetration in the coronal and apical thirds. These findings align with previous reports showing superior flow and tubule infiltration of BioRoot RCS compared with AH Plus and other resin-based sealers [22–24]. The enhanced penetration of BioRoot RCS can be attributed to its hydrophilic nature, nanometric particle size, and ability to form calcium silicate hydrogel and hydroxyapatite compounds that chemically bond with dentin [25]. The process of biomineralization within the dentinal tubules produces a mineral infiltration zone and tag-like structures, reinforcing adhesion and sealing [27]. MTA Fillapex, in contrast, showed the highest penetration in the middle third, which was not significantly different from BioRoot RCS. Its higher flow, pseudoplasticbehavior, and alkaline pH may explain this result [22,28]. Similar findings were reported by Cruz et al. [26] and Kuç IA et al., who observed better flow and adaptation of MTA Fillapex compared with AH Plus. The resin-based AH Plus showed the least penetration across all levels, consistent with prior studies [23,24], due to its hydrophobic epoxy resin matrix and larger particle size that limit penetration into narrow apical tubules [12]. Residual sealer after retreatment The retreatability analysis showed that none of the sealers could be completely removed from the root canal system, in agreement with previous literature [16,19]. Residual remnants were highest in the coronal third, decreasing toward the apex. BioRoot RCS showed greater remnants in the coronal and middle thirds than MTA Fillapex and AH Plus, which corresponds with its deeper dentinal penetration and formation of a mineral infiltration layer that increases bonding to dentin [25,27]. Similar findings were reported by Uzunoglu-Özyürek et al. [23] and Dsouza et al. [24]. AH Plus exhibited the greatest residual material in the apical third, possibly due to its strong covalent bonding between epoxide groups and dentinal amino sites, which makes mechanical removal difficult [12,24]. MTA Fillapex showed moderate remnants across all thirds, likely due to its higher solubility and resin component, which make it comparatively easier to remove [26]. FESEM correlation FESEM analysis qualitatively confirmed the CLSM findings. BioRoot RCS specimens displayed dense, continuous sealer tags in the coronal third, whereas MTA Fillapex and AH Plus presented cleaner canal walls, particularly in the apical regions. This correlates with previous microscopic observations that calcium silicate–based sealers exhibit superior dentinal adaptation and mineralized tag formation compared to resin-based sealers [25,27,28]. Clinical implications Deeper dentinal tubule penetration improves the mechanical interlocking of the sealer and its sealing ability, reducing microleakage and reinfection risk [13,15]. However, it may also complicate retreatment due to the formation of mineralized sealer-dentin interfaces [24]. Bioceramic sealers such as BioRoot RCS and MTA Fillapex show promising bioactivity and sealing potential, but their retrievability remains a clinical concern during non-surgical retreatment [14,17,18]. Limitations and future scope The present study was conducted under in-vitro conditions with a limited sample size. Factors such as variations in root canal morphology, moisture conditions, and clinical operator variability could influence sealer behaviorin vivo. Further studies using micro-computed tomography (micro-CT) and dynamic fluid simulation models are recommended to assess three-dimensional sealer penetration and retreatability under clinical conditions.

Within the limitations of this in-vitro study, BioRoot RCS exhibited the deepest dentinal tubule penetration and the highest residual sealer remnants after retreatment, followed by MTA Fillapex and AH Plus. All sealers showed greater penetration and residual material in the coronal third, decreasing toward the apical third. None of the sealers were completely removable from the canal system, indicating that complete sealer elimination during retreatment remains challenging. The null hypothesis was therefore rejected.

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