Effect of final irrigation protocols on the bond strength and dentinal tubule penetration of root canal sealers: an ex vivo laboratory study

Abstract

Background

Effective disinfection and sealing of the root canal system are essential for the long-term success of endodontic treatment. Final irrigation solutions can influence the interactivity between dentin and sealers. This study aimed to evaluate the effect of different final irrigation protocols on the bond strength and dentinal tubule penetration of an epoxy resin-based and a bioceramic-based root canal sealers.

Methods

One hundred and twenty extracted single-rooted human teeth were prepared and randomly assigned to five final irrigation regimens: distilled water (DW), NaOCl + DW + EDTA, NaOCl + DW + EDTA + DW + OCT, NaOCl + DW + EDTA + DW + CHX, and NaOCl + DW + EDTA + DW + NaOCl. Teeth were obturated using a single-cone technique with either an epoxy resin-based or a bioceramic-based sealer. Push-out bond strength was measured using a universal testing machine, and dentinal tubule penetration was analyzed by confocal laser scanning microscopy. Data were analyzed with two-way analysis of variance and Tukey post hoc tests (p = 0.05).

Results

Both the type of sealer and the irrigation protocol significantly influenced bond strength, whereas only the sealer type affected dentinal tubule penetration. The epoxy resin-based sealer exhibited higher bond strength across all groups, while the bioceramic sealer demonstrated greater dentinal tubule penetration regardless of the irrigation protocol. Among the irrigation protocols NaOCl + EDTA and NaOCl + EDTA + CHX resulted in higher bond strength compared with distilled water. The inclusion of octenidine did not enhance adhesion or penetration.

Conclusions

The epoxy resin based sealer showed superior adhesion, whereas the bioceramic sealer achieved deeper dentinal tubule penetration. Final irrigation protocols influenced bond strength but did not affect tubular penetration. Octenidine showed no additional benefit under the tested conditions.

Background

The success of endodontic treatment relies on the effective disinfection, shaping, and three-dimensional obturation of the root canal system to prevent reinfection and promote periapical healing [1]. Root canal irrigation plays a pivotal role in endodontic treatment by facilitating the removal of microorganisms, debris and smear layer from areas that are inaccessible to mechanical instrumentation.

Various chemical agents have been used for root canal irrigation to enhance disinfection and tissue dissolution. Sodium hypochlorite (NaOCl) remains the most widely used irrigant because of its strong antimicrobial activity, ability to dissolve organic tissue, and effectiveness against biofilms [2, 3]. However, its collagen-degrading effect may negatively influence the adhesion of resin-based sealers to root dentin [4]. Chlorhexidine (CHX) is frequently used as an alternative or final irrigant due to its substantivity, prolonged antimicrobial action, and relatively low cytotoxicity, particularly in situations where apical extrusion is a concern [5,6]. Ethylenediaminetetraacetic acid (EDTA), a commonly used chelating agent, facilitates smear layer removal and alters the chemical and structural properties of dentin, which may affect sealer adhesion [7, 8].

Octenidine hydrochloride (OCT), a cationic bispyridine compound traditionally used as a mucosal antiseptic in the treatment of severe burns, has recently gained attention as an alternative endodontic irrigant due to its low cytotoxicity and broad-spectrum antimicrobial efficacy [9–11]. Previous studies have reported that OCT may exhibit superior antimicrobial activity compared to CHX [12, 13]. In addition, OCT has demonstrated potential in smear layer removal and improving dentinal tubule cleanliness, which may influence the interaction between the dentin surface and root canal sealers [11]. However the effects of OCT on the bond strength and tubular penetration of different types of sealers, particularly resin and bioceramic-based materials, remain largely unexplored.

Root canal sealers are used in combination with gutta-percha to achieve a hermetic seal within the root canal system [14, 15]. The ability of sealers to penetrate into dentinal tubules is clinically significant, as it enhances sealer adaptation and may contribute to the formation of a micromechanical interlocking system [16]. Tubular penetration may also help to eliminate residual bacteria and reduce the risk of reinfection by limiting bacterial colonization [17, 18]. Epoxy resin-based sealers are known for their favorable physical properties, apical sealing ability, and strong microretention to dentin [19]. Recently introduced bioceramic-based sealers consist of particles such as tricalcium silicate, dicalcium silicate, monobasic calcium phosphate, amorphous silicon dioxide, and tantalum pentoxide [20]. These sealers have shown good penetration into dentinal tubules, are minimally affected by intratubular moisture, and demonstrate minimal shrinkage [21].

Therefore, the aim of this study was to investigate the effects of different final irrigation protocols on the tubular penetration ability of bioceramic and resin-based root canal sealers and on the bond strength to root dentin.

The null hypotheses of this study were that different final irrigation protocols would not affect the push-out bond strength or dentinal tubule penetration of root canal sealers, and there would be no difference between epoxy resin-based and bioceramic-based sealers regarding these outcomes.

Methods

The manuscript of this laboratory study has been written according to Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021 guidelines. The experimental workflow is illustrated in Fig. 1 according to the PRILE guidelines.

Fig. 1.

PRILE 2021 flowchart summarising the ex vivo evaluation of the effect of different final irrigation protocols on the push-out bond strength and dentinal tubule penetration of epoxy resin and bioceramic-based root canal sealers

Sample selection and preparation

Sample size calculation was performed before the experiments using statistical software (GPower v.3.1, Düsseldorf, Germany). An alpha-type error of 0.05 and a beta power of 0.80 were assumed. The analysis calculated a total sample size of 120 specimens. This study was approved by the Hacettepe University Health Health Sciences Research Ethics Committee (Approval number: GO 22/197)). A total of 120 freshly extracted, single-rooted human teeth with fully formed apices and single round canals were collected from patients requiring extraction due to periodontal or deep carious lesions. The extracted teeth were obtained from adult patients aged 18–60 years. Teeth with open apices, root caries, cracks, or resorptions were excluded. Periapical radiographs were taken from different angles to confirm the single canal anatomy. The crowns were removed using a diamond disc under water cooling, and root lengths were standardized to 15 mm. Working length was determined by subtracting 0.5 mm from the canal length, which was initially confirmed with a size #15 K-file. Root canals were shaped using the ProTaper rotary file system (Dentsply Sirona, Ballaigues, Switzerland) up to size F4 using an endomotor (Dentsply Sirona, Ballaigues, Switzerland). After standardization of root length and canal preparation, specimens were randomly allocated to the experimental groups according to sealer type and final irrigation protocol. The samples were first randomly divided into two main groups (n = 60) according to the type of root canal sealer used: epoxy resin-based or bioceramic-based. Each main group was further divided into five subgroups (n = 12) based on the final irrigation protocol (Table 1).

Table 1.

Final irrigation protocols applied in the experimental groups

Group	Final irrigation protocol
G1	Distilled water (DW), 5 mL for 2 min
G2	5.25% NaOCl (5 mL, 2 min) + DW + 17% EDTA (5 mL, 2 min)
G3	5.25% NaOCl (5 mL, 2 min) + DW + 17% EDTA (5 mL, 2 min) + DW + 0.1% OCT (5 mL, 2 min)
G4	5.25% NaOCl (5 mL, 2 min) + DW + 17% EDTA (5 mL, 2 min) + DW + 2% CHX (5 mL, 2 min)
G5	5.25% NaOCl (5 mL, 2 min) + DW + 17% EDTA (5 mL, 2 min) + DW + 5.25% NaOCl (5 mL, 2 min)

Distilled water (3 mL) was used as an intermediate flush between irrigants to prevent chemical interactions

The irrigation protocols included 5.25% sodium hypochlorite and 17% EDTA (Promida Co., Eskisehir, Turkey), 0.1% octenidine hydrochloride (Octenisept, Schülke & Mayr GmbH, Norderstedt, Germany), 2% chlorhexidine (Microvem, Istanbul, Turkey), and distilled water.

All irrigants were delivered using conventional syringe and needle irrigation. Each solution was applied in a volume of 5 mL for 2 min with intermediate flushing using 3 mL of distilled water between irrigants to prevent chemical interactions.

All canals were finally irrigated with 5 mL of DW for 1 min and dried with paper points (Sure-Endo, Sure Dent Corp., Gyeonggi-do, Republic of Korea). The exact sequence, volume, and application time of irrigants for each group are summarized in Table 1. The root canals were obturated with gutta-percha Sure-Endo, Sure Dent Corp., Gyeonggi-do, Republic of Korea) and either an epoxy resin-based sealer (AH Plus, Dentsply Sirona, Charlotte, NC, USA) or a bioceramic-based sealer (AH Plus Bioceramic Sealer; Dentsply Sirona, Charlotte, NC, USA) using single cone technique. The sealers were mixed with 0.1% Rhodamine B fluorescent dye (Sigma-Aldrich, St. Louis, MO, USA) before the obturation. Excess gutta-percha was removed with a heated plugger. Samples were stored at 37 °C and 100% relative humidity for 1 week to allow complete setting of the sealers before testing. Each root was embedded in epoxy resin (Meliodent, Kulzer GmbH, Hanau, Germany) using a custom-fabricated split copper mold. Once the resin had fully set, horizontal sectioning was performed at 5 mm coronally from the root apex. Two horizontal slices each measuring 1 mm in thickness representing the middle third of the root was obtained using a water-cooled precision cutting machine (Isomet 2000; Buehler, Lake Bluff, IL, USA).

Push-out bond strength test

One slice of each sample was used to measure push-out bond strength by applying force in an apical-to-coronal direction using a universal testing machine (Instron, Canton, MA, USA) until bond failure occurred. Samples were secured to a metal holder with the coronal surface positioned downward. A vertical compressive load was applied in an apicocoronal direction using a flat metal plunger (0.6–0.8 mm in diameter). Plunger diameter was selected according to the canal diameter of each specimen to ensure contact exclusively with the filling material and to avoid stress transmission to the canal walls. The load was applied at a crosshead speed of 1 mm/min, and the peak force (in Newtons) at the point of dislodgement was recorded. The push-out bond strength (MPa) was calculated using the formula:

The bonded area was calculated using the equation:

where π = 3.14, R is the coronal radius, r is the apical radius, and h is the slice thickness.

Confocal laser scanning microscopic analysis

Confocal laser scanning microscopy (CLSM Pascal; Carl Zeiss, Jena, Germany) was used to evaluate the depth of sealer penetration into dentinal tubules. The specimens were mounted on microscope slides, and images were obtained using a confocal laser scanning microscope (CLSM; LSM Pascal, Carl Zeiss, Jena, Germany). To assess the extent of sealer penetration, measurements were performed at 200 μm from the root canal wall toward the cementum. The perimeter of the penetrated sealer was outlined using specialized software (LSM Image Examiner, Carl Zeiss, Germany) (Fig. 2). The sealer penetration percentage for each depth level was then calculated using the following formula.

Fig. 2.

CLSM image illustrating the method used to calculate sealer penetration. Penetrated areas were outlined at 200 μm from the canal wall toward the cementum, and the percentage of dentinal tubule penetration was determined accordingly

Statistical analysis

Statistical analysis was performed using SPSS (IBM SPSS Statistics). The Shapiro–Wilk test was used to assess data normality. Data was statistically analysed using two-way ANOVA and Tukey test (p = 0.05).

Results

Two-way ANOVA revealed significant effects of the sealer type (p < 0.001) and irrigation protocol (p = 0.014) on push-out bond strength, while the interaction between these factors was not statistically significant (p = 0.072) (Table 2). Post-hoc Tukey tests indicated that the DW group showed significantly lower bond strength compared with NaOCl + EDTA group (p = 0.001) and NaOCl + EDTA + CHX group (p = 0.014). No other pairwise comparisons among irrigation protocols demonstrated statistically significant differences (p > 0.05) (Table 2).

Table 2.

Push-out bond strength values (mean ± SD, MPa) of sealers according to irrigation protocol

Irrigation protocol	AH Plus	AH Plus Bioceramic
DW	4.29 ± 1.00ᵇ	3.26 ± 0.39ᵇ
NaOCl + EDTA	8.18 ± 2.63ᵃ	4.14 ± 0.69ᵃᵇ
NaOCl + EDTA + 0.1% OCT	5.91 ± 2.39ᵃᵇ	4.36 ± 1.41ᵃᵇ
NaOCl + EDTA + CHX	6.36 ± 1.86ᵃ	5.76 ± 1.88ᵃ
NaOCl + EDTA + NaOCl	6.70 ± 1.96ᵃᵇ	3.78 ± 2.06ᵃᵇ

* Different superscript letters within the same column indicate significant differences among irrigation protocols (p < 0.05). Across all irrigation protocols, AH Plus exhibited significantly higher push-out bond strength than AH Plus Bioceramic (p < 0.001)

For dentinal tubule penetration percentage, the two-way ANOVA demonstrated a significant effect of the sealer type (p < 0.001), whereas the irrigation protocol (p = 0.257) and the sealer × irrigation interaction (p = 0.396) were not significant (Fig. 3). AH Plus Bioceramic sealer showed significantly higher penetration percentages than AH Plus sealer across all irrigation protocols (Table 3) (Fig. 3). No statistically significant differences in penetration were detected among the irrigation protocols within the same sealer group (p > 0.05).

Fig. 3.

Representative CLSM images showing dentinal tubule penetration of AH Plus and AH Plus Bioceramic sealers under all final irrigation protocols

Table 3.

Dentinal tubule penetration percentage (mean ± SD) of sealers according to irrigation protocol

Irrigation protocol	AH Plus	AH Plus Bioceramic
DW	0.359 ± 0.180a	0.678 ± 0.201b
NaOCl + EDTA	0.538 ± 0.149a	0.702 ± 0.260b
NaOCl + EDTA + 0.1% OCT	0.500 ± 0.136a	0.721 ± 0.183b
NaOCl + EDTA + CHX	0.607 ± 0.309a	0.631 ± 0.220b
NaOCl + EDTA + NaOCl	0.585 ± 0.155a	0.772 ± 0.216b

* Different superscript letters within the same row indicate significant differences between the sealers (p < 0.05)

Discussion

This ex vivo study evaluated the effect of different final irrigation protocols on sealer dentin interaction using push-out bond strength and dentinal tubule penetration as outcome measures. These two parameters reflect different aspects of sealer performance and should not be considered interchangeable. Bond strength primarily represents the resistance of the sealer-dentin interface to dislodgement forces, whereas dentinal tubule penetration is mainly influenced by sealer flow properties and dentin permeability. Therefore, the influence of irrigation protocols and sealer type on these outcomes should be interpreted within their distinct underlying mechanisms. The present findings revealed that sealer type and irrigation solution significantly affected bond strength, whereas only the sealer type influenced dentinal tubule penetration.

The superior bond strength of AH Plus observed in this study is in agreement with earlier reports that highlight the strong adhesion of epoxy resin-based sealers to dentin [22]. This property is attributed to covalent bonding between epoxide rings and amino groups in dentin collagen, as well as micromechanical interlocking with partially demineralized dentin [8, 23]. In contrast, bioceramic sealers rely on calcium ion release and subsequent hydroxyapatite formation for bonding, which enhances long-term sealing but may not provide the same immediate dislodgement resistance [21]. This mechanistic difference explains the consistently higher push-out bond strength values for the epoxy resin-based sealer.

The present results also confirmed the beneficial role of chelating agents. NaOCl followed by EDTA produced significantly higher bond strength than DW. According to previous studies, EDTA effectively removes the smear layer, exposes dentin collagen fibrils, and increases the surface area available for sealer infiltration [23, 24]. The present data reinforce that EDTA remains a reliable choice for optimizing the adhesion of both sealer types. It also should be noted that the duration of EDTA application may affect dentin demineralization and consequently sealer-dentin interaction, which should be considered when interpreting the present findings.

Chlorhexidine as a final irrigant also resulted in significantly higher bond strength compared to DW. This finding aligns with the results of Prado et al. (2013), who reported improved adhesion of AH Plus following CHX irrigation [25]. The mechanism has been attributed to the ability of CHX to inhibit dentinal collagen degradation by matrix metalloproteinases, thereby preserving the bonding substrate. Razmi et al. (2016) further demonstrated that CHX may enhance the stability of adhesive interfaces, although its effects on bioceramic sealers remain inconsistent [26]. In the present study, CHX enhanced bond strength for AH Plus, whereas the improvement for the bioceramic sealer was less pronounced. Although OCT has shown superior antimicrobial and cleaning activity in vitro [11, 27], this benefit did not translate into improved adhesion in the current study.

The middle third of the root was selected for dentinal tubule penetration analysis because it provides a more standardized and reproducible region with relatively uniform tubule density and diameter compared with the coronal and apical thirds. However dentinal tubule density and diameter are known to vary significantly along the root length, with a marked reduction toward the apical third [28] This may influence sealer penetration and bonding behavior. Therefore the results should be interpreted within this anatomical limitation. Based on the present findings, AH Plus Bioceramic exhibited significantly greater dentinal tubule penetration than AH Plus, independent of the irrigation protocol. These results suggest that the inherent physicochemical properties of the bioceramic sealer such as nanoparticle size, hydrophilicity, and favorable flow characteristics are the primary determinants of its superior tubular penetration, rather than the choice of final irrigant. Previous studies have frequently emphasized the role of irrigation solutions and activation methods in enhancing sealer penetration. Chelating agents such as EDTA, maleic acid, and citric acid have been reported to increase penetration by removing the smear layer and enlarging dentinal tubule orifices [29]. Similarly, CHX following chelation has been suggested to improve penetration [30]. The discrepancy between the present findings and earlier studies may be attributed to methodological variations, such as differences in irrigant concentration, activation protocols, or regional analysis of tubule penetration. Importantly, our results underscore that when standardized irrigation without activation is applied, the material properties outweigh the influence of irrigant type. Additionally, penetration was evaluated at the middle third of the root, where tubule density and diameter are relatively larger, potentially reducing the sensitivity to irrigant related differences that may be more evident in the apical third. This highlights that while irrigation strategies can play a role under certain conditions, the intrinsic formulation of the sealer remains the dominant factor for penetration.

OCT has attracted attention as an alternative endodontic irrigant due to its broad-spectrum antimicrobial efficacy, ability to disrupt biofilms, and relatively low cytotoxicity compared with NaOCl [9, 10]. Furthermore, OCT has been proposed as a safer option due to its lower tissue toxicity profile [27]. However, despite these promising antimicrobial properties, the present study did not demonstrate any improvement in dentinal tubule penetration when OCT was used as the final irrigant. This is consistent with previous observations that OCT alone has limited smear layer removal capacity [11] and may therefore fail to adequately modify the dentin substrate for enhanced sealer bonding. Although OCT can enhance dentin cleanliness at a microscopic level, this effect did not translate into improved adhesion in the current study. The lack of improvement observed with octenidine hydrochloride may be related to the irrigation sequence used in the present study. OCT was applied after EDTA and followed by a final distilled water flush, which may have reduced its interaction with the dentin surface. As OCT does not exhibit strong chelating properties, its potential effects on dentin sealer interaction may be limited when applied after smear layer removal and subsequent rinsing. Further investigations are warranted to clarify whether combining OCT with chelating agents or using it with irrigant activation techniques may improve its impact on the adhesive interface.

The findings of the present study support the notion that penetration and adhesion represent related but distinct phenomena. Similarly reported that although smear layer removal increased the penetration of certain bioceramic sealers, this did not consistently translate into higher bond strength, emphasizing that tubular penetration alone may have a limited impact on sealer retention [31].

The present ex-vivo study has certain limitations that should be considered when interpreting the results. Age related variations in dentinal tubule morphology and sclerosis represent an inherent limitation of ex vivo studies. Although a defined adult age range was used, such variations may have influenced sealer penetration and bonding behavior, and the results should be interpreted accordingly. Moreover, penetration in the apical third where tubule density is lower but sealing ability is clinically critical was not assessed. Therefore, the outcomes should be regarded as specific to the materials and conditions tested, and further studies are needed to confirm their applicability to other sealers and clinical situations.

Conclusions

Within the limitations of this study, AH Plus demonstrated higher bond strength, whereas AH Plus Bioceramic showed superior dentinal tubule penetration. Final irrigation protocols, including EDTA and CHX, influenced bond strength, but none of the irrigation protocols affected tubular penetration. The role of OCT in enhancing adhesion requires further investigation.

Abbreviations

CHX

Chlorhexidine

CLSM

Confocal Laser Scanning Microscopy

DW

Distilled Water

EDTA

Ethylenediaminetetraacetic Acid

NaOCl

Sodium Hypochlorite

OCT

Octenidine Hydrochloride

Authors’ contributions

AK and SKE conceived and designed the study. AK, SAÖ, NS, and GAY performed the experimental procedures and data acquisition. SAÖ and SKE conducted data analysis, interpretation, and manuscript preparation. All authors read and approved the final manuscript and agree to be accountable for its contents.

Funding

This study was supported by the Hacettepe University Scientific Research Projects Coordination Unit (HÜ-BAP) under project code THD-2023-20689.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the Hacettepe University Health Sciences Research Ethics Committee (Approval No: GO 22/197). This study was conducted in accordance with the Declaration of Helsinki. Extracted human teeth were obtained with informed consent from donors in accordance with institutional ethical standards. No experiments involving animals were conducted.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.