Comparative analysis of dentinal tubule penetration: effects of irrigation activation methods and root canal sealers: in-vitro study

Abstract

Background

To seal the entire root canal system is the main objective of root canal treatment. While obturating the main root canal, sealing the dentinal tubules that are known to open to the root canal system plays an important role. Present study investigated the dentine tubule penetration of different root canal sealers combined with different irrigation activation methods using an in-vitro model.

Methods

The root canal preperation of the 144 samples was completed using the Ni-Ti rotary K7 system. All samples were divided into 3 groups (n = 48) according to irrigation activation(sonic activation, passive ultrasonic activation and needle activation). Activation groups were divided into 4 subgroups (n = 12) according to the root canal sealer used (GuttaFlow, Bioroot RCS, AH Plus Bio, AH Plus Jet). Horizontal sections taken at the 6 mm level from the apex were examined with CLSM to evaluate the maximum penetration depth and penetration percentage. The normal distribution of numerical variables was assessed using the Shapiro-Wilk normality test. The homogeneity of variances among groups was analyzed with the Levene test. Comparisons for log penetration depth and penetration percentage were made using two-way analysis of variance.

Results

In terms of penetration area, sonic activation showed significantly higher values compared to needle activation, while ultrasonic activation did not differ significantly from these two methods (p = 0.048). The Bioroot RCS group had a significantly higher penetration area percentage compared to the GuttaFlow group, with no significant differences among the other sealer groups (p = 0.017). In the AH Plus Jet group, the maximum penetration depth with ultrasonic activation was found to be significantly higher compared to needle activation (p = 0.036).

Conclusion

It was found that sonic activation and BioRoot RCS were superior in dentinal tubule penetration compared to other groups. All the groups have demonstrated penetration. It is believed that effective dentinal tubule penetration can be achieved with continuous irrigation, properly applied activation systems, and adherence to manufacturer instructions when using root canal sealers.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12903-025-06419-y.

Background

One of the aims of root canal treatment is to prevent reinfection by inhibiting the proliferation of residual microorganisms and the entry of external microorganisms into the root canal system [1]. Studies have demonstrated that in cases of root canal infections, microorganisms are present not only in the root canal lumen but also within the dentinal tubules, adding to the complexity of endodontic infections [2, 3]. The penetration of root canal sealers into the dentinal tubules is crucial for inactivating residual microorganisms that have infiltrated the tubules [1, 4]. This enhanced penetration positively impacts the treatment prognosis by increasing the overall sealing efficacy [5]. The two main factors influencing the penetration of root canal sealers into the dentinal tubules are the presence of the empty dentinal tubules and the physicochemical properties of the root canal sealers.

The smear layer formed as a result of mechanical preparation on the root canal walls affects the sealer penetration into the dentinal tubules [6]. This drawback can be neutralized by successfully removing the smear layer, necrotic tissue residues, and microorganisms inside the dentinal tubules with the appropriate irrigation and irrigation activation techniques [7]. The efficacy of chemical irrigation is enhanced by the use of irrigation activation techniques in narrow and thin isthmuses and lateral root canals that cannot be cleaned mechanically. But it remains unclear which activation method is superior.

As for the root canal sealers, their penetration into the dentinal tubules depends on their physical and chemical properties, including surface tension, viscosity, solubility, and particle size [7]. The dental market currently offers a wide variety of root canal sealers. Each one has unique physical, chemical, and biological properties. These properties are made to meet the needs of dental professionals. One of the novel products launched into the market is AH Plus Bioceramic (Dentsply Sirona USA) which contains tricalcium silicate and dimethyl sulfoxide as filler. The absence of dicalcium silicate in AH Plus Bioceramic results in a lower calcium silicate ratio compared to other pastes. Another example of a root canal sealer is Guttaflow Bioseal (Coltene, Langeneu, Germany), which combines a canal sealer and gutta-percha in one product. It consists of ground gutta-percha incorporated into a polydimethylsiloxane matrix. This formulation is reported to provide high radiopacity and biocompatibility. Bioroot RCS (Septodont, Saint-Maur-des-Fossés, France) is a calcium silicate-based material consisting of a powder and liquid structure. It is a bioactive material with antimicrobial properties due to the release of calcium hydroxide. The manufacturer reports that this root canal sealer does not exhibit polymerization shrinkage.

Among the various available options, AH Plus, a resin-based sealer (Dentsply De Trey, Konstanz, Germany), is widely regarded as the gold standard for comparing root canal sealers due to its high radiopacity, biocompatibility, and favorable fluidity. This sealer has been widely used in numerous studies and is a preferred choice among clinicians. AH Plus Jet, a newer product based on the established AH Plus sealer, retains the same chemical composition, offering similar advantages for clinical applications. AH Plus Jet is available as an auto-mixing syringe equipped with disposable tips, designed to minimize operator-induced variability. This feature ensures a consistent mix, enhancing the ease of application and maintaining the desired material properties throughout the procedure [8].

Various microscopy techniques are available for evaluating root canal sealer penetration. Among these, confocal laser scanning microscopy (CLSM) is particularly effective for assessing dentinal tubule penetration [9]. It offers standardized and detailed imaging at lower magnifications (10x) through the use of a fluorescent dye [10, 11].

Fewer studies have examined the impact of different irrigation activation methods on various root canal sealers. Therefore, this study aims to compare the penetration of several types of root canal sealers, including GuttaFlow, BioRoot RCS, AH Plus Bio, AH Plus Jet, and after activation with sonic and passive ultrasonic, and needle activation techniques using confocal laser scanning microscopy (CLSM). The null hypothesis is that there will be no difference in dentinal tubule penetration depth among the four root canal sealers with (1) sonic activation, (2) passive ultrasonic activation, and (3) needle activation techniques.

Methods

The study protocol was approved by the local ethics committee. The manuscript of this laboratory study has been written according to Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021 guidelines. A power analysis was conducted based on data from a previous study [12]using variance analysis in G*Power 3.1 software (Heinrich Heine University, Düsseldorf, Germany). The calculation indicated a minimum sample size of 10 per group (α = 0.05, β = 0.95). To account for potential sample loss, 12 samples per group were deemed sufficient.

One hundred forty-four extracted mandibular premolars with closed root apices and a single root canal were selected for the study. The teeth were shortened under water cooling to standardize the root length to 13 mm. Apical patency was established with a size 10 K-file (Dentsply Sirona, York, PA), and the working length was set 1 mm short of the apical foramen.

Root canal preparation was performed using the X-smart Plus endodontic motor (Dentsply Sirona, Ballaigues, Switzerland) and a Ni-Ti rotary K7 system (ScopeEndo) following the manufacturer’s instructions (#10.04, #15.04, #20.04, #25.06, #30.06, #35.06, #40.06). Canals were irrigated with 2 mL of 2.5% sodium hypochlorite (NaOCl) between each file. Final irrigation was completed with 2 mL of 17% EDTA followed by 2 mL of 2.5% NaOCl. Samples were embedded in floral foam sponges during the irrigation process to ensure retention of the irrigation solution within the canal. Subsequently, the samples were categorized into three main irrigation activation groups (n = 48) (Fig. 1).

Fig. 1.

Figure shows that study groups

Group 1: Sonic Activation (SA): The EndoActivator (Dentsply, New York, USA) was operated at a frequency of 167 Hz using a size 25.04 polymer tip. A total of 5 mL of 2.5% NaOCl was delivered into the canal, followed by irrigation activation with the EndoActivator for 20 s per cycle, repeated for three cycles (totaling 15 mL of NaOCl and 1 min of activation), employing a back-and-forth motion.

Group 2: Passive Ultrasonic Activation (PUA): The Ultra X device (Eighteeth, Changzhou City, China) was operated with an ISO #25 tip size at a frequency of 30 kHz. A total of 5 mL of 2.5% (NaOCl) was introduced into the canal, followed by irrigation activation using the PUA tip positioned 1–3 mm behind the working length. Activation was performed using back-and-forth movements for 20 s over three cycles (totaling 15 mL of NaOCl and 1 min of activation).

Group 3: Needle Activation (NA): A 27-G side-perforated irrigation needle (NaviTip; Ultradent, South Jordan, UT) was utilized to deliver a total volume of 15 mL of 2.5% NaOCl through back-and-forth movements.

All samples were irrigated with 1 mL of distilled water, and activation was performed similarly within each group following 2 mL of 17% EDTA irrigation. After the irrigation activation procedures, the root canals were irrigated again with 1 mL of distilled water and dried using paper points. Each irrigation activation group was further divided into four subgroups (n = 12) for the application of different root canal sealers: GuttaFlow (Coltene, Langeneu, Germany), BioRoot RCS (Septodont, Saint-Maur-des-Fossés, France), AH Plus Bio (Dentsply Sirona, New York, USA) and AH Plus Jet (Dentsply De Trey, Konstanz, Germany). Fluorescein C dye (0.1%) was mixed with the sealers, and a standardized volume of 0.1 cc of the Fluorescein-Sealer mixture was placed in each canal. Root canal obturation was performed using the lateral compaction technique with a #40.06 master gutta-percha cone and ISO 0.02 taper accessory cones (Diadent, Korea). Excess gutta-percha was removed to a depth of 1 mm below the canal orifice using a heated instrument, and the orifice was subsequently sealed with a temporary filling material (Cavit, 3 M ESPE, Germany). To allow for the proper setting of the sealant, the samples were maintained in a dark environment at 100% humidity for one week.

The samples were horizontally sectioned at a 6-mm level from the apex using a low-speed saw (IsoMet; Buechler, Lake Bluff, IL, USA) under water cooling, resulting in sections with a thickness of 1 mm. The coronal surfaces of these sections were polished and smoothed using 600, 800, and 1200 grit water sandpapers, respectively. The samples were then examined using a CLSM, and the resulting images were analyzed with ImageJ image processing software (version 1.48, National Institutes of Health, Wisconsin, USA) to measure the maximum penetration depth (MPD) and the penetration area (PA). The PA was calculated using the formula outlined in the study by Keleş et al. [13] [(penetration area / total root canal area) x 100].

Statistical analysis

Data were evaluated using IBM SPSS Statistics Standard Concurrent User Version 29 (IBM Corp., Armonk, New York, USA). Descriptive statistics are presented as mean ± standard error. The normality of numerical variables was assessed using the Shapiro-Wilk normality test, while the homogeneity of variances among groups was analyzed with the Levene test. For MPD that did not exhibit a normal distribution, a base-10 logarithmic transformation was applied. Comparisons for log-transformed MPD and PA were conducted using two-way analysis of variance. Bonferroni correction was applied to all pairwise comparisons, and a p-value of < 0.05 was considered statistically significant.

Results

Figure 2 illustrates the CLSM images of the experimental groups.

Fig. 2.

Confocal laser microscope images of groups. A: Sonic activation/GuttaFlow B: Sonic activation/Bioroot RCS C: Sonic activation/AH Plus BIO D: Sonic activation/AH Plus E: Neddle activation/GuttaFlow F: Neddle activation/Bioroot RCS G: Neddle activation/AH Plus BIO H: Neddle activation/AH Plus I: Ultrasonic activation/GuttaFlow J: Ultrasonic activation/Bioroot RCS K: Ultrasonic activation/AH Plus BIO L: Ultrasonic activation/AH Plus (Red lines show penetration area)

Evaluation of maximum penetration depth

Analyzing the MPD considering only the irrigation activation methods or root canal sealers, no significant differences were observed (Table 1). Pairwise comparisons among activation methods revealed no significant differences in MPD across the different root canal sealers for sonic, passive ultrasonic and needle activation method. When each activation method is analyzed independently, there is no statistical difference in MPD between different root canal sealer groups (p = 0.396, 0.093, and 0.123 for SA, PUA, and NA respectively). Within the sealer groups, only AH Plus Jet showed statistical differences regarding MPD (p = 0.036). In the AH Plus Jet group, the passive ultrasonic activation method demonstrated a significantly higher MPD compared to needle activation. The MPD values for the sonic activation in the AH Plus Jet group were not statistically different from those of the other two methods (Table 2).

Table 1.

This table show that MPD and PA between main group. The data is provided as mean (standard error). F and p values for each group are presented. Comparisons were made on logarithm transformed to base 10 data. F and p values for each group are presented. Superscripts a and b indicate differences between methods. There is no statistically significant difference between methods containing the same superscripts

		MPD	PA
Comparison of activation methods
Sonic Activation		162.31 ± 17.83	0.558 ± 0.035a
Passive Ultrasonic Activation		155.18 ± 18.03	0.456 ± 0.035ab
Neddle Activation		125.14 ± 18.03	0.447 ± 0.035b
	F; p	2.051; 0.133	3.112; 0.048
Comparison of root canal sealers
GuttaFlow		141.27 ± 20.58	0.404 ± 0.040b
Bioroot RCS		179.91 ± 20.58	0.586 ± 0.040a
AH Plus BIO		143.13 ± 20.58	0.495 ± 0.040ab
AH Plus Jet		125.84 ± 21.20	0.464 ± 0.042ab
	F; p	1.440; 0.234	3.523; 0.017

Table 2.

This table show that MDP between group. The data is provided as mean (standard error). Comparisons were made on logarithm transformed to base 10 data. F and p values for each group are presented. Superscripts a and b indicate differences between methods. There is no statistically significant difference between methods containing the same superscripts

Pairwise comparison of maximum penetration depth (MPD) (µm mean. Std. Err)
	SA	PUA	NA	F; p
GuttaFlow	180.02 ± 35.66	103.02 ± 35.66	140.77 ± 35.66	2.804; 0.064
Bioroot RCS	229.79 ± 35.66	162.71 ± 35.66	147.25 ± 35.66	0.687; 0.505
AH Plus BIO	124.89 ± 35.66	168.82 ± 35.66	135.71 ± 35.66	0.693; 0.502
AH Plus Jet	114.53 ± 35.66ab	186.15 ± 37.24a	76.84 ± 37.24b	3.416; 0.036
F; p	0.998; 0.396	2.182; 0.093	1.963; 0.123

Evaluation of penetration area

Significant differences were observed in PA between the irrigation activation methods (p = 0.048) and among the root canal sealers (p = 0.017). The PA in the sonic activation group were statistically higher compared to needle activation. However, the passive ultrasonic activation method did not show significant differences from the other two methods. Additionally, the PA in the BioRoot RCS group was statistically higher compared to the GuttaFlow group, while no statistically significant differences were found among the other groups (Table 1). The analysis of each activation method revealed no statistically significant difference in the level of PA between the various root canal sealers (p = 0.362, 0.459, and 0.052 for SA, PUA, and NA respectively). Similarly, when each root canal sealers were analyzed separately, no statistically significant difference in PA was observed between the different activation groups; GuttaFlow, Bioroot RCS, AH Plus BIO, AH Plus Jet, respectively (p = 0.265; p = 0.296; p = 0.691; p = 0.177) (Table 3).

Table 3.

This table show that PA between subgroup. The data is provided as mean (standard error). F and p values for each group are presented

Pairwise comparison of penetration area(PA) (mean. Std. Err)
	SA	PUA	NA	F; p
GuttaFlow	0.497 ± 0.07	0.365 ± 0.070	0.349 ± 0.07	1.342; 0.265
Bioroot RCS	0.663 ± 0.07	0.508 ± 0.070	0.585 ± 0.07	1.229; 0.296
AH Plus BIO	0.536 ± 0.07	0.451 ± 0.070	0.498 ± 0.070	0.371; 0.691
AH Plus Jet	0.538 ± 0.07	0.499 ± 0.073	0.356 ± 0.073	1.754; 0.177
F; p	1.076; 0.362	0.870; 0.459	2.639; 0.052

Discussion

The varied physical and chemical properties of sealers [14] and the different irrigant activation or agitation system [15, 16] used during the irrigation procedure can influence the penetration depth of root canal sealers into the dentin tubules. Consequently, this study aimed to compare the effects of different activation methods—sonic activation, passive ultrasonic activation and needle activation—on the penetration of four different sealers into dentin tubules. The results of the present study indicated significant differences in the PA of the sealers among all experimental groups; thus, the null hypothesis tested in this study can be rejected. The present study investigates the combined effects of root canal sealer types and irrigation activation techniques, which are widely used in clinical practice, on MPD and PA. Conducting an in vitro evaluation of the combined effect will facilitate clinicians’ development of a roadmap to increase clinical success.

According to the literature, the number of infected tubules and the depth of bacterial penetration into the dentinal tubules due to pulpal infection are highly variable [2, 3]. While it is recognized that effective instrumentation and irrigation procedures play a crucial role in reducing the bacterial load in root canals [17]the penetration of root canal sealers into dentinal tubules is also essential for suppressing residual bacteria that may remain within the tubules [18, 19]. Furthermore, the penetration of root canal sealers into dentin tubules enhances the bonding between the sealer and dentin, thereby decreasing microleakage, preventing reinfection, and positively impacting the overall prognosis [5, 7]. Various factors influence the extent of penetration, including the effectiveness of smear layer removal, the diameter of dentinal tubules, the physicochemical properties of the root canal sealers, surface activity, and the contact angle with dentin [7, 20].

To date, parameters such as maximum penetration depth, average penetration depth, and penetration area have been used to evaluate the penetration of sealers into dentinal tubules. The literature suggests that clinical significance extends beyond MPD alone, highlighting the importance of circumferential penetration rate relative to other parameters [20–22]. Therefore, in our study, the percentage of circumferential penetration around the root canal was calculated as a penetration area (PA) in addition to measuring the maximum penetration depth (MPD).

The penetration of root canal sealer into dentinal tubules also depends on the diameter of the tubules [23]. This diameter is generally smaller in the apical region due to the presence of sclerotic dentin, which may limit sealer penetration. Conversely, sealer penetration tends to be higher in the coronal region, which is more exposed to instrumentation and irrigation during chemo-mechanical disinfection procedure [20, 24]. Therefore, in our study, evaluation was conducted on sections taken from the middle third region (6 mm from the apical to coronal).

The primary aim of irrigation activation procedures is to enhance the efficacy of the irrigation solution without increasing its toxicity, ensuring effective smear layer removal and thorough disinfection in areas that cannot be mechanically cleaned, such as the isthmus and lateral canals [21, 25]. The smear layer, which obstructs the dentinal tubules, significantly impacts tubule penetration. Studies indicate that the use of activation devices in root canals enhances smear layer removal compared to canals without activation, regardless of activation duration [26, 27]. Moreover, multiple studies indicate no statistically significant differences in smear layer removal, dentinal tubule penetration depth, or the penetration area when comparing sonic and passive ultrasonic activation of irrigation solutions during root canal disinfection [28–32].

In our study, the sonic activation method showed a significantly higher PA for root canal sealers compared to needle activation, while the passive ultrasonic activation method did not demonstrate a significant difference from the other methods. Wigler et al. [33, 34] also found needle activation to be the least effective, with no significant difference compared to the passive ultrasonic activation system, aligning with our findings. Conversely, other studies [29, 35] comparing needle activation with activation systems reported that needle activation was associated with significantly less smear layer removal and reduced dentinal tubule penetration.

In this context, it is hypothesized that narrow root canals may restrict the oscillation of ultrasonic tips, and that during oscillation, contact between the dentin wall and metal tips could produce a new smear layer, even when non-cutting tips are used [33, 34]. This effect could impair the cleaning of dentinal tubules. In our study, needle activation resulted in a lower MPD, potentially indicating less smear removal; however, this difference was not statistically significant. This outcome may be related to the continuous characteristic of needle activation, unlike other methods, as well as the high concentration of solutions used in this study (5.25% NaOCl and 17% EDTA).

Comparing to the studies that used the similar methodology of CLSM, two studies [32, 36] have found no difference between resin based and bioceramic sealers on MPD and PA. One of the studies [36] showed no difference between conventional irrigation and passive ultrasonic irrigation, and the other one [32] found sonic and passive ultrasonic activation to have similar MPD and PA while both have demonstrated laser driven methods to have superior MPD and PA values. Unlike these studies, our study did not utilize laser devices to compare basic clinical situations.

Root canal sealers containing calcium silicate, such as AH Plus Bio and BioRoot RCS, have gained prominence in endodontic practice in recent years due to their bioactive properties [37, 38]. Upon contact with dentinal fluid, these sealers facilitate biomineralization within the dentinal tubules owing to their hydrophilic characteristics, thereby forming a plug that effectively prevents microleakage [38]. GuttaFlow, which incorporates a silicone base and contains ground gutta-percha particles with a particle size of approximately 30 microns, raises concerns regarding its ability to penetrate dentin. Specifically, the increased viscosity of GuttaFlow relative to other root canal sealers may limit its infiltration into the dentinal tubules. However, due to its thixotropic properties, the viscosity of GuttaFlow decreases under pressure, enhancing its penetration capabilities. The study’s results that BioRoot RCS demonstrated the highest PA, whereas GuttaFlow exhibited the lowest, with both results achieving statistical significance. Consistent with previous studies [39, 40]the penetration levels of AH Plus Jet and AH Plus Bio were found to be comparable to those of BioRoot RCS and GuttaFlow.

Calcium silicate-based root canal sealers have gained popularity due to their bioactivity, sealing ability, and biocompatibility. Unlike traditional resin-based or zinc oxide-eugenol sealers, calcium silicate formulations promote the formation of hydroxyapatite and stimulate periapical healing, contributing to favorable clinical outcomes [41, 42]. Their intrinsic alkalinity and antimicrobial properties further support their use, especially in challenging endodontic conditions.

Recent advancements have also focused on improving handling characteristics and reducing setting time without compromising biological properties. These improvements, combined with their favorable interaction with dentin and periapical tissues, position calcium silicate sealers as a viable long-term option for root canal obturation [43].

The future of obturation may shift toward simplified, biologically-driven techniques that rely more heavily on bioactive sealers and less on core materials. When paired with bioactive sealers, techniques such as the single-cone method can challenge traditional warm vertical compaction with reduced procedural complexity. As materials evolve, emphasis will likely remain on improving long-term periapical health through minimally invasive yet biologically compatible solutions.

It is well-established that pulpal and periodontal diseases can alter the diameter of dentinal tubules. This study has several limitations, including the use of extracted teeth for which the reasons for extraction are unknown and the inability to assess the initial diameter of the dentinal tubules. Furthermore, due to the standardization procedures employed, which included decoronation, the absence of a coronal cavity structure that typically serves as an irrigant reservoir may affect the irrigation effectiveness. Additionally, given that the horizontal cross-sectional shape of the root canal influences sealer penetration, teeth with an oval canal cross-section were excluded from this study to mitigate this limitation.

Conclusion

In the context of this study, the impact of three prevalent irrigation activation methods and four distinct root canal sealers on the penetration of dentinal tubules was examined. While all the groups have demonstrated sealer penetration, the results indicated that sonic activation and BioRoot RCS provided superior dentinal tubule penetration compared to the other groups. The findings of this study suggest that effective dentinal tubule penetration can be achieved through continuous irrigation, proper application of activation systems, and strict adherence to manufacturer instructions when utilizing root canal sealers. Further studies are recommended to explore the long-term clinical implications of these findings on sealing efficacy and reinfection prevention.

Electronic supplementary material

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Abbreviations

CLSM

Confocal laser scanning microscopy

NaOCl

Sodium hypochlorite

MPD

Maximum penetration depth

PA

Penetration area

Author contributions

T.T. and Y.K. contributed to conception and design of the work. Y.K. and M.M.T. conducted the experiment, contributed on acquisition of the data, prepared figures and wrote the main manuscript text. E.K. contributed on interpretation the data. All the authors reviewed the manuscript.

Funding

The study is not funded by any company or institute.

Data availability

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Katip Çelebi University Non-Interventional Clinical Studies Institutional Review Board (approval number: 2023-GOKAE-0490) and conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants prior to the extraction of their teeth for inclusion in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.