Clinically relevant radiation therapy doses and obturation techniques modulate the adhesive performance of a resin-based sealer: interfacial integrity and bond strength analyses
Mariana Travi Pandolfo, Luíz Carlos de Lima Dias-Júnior, Maria Eduarda Paz Dotto, Mariana Comparotto Minamisako, Paulo Marcelo Rodrigues, Nayara Cardoso Cábia, Bianca de Sousa Veiga, Maíra do Prado, Cleonice da Silveira Teixeira, Lucas da Fonseca Roberti Garcia
TL;DR
This study shows that radiation therapy used in cancer treatment can weaken the bond of dental sealers in teeth, but certain filling techniques can help maintain their effectiveness.
Contribution
The study demonstrates that radiation therapy doses and obturation techniques significantly affect the adhesive performance of resin-based endodontic sealers.
Findings
Radiation doses of 30 Gy and 70 Gy significantly reduced bond strength of the sealer compared to non-irradiated teeth.
Tagger’s hybrid obturation technique produced higher bond strength than lateral condensation across all irradiation conditions.
Higher radiation doses caused more severe interfacial degradation and reduced sealer penetration into dentin.
Abstract
To evaluate the effects of clinically relevant radiation therapy doses and obturation techniques on the adhesive performance of a resin-based endodontic sealer. Sixty human mandibular premolars were allocated to 3 groups according to radiation exposure: non-irradiated (NoRT), 30 Gy (clinically absorbed dose for mandibular premolars during nasopharyngeal cancer radiotherapy), and 70 Gy (tumor-target dose). Irradiation was performed using intensity-modulated radiotherapy with fractionated doses of 2 Gy. Root canals were prepared and obturated using lateral condensation (LC) or Tagger’s hybrid technique (HB) with an epoxy resin-based sealer. Push-out bond strength was evaluated in cervical, middle, and apical thirds. Failure modes were analyzed by stereomicroscopy and scanning electron microscopy (SEM). Sealer-dentin interface characteristics and sealer penetration were assessed using SEM…
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Figure 6- —Universidade Federal De Santa Catarina
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Taxonomy
TopicsEndodontics and Root Canal Treatments · Dental materials and restorations · Surgical Sutures and Adhesives
Introduction
Head and neck cancer (HNC) is a prevalent malignancy that affects several anatomical regions, including the oral cavity, nasal cavity, paranasal sinuses, salivary glands, pharynx, and larynx [1]. Among HNC subtypes, oral cavity cancer is one of the most common and may involve the lips, buccal mucosa, palate, tongue, and floor of the mouth [2]. The management of HNC typically includes surgery, chemotherapy, radiotherapy, immunotherapy, or combinations of these modalities [3, 4], with radiotherapy playing a fundamental role in disease control [5, 6].
Despite its therapeutic efficacy, ionizing radiation affects not only neoplastic cells but also adjacent healthy tissues [2, 7, 8]. Consequently, radiotherapy is associated with several oral adverse effects, including mucositis, trismus, hyposalivation, radiation-related caries, and osteoradionecrosis [9, 10]. In addition, radiation-induced structural changes in enamel and dentin have been documented and appear to intensify with increasing radiation doses [5, 11].
Initially, dental degradation following radiotherapy was considered an indirect consequence of salivary gland damage and subsequent hyposalivation [12]. However, accumulating evidence indicates that radiation also exerts direct effects on dental tissues, with the severity of structural alterations being dose dependent [5, 11–13].
In intraradicular dentin specifically, ionizing radiation has been associated with disorganization of the collagen matrix [10], fragmentation of the fibrillar network [10], reduction in intermolecular cross-linking [14], and alterations in the mineral-to-organic ratio [8], collectively compromising the structural integrity of the dentin substrate. Additionally, radiation-induced dehydration and microcrack formation may further modify dentin permeability and micromechanical properties [10]. These physicochemical alterations may directly affect the conditions required for effective micromechanical interlocking and endodontic sealer adaptation within root canal walls [15].
Because epoxy resin-based endodontic sealers rely primarily on tubular penetration and interaction with the organic matrix to enhance retention and minimize interfacial gaps [15], disruption of collagen architecture and mineral balance may impair the stability of the sealer-dentin interface [12, 14]. Thus, beyond generalized dentin degradation, radiation-induced substrate modification may compromise a critical interface that underpins obturation quality and interfacial integrity under compromised biological conditions [15].
Recent dosimetric studies have demonstrated that the radiation dose absorbed by teeth may differ substantially from the dose delivered to the tumor site [16]. In radiotherapy protocols for nasopharyngeal cancer, while tumor doses typically range from 50 to 70 Gy, mandibular premolars may receive approximately 30 Gy [16], suggesting that radiation levels used in previous in vitro investigations may have been overestimated [10, 12, 14, 17, 18]. Therefore, a comprehensive understanding of the effects of clinically realistic radiation therapy doses on dentin is essential for evidence-based clinical decision-making.
Accordingly, this in vitro study aimed to evaluate the effects of clinically relevant doses of ionizing radiation (30 Gy and 70 Gy) on the bond strength of an endodontic sealer to intraradicular dentin, considering different root canal obturation techniques (lateral condensation and Tagger’s hybrid technique). Additionally, the effects of these radiation doses on the sealer-dentin interface and on sealer penetration into dentinal tubules were assessed. The null hypotheses tested were that radiation doses would not affect (i) the bond strength of the sealer to root dentin, (ii) the integrity of the sealer-dentin interface, or (iii) sealer penetration into dentinal tubules, regardless of the root canal obturation technique employed.
Materials and methods
Ethical approval and sample size calculation
This study was approved by the Human Research Ethics Committee of the of the Federal University of Santa Catarina (No. 5.893.644) and by the Department of Radiotherapy of the Oncology Research Center (No. 6.013.576). Sample size calculation was performed using G*Power software (version 3.1.9.7; Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany), based on data from Jordani et al. [2], to determine the minimum number of specimens required per experimental group to detect a statistically significant difference of 5% among groups. The parameters adopted were α = 0.05, statistical power (1-β) = 0.80, and effect size = 0.5. An a priori power analysis was conducted considering comparisons between independent groups. Based on this calculation, ten specimens per experimental group were required for the bond strength test. Post hoc evaluation of the observed effect sizes derived from the final three-way ANOVA model confirmed that the achieved statistical power exceeded 80% for the primary comparisons.
Sample selection, preparation, and group allocation
Sixty freshly extracted human mandibular premolars with a single, fully formed root canal were selected. Teeth presenting caries, hypoplastic or hypomineralized areas, resorptions, calcifications, or previous endodontic treatment were excluded. Sample standardization was performed through visual inspection (4× magnification) and digital periapical radiography (Digital Sensor Fit; Acteon, Indaiatuba, SP, Brazil) in the mesiodistal and buccolingual directions. Only teeth with a standardized root length of 16 mm and a buccolingual-to-mesiodistal dimension ratio ≤ 1.5 were included [2, 17]. Specimens were ultrasonically cleaned (Profi Neo; Dabi Atlante, Ribeirão Preto, SP, Brazil), disinfected in 0.1% thymol for 48 h, and stored in distilled water at 37 °C until use.
The crowns of the teeth were sectioned 1 mm above the cementoenamel junction using a double-sided diamond disc (Fava, São Paulo, SP, Brazil) under water cooling, and root length was confirmed with a digital caliper (Mitutoyo, Kawasaki, Japan). The specimens were randomly allocated (www.random.org) into three groups (n = 20): non-irradiated teeth (NoRT - control groups), teeth irradiated with 30 Gy (clinically absorbed dose for mandibular premolars during nasopharyngeal cancer radiotherapy), and teeth irradiated with 70 Gy (tumor-target dose during nasopharyngeal cancer radiotherapy) [16]. Each group was further subdivided (n = 10) according to the obturation technique: lateral condensation (LC) or Tagger’s hybrid technique (HB) (Fig. 1).
Fig. 1. Flowchart of groups distribuition
Teeth irradiation
Teeth were irradiated prior to the experimental procedures using a linear accelerator (Clinac 2100 C; Varian Medical Systems, Palo Alto, CA, USA) with the intensity-modulated radiation therapy (IMRT) technique and a dynamic multileaf collimator. Specimens were positioned in a plastic holder containing distilled and deionized water and aligned equidistantly from the radiation source to ensure uniform dose distribution (400 MU/min) [19]. Total radiation doses of 30 Gy and 70 Gy were delivered in daily fractions of 2 Gy, five days per week, over three and seven weeks, respectively. After each irradiation cycle, specimens were stored in artificial saliva (Dermus Pharmacy, Florianópolis, SC, Brazil) at 37 °C to simulate intraoral conditions; the artificial saliva was replaced with distilled water before the next irradiation session. The artificial saliva formulation consisted of a viscosifying agent (sodium carboxymethylcellulose), mineral salts (potassium chloride, sodium chloride, magnesium chloride, and dibasic calcium phosphate), a humectant (sorbitol), distilled water as vehicle, and preservatives (sodium benzoate) to ensure product stability. All irradiation procedures were performed at the Department of Radiotherapy of the Oncology Research Center (CEPON; Florianópolis, SC, Brazil) under the supervision of a medical physicist and a radiation oncologist. After completion of the protocol, the teeth were stored in artificial saliva at 37 °C until testing.
Root canal preparation
Root canals were negotiated with a size 10 K-file (Dentsply-Maillefer, Ballaigues, Switzerland), and the working length was established 1.0 mm short of the apical foramen. Canal preparation was performed using a reciprocating single-file system (40/0.06) (Reciproc R40; VDW, Munich, Germany) operated with a 6:1 reduction contra-angle handpiece (VDW Silver Reciproc; Dentsply Sirona, Bensheim, Germany). The file was advanced apically with a gentle pecking motion and, after every three strokes, was removed, cleaned with gauze, and reinserted until the working length was reached. Canal patency was maintained throughout preparation using a size 10 K-file (Dentsply-Maillefer). Irrigation was performed with 2 mL of 2.5% sodium hypochlorite solution (Biodinâmica, Ibiporã, PR, Brazil), delivered through a disposable syringe and a NaviTip needle (Ultradent Products, South Jordan, UT, USA) positioned 2 mm short of the working length. Simultaneous aspiration was performed using a metallic suction cannula. Final irrigation consisted of 3 mL of 17% EDTA (Biodinâmica, Ibiporã, PR, Brazil) for 3 min, followed by 2 mL of 2.5% NaOCl solution. Finally, the canals were dried with R40 paper points (Reciproc R40; VDW).
Root canal obturation
Root canals were obturated using an epoxy resin-based sealer (AH Plus Jet; Dentsply DeTrey, Konstanz, Germany), an R40 gutta-percha master cone (Reciproc; VDW), and B7 accessory cones (Tanariman, Manaus, AM, Brazil). Specimens were obturated as follows. LC technique: The master cone was coated with endodontic sealer and inserted into the root canal with circumferential motions until reaching the working length. A 25 mm digital spreader (Dentsply-Maillefer) was then applied laterally to the master cone at the working length, followed by the insertion of sealer-coated accessory cones until the root canal was completely filled. Excess material was removed, and vertical compaction was performed using a Paiva plugger (Golgran, São Paulo, Brazil) heated to the level of the cementoenamel junction. HB technique: The master cone was coated with sealer and inserted to the working length with gentle circumferential motions. A 25 mm digital spreader (Dentsply-Maillefer) was then placed laterally to the master cone, followed by the insertion of two sealer-coated accessory cones. A size 60 McSpadden compactor (Dentsply-Maillefer), calibrated 3 mm short of the working length and mounted on a low-speed handpiece (Model 605; Kavo, Joinville, SC, Brazil), was positioned parallel to the tooth’s long axis. A back-and-forth motion was performed until the predetermined working length was reached. The quality of obturation was confirmed by digital periapical radiographs obtained in the buccolingual and mesiodistal directions.
For confocal laser scanning microscopy (CLSM) analysis, two specimens from each experimental group were obturated with the epoxy resin-based sealer containing 0.001 g of 0.1% Rhodamine B (Sigma-Aldrich, St. Louis, MO, USA), diluted in 0.05 mL of distilled water and buffered with a 0.5% sodium phosphate solution per gram of sealer, following a previously described protocol [20]. Access cavities were sealed with an adhesive system (Adper Single Bond 2; 3 M ESPE, St. Paul, MN, USA) and a composite resin (Filtek Z350 XT; 3 M ESPE). Specimens were stored at 37 °C and 100% humidity for a period corresponding to three times the sealer setting time.
Push-out bond strength test
Roots were sectioned perpendicular to their long axis using a double-sided diamond disc (Buehler, Lake Bluff, IL, USA) under water cooling to obtain dentin slices (1.0 ± 0.1 mm thick) from the cervical, middle, and apical thirds. Push-out tests were performed using a universal testing machine (OM150; Odeme Dental Research, Luzerna, SC, Brazil). Plungers with diameters of 0.6, 0.8, 1.0, or 1.2 mm were used and were previously selected according to the root canal diameter in the cervical, middle, or apical third. Plunger diameter was chosen individually for each specimen and was at least 0.2 mm smaller than the corresponding canal lumen to ensure contact exclusively with the filling material and to prevent friction with dentin walls. To ensure accurate positioning and alignment of the plunger tip over the filling material, a meticulous visual examination was performed at 4× magnification using a magnifying lens (Optima Dart LED Hand Magnifier). The load was applied in an apical-to-coronal direction at a crosshead speed of 0.5 mm/min until bond failure occurred. Bond strength values were calculated in MPa by dividing the maximum load (N) by the bonded surface area, which was determined using the following formula, BS = π (R + r) √(h² + (R − r)²), where BS represents the bonded surface area, h is the dentin slice thickness (measured with a digital caliper), and R and r correspond to the cervical and apical radii of the canal space, respectively.
Failure mode analysis
After push-out testing, each dentin slice was inspected under a stereomicroscope (SteREO Discovery V12; Carl Zeiss, Jena, Germany) at 15× and 40× magnifications to determine the failure mode. Representative specimens were then selected and examined under scanning electron microscopy (SEM) (TESCAN VEGA, Brno, Czech Republic). Specimens were affixed to aluminum stubs with carbon tape, sputter-coated with a 300–500 Å gold-palladium layer (Desk II; Denton Vacuum, Moorestown, NJ, USA) for 120 s, and imaged at 500×, 1000×, and 1500× magnifications. Failures were classified as follows: adhesive (root canal walls free of endodontic sealer or disruption of the cementation line), cohesive in the sealer (fracture of the filling material while dentin remained covered by sealer), cohesive in dentin (fracture of the dentin substrate), and mixed (a combination of different failure types occurring within the same specimen) [2].
Adhesive interface analysis
Non-tested dentin slices were reserved for analysis of the adhesive interface under SEM (JSM-5410; JEOL, Tokyo, Japan). The slices were immersed in a 2.5% glutaraldehyde solution buffered to pH 7.4 with 0.1 M sodium cacodylate (Dermus, Florianópolis, SC, Brazil) for 12 h at 4 °C. Subsequently, the slices were rinsed with deionized water and dehydrated through ascending concentrations of ethyl alcohol (25% for 20 min, 50% for 20 min, 75% for 20 min, 95% for 30 min, and 100% for 1 h). Each slice was then dried and mounted with double-sided adhesive tape onto Teflon rings (5 mm thickness × 2.0 cm diameter), with the cervical surface facing downward, and embedded in epoxy resin. After resin polymerization, both sides of the samples were polished using a polishing machine (DP-10; Panambra, São Paulo, SP, Brazil) with abrasive papers (Norton, São Paulo, SP, Brazil) applied under light pressure and constant water irrigation in a decreasing grit sequence (#400, #600, and #1200), followed by polishing with felt discs and alumina pastes with particle sizes of 0.3 μm and 0.1 μm (Arotec, São Paulo, SP, Brazil). After thorough rinsing with running water, the samples were demineralized with 6 mol/L hydrochloric acid (Dermus) for 30 s, followed by deproteinization with a 2% sodium hypochlorite solution (Dermus) for 10 min [21]. The specimens were then placed in a vacuum chamber and sputter-coated with a 300 Å layer of gold-palladium alloy (80 wt% / 20 wt%) (Desk II; Denton Vacuum). Images of different areas of each dentin slice were obtained at magnifications of 100×, 500×, 1000×, and 2000×.
The integrity of the adhesive interface between dentin and the endodontic sealer (continuous and homogeneous interface and/or presence of gaps), as well as the density and depth of sealer penetration into the dentinal tubules [21].
Sealer penetration analysis
Sealer penetration into dentinal tubules was assessed by CLSM (Leica SP8; Leica Microsystems, Mannheim, Germany) using specimens labeled with 0.1% Rhodamine B, a concentration selected based on previous evidence demonstrating fluorescence stability and minimal interference with the physicochemical properties of epoxy resin-based sealers [20].
Each dentin slice was polished on both surfaces for 1 min using silicon carbide abrasive papers (Norton) in ascending grit sequence (#800, #1200, and #1500) to obtain a standardized, smooth, and uniform surface. The slices were then mounted on 0.17-mm-thick glass coverslips.
CLSM imaging was performed in epifluorescence mode using excitation and emission parameters corresponding to Rhodamine B (552 nm excitation laser; 548–588 nm emission range) with a red-light filter. Imaging parameters, including laser intensity, detector gain, pinhole aperture, and optical section thickness, were standardized for all specimens to minimize signal variability and reduce potential optical artifacts. Analyses were conducted at a standardized depth of 10 μm below the surface to limit superficial scattering effects. Images were acquired under 10× and 20× magnifications using Leica Application Suite-Advanced Fluorescence software (Leica Microsystems) for evaluation of sealer penetration patterns.
All image acquisitions and analyses were performed under blinded conditions by calibrated examiners to reduce observational bias.
Statistical analysis
The statistical analysis was performed using SPPS Statistics version 25 software (IBM Corp., Armonk, EUA). As bond strength values were highly skewed (non-normal distribution, Shapiro-Wilk test) data underwent log^− 10^ transformation (LG10 function, SPSS Statistics, IBM). After transformation to approximate normally distributed residuals, the influence of obturation technique (LC and HB), radiotherapy regimen (no radiotherapy, 30 Gy, and 70 Gy), and root thirds (cervical, middle and apical) on the bond strength values were analyzed using a three-way ANOVA test with Bonferroni’s post-hoc test. Failure mode distribution was analyzed using Fisher’s exact test. Qualitative analyses of sealer–dentin interface characteristics and sealer penetration by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were conducted by calibrated and blinded examiners. Interexaminer agreement was assessed using the Kappa statistic (κ > 0.75 indicating excellent agreement). The significance level was set at 5% for all analyses.
Results
Bond strength
All specimens were successfully tested. Three-way ANOVA revealed that bond strength values were significantly affected by the radiotherapy regimen (P < .001), and obturation technique (P < .001). The root thirds did not affect the bond strength values in any of the experimental settings (P > .05). Also, the interaction among radiotherapy regimen, obturation techniques, and root thirds was not significant (P = .899).
The bond strength of the specimens irradiated with either 30–70 Gy were significantly lower than the non-irradiated specimens, in both CL and HB groups, regardless of the root third (P < .05).
Overall, the HB technique showed significantly higher values of bond strength, compared to the LC, in both non-irradiated specimens, or irradiated with 30–70 Gy (P < .05). The bond strength values were back-transformed to the original scale and expressed as mean and standard deviation. The complete results of the pairwise comparisons with their respective p-values are demonstrated in Table 1; Fig. 2.
Table 1. Mean and standard deviation values of bond strength of each experimental setting, according to the obturation technique, radiotherapy regimen, and root thirdsRoot ThirdsGroupRadiotherapy NoRT
30 Gy
70 Gy P-value Cervical
LC 3.18 ± 1.16^A, a^1.34 ± 0.87^B, a^1.55 ± 0.75^B, a^< 0.001 HB 4.01 ± 0.64^A, b^2.43 ± 0.50^B, b^2.02 ± 0.44^B, b^< 0.001P-value0.0220.0030.047 Middle
LC 3.08 ± 0.48^A, a^1.59 ± 0.86^B, a^1.81 ± 0.39^B, a^< 0.001 HB 4.07 ± 0.99^A, b^2.57 ± 0.52^B, b^2.69 ± 0.89^B, b^< 0.001P-value0.0060.0070.015 Apical
LC 3.42 ± 1.23^A, a^1.91 ± 0.50^B, a^1.73 ± 0.64^B, a^< 0.001 HB 4.19 ± 1.18^A, b^2.76 ± 1.06^B, b^2.23 ± 0.37^B, b^< 0.001P-value0.0340.0180.039 Overall
LC 3.23 ± 0.99^A, a^1.61 ± 0.78^B, a^1.69 ± 0.60^B, a^< 0.001 HB 4.09 ± 0.93^A, b^2.59 ± 0.73^B, b^2.31 ± 0.66^B, b^< 0.001P-value0.001< 0.0010.001Different uppercase letters in a row indicate statistical differences regarding the radiotherapy regimen. Different lowercase letters in a column indicate statistical difference between groups. Three-way ANOVA test with Bonferroni’s post-hoc test (α = 0.05)
Fig. 2. Boxplots of push-out bond strength values according to radiation regimen, obturation technique, and root third
Failure mode
Table 2; Fig. 3 present the distribution and frequency, respectively, of failure modes in each experimental group. Representative SEM images of the failure modes may be seen in Fig. 4. Failure mode analysis revealed no significant differences among the experimental groups (P > .05). Cohesive failure within the sealer occurred more frequently than mixed failures or adhesive failures at the dentin-sealer interface. No cohesive failures within dentin were observed.
Table 2. Distribution of failure modes among the experimental groupsRadiotherapyGroupFailure modeAdhesiveCohesive - SealerCohesive - DentinMixedNoRTLC32205HB2210730 GyLC52005HB2230570 GyLC02901HB42006
Fig. 3. Bar chart of the failure modes frequency, according to the obturation technique and radiotherapy regimen
Fig. 4. Representative SEM images of the failure modes after the bond strength test. (A) Adhesive failure (×150). (B) Higher magnification showing disruption of the cementation line (arrow) (×1000). (C) Cohesive failure within the sealer (box) (×150). (D) Higher magnification showing fracture of the filling material (box) (×500). (E) Mixed failure, showing a combination of adhesive (white arrow) and cohesive failure at the sealer (black arrow) within the same specimen (×150). Images were acquired at 15 kV. Scale bar = 50, 100 and 500 µm
Adhesive interface analysis
Representative SEM images are shown in Fig. 5. SEM analysis revealed distinct morphological patterns at the adhesive interface among the experimental groups. In the NoRT-LC specimens, a continuous and compact cementation line was observed, without voids or interfacial gaps, and characterized by the presence of long, well-defined sealer tags. Similarly, the NoRT-HB group showed dense and elongated tags, although occasional discontinuities were evident along the cementation line. In the 30 Gy-LC group, the interface appeared generally uniform; however, small interfacial gaps and partial disruption of some sealer tags were visible. The 30 Gy-HB group presented a denser arrangement of sealer tags compared with the LC subgroup, indicating enhanced adaptation of the sealer to the dentinal surface. In contrast, specimens from the 70 Gy groups demonstrated pronounced interfacial degradation. The 70 Gy-LC samples exhibited evident gaps and severe disruption of the sealer tags, while the 70 Gy-HB samples showed long but irregular tag morphology and discontinuous interfaces. These findings suggest that higher radiation doses adversely affect the integrity of the sealer-dentin interface, compromising both tag formation and interfacial continuity.
Fig. 5. Representative SEM images of the adhesive interface. (A) NoRT-LC: Continuous and compact cementation line (), without voids or interfacial gaps; long and well-defined sealer tags are observed within dentinal tubules. (×500). (B) NoRT-HB: Discontinuous cementation line (boxed area); however, sealer tags appear denser and longer compared with NoRT-LC (×500). (C) 30 Gy-LC: Predominantly uniform interface; the white arrow indicates an interfacial microgap, while black arrows denote disrupted or shortened sealer tags (×500). (D) 30 Gy-HB: Increased density and length of sealer tags compared with 30 Gy-LC, with improved tubular adaptation (×500). (E) 70 Gy-LC: Presence of interfacial gaps (); circles highlight evident disruption and fragmentation of sealer tags (×500). (F) 70 Gy-HB: Interfacial gaps are present; circles indicate long but irregular and disorganized sealer tags (×500). Overall, specimens irradiated with 70 Gy exhibited a reduced number and structural integrity of sealer tags compared with the 30 Gy and non-irradiated (NoRT) groups. Images were acquired at 15 kV. Scale bar = 50 µm.(Asterisk: cementation line or interfacial gap; box: discontinuity of the cementation line; white arrow: interfacial microgap; black arrows: disrupted sealer tags; circle: irregular or fragmented sealer tags)
Sealer penetration into dentinal tubules
Representative CLSM images for all groups and root thirds are shown in Fig. 6. CLSM analysis also revealed distinct patterns of sealer penetration into dentinal tubules among the experimental groups. Overall, the 30 Gy and 70 Gy groups exhibited a less pronounced pattern of sealer penetration compared with the NoRT group, regardless of the obturation technique employed (LC or HB). In the NoRT-LC specimens, the sealer showed deep and continuous penetration, with long and uniformly distributed fluorescent tags extending along the entire circumference of the root canal. The NoRT-HB group demonstrated a similar pattern, with even greater tag density, indicating effective sealer adaptation and homogeneous penetration along the dentinal walls. In the 30 Gy-LC specimens, sealer penetration was still evident but appeared slightly reduced in both depth and density, with areas of partial discontinuity in tag distribution. The 30 Gy-HB specimens exhibited moderately intense fluorescence, with long sealer tags extending along the tubule paths, although small regions of irregular distribution were observed. In contrast, the 70 Gy-LC group displayed a markedly reduced fluorescence signal, suggesting limited sealer penetration and disruption of tag continuity, while the 70 Gy-HB group exhibited sparse and irregular fluorescent tags concentrated mainly near the cementation line.
Fig. 6. Representative CLSM images of the experimental groups according to root level (cervical, middle, and apical thirds). Rhodamine B-labeled sealer penetration is visualized as red fluorescence within dentinal tubules. NoRT-LC and NoRT-HB specimens show deep and continuous sealer penetration, characterized by long, uniformly distributed fluorescent tags along the dentinal walls.The 30 Gy-LC and 30 Gy-HB groups display reduced penetration depth and tag density, with areas of partial discontinuity and less homogeneous fluorescence distribution. In contrast, the 70 Gy-LC and 70 Gy-HB specimens exhibit markedly decreased fluorescence intensity, with sparse, irregular, and shorter sealer tags predominantly concentrated near the cementation line.Fluorescent signal intensity corresponds to the extent of sealer penetration within dentinal tubules. Images are representative of each experimental condition
Discussion
Radiotherapy is known to induce significant alterations in dental tissues, especially dentin, primarily due to the interaction of ionizing radiation with water molecules, which generates highly reactive free radicals through radiolysis [10, 22, 23]. This in vitro study evaluated the effects of two clinically realistic radiation doses used in the treatment of nasopharyngeal cancer on the bond strength of an epoxy resin-based endodontic sealer to intraradicular dentin, considering different obturation techniques.
A radiation dose of 30 Gy was selected to represent the clinically absorbed dose by mandibular premolars during nasopharyngeal cancer radiotherapy, whereas a dose of 70 Gy corresponded to the tumor-target dose typically delivered during nasopharyngeal cancer radiotherapy. Our results demonstrated that exposure to both radiation doses was associated with a significant reduction in bond strength, along with compromised sealer-dentin interfacial integrity and reduced sealer penetration into dentinal tubules, leading to rejection of all null hypotheses. In addition, the obturation technique significantly influenced bond strength outcomes, independent of the radiotherapy regimen. These findings suggest that radiation-induced alterations in dentin may negatively affect the adhesive performance of epoxy resin-based sealers, while the obturation technique appears to play a relevant role in mitigating, at least partially, these adverse effects.
Recent dosimetric studies have demonstrated that radiation is heterogeneously distributed across dental structures during HNC radiotherapy, and that the absorbed dose by teeth frequently differs from the dose prescribed to the tumor [16, 24]. Consequently, in vitro studies that exclusively employ high radiation doses (60–70 Gy) may overestimate the extent of radiation-induced dental damage [16, 24]. Clinically, although total prescribed doses for head and neck radiotherapy typically range from 50 to 70 Gy [8], the radiation dose effectively absorbed by mandibular premolars in nasopharyngeal cancer may be substantially lower, reaching approximately 30 Gy, depending on tumor location and extent [16]. Therefore, the radiation doses of 30 Gy and 70 Gy adopted in the present study were selected to represent clinically absorbed dental doses and tumor-target doses, respectively. Fractionation in daily 2-Gy doses followed conventional radiotherapy protocols for HNC [25].
To better simulate clinical conditions, specimens were stored in artificial saliva between irradiation cycles [2, 22]. Although artificial saliva cannot fully replicate the complex biological and dynamic properties of natural saliva in irradiated patients, it remains the most suitable experimental medium for in vitro studies [15]. During irradiation, teeth were immersed in distilled water to ensure homogeneous radiation delivery, as the viscosity and ionic composition of artificial saliva may compromise dose uniformity [2, 15, 22].
Root canal obturation was performed using lateral condensation, considered a reference technique for comparison with alternative obturation methods [26], and Tagger’s hybrid technique, which has been shown to promote superior sealer adaptation to the complex internal anatomy of the root canal system [27]. Despite its widespread clinical use, lateral condensation has been associated with void formation, uneven sealer distribution, and reduced apical density [28], factors that may compromise the mechanical integrity of the filling. These limitations may help explain our findings, as specimens obturated with the hybrid technique exhibited significantly higher bond strength values than those filled using lateral condensation, regardless of the irradiation regimen. From a methodological perspective, canal anatomy was carefully standardized by selecting specimens with comparable buccolingual and mesiodistal dimensions (ratio ≤ 1.5) [2, 17], resulting in similar cementation line geometries among groups. This approach minimized anatomical variability and strengthened the internal validity of the push-out bond strength outcomes.
Radiation exposure significantly reduced the bond strength of the epoxy resin-based sealer to root dentin, even at the lower dose of 30 Gy, indicating that clinically absorbed radiation levels are sufficient to induce deleterious alterations in dentin structure and substrate properties [11, 14, 15, 18, 23]. These quantitative findings are consistent with the qualitative observations obtained by SEM, which demonstrated progressive disruption of the sealer-dentin interface and reduced sealer penetration into dentinal tubules with increasing radiation doses. Non-irradiated specimens exhibited continuous cementation lines and dense, well-defined sealer tags, whereas specimens exposed to 30 Gy already showed localized interfacial gaps and partial tag disruption. At 70 Gy, these alterations became more pronounced, with marked interfacial discontinuities and irregular or sparse sealer tags, particularly when lateral condensation was employed. These effects are consistent with radiation-induced dentin damage, which collectively impair micromechanical interlocking and sealer adhesion [8, 11, 14, 15, 18, 23]. Conversely, Tagger’s hybrid technique promoted more continuous cementation lines and deeper, more homogeneous sealer penetration, likely due to enhanced sealer flow associated with thermoplasticized gutta-percha [29], which may partially compensate for radiation-related dentin alterations [2].
CLSM analysis supported these observations by demonstrating a dose-dependent reduction in both the depth and homogeneity of sealer penetration, which may reflect compromised micromechanical interlocking between the sealer and irradiated dentin. Rhodamine B at a concentration of 0.1% was selected due to its fluorescence stability and minimal interference with the physicochemical properties of the sealer [20]. Importantly, this concentration was adopted according to a previously validated protocol [20] demonstrating that 0.1% Rhodamine B does not significantly alter critical physicochemical properties, including flow and viscosity, of epoxy resin-based sealers, thereby preserving their penetration behavior. This concentration has been widely employed in endodontic research to enable reliable visualization of sealer penetration without significantly altering material performance [20].
CLSM was performed because it allows non-destructive optical sectioning, high-resolution imaging, and three-dimensional assessment of fluorescently labeled materials within dentinal tubules, making it one of the most suitable techniques for evaluating sealer penetration patterns [20, 30]. Nevertheless, CLSM is subject to inherent optical limitations, including the so-called “butterfly effect,” whereby fluorescence intensity and apparent penetration patterns can be influenced by a lower density of dentinal tubules mesiodistally [30]. To minimize such bias, imaging parameters (laser intensity, gain, pinhole size, and optical section thickness) were standardized for all specimens, and analyses were performed at consistent depths under blinded conditions.
Importantly, the qualitative CLSM findings were corroborated by SEM observations, which demonstrated reduced tag formation and interfacial adaptation in irradiated specimens. Therefore, although optical artifacts cannot be entirely eliminated [30], the convergence of CLSM and SEM findings strengthens the validity of the observed dose-dependent interfacial degradation. These limitations should be considered when extrapolating laboratory findings to clinical scenarios, but they do not invalidate the overall trend demonstrated in the present study.
While the present investigation focused on an epoxy resin-based sealer, it is relevant to consider how other sealer categories may behave under irradiated dentin conditions. Previous research [15] comparing an epoxy resin-based sealer and a calcium silicate-based sealer demonstrated that irradiation significantly reduced push-out bond strength for both materials, with the calcium silicate-based sealer showing lower bond strength values overall and a higher percentage of adhesive failures after radiation exposure. SEM also revealed increased interfacial gaps and reduced tag formation in irradiated specimens, particularly for the calcium silicate-based sealer [15]. These findings suggest that radiation-induced dentin alterations negatively affect sealer-dentin interaction regardless of material type; however, the magnitude and failure pattern may vary according to the material’s bonding mechanism [15]. Epoxy resin-based sealers primarily rely on micromechanical interlocking and potential chemical interaction with exposed collagen, whereas calcium silicate-based sealers depend more on biomineralization and interfacial apatite formation [15]. In substrates compromised by radiation-induced collagen disorganization and mineral imbalance, such differences in adhesion strategy may influence interfacial stability [15]. Nevertheless, direct material comparisons under identical experimental conditions are required before definitive conclusions can be drawn.
Recent investigations have reinforced the critical role of substrate condition in determining adhesive performance in endodontically treated teeth [31, 32]. Although those studies focused on coronal dentin and restorative adhesive strategies, their findings underscore how alterations in the mineral-organic balance and collagen integrity directly impact restorative materials-dentin interactions [31, 32]. In the present study, irrigation was performed using 2.5% sodium hypochlorite solution followed by 17% EDTA, which is widely considered a standard approach for smear layer removal and canal disinfection [33]. Sodium hypochlorite solution promotes collagen degradation and increased mineral exposure, while EDTA induces demineralization and partial tubular opening-substrate changes that may influence micromechanical interlocking [31]. However, unlike chemically induced modifications described in coronal dentin, the present findings reflect the additional impact of ionizing radiation on intraradicular dentin, suggesting a compounded alteration of the organic matrix and mineral framework. From a mechanistic standpoint, degradation pathways, including collagen destabilization, hydrolytic breakdown, and vulnerability of the hybridized interface, provide a conceptual framework to understand how radiation-induced structural disruption may further compromise endodontic sealer-dentin interaction [34]. Together, these considerations reinforce that adhesive performance in root canal systems is highly substrate-dependent and sensitive to both chemical and physical insults [31–34], although direct extrapolation between restorative adhesive systems and epoxy resin-based endodontic sealers should be made cautiously.
The influence of obturation technique on adhesive performance observed in the present study is consistent with previous micro-CT evidence indicating that different compaction approaches may affect the internal porosity and overall quality of root canal fillings [35]. In severely curved canals, manual compaction and the Auger technique (using NiTi rotary instruments for material compaction) demonstrated comparable total porosity, although open porosity predominated in both methods. These findings highlight that the mechanical aspects of material placement and condensation can influence void distribution within the filling mass. In the present investigation, Tagger’s hybrid technique consistently yielded higher bond strength values, which may be partially attributed to improved sealer distribution and interfacial adaptation, potentially minimizing gap formation and enhancing micromechanical interlocking with dentin [35].
Failure mode analysis revealed a predominance of cohesive failures within the sealer, regardless of radiation dose or obturation technique. This pattern suggests that, under the present experimental conditions, the adhesion to dentin may have exceeded the internal cohesive strength of the epoxy resin-based material. Such behavior may be related to intrinsic mechanical characteristics of AH Plus Jet, including its stiffness and potential brittleness under shear stress [15, 18, 36]. In addition, radiation-induced alterations in dentin structure may influence stress distribution at the adhesive interface, potentially shifting the locus of failure toward the endodontic sealer mass rather than the dentin substrate itself [18, 36].
The findings of this study should be interpreted in light of certain limitations. As an in vitro investigation, the experimental model does not fully replicate the complex biological, mechanical, and environmental conditions present in the oral cavity, including dynamic loading, pulpal pressure, and long-term aging effects [24]. Nevertheless, the controlled laboratory conditions allowed isolation of the variables under investigation, providing valuable insights into the effects of radiation therapy doses and obturation techniques on sealer-dentin interaction.
Gradual exposure to ionizing radiation at doses up to 70 Gy was associated with adverse effects on the evaluated outcomes. Although no statistically significant differences were detected among the radiation doses, exposure to 30 Gy was sufficient to reduce the bond strength of the sealer to root dentin. These findings indicate that radiation exposure, even at clinically relevant levels, may negatively influence dentin-sealer adhesion under controlled laboratory conditions. However, the results should be interpreted as mechanistic evidence of altered adhesive behavior rather than direct predictors of the durability of endodontic obturation. Further translational and clinical investigations are required to determine the extent to which these laboratory findings may be reflected in long-term clinical performance.
Conclusions
Radiation-induced changes in dentin were associated with reduced bond strength and decreased sealer penetration, indicating impairment of micromechanical interlocking and modification of the adhesive interface under laboratory conditions. The obturation technique remained a determinant of adhesive performance across all irradiation conditions, with Tagger’s hybrid technique consistently promoting improved sealer adaptation and penetration. Within the limitations of this in vitro design, these findings underscore the influence of substrate condition and obturation strategy on sealer-dentin interaction in irradiated teeth, without implying direct clinical outcomes.