Influence of moist dentin and dry dentin on sealing ability of bioceramic sealer using confocal laser scanning microscope: An in vitro study

Abstract

Background:

The quality of adhesion between root canal dentin and sealer is influenced by the moisture condition of the root canal before obturation, which interferes with sealer setting and prevents it from entering dentinal tubules, thereby compromising the seal and leading to microleakage.

Aim:

This study aims to evaluate the effect of root canal moisture on bioceramic sealer penetration into dentinal tubules at different levels using a confocal laser scanning microscope.

Materials and Methods:

Forty single-rooted teeth were selected, and the crowns were removed at the cementoenamel junction. Working length was determined using the K file; further instrumentation with rotary files. The root canals were irrigated with 10 mL of 5.25% NaOCl, followed by 17% ethylenediaminetetraacetic acid for 1 min. The roots were randomly divided into two groups (n = 20) based on the amount of remaining moisture in the canal. Group 1 (Dry dentin): The root canals were dried with paper points until the last paper point was totally dry. Group 2 (Moist dentin): Two paper points were used to blot dry the canal. The root canal sealer was labeled with rhodamine B isothiocyanate. After obturation, teeth were sectioned perpendicularly of 1 mm in thickness at 1 mm, 3 mm, and 5 mm intervals in thickness from every root sample and were observed under confocal laser scanning microscopy for sealer penetration.

Results:

The Group-2 moist dentin group exhibited greater penetration of bioceramic sealer at all three levels. Greater sealer penetration was observed in the cervical third in both groups, followed by the middle and apical thirds.

Conclusion:

Maintaining slightly controlled moisture through blot drying significantly enhances the intratubular penetration of bioceramic sealers across all root canal levels.

INTRODUCTION

Achieving a fluid-tight seal along the entire root canal system remains a cornerstone of successful endodontic treatment. The quality of the seal is essential for preventing microleakage and reinfection of the periapical tissues, which are primary causes of endodontic failure.[1] The integrity of this seal depends on multiple factors, including the physical and chemical properties of the root canal sealer, its interaction with the dentinal substrate, and the moisture condition of dentin during obturation.[2] While modern obturation materials and techniques have evolved considerably, the effect of dentin moisture on the sealing performance of bioceramic sealers remains a subject of clinical and scientific interest.

Root canal sealers serve not only to fill voids and irregularities between the gutta-percha and dentinal walls, but also to establish a strong interfacial bond with dentin, thereby preventing fluid and bacterial percolation.[3] Traditional sealers such as zinc oxide–eugenol and epoxy resin-based sealers are relatively hydrophobic and tend to perform best when applied to dry canal walls. However, the advent of hydraulic calcium silicate-based or bioceramic sealers has introduced a paradigm shift in obturation materials. These sealers are inherently hydrophilic, capable of utilizing moisture from the dentinal tubules and surrounding tissues to initiate and sustain their hydration reaction.[4] Their bioactive nature allows for hydroxyapatite formation and potential chemical bonding with dentin, which enhances sealing integrity and biocompatibility.[5]

The degree of moisture in root dentin, however, is a critical determinant of the performance of these hydrophilic sealers. Overly dry dentin may impede hydration, leading to incomplete setting and reduced interfacial bonding. Conversely, excessive moisture may dilute the sealer, alter its viscosity, and interfere with its adaptation and penetration into the dentinal tubules.[6] Maintaining an optimal “moist but not wet” environment has been advocated for the practical application of bioceramic sealers. However, achieving and quantifying such optimal moisture clinically is challenging, as variations in irrigation, paper point drying, and the canal’s anatomical configuration may affect the final dentin surface condition.[7]

Confocal laser scanning microscopy (CLSM) has emerged as a valuable analytical tool for studying sealer penetration and adaptation at the dentin–sealer interface. Compared to conventional scanning electron microscopy (SEM), CLSM offers the advantage of evaluating specimens without inducing dehydration artifacts. It enables visualization of fluorescently labeled sealers within dentinal tubules, allowing quantification of penetration depth and interfacial gaps.[8] Such microscopic insights are critical for understanding how dentin moisture levels affect the interaction of bioceramic sealers with the dentinal surface and, consequently, their sealing ability.[9]

Recent studies have shown that bioceramic sealers demonstrate superior sealing and dentin adaptation under moist conditions compared to epoxy resin-based sealers, owing to their hydrophilic chemistry.[10] However, inconsistent findings have been reported when the dentin is excessively dried or inadequately moist, suggesting that deviations from the optimal hydration state could significantly alter sealer behavior. Understanding this relationship is clinically significant because endodontic irrigation protocols typically involve the use of agents such as sodium hypochlorite and ethylenediaminetetraacetic acid (EDTA), followed by drying steps that may unintentionally render the dentin either too wet or too dry.[11] Therefore, a scientific evaluation of sealer performance under controlled moist and dry dentin conditions can help clinicians standardize the obturation protocol for bioceramic sealers.

Given these considerations, the present study aims to evaluate the influence of moist and dry dentin conditions on the sealing ability of a bioceramic root canal sealer using CLSM. This investigation aims to provide a microscopic understanding of how dentin moisture affects sealer penetration, adaptation, and sealing efficacy, thereby offering evidence-based recommendations to optimize clinical outcomes in endodontic obturation.

MATERIALS AND METHODS

Study design

An in vitro experimental study was designed to evaluate the influence of dentinal moisture on the sealing ability and tubular penetration of a bioceramic sealer using CLSM. The study compared sealer penetration under two drying protocols: Complete drying and blot drying of the root canal.

Research question and hypothesis

Does canal moisture content influence the penetration of a bioceramic sealer into dentinal tubules?

According to the PICO framework:

• P (Population): Root canals obturated with a bioceramic sealer

• I (Intervention): Blot drying of the root canal

• C (Comparison): Complete drying of the root canal

• (Outcome): Sealer penetration assessed using CLSM

• S (Study design): In vitro study.

The null hypothesis (H₀) stated that there would be no significant difference in the penetration of the bioceramic sealer into dentinal tubules with respect to the moisture condition of the canal.

Institutional Ethical Committee approval

The study received approval from the Institutional Ethical Committee, IEC/NDCH/2025/JAN-MAR P-24.

Sample size calculation

Based on previous CLSM studies[12] evaluating sealer penetration, a minimum sample size of 18 specimens per group was calculated at 80% power and a 5% significance level. To account for potential specimen loss during preparation and sectioning, the sample size was increased to 20 specimens per group, resulting in a total sample size of 40 teeth.

Sample selection and preparation

Forty freshly extracted, single-rooted human teeth with fully formed apices and similar root lengths were selected. Teeth with caries, cracks, restorations, resorptive defects, or previous endodontic treatment were excluded. The teeth were cleaned of debris and calculus, stored in a 0.1% thymol solution until use. After the teeth were cleaned using a periodontal ultrasonic tip, the crowns were removed using a 0.3 mm low-speed diamond disc at the cementoenamel junction to standardize root length at 15 mm.

Root canal preparation

The working length was determined by inserting a size #10 K-file until the tip was visible at the apical foramen, then subtracting 1 mm. Root canals were prepared using rotary NiTi files (ProTaper Universal, Dentsply Maillefer, Ballaigues, Switzerland) up to F3 size. During instrumentation, canals were irrigated with 3% sodium hypochlorite after each file. A final rinse with 17% EDTA for 1 min removed the smear layer, followed by distilled water to eliminate irrigant residues.

Grouping and drying protocols

The specimens were randomly assigned to two groups (n = 20 each) based on the drying protocol:

• Group I – Complete drying: Root canals were dried with sterile paper points until no visible moisture was observed

• Group II – Blot drying: Excess irrigant was removed using two paper points, and the canal walls were left visibly moist by gently blotting without complete desiccation.

Sealer preparation and obturation

A premixed bioceramic root canal sealer (BIO-C Sealer, Angelus Odontologia, Brazil) was mixed with 0.1% rhodamine B (HIMEDIA) dye to allow fluorescence under CLSM. The sealer was introduced into the canal using a lentulo spiral at low speed, followed by placement of a size-matched gutta-percha cone coated with the same sealer. Access cavities were sealed with temporary restorative material, and the specimens were stored at 37°C in 100% humidity for 7 days to ensure complete setting of the material.

Sample preparation for confocal laser scanning microscopy analysis

Each root was horizontally sectioned at coronal, middle, and apical thirds using a low-speed diamond saw under water cooling to obtain 1-mm-thick slices. The sections were polished, mounted on glass slides, and examined under a confocal laser scanning microscope (Leica Stellaris 5 White Light Laser [WLL]) at ×10 magnification. Excitation was performed using a 540-nm laser. Digital images were captured, and the sealer penetration depth was measured at four standardized points per section using image analysis software. The maximum, mean, and percentage depth of penetration were recorded for each specimen.

After obturation, all specimens were stored at 37°C and 100% humidity for 7 days to ensure complete setting of the bioceramic sealer. Each root was sectioned horizontally using a low-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) under continuous water cooling. Sections were made at regular 3 mm intervals from the root apex, obtaining three slices representing the apical, middle, and cervical thirds of each specimen. Each section was approximately 1 mm in thickness, and the total root length evaluated was 13 mm. The root slices were gently polished with 600- and 1200-grit silicon carbide abrasive papers to achieve a uniform surface. Each section was mounted on a glass slide with a drop of distilled water and covered with a coverslip. The samples were then examined under a confocal laser scanning microscope (Leica Stellaris 5 WLL). A rhodamine B fluorescent dye (0.1%) was incorporated into the sealer before obturation to allow clear visualization under CLSM, using an excitation wavelength of 540 nm and emission wavelength of 590 nm.

Digital images were obtained at ×10 magnification, and the depth of sealer penetration into dentinal tubules was measured using Leica Application Suite X image analysis software. Measurements were taken at four standardized points (0°, 90°, 180°, and 270°) around the circumference of each section of the canal. For each specimen, the maximum, mean, and percentage depth of penetration were recorded. This technique enabled high-resolution, nondestructive quantitative assessment of sealer penetration into dentinal tubules under different canal moisture conditions.

Statistical analysis

Data were compiled and analyzed using SPSS software (Version 25.0; IBM Corp., Armonk, NY, USA). The Shapiro–Wilk test was used to assess normality. Intergroup comparisons were performed using the independent t-test for normally distributed data or the Mann–Whitney U-test for nonparametric data. The statistical significance level was set at P < 0.05.

RESULTS

The comparison of dentinal tubule penetration depth between the two groups revealed consistently higher values for Group 2 across all root regions. Independent t-tests [Table 1] showed that Group 2 exhibited greater penetration in the coronal (1509.96 ± 92.82 μm vs. 1319.70 ± 54.36 μm; P = 0.000), middle (1426.50 ± 92.82 μm vs. 697.63 ± 51.34 μm; P = 0.000), and apical thirds (524.16 ± 156.30 μm vs. 400.71 ± 49.02 μm; P = 0.000). These findings were further supported by the one-way ANOVA [Table 2], which confirmed statistically significant intergroup differences (P < 0.05) in all regions. Within-group multiple comparisons using the Tukey post hoc test [Table 3] showed a progressive decrease in penetration depth from the coronal to the apical third in both groups. In Group 1, all regional comparisons were statistically significant (coronal–middle: mean difference 622.07 μm; middle–apical: 918.99 μm; coronal–apical: 296.92 μm; all P = 0.000) except the middle–coronal reverse comparison (P = 0.063). In Group 2, the regional differences were more pronounced, with the coronal–apical comparison showing the highest mean difference (985.80 μm; P = 0.000), indicating substantially enhanced penetration capability in this group. Overall, the results confirm that Group 2 exhibited superior intratubular penetration across all dentinal levels, with the highest difference observed in the middle third, reflecting the enhanced dentin interaction and diffusion characteristics of the material/agent used under dry and moist canal conditions.

Table 1.

Intergroup comparison of penetration depth

Area	VAR00004	Mean±SD	t	P
Coronal	Group 1	1319.7024±54.35999	−7.91	0.000*
	Group 2	1509.9600±92.81938
Middle	Group 1	697.6277±51.34407	−34.723	0.000*
	Group 2	1426.5003±92.81938
Apical	Group 1	400.7108±49.01588	−3.37	0.000*
	Group 2	524.1617±156.29996

*Independent t-test P<0.05 significant. SD: Standard deviation

Table 2.

Comparison of penetration depth of sealers

Areas	Group 1	Group 2
Coronal	1319.7024±54.35999	1509.9600±92.81938
Middle	697.6277±51.34407	1426.5003±92.81938
Apical	400.7108±49.01588	524.1617±156.29996
P	0.000*	0.000*

*Oneway ANOVA P<0.05 significant

Table 3.

Multiple comparison using Post hoc Tukey

Groups	Areas	Mean difference	P
Group 1	Coronal
	Middle	622.07465	0.000*
	Apical	918.99160	0.000*
	Middle
	Apical	296.91695	0.000*
Group 2	Coronal
	Middle	83.45965	0.063 (NS)
	Apical	985.79830	0.000*
	Middle
	Apical	902.33865	0.000*

*Post hoc Tukey P<0.05 significant. NS: Not significant

Confocal laser scanning microscopy evaluation

A direct visual comparison of CLSM images between the subgroups revealed distinct differences in intratubular sealer penetration. Group 1, as shown in Figure 1, exhibited limited and discontinuous penetration across all thirds, with shorter fluorescent streaks and lower intensity, particularly in the middle and apical regions. Group 1 demonstrated greater penetration depth, more uniform radial spread, and brighter fluorescence signals, consistent with enhanced sealer–dentin interaction.

Figure 1.

Representative confocal laser scanning microscopy images illustrating dentinal tubule penetration of Rhodamine B-tagged bioceramic sealer across coronal (C), middle (M), and apical (A) levels in Group 1 (sub group 1 - left panel and sub group 2 - right panel). In each group, Subgroup 1 appears in the left column and Subgroup 2 in the right column. Brighter and longer red fluorescent projections indicate deeper and more continuous sealer penetration. Group 1 Subgroup 1 shows limited and discontinuous fluorescence, whereas Subgroup 2 exhibits greater radial spread

Group 2 [Figure 2] showed substantially higher penetration than in Group 1. Group 2 displayed the most significant overall penetration, with long, well-defined fluorescent extensions in the coronal and middle thirds and markedly deeper apical infiltration. Group 2 also demonstrated strong and continuous penetration, although with greater variability in the apical third. Across all groups, sealer penetration consistently followed the pattern coronal > middle > apical, with Group 2 outperforming Group 1 at as shown in Graph 1. These qualitative CLSM observations corroborate the quantitative results, confirming significantly enhanced intratubular penetration in the subgroups with optimal canal moisture.

Figure 2.

Representative confocal laser scanning microscopy images illustrating dentinal tubule penetration of Rhodamine B-tagged bioceramic sealer across coronal (C), middle (M), and apical (A) levels in Group 2 (sub group 1 - left panel and sub group 2 - right panel). Group 2 demonstrates markedly higher penetration in both subgroups, especially in the coronal and middle thirds, with Subgroup 1 showing the deepest tubular infiltration. Apical sections show reduced penetration in all groups, reflecting decreased tubule density.

Graph 1.

Comparison of sealer penetration between the two groups at three levels

DISCUSSION

The present in vitro study evaluated the effect of canal moisture on the intratubular penetration of a bioceramic sealer using CLSM. The quantitative analysis revealed that canals in the blot-dried (Group 2) condition exhibited significantly greater sealer penetration compared with conventionally dried canals (Group 1) at all dentinal levels – coronal, middle, and apical thirds. This was consistently demonstrated through independent t-tests and one-way ANOVA, with Group 2 showing markedly higher mean penetration depths, particularly in the middle third. The post hoc Tukey analysis further confirmed that penetration depth progressively decreased from the coronal toward the apical region in both groups, with the most remarkable inter-regional differences observed in Group 2. These findings highlight the critical role of canal moisture in the performance of calcium silicate-based sealers.[12]

Bioceramic sealers, such as BIO-C Sealer (Angelus, Brazil), depend on moisture to initiate and sustain the hydration reaction of calcium silicate phases. This hydration process forms calcium silicate hydrate gel and calcium hydroxide, facilitating the subsequent release of calcium and hydroxyl ions, which are responsible for bioactivity and mineral interlocking with dentin.[13] When canals are excessively dried–such as during prolonged paper-point drying–the limited moisture availability may inhibit proper hydration, reduce flowability, and ultimately impede dentinal tubule infiltration.[14,15] The significantly lower penetration observed in the conventionally dried group in the present study reinforces this mechanistic explanation.

The superior penetration in the blot-dried group aligns with previous studies by Viapiana et al. (2014)[16] and Gandolfi et al. (2015),[17] who reported that maintaining a minimally moist canal enhances the adaptation, biomineralization, and interfacial integrity of calcium silicate sealers. Moisture improves the rheological properties of bioceramic materials, promoting deeper intratubular diffusion and improved interaction with the dentin substrate. Conversely, Hegde VR, et al. (2025)[18] demonstrated that over-drying detrimentally affects the bonding and sealing ability of these materials, consistent with the reduced penetration observed in Group 1.

In both groups, the coronal and middle thirds demonstrated significantly higher sealer penetration than the apical third. This pattern is well supported by dentin morphology: tubules in the coronal and middle regions are more numerous, wider, and less mineralized than those in the apical region.[18,19] The apical third is characterized by reduced tubule density, smaller diameters, and potentially more smear-layer remnants–factors that collectively restrict sealer penetration.[19] The post hoc Tukey test confirmed these anatomical differences, showing significant inter-regional variation, particularly between middle and apical sections in both groups.

Moisture levels within the canal also influence the sealing performance and setting characteristics of hydrophilic sealers. Improper moisture can impede or prolong setting, alter dimensional stability, and increase the risk of microleakage.[20] Shokouhinejad N, et al. (2013)[21] emphasized that adequate moisture maintains the hydration of collagen fibrils, increasing interfibrillar spaces and facilitating deeper penetration of hydrophilic sealers. The enhanced penetration observed in the blot-dried group corroborates this concept, reinforcing the notion that controlled residual moisture optimizes bioceramic sealer performance.[22,23]

The findings of this study also demonstrate the hydrophilic nature of BIO-C Sealer. The blot-drying technique maintained sufficient moisture to support hydration and biomineralization reactions without excessively diluting the sealer.[24] This aligns with the observations of Al-Haddad and Che Ab Aziz, who reported improved adaptation and sealing efficiency of calcium silicate sealers in moist dentin compared with epoxy resin-based sealers.[25]

CLSM was chosen as the evaluation technique due to its ability to provide high-resolution, nondestructive visualization of Rhodamine B-labeled sealer within dentinal tubules. Unlike SEM, CLSM minimizes specimen preparation artifacts and enables three-dimensional reconstruction of penetration patterns, providing more accurate and comprehensive quantitative measurements.[26,27,28,29,30] This makes CLSM highly suitable for assessing intratubular penetration behavior.

Clinical significance

Maintaining controlled residual moisture within the root canal before obturation significantly enhances the intratubular penetration of bioceramic sealers. Blot drying, rather than complete desiccation, creates the optimal environment for calcium silicate-based sealers to hydrate, flow, and interact with dentin. Improved penetration–especially in the middle and coronal thirds–promotes better sealer–dentin adaptation, reduces interfacial gaps, and may enhance long-term sealing ability. Clinically, this suggests that routine over-drying of canals with multiple paper points may compromise the performance of bioceramic sealers. In contrast, a simple blot-drying step can improve obturation quality, potentially decreasing microleakage and improving treatment outcomes in endodontic therapy.

Strengths and limitations of the study

The strengths of this study include standardized sample selection, clinically relevant drying protocols, and high-resolution CLSM evaluation at different root levels. Limitations include its in vitro design, qualitative moisture assessment, use of a single sealer, and absence of long-term aging or clinical outcome evaluation.

CONCLUSION

Within the limitations of this in vitro study, it can be concluded that the presence of residual moisture in the root canal enhances the penetration of bioceramic sealer into dentinal tubules. Blot drying of the canal before obturation resulted in significantly greater sealer penetration at all root levels compared with conventional drying. This suggests that maintaining a slightly moist canal environment optimizes the hydration and sealing ability of BIO-C Sealer (Angelus, Brazil), thereby improving its potential for achieving a hermetic apical seal. The enhanced penetration observed in Group 2 suggests improved potential for interfacial sealing and long-term clinical performance. Future in vivo studies and microleakage analyses are recommended to corroborate these findings under clinical conditions.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.