Abstract
Objective
This study evaluated the effects of sonic activation and graphene-functionalized methylene blue on the push-out bond strength of two endodontic sealers, AH Plus and Bio C, following Photodynamic therapy (PDT).
Methods
Sixty extracted single-rooted human teeth were divided into six groups (n = 10), treated with either 5% NaOCl (Groups 1 & 2), MB-based PDT (Groups 3 & 4), or MB-rGO-based PDT (Groups 5 & 6). In Groups 2, 4, and 6, sonic activation was applied prior to laser irradiation. Each group was further divided based on the sealer used: AH Plus or Bio-C. One mm-thick root slices from the coronal and middle thirds were subjected to push-out bond strength testing. Statistical analysis included one-way ANOVA, Tukey’s post-hoc, and independent t-tests.
Results
Group 6 (MB-rGO + Sonic Activation + PDT) showed the highest bond strength for both AH Plus (6.07 ± 0.99 MPa) and Bio-C (5.54 ± 0.76 MPa), while Group 3 (MB + PDT) showed the lowest. Significant differences were observed between groups for both sealers (p < 0.05). Sonic activation significantly enhanced bond strength in Bio-C Sealer (p = 0.026) but not in AH Plus (p = 0.124). AH Plus showed predominantly cohesive failures, whereas Bio-C exhibited more adhesive failures.
Conclusion
Sonic activation and graphene functionalization significantly enhanced the bond strength of both sealers, with the highest values in the MB-rGO with activation group. AH Plus showed superior adhesion, attributed to its chemical interaction with dentin collagen, while Bio-C Sealer benefited from hydroxyapatite bonding and improved mechanical retention. These findings suggest that optimizing photodynamic therapy with nanotechnology and activation techniques can improve the adhesion and clinical performance of endodontic sealers.
Keywords: Graphene oxide functionalization, Methylene blue, Photodynamic therapy, Push-out bond strength, Root canal sealer
Introduction
The long-term success of root canal treatment largely depends on the ability of endodontic sealers to establish a durable bond to the dentin, creating a secure seal that prevents bacterial reinfection. Push-out bond strength (PBS) is a critical parameter for assessing the adhesive integrity between the root canal sealer and the dentinal walls. Numerous factors influence PBS, for instance the type of sealer used, the status of the dentin, and the pretreatment procedures employed prior to obturation [1, 2]. Effective disinfection of the root canal system is essential for ensuring durable adhesion, as prolonged microbial contamination can weaken the quality of the obturation seal.
Photodynamic therapy (PDT) has emerged as a promising antimicrobial technique in endodontics, using a photosensitizer that, upon activation by a particular laser wavelength, produces reactive oxygen species (ROS) to eliminate bacteria [3]. Methylene blue (MB) is one of the most extensively studied photosensitizers owing to its effectiveness against the commonly encounter root canal pathogens in endodontic treatment failure cases like Enterococcus faecalis [4]. Despite its strong antimicrobial potential, the impact of PDT on dentin’s mechanical properties and the adhesion of sealers remains controversial. Some research suggests that PDT may change dentin structure and reduce bond strength, predominantly at higher concentrations [5]. However, optimizing PDT protocols could mitigate these effects and potentially enhance sealer bonding.
One such method to enhance the effectiveness of aPDT is to sonically or ultrasonically activate PSs before the laser exposure, which can enhance the penetration of PS into the dentinal tubules and also help to remove attached bacterial biofilm from the dentinal wall, hence may enhance the adhesion ability of dentinal sealers [6]. However, the role of activation on the PBS of different root canal sealers remains largely unknown.
Recent attention has also turned to the incorporation of nanomaterials to further enhance aPDT efficacy. Among them are graphene oxide (GO) and reduced graphene oxide (rGO) which have large surface area and biocompatibile with human tissue [7, 8]. Graphene-based carriers not only support improved antimicrobial action but may also reinforce the dentin–sealer interface by stabilizing the collagen and improving the material interaction. Functionalizing MB with rGO has the potential to reduce the negative effects of PDT while simultaneously enhancing sealer adhesion and mechanical properties of the obturation [9].
Although a few studies have assessed the effect of PDT using MB on the bond strength of sealers [10, 11], further investigation is needed, particularly regarding newer modifications such as sonic activation and nanoparticle incorporation. Adhesion and penetration of sealers are critical for the long-term success. While adhesion ensures the stability of the filling by bonding gutta-percha to dentin, penetration into tubules helps entomb the residual bacteria and reinforce the seal [12–14]. Both factors are influenced by dentin condition and sealer composition [13, 15]. Scientific literature indicates that while PDT does not significantly affect the bond strength of epoxy resin-based sealers like AH Plus, it may impair the adhesion of bioceramic sealers such as MTA Fillapex [16].
Advancements in endodontic disinfection, including both PDT and photobiomodulation therapy (PBM) have also contributed to smear layer removal, further enhancing sealer bonding [17]. It is crucial to understand how these therapies interact with different types of sealers, given the differences in material properties. For example, AH Plus bonds via chemical interaction and micromechanical interlocking, while Bio C, a bioceramic sealer, mostly forms a chemical bond with hydroxyapatite crystals [5]. Since bioceramic sealers are more sensitive to dentin surface changes, their response to PDT, activation methods, and graphene-based modifications may differ significantly from resin-based sealers.
This study hypothesizes that aPDT alone may have a negative effect on PBS of root canal filling material cemented with two different type of sealers due to alterations in dentin surface, while activation of the PS will possibly improve its penetration and counteract this effect. Furthermore, MB functionalized with rGO (MB-rGO) contributed to increase dentin interaction and hence sealer adhesion. The activation of MB-rGO demonstrated improved outcomes in terms of bond strength. Therefore, this study aims to evaluate the effect of sonic activation and graphene-functionalized methylene blue on the push-out bond strength of two endodontic sealers, AH Plus and Bio C, following PDT.
Materials and methods
This in-vitro investigation was conducted from September 2024 to March 2025. Ethical approval was obtained from Universiti Sains Malaysia (USM/JEPeM/KK/24030230). Sample size was estimated using Open Epi software with an effect size of 0.40, α = 0.05, and power = 80%, indicating that 10 specimens per group were suggested sufficient to detect significant differences with 95% confidence [10].
Synthesis and functionalization of rGO with methylene blue
The previous research confirmed the successful synthesis and functionalization of rGO with MB, demonstrating the effective incorporation of MB onto the rGO surface [3]. In summary, rGO was produced from high-purity graphene oxide (GO, 30–50 nm) by dispersing 100 mg of GO in ethanol, followed by NaOH treatment under continuous stirring. The material was then centrifuged and washed sequentially with deionized water, ethanol, and HCl before being dried at 80 °C. FTIR analysis verified the reduction process, highlighting characteristic —OH (3750–3000 cm⁻¹) and residual oxygen peaks (1640 cm⁻¹).
For functionalization, 50 mg of rGO was sonicated in ethanol, after which an equal amount (50 mg) of MB was added under continuous stirring at 40–45 °C for six hours, maintaining a 1:1 MB-rGO ratio. FTIR spectra verified the successful adsorption of MB onto rGO, indicated by distinct peaks corresponding to C = N⁺(CH₃)₂, C—N, C—C, C = S⁺, and C—H vibrations. Finally, a 200 ppm stock solution of MB and MB-rGO was prepared in deionized water for this study.
Sample selection and Preparation
A total of 60 freshly extracted single-rooted human teeth were obtained from patients undergoing orthodontic treatment or extraction due to periodontal reasons. The teeth were collected from patients who consented to their teeth being collected for this study. The teeth were kept in a 0.1% thymol solution until further processing. To ensure consistency, teeth with root caries, restorations, cracks, fractures, or curved roots were excluded from the study. The coronal portions were removed using a diamond disc, standardizing the remaining root length to 13 mm for all samples.
Root canal instrumentation was performed using ProTaper rotary files (Dentsply, Maillefer, Switzerland) up to size F3, maintaining a working length 1 mm short of the apex. The smear layer was removed by treating the canals with EDTA solution for 30 s. To prevent apical leakage, the apical foramen of each specimen was sealed with composite resin (Filtek™ Z250, 3 M™, New Zealand).
Experimental grouping, irrigation protocols, and obturation
Randomization was performed using a computer-generated random number sequence to assign specimens to the six experimental groups. Additionally, the operator conducting the push-out bond strength testing and failure mode analysis was blinded to the group allocations to minimize potential bias. The specimens were randomly distributed into six experimental groups (n = 10 per group), each subjected to distinct irrigation and activation protocols:
Group 1 (Control): Irrigation with 5% sodium hypochlorite (NaOCl).
Group 2: Irrigation with 5% NaOCl with sonic activation.
Group 3: Photodynamic therapy (PDT) using MB and laser light activation.
Group 4: Modified PDT with sonic activation of MB before laser light exposure.
Group 5: PDT using methylene blue functionalized with reduced graphene oxide (MB-rGO).
Group 6: Modified PDT with sonic activation of MB-rGO before laser light activation.
In Group 1, root canals were irrigated with 3 mL of 5% NaOCl using a 27-gauge needle, followed by manual agitation with a size 10 K-file for 60 s to eliminate air entrapment, then left for a 5-minute retention period. Group 2 followed the same irrigation protocol as Group 1 but included an additional 2-minute sonic activation step using a 25/04 tip operating at 6000 cycles per minute (cpm).
For Group 3, 200 µg/mL of MB was introduced into the root canals, manually agitated for 60 s with a size 10 K-file, and then activated with laser light (650 ± 5 nm, 200 mW) for 2 min to facilitate PDT. Group 4 followed the same protocol as Group 3, with the addition of a 2-minute sonic activation step before laser light exposure.
Group 5 replaced MB with MB functionalized with reduced graphene oxide (MB-rGO) at a 200 µg/mL concentration (1:1 ratio) while maintaining the same PDT protocol as Group 3. Group 6 followed the same protocol as Group 4 but substituted MB with MB-rGO, incorporating both sonic activation and laser light exposure to evaluate the combined effect on sealer adhesion.
To maintain consistency across all groups and remove any residual PS, all root canals were finally flushed with 5 mL of deionized water before obturation. Following irrigation, each group was further divided into two subgroups (n = 5 per subgroup) based on the sealer used for obturation. Root canals were obturated using the cold lateral condensation technique with F3 gutta-percha cones as the primary cone, supplemented with accessory cones for consistency. Subgroup 1 was obturated with AH Plus sealer (Dentsply, Germany), while Subgroup 2 used Bio-C Sealer (Angelus, Brazil). All specimens were then stored at 37 °C in 99% humidity for 72 h to ensure complete sealer setting under controlled conditions.
Push-Out bond strength testing
Two 1 mm-thick slices were sectioned from the coronal to middle thirds of each root using a low-speed diamond saw (IsoMet™ 1000; Buehler Ltd., Illinois, USA) under continuous water cooling at 200 rpm. These slices were then subjected to PBS testing using a universal testing machine (Instron 5965, Canton, MA, USA) at a crosshead speed of 1 mm/min until bond failure occurred (Fig. 1).
Fig. 1.
Push-out bond test setup showing (left) the Universal Testing Machine (Instron®) used for applying force and (right) a root dentin slice positioned for testing, with the plunger aligned to dislodge the filling material
To ensure uniform force application, the diameter of the loading rod was maintained 0.2 mm smaller than the inner canal diameter. The large radius (R), small radius (r), and height (h) of each sample were precisely measured using a digital caliper (Mitutoyo, Suzano, Brazil) with a measurement precision range of 0–150 mm.
The push-out bond strength (PBS) in megapascals (MPa) was calculated using the formula:
 |
Where the bonded surface area (A) was determined using the equation:
 |
Where π = 3.14, R = coronal radius, r = apical radius, and h = sample height.
Following the push-out bond strength test, all specimens were meticulously examined under a stereomicroscope (Swift S7-TL, Xiamen, Fujian, China) at 20X magnification to assess the mode of failure. The failure types were categorized as follows:
Adhesive failure: Separation at the sealer-dentin interface.
Cohesive failure: Fracture occurring within the sealer.
Mixed failure: A combination of adhesive and cohesive failure.
Each sample’s failure mode was documented to evaluate the bonding characteristics across the different experimental groups.
Statistical analysis
Statistical analysis was performed using SPSS version 23.0 (SPSS Inc., IL, USA). The mean and standard deviation of PBS values were calculated for each experimental group and sealer type. The assumption of normality was assessed using the Shapiro–Wilk test. A one-way ANOVA test was performed to determine significant differences among the six groups; subsequently, Tukey’s post-hoc test was performed for pairwise comparisons. An independent t-test was applied to evaluate the pool group analysis between the two sealers. A p-value of less than 0.05 was taken as statistically significant.
Results
The mean push-out bond strength (PBS) values for AH Plus and Bio-C Sealers across the six experimental groups are presented in Table 1. Notably, Group 6 (MB-rGO + Sonic Activation + Laser PDT) show the highest PBS for both sealers, 6.07 ± 0.99 MPa for AH Plus and 5.54 ± 0.76 MPa for Bio-C. Conversely, the lowest bond strength was observed in Group 3 (MB + Laser PDT) with values of 4.82 ± 1.03 MPa for AH Plus and 3.92 ± 1.07 MPa for Bio-C. One-way ANOVA confirmed statistically significant differences among groups for both sealers (p = 0.024 for AH Plus; p = 0.003 for Bio-C).
Table 1.
Comparison of mean push-out bond strength (in MPa) between AH plus and Bio-C sealers across treatment groups. Values are presented as mean ± standard deviation [95% confidence interval]
| Group | AH Plus (Mean ± SD) [95% CI] |
Bio C Sealer (Mean ± SD) [95% CI] |
p-value |
|---|---|---|---|
| 1 (NaOCl only - Control) | 5.25 ± 0.94 [4.58, 5.93] | 4.20 ± 0.80 [3.63, 4.77] | 0.015* |
| 2 (NaOCl + Sonic Activation) | 5.87 ± 0.86 [5.26, 6.49] | 4.78 ± 0.88 [4.14, 5.40] | 0.011* |
| 3 (Methylene Blue (200 µg/mL) + Laser PDT) | 4.82 ± 1.03 [4.08, 5.55] | 3.92 ± 1.07 [3.15, 4.68] | 0.071* |
| 4 (Methylene Blue (200 µg/mL) + Sonic Activation + Laser PDT) | 5.27 ± 1.00 [4.55, 5.99] | 4.45 ± 0.90 [3.81, 5.10] | 0.071* |
| 5 (Methylene Blue (200 µg/mL) + Graphene Functionalization + Laser PDT) | 5.95 ± 0.73 [5.42, 6.47] | 4.93 ± 0.91 [4.28, 5.58] | 0.013* |
| 6 (Methylene Blue + Graphene Functionalization + Sonic Activation + Laser PDT) | 6.07 ± 0.99 [5.36, 6.77] | 5.54 ± 0.76 [5.00, 6.08] | 0.198* |
| p-value | 0.024 | 0.003 |
One-way ANOVA
Independent Sample t-test*
Additional intergroup comparisons indicated that AH Plus consistently outperformed Bio-C in most groups, with statistically significant differences in Groups 1, 2, and 5 (p = 0.015, 0.011, and 0.013, respectively). However, no significant differences were found between sealers in Groups 3, 4, and 6.
Pairwise comparisons (Table 2) revealed significant differences, particularly between Group 3 and Group 6 for both sealers, indicating the strong enhancing effect of combining sonic activation with graphene-functionalized MB. Specifically, Specifically, AH Plus in Group 6 showed significantly higher PBS than in Group 3 (p = 0.045). The effect size for this comparison, calculated using Cohen’s d, was 1.35, indicating a large effect, while Bio-C exhibited significant differences between Group 6 and both Groups 1 and 3 (p = 0.017 and p = 0.002, respectively). These results underscore the potential of sonic activation and graphene integration to improve sealer adhesion.
Table 2.
Pairwise comparison of push-out bond strength (MPa) among experimental groups for AH Plus and Bio-C Sealers
| Variables | Push-out bond strength | ||
|
Mean ± SD MP |
p -value | ||
| AH Plus Sealer | Group 1 vs. Group 2 | 5.25 ± 0.94 vs. 5.87 ± 0.86 | 0.672 |
| Group 1 vs. Group 3 | 5.25 ± 0.94 vs. 4.82 ± 1.03 | 0.901 | |
| Group 1 vs. Group 4 | 5.25 ± 0.94 vs. 5.27 ± 1.00 | 1.000 | |
| Group 1 vs. Group 5 | 5.25 ± 0.94 vs. 5.95 ± 0.73 | 0.558 | |
| Group 1 vs. Group 6 | 5.25 ± 0.94 vs. 6.07 ± 0.99 | 0.380 | |
| Group 2 vs. Group 3 | 5.87 ± 0.86 vs. 4.82 ± 1.03 | 0.132 | |
| Group 2 vs. Group 4 | 5.87 ± 0.86 vs. 5.27 ± 1.00 | 0.701 | |
| Group 2 vs. Group 5 | 5.87 ± 0.86 vs. 5.95 ± 0.73 | 1.000 | |
| Group 2 vs. Group 6 | 5.87 ± 0.86 vs. 6.07 ± 0.99 | 0.997 | |
| Group 3 vs. Group 4 | 4.82 ± 1.03 vs. 5.27 ± 1.00 | 0.882 | |
| Group 3 vs. Group 5 | 4.82 ± 1.03 vs. 5.95 ± 0.73 | 0.089 | |
| Group 3 vs. Group 6 | 4.82 ± 1.03 vs. 6.07 ± 0.99 | 0.045 | |
| Group 4 vs. Group 5 | 5.27 ± 1.00 vs. 5.95 ± 0.73 | 0.589 | |
| Group 4 vs. Group 6 | 5.27 ± 1.00 vs. 6.07 ± 0.99 | 0.408 | |
| Group 5 vs. Group 6 | 5.95 ± 0.73 vs. 6.07 ± 0.99 | 1.000 | |
| Bio-C Sealer | Group 1 vs. Group 2 | 4.20 ± 0.80 vs. 4.78 ± 0.88 | 0.703 |
| Group 1 vs. Group 3 | 4.20 ± 0.80 vs. 3.92 ± 1.07 | 0.980 | |
| Group 1 vs. Group 4 | 4.20 ± 0.80 vs. 4.45 ± 0.90 | 0.988 | |
| Group 1 vs. Group 5 | 4.20 ± 0.80 vs. 4.93 ± 0.91 | 0.452 | |
| Group 1 vs. Group 6 | 4.20 ± 0.80 vs. 5.54 ± 0.76 | 0.017 | |
| Group 2 vs. Group 3 | 4.78 ± 0.88 vs. 3.92 ± 1.07 | 0.277 | |
| Group 2 vs. Group 4 | 4.78 ± 0.88 vs. 4.45 ± 0.90 | 0.965 | |
| Group 2 vs. Group 5 | 4.78 ± 0.88 vs. 4.93 ± 0.91 | 0.999 | |
| Group 2 vs. Group 6 | 4.78 ± 0.88 vs. 5.54 ± 0.76 | 0.400 | |
| Group 3 vs. Group 4 | 3.92 ± 1.07 vs. 4.45 ± 0.90 | 0.759 | |
| Group 3 vs. Group 5 | 3.92 ± 1.07 vs. 4.93 ± 0.91 | 0.128 | |
| Group 3 vs. Group 6 | 3.92 ± 1.07 vs. 5.54 ± 0.76 | 0.002 | |
| Group 4 vs. Group 5 | 4.45 ± 0.90 vs. 4.93 ± 0.91 | 0.833 | |
| Group 4 vs. Group 6 | 4.45 ± 0.90 vs. 5.54 ± 0.76 | 0.086 | |
| Group 5 vs. Group 6 | 4.93 ± 0.91 vs. 5.54 ± 0.76 | 0.650 | |
Statistical analysis performed using Tukey’s post hoc test following one-way ANOVA. Comparisons with p < 0.05 were considered statistically significant
Pooled analysis based on sonic activation effect (Table 3) showed that sonic activation resulted in higher bond strength values for both sealers. While AH Plus showed an increase from 5.33 ± 0.99 MPa (non-sonic) to 5.73 ± 0.98 MPa (sonic), the difference was not statistically significant (p = 0.124). In contrast, Bio-C Sealer exhibited a significant improvement with sonic activation (4.35 ± 0.99 MPa to 4.92 ± 0.94 MPa, p = 0.026), emphasizing its enhanced response to activation techniques. Additionally, AH Plus showed significantly higher bond strength in comparison to Bio-C Sealer in both non-sonic and sonic activation pooling group conditions (p < 0.001 and p = 0.002, respectively).
Table 3.
Pooled Group Analysis of Push-Out Bond Strength for AH Plus and Bio-C Sealer
| Pool Groups | AH Plus (Mean ± SD) |
Bio C Sealer (Mean ± SD) |
p-value |
|---|---|---|---|
| Non-sonic Activation | 5.33 ± 0.99 | 4.35 ± 0.99 | < 0.001 |
| Sonic Activation | 5.73 ± 0.98 | 4.92 ± 0.94 | 0.002 |
| p-value | 0.124 | 0.026 |
Independent Sample t-test
Failure mode analysis (Table 4, Fig. 2) provided further insights into the adhesion characteristics of the two sealers. AH Plus predominantly exhibited cohesive failures, especially in Groups 5 (60%) and 6 (70%), suggesting strong internal bonding. In contrast, Bio-C Sealer showed more adhesive failures, particularly in Group 3 (70%), indicating weaker interaction with dentin in the absence of sonic activation. Mixed failures were present in all groups but remained less frequent (10–20%), with slightly higher incidence in Groups 2 and 5 for Bio-C.
Table 4.
Failure modes in Percentage with photodynamic therapy
| Groups | Failure Mode (%) | |||||
|---|---|---|---|---|---|---|
| AH-Plus Sealer | Bio-C Sealer | |||||
| Adhesive | Cohesive | Mixed | Adhesive | Cohesive | Mixed | |
| Group 1 | 30 | 50 | 20 | 50 | 30 | 20 |
| Group 2 | 30 | 50 | 10 | 40 | 30 | 30 |
| Group 3 | 50 | 30 | 20 | 70 | 20 | 10 |
| Group 4 | 40 | 40 | 20 | 50 | 30 | 20 |
| Group 5 | 20 | 60 | 20 | 30 | 40 | 30 |
| Group 6 | 10 | 70 | 20 | 30 | 50 | 20 |
Fig. 2.
Representative stereomicroscopic images (20× magnification) showing failure modes after push-out bond strength testing. Images A–C depict AH Plus sealer specimens, illustrating adhesive (A), mixed (B), and cohesive (C) failures, respectively. Images D–F represent Bio-C Sealer specimens, also showing adhesive (D), mixed (E), and cohesive (F) failures. A scale bar of 100 μm is included for dimensional reference
Discussion
This study investigated the effects of sonic activation and graphene-functionalized methylene blue (MB-rGO) on the push-out bond strength (PBS) of two endodontic sealers, AH Plus and Bio-C, following photodynamic therapy (PDT). The key finding was that Group 6, which incorporated both MB-rGO and sonic activation prior to laser PDT, demonstrated the highest adhesive performance among all groups for both sealers. In contrast, Group 3 (MB + laser PDT without activation) showed the lowest PBS values, highlighting the potential negative effect of unactivated PDT. These results support the hypothesis that activation and nanoparticle functionalization can significantly improve the interaction between sealer and dentin.
Photodynamic therapy, while effective in bacterial reduction via reactive oxygen species (ROS) generation, may leave residual photosensitizer (PS) within the canal, interfering with sealer bonding and reducing mechanical integrity, hence increasing the risk of microbial leakage [18, 19]. This aligns with previous findings indicating that residual PS can reduce adhesion by occupying bonding sites or altering dentin surface properties [20]. The current results confirm that proper activation protocols are necessary to mitigate such adverse effects.
The significantly improvement in interfacial strength observed in the sonic-activated MB-rGO group suggests a synergistic effect. Graphene functionalization demonstrated to enhance PS penetration, stabilize collagen, and improve mechanical interlocking [9]. Additionally, rGO’s high surface area and π-π stacking interactions may aid in better adhesion to dentinal walls, potentially modifying surface energy and promoting more uniform sealer distribution. Furthermore, rGO may improve dentin wettability and facilitate better adaptation of sealer through physicochemical interactions with both the dentin substrate and the sealer material [7, 9].
Sonic activation facilitates better distribution and dentinal tubule penetration of the photosensitizer by disrupting surface tension and agitating fluids [6, 21]. This enhanced diffusion and interaction with the dentin surface may contributed to the superior adhesive behavior observed in Group 6. MB was selected due to its established effectiveness as a photosensitizer in endodontics and compatibility with diode lasers in the 600–700 nm range, which matches the wavelength used in this study [21].
AH Plus consistently demonstrated higher mechanical retention than Bio-C, regardless of treatment group, which may be attributed to its superior micromechanical and chemical bonding properties [2, 10]. Cohesive failure modes dominated AH Plus groups, indicating stronger internal sealer integrity. Conversely, Bio-C exhibited more adhesive failures, particularly in less favorable conditions (e.g., Group 3), reflecting its sensitivity to dentin surface alterations. This increased failure rate may be attributed to the effects of photodynamic therapy (PDT), which can alter the moisture levels within dentin tubules. The penetration of the photosensitizer, followed by light activation, may lead to partial dehydration or changes in dentinal fluid dynamics, ultimately affecting the bonding efficacy of the sealer [22, 23]. Bioceramic sealers exhibit good wettability due to their hydrophilic nature [24]. However, a recent study by Ramirez et al. contrasts with our findings. They proposed that bioceramic root canal sealers interact with dentin by forming a mineral infiltration zone (MIZ), characterized by the dissolution of sealer components, ion exchange, and the precipitation of calcium phosphate minerals within the dentin matrix. This process is supported by the sealer’s alkaline pH and the presence of moisture in the dentinal tubules, resulting in a mineralized layer that enhances both adhesion and biocompatibility [25]. Additionally, some studies indicate that bioceramic sealers may facilitate the formation of a more uniform and consistent hybrid layer [26, 27].
Comparative studies corroborate our findings. Hashem et al. [11] and Souza et al. [10] reported that advanced irrigation protocols enhance adhesive properties, with AH Plus outperforming MTA Fillapex. Similarly, Çakici [28], and Yavari et al. [29] found AH Plus to be more resistant to PDT-related bond strength reduction than calcium silicate-based sealers. However, studies like Menezes et al. [20] reported decreased PBS following PDT, which was attributed to unremoved PS residues, a limitation addressed in our protocol by incorporating activation and adequate rinsing. Notably, Menezes et al. also observed that non-thermal plasma (NTP) had a lesser effect on AH Plus bond strength than PDT, reinforcing the need to optimize PDT protocols to preserve adhesion [20].
The push-out bond strength (PBS) test was used to evaluate sealer adhesion because it generates parallel fractures at the sealer-dentin interface and is considered more reliable than other methods. Specimen thickness was standardized at 1 mm to reduce the risk of stress concentration and ensure uniform force distribution during testing [16].
The pooled analysis revealed a statistically significant improvement in Bio-C bond strength with sonic activation (p = 0.026), while AH Plus showed an increasing trend that did not reach significance (p = 0.124). This suggests that bioceramic sealers may be more responsive to physical enhancement techniques, perhaps due to their reliance on hydroxyapatite-mediated bonding [5, 14].
Failure mode analysis further supports the mechanical data, as groups with higher PBS showed more cohesive failures, while groups with lower PBS, particularly Bio-C in Group 3, showed more adhesive failures. These results align with Zadsirjan et al. [22], Yavari et al. [29], and Banci et al. [30], who also observed failure mode shifts corresponding to bonding strength and material type. These findings suggest that higher bond strength tends to result in cohesive failures, while lower adhesion is associated with more adhesive or mixed failures.
Clinically, these findings suggest that combining graphene-functionalized photosensitizers with sonic activation prior to PDT can optimize the sealer–dentin interface, potentially improving the long-term performance of root canal obturation. This is particularly relevant for bioceramic sealers, which may benefit more from such enhancements due to their surface bonding dependency [7, 12]. Sealer flow and adhesion are critical for dentinal tubule penetration, interface adaptation, and prevention of microleakage—all of which contribute to clinical success [31, 32].
The outcome of this study should be considered within the context of specific limitations. Despite its valuable insights, this study was conducted in vitro and does not replicate the complex intraoral environment. Variables such as thermal cycling, occlusal/dynamic loading, and long-term sealer aging were not simulated. Additionally, assessments of sealer penetration into dentinal tubules, which could impact adhesion and leakage resistance, were not performed. These factors should be explored in future in vivo studies or through aging protocols to better predict clinical outcomes.
Conclusion
This study concluded that sonic activation and graphene functionalization significantly enhance the push-out bond strength of both AH Plus and Bio-C Sealer, with the greatest improvement observed in Group 6 (Methylene Blue + Graphene Functionalization + Sonic Activation + Laser PDT). AH Plus Sealer exhibited stronger chemical bonding with dentin, resulting in higher cohesive failures, while Bio-C Sealer relied more on mechanical interlocking, leading to increased adhesive failures. These findings suggest that sonic activation and graphene functionalization can improve the adhesion and mechanical performance of endodontic sealers, hence may enhance the longevity of root canal treatments.
Acknowledgements
The author thanks Universiti Sains Malaysia, Malaysia and King Faisal University, Saudi Arabia for supporting this project.
Abbreviations
- MB
Methylene Blue
- MB
rGO-Functionalized Methylene Blue with Reduced Graphene Oxide
- NaOCl
Sodium Hypochlorite
Authors’ contributions
Conceptualization, R.J. and M.S.H.; methodology, R.J. and M.S.H.; software, R.J. and M.S.H.; validation, R.J., M.S.H., M.A.A. and Z.S.; formal analysis, R.J. and M.S.H.; investigation, R.J.; resources, R.J. and M.S.H.; data curation R.J., M.S.H. and M.A.A..; writing—original draft preparation, R.J.; writing—review and editing, M.S.H., M.A.A. and Z.S.; visualization, M.S.H., M.A.A. and Z.S.; supervision, M.S.H. and M.A.A; project administration, M.S.H; funding acquisition, M.A.A. All authors reviewed the manuscript.
Funding
This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia [Grant No. KFU253020].
Data availability
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
Ethical approval was obtained from Universiti Sains Malaysia (USM/JEPeM/KK/24030230). All experiments were performed following relevant guidelines and regulations regarding the experiments on the use of human tissue samples. Informed consent to participate was obtained from all of the participants (owners of the extracted teeth) in the study.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Mohamad Syahrizal Halim, Email: drsyah@usm.my.
Muhammad Adeel Ahmed, Email: mshakeel@kfu.edu.sa.
References
- 1.Dudulwar D, Patil S, Bandekar S, Patil M, Gupta D, Gupta R. A comparative evaluation of push-out bond strength of six different root canal sealers: an in-vitro study. Cureus. 2024;16(3):e56481. 10.7759/cureus.56481. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Sfeir G, Bukiet F, Hage W, El Hachem R, Zogheib C. Impact of final irrigation protocol on the push-out bond strength of two types of endodontic sealers. Materials. 2023;16(5):1761. 10.3390/ma16051761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Jouhar R, Halim MS, Ahmed MA, Shah F, Quadri SA. Synthesis, characterization, and application of methylene blue functionalized reduced graphene oxide for photodynamic therapy in root canal treatment. Pak J Med Sci. 2025;41(2):519–24. 10.12669/pjms.41.2.11001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Chrepa V, Kotsakis GA, Pagonis TC, Hargreaves KM. The effect of photodynamic therapy in root canal disinfection: a systematic review. J Endod. 2014;40:891–8. [DOI] [PubMed] [Google Scholar]
- 5.de Oliveira BP, Aguiar CM, Câmara AC. Photodynamic therapy in combating the causative microorganisms from endodontic infections. Eur J Dent. 2014;8(3):424–30. 10.4103/1305-7456.137662. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Liu C, Li Q, Yue L, Zou X. Evaluation of sonic, ultrasonic, and laser irrigation activation systems to eliminate bacteria from the dentinal tubules of the root canal system. J Appl Oral Sci. 2022;30:e20220199. 10.1590/1678-7757-2022-0199. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Mariano S, Carata E, Calcagnile L, Panzarini E. Recent advances in photodynamic therapy: metal-based nanoparticles as tools to improve cancer therapy. Pharmaceutics. 2024;16(7):932. 10.3390/pharmaceutics16070932. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Joudeh N, Linke D. Nanoparticle classification, physicochemical properties, characterization, and applications: a comprehensive review for biologists. J Nanobiotechnol. 2022;20(1):262. 10.1186/s12951-022-01477-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Li J, Zeng H, Zeng Z, Zeng Y, Xie T. Promising graphene-based nanomaterials and their biomedical applications and potential risks: a comprehensive review. ACS Biomater Sci Eng. 2021;7(12):5363–96. 10.1021/acsbiomaterials.1c00875. [DOI] [PubMed] [Google Scholar]
- 10.Souza MA, Padilha Rauber MG, Zuchi N, Bonacina LV, Ricci R, Dias CT, et al. Influence of final irrigation protocols and endodontic sealer on bond strength of root filling material with root dentin previously treated with photodynamic therapy. Photodiagnosis Photodyn Ther. 2019;26:137–41. 10.1016/j.pdpdt.2019.03.013. [DOI] [PubMed] [Google Scholar]
- 11.Hashem M, El-Hejazi AA, Niaz MO, Alsadon O, Alolayani BM, Fouad H. Bond strength of root canal filling with radicular dentin previously treated with either photobiomodulation or photodynamic therapy: effect of disinfection protocols. Photodiagnosis Photodyn Ther. 2020;30:101733. 10.1016/j.pdpdt.2020.101733. [DOI] [PubMed] [Google Scholar]
- 12.Gutmann JL, Dees LA. Historical perspectives on Core-Carrier Gutta-Percha obturation. J Hist Dent. 2016;64(1):22–5. [PubMed]
- 13.Alfredo E, Silva SR, Ozório JE, Sousa-Neto MD, Brugnera-Júnior A, Silva-Sousa YT. Bond strength of AH plus and epiphany sealers on root dentine irradiated with 980 Nm diode laser. Int Endod J. 2008;41(9):733–40. 10.1111/j.1365-2591.2008.01418.x. [DOI] [PubMed] [Google Scholar]
- 14.Akcay M, Arslan H, Durmus N, Mese M, Capar ID. Dentinal tubule penetration of AH plus, iRoot SP, MTA fillapex, and guttaflow bioseal root canal sealers after different final irrigation procedures: a confocal microscopic study. Lasers Surg Med. 2016;48(1):70–6. 10.1002/lsm.22446. [DOI] [PubMed] [Google Scholar]
- 15.Leal F, Simão RA, Fidel SR, Fidel RA, Prado M. Effect of final irrigation protocols on push-out bond strength of an epoxy resin root Canal sealer to dentin. Aust Endod J. 2015;41(3):135–9. 10.1111/aej.12114. [DOI] [PubMed] [Google Scholar]
- 16.Ok E, Ertas H, Saygili G, Gok T. Effect of photo-activated disinfection on bond strength of three different root canal sealers. Eur J Dent. 2014;8(1):85–9. 10.4103/1305-7456.126252. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Souza MA, Tumelero Dias C, Zandoná J, Paim Hoffmann I, Sanches Menchik VH, Palhano HS, et al. Antimicrobial activity of hypochlorite solutions and reciprocating instrumentation associated with photodynamic therapy on root canals infected with Enterococcus faecalis - an in vitro study. Photodiagnosis Photodyn Ther. 2018;23:347–52. 10.1016/j.pdpdt.2018.07.015. [DOI] [PubMed] [Google Scholar]
- 18.Sheng T, Ong YH, Guo W, Zhu T. Reactive oxygen species explicit dosimetry to predict tumor growth for benzoporphyrin derivative-mediated vascular photodynamic therapy. J Biomed Opt. 2020;25(6):1–13. 10.1117/1.jbo.25.6.063805. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Souza MA, Pazinatto B, Bischoff KF, Palhano HS, Cecchin D, de Figueiredo JAP. Influence of ultrasonic activation over final irrigants in the removal of photosensitizer from root canal walls after photodynamic therapy. Photodiagnosis Photodyn Ther. 2017;17:216–20. 10.1016/j.pdpdt.2016.12.011. [DOI] [PubMed] [Google Scholar]
- 20.Menezes M, Prado M, Gomes B, Gusman H, Simão R. Effect of photodynamic therapy and non-thermal plasma on root canal filling: analysis of adhesion and sealer penetration. J Appl Oral Sci. 2017;25(4):396–403. 10.1590/1678-7757-2016-0498. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Sebrão CC, Bezerra AG Jr, de França PH, Ferreira LE, Westphalen VP. Comparison of the efficiency of Rose Bengal and methylene blue as photosensitizers in photodynamic therapy techniques for Enterococcus faecalis inactivation. Photomed Laser Surg. 2017;35(1):18–23. 10.1089/pho.2015.3995. [DOI] [PubMed] [Google Scholar]
- 22.Zadsirjan S, Asnaashari M, Yazdani A, Heidari S, Estarami T. Effect of photodynamic therapy on the pushout bond strength of resin-based and calcium silicate-based endodontic sealers. J Lasers Med Sci. 2023;14:e33. 10.34172/jlms.2023.33. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Donnermeyer D, Dornseifer P, Schäfer E, Dammaschke T. The push-out bond strength of calcium silicate-based endodontic sealers. Head Face Med. 2018;14(1):13. 10.1186/s13005-018-0170-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Joshi P, Shetty R, Banpurkar A, et al. In vitro comparison of the wettability of a bioceramic root canal sealer on dentin with and without Erbium-doped yttrium aluminum Garnet (Er:YAG) laser irradiation. Cureus. 2022;14(3):e23715. 10.7759/cureus.23715. Published 2022 Mar 31. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Ramirez I, dos Santos GN, de Castro GP, et al. How can calcium silicate–based sealers impact the mineral phase of root dentin after the use of intracanal medications? A chemical and spectroscopic analysis. Int J Adhes Adhes. 2025;103991. 10.1016/j.ijadhadh.2025.103991.
- 26.Chisnoiu RM, Moldovan M, Prodan D, Chisnoiu AM, Hrab D, Delean AG, et al. In-vitro comparative adhesion evaluation of bioceramic and dual-cure resin endodontic sealers using SEM, AFM, push-out and FTIR. Appl Sci. 2021;11(10):4454. 10.3390/app11104454. [Google Scholar]
- 27.Costa JLSG, Barros APO, Junior AJ, Gelio MB, Dantas AAR, Kuga MC. Influence of residues from bioceramic sealer on the bond interface with universal adhesive in different application modes. Eur J Oral Sci. 2024;132(4):e12994. 10.1111/eos.12994. [DOI] [PubMed] [Google Scholar]
- 28.Çakici F, Çakici EB, Ilker A, Topçuoğlu H. Evaluation of effect of disinfection systems on bond strength of root canal sealers. Middle Black Sea J Health Sci. 2020;6(1):92–8. 10.19127/mbsjohs.706381. [Google Scholar]
- 29.Yavari H, Ghasemi N, Divband B, Rezaei Y, Jabbari G, Payahoo S. The effect of photodynamic therapy and polymer solution containing nano-particles of ag/zno on push-out bond strength of the sealers AH-Plus and MTA fillapex. J Clin Exp Dent. 2017;9(9):e1109–14. 10.4317/jced.54069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Banci HA, Strazzi-Sahyon HB, Duarte M, Cintra L, Gomes-Filho JE, Chalub LO, et al. Influence of photodynamic therapy on bond strength and adhesive interface morphology of MTA based root canal sealer to different thirds of intraradicular dentin. Photodiagnosis Photodyn Ther. 2020;32:102031. 10.1016/j.pdpdt.2020.102031. [DOI] [PubMed] [Google Scholar]
- 31.Silva RV, Silveira FF, Horta MC, Duarte MA, Cavenago BC, Morais IG, et al. Filling effectiveness and dentinal penetration of endodontic sealers: a stereo and confocal laser scanning microscopy study. Braz Dent J. 2015;26(5):541–6. 10.1590/0103-6440201300138. [DOI] [PubMed] [Google Scholar]
- 32.Almadi KH. Impact of antimicrobial photodynamic therapy on the bond-strength and penetration of endodontic sealers: a systematic review. Photodiagnosis Photodyn Ther. 2023;41:103249. 10.1016/j.pdpdt.2022.103249. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.