Microscopic evaluation of sealer penetration and interfacial adaptation of three different endodontic sealers: An in vitro study.

Background: The dentinal tubular penetration depth and interfacial adaptation of sealer are important factors for successful root canal treatment. Aims: This study aims to assess and evaluate the sealer penetration depth and interfacial adaptation of AH Plus, Apexit Plus, and GuttaFlow Bioseal sealer to root dentin. Materials and
Methods: After decoronating sixty extracted single-rooted premolar teeth, preparation was done using ProTaper Gold rotary files until F3. The samples were randomly divided into three groups (n = 20 each) consistent with the sort of sealer used for obturation. After obturation with lateral compaction technique, half of the samples (n = 10 each) were sectioned transversely for measuring tubular depth penetration under a confocal laser scanning microscope. Longitudinal sections were obtained for the rest half the samples to gauge the difference of sealer using the scanning electron microscope. Statistical Analysis Used: Data were analyzed using one-way analysis of variance and Tukey's post hoc tests.
Results: GuttaFlow Bioseal has shown a significantly higher depth of sealer penetration and minimum interfacial adaptation than resin-based sealer and calcium hydroxide-based sealer (P < 0.05).
Conclusion: At all root regions, the GuttaFlow Bioseal sealer exhibited more sealer penetration and minimum interfacial adaptation whereas the Apexit Plus sealer exhibited less sealer penetration and maximum interfacial adaptation. Copyright:

INTRODUCTION

Long term success of the root canal treatment majorly depends on the three dimensional obturation of the properly cleaned and shaped root canal with an inert, biocompatible sealer material.[12] Because of the complexity of the root canal system, sealers need to be used to fill the anatomical irregularities, isthmuses, ramifications, deltas, accessory, and lateral canals and to penetrate dentinal tubules to obtain a fluid-tight seal and improve the adaptation of root filling at the sealer–dentin interface.[3]

AH Plus is a widely used epoxy resin-based sealer with good physicochemical properties and antibacterial effects.[45] AH Plus does not resorb easily and can produce a short-term inflammatory response if extruded into the periapical tissues. AH Plus also exhibits slight shrinkage after being immersed in water for 30 days, albeit it meets the standards of ISO 6876/2001.[67]

Apexit Plus, one of the calcium hydroxide root canal sealers, was introduced for periapical tissue stimulation. Its antibacterial activity stabilizes health or enhances healing.[8]

GuttaFlow Bioseal is a recently developed, silicone-based, cold-filling sealer containing GP powder and bioactive glass that can induce an osteogenic and osteoconductive response. GuttaFlow Bioseal has adequate physical and biological properties, good dentin penetrability, and a higher cytocompatibility than AH Plus. Moreover, GuttaFlow Bioseal sets in vivo when water diffuses into the sealer.[9101112]

The analysis of the different adaptation of sealers to canal walls and marginal gaps is often assessed with scanning electron microscope (SEM) because the defects at the submicron level are often observed at high magnification. Confocal laser scanning microscopy (CLSM) provides detailed information about the presence and distribution of sealers inside dentinal tubules at relatively low magnification through the use of fluorescent Rhodamine-marked sealers.[13]

Although several silicone-based sealers have been recently developed, none fulfill the necessary criteria of stability and interaction with dentin, and to the best of our knowledge, no study has assessed and compared the tubule penetration and interfacial adaptation of these three different sealers.

The purpose of this study was to assess and compare the sealer penetration depth and interfacial adaptation of AH Plus, Apexit Plus, and GuttaFlow Bioseal under confocal laser scanning microscope and SEM, respectively. The null hypothesis tested was that there should be no significant differences within the sealer penetration depth and interfacial adaptation among the three experimental groups at all root regions.

MATERIALS AND METHODS

Preparation of specimens

Ethical clearance for this current in vitro study was attained from the institutional ethical committee (GNIDSR/IEC/19-25). Sixty human mandibular premolar teeth freshly extracted for orthodontic reasons within the age group of 18–30 years irrespective of the position in an arch with no cracks, caries, restoration, internal or external root resorption, aberrant canal morphology and exhibiting single root canal, single apical foramen on buccal and proximal radiographic examinations were chosen. The sample size was decided based on the pilot study done with ten samples used per group using OpenEpi, version 3 (Copyright (c) 2003, 2008 Andrew G. Dean and Kevin M. Sullivan, Atlanta, GA, USA). The samples utilized in the pilot study were discarded.

The extracted teeth were cleaned of calculus and soft-tissue remnants employing a hand curette and were disinfected using 0.5% sodium hypochlorite for at least 30 min and rinsed with distilled water as per the Occupational Safety and Health Administration and Centers for Disease Control and Prevention recommendation guidelines. The teeth were stored in normal saline at 4°C until use in a beaker.

All the samples were standardized to 14-mm length measured by Vernier caliper, decoronation using a diamond disc by a single operator. Access cavities were prepared using endo access bur, 21 mm size 2 (Dentsply Maillefer, Switzerland) was used and initial canal patency to full working length was established with ISO 10K file (Dentsply Maillefer, Ballaigues, Switzerland), visible at the apical foramen, and the working length was established 0.5 mm short of working length.

The root canals were prepared with the crown-down technique, using ProTaper® Gold NiTi system (Dentsply Maillefer, Switzerland) incrementally up to size F3 (30/09) to working length. The Canal Pro CL2 Endomotor (Coltene, Switzerland) was used at 250 rpm and torque 3 N-m. All files were discarded after using three times after biomechanical preparation of canals.

The root canals were irrigated with 2-ml 3% sodium hypochlorite at the change of each instrument. Final irrigation was performed by 2-ml 17% ethylenediaminetetraacetic acid for 1 min followed by a final rinse of 2-ml normal saline. All root canals were irrigated using 30-gauge side-vented needles (Max-I-Probe; Dentsply Maillefer, Switzerland) and placed before the binding point with a distance of 3 mm from the working length. All root canals were manually agitated with the F3 gutta-percha master cone (Dentsply Maillefer, Switzerland). After completion of the biomechanical preparation of root canals, the samples were dried with F3 paper points (Dentsply Maillefer, Switzerland) and randomly divided into three experimental groups of n = 60 according to the sealer placed:

Group 1: AH Plus sealer (n = 20),

Group 2: Apexit Plus sealer (n = 20),

Group 3: GuttaFlow Bioseal sealer (n = 20).

Obturation of the root canals

The sealers were manipulated consistently with the manufacturer's instructions. For analysis under the CLSM, each sealer was labeled with Rhodamine B dye to an approximate concentration of 0.1%. The sealer was placed with size 30 Lentulo spiral within the root canals keeping it 1 mm above the working length, and size F3 master gutta-percha cone was coated with sealer and placed within canal at working length. The root canals were obturated with the lateral compaction technique using an endodontic finger spreader and accessory gutta-percha cones with a 0.02 taper until the entire length of the root canal was filled. Excess gutta-percha was removed employing a heated plugger, and vertical compaction was performed 1 mm below the orifice level; then, the teeth were sealed with a temporary cement barrier and stored in an incubator at 37°C at 100% humidity to allow the complete set of the sealer.

The sectioning of the samples was done after 1 week. For examining of sealer penetration depth, each specimen was sectioned perpendicular to its long axis with a diamond disc at 7 mm, 5 mm, and 3 mm from the root apex using a diamond disk (Horico Dental, Berlin) with a slow-speed (25,000 rpm) handpiece under the copious amount of water coolant to avoid friction.

The specimens were then mounted onto glass slides, and the sample thickness submitted to CLSM was approximately 1.0 ± 0.1 mm thick. The rest 30 samples (n = 10 each/group) were sectioned vertically for measuring the sealer adaptation to the root dentin perimeter under SEM.

Penetration depth measurement

All the sections were examined under CLSM (Leica TCS SP8, Germany) at ×20 magnification with a wavelength of 514 nm. Sealer penetration depth into the dentinal tubules was measured by Adobe Photoshop CS3 using the method described by Bolles et al.[14] Using a ruler tool on the LSM Image Browser software, the sealer penetration depth was measured in micrometers (μm) and the depth of sealer penetration was recorded at four standardized points (mesial, distal, buccal, and lingual) on each section. The measured readings were averaged to obtain a single mean value for each section and tabulated [Figure 1].

Figure 1

Confocal laser scanning microscope images: Group 1 (AH plus) at 7 mm (a), 5 mm (b), 3 mm (c); Group 2 (Apexit plus) 7 mm (d), 5 mm (e), 3 mm (f); Group 3 (GuttaFlow Bioseal) 7 mm (g), 5 mm (h), 3 mm (i) from the root apex

Assessment of adaptation

The samples were mounted on an aluminum stub, placed in a vacuum chamber, and targeted sputter coated with gold and viewed under SEM (S-3700N, Hitachi, Japan) at ×2000 magnification, 7 mm, 5 mm, and 3 mm from the root apex by taking photomicrographs. For each section, the maximum gap in micrometers (μm) was recorded [Figure 2].

Figure 2

Scanning electron microscope images: Group 1 (AH plus) at 7 mm (a), 5 mm (b), 3 mm (c); Group 2 (Apexit plus) 7 mm (d), 5 mm (e), 3 mm (f); Group 3 (GuttaFlow Bioseal) 7 mm (g), 5 mm (h), 3 mm (i) from the root apex

Statistical analysis

Statistical analysis was conducted using IBM SPSS Software version 22 (IBM SPSS Inc., Chicago, IL, USA). The normality distribution of continuous variables was assessed with the Shapiro–Wilk test. A one-way analysis of variance was used to compare the depth of penetration and interfacial gap formation among the three experimental groups at three root levels. Post hoc Tukey's test was done for pairwise comparison between the experimental groups at a 95% confidence level.

RESULTS

A significant difference was observed between AH Plus and Apexit Plus at all root levels (P < 0.05), and a significant difference was found between Apexit Plus and GuttaFlow Bioseal at all root levels (P < 0.05). No statistical significance in the sealer penetration depth was found between AH Plus and GuttaFlow Bioseal at 3-mm level (P > 0.05), but a significant difference was observed at 7-mm and 5-mm level (P < 0.05), with GuttaFlow Bioseal showing greater sealer penetration [Table 1]. The overall penetration of the sealers was deeper at the 7-mm level as compared to the 3-mm level [Figure 1].

Table 1

The mean±standard deviation of the deepest tubular penetration of AH Plus, Apexit Plus, and GuttaFlow Bioseal at 7 mm, 5 mm, and 3 mm from the root apex (µm)

Root section	Sealer, mean±SD			P
	AH Plus (Group 1)	Apexit Plus (Group 2)	GuttaFlow Bioseal (Group 3)
7 mm (coronal)	1021.245±271.01	991.28±261.01	1342.08±247.41	<0.001
5 mm (middle)	884.131±251.51	854.28±256.89	1052.52±267.51	<0.001
3 mm (apical)	518.96±222.89	408.95±232.79	551.96±249.91	<0.001

SD: Standard deviation

A significant difference was found between the sealers regarding dentin sealer interfacial gaps [Table 2], whereas GuttaFlow Bioseal sealer exhibited minimum gaps and the Apexit Plus sealer showed maximum gap formation. Higher gap widths were observed at 3-mm and 5-mm root levels than 7-mm root levels [Figure 2].

Table 2

The mean±standard deviation of the interfacial gaps of AH Plus, Apexit Plus, and GuttaFlow Bioseal at 7 mm, 5 mm, and 3 mm from the root apex (µm)

Root section	Sealer, mean±SD			P
	AH Plus (Group 1)	Apexit Plus (Group 2)	GuttaFlow Bioseal (Group 3)
7 mm (coronal)	1.98±0.35	2.26±0.33	1.08±0.36	<0.001
5 mm (middle)	2.36±0.14	3.96±0.24	2.05±0.28	<0.001
3 mm (apical)	5.15±0.25	7.01±0.36	4.94±0.36	<0.001

SD: Standard deviation

DISCUSSION

In the current study, the null hypothesis has been rejected. The ideal outcome in root canal obturation is to possess a high volume of gutta-percha and a minimal volume of sealer with enhanced penetration into the canal irregularities and dentinal tubules. The degree of adhesion and penetration of sealers into dentinal tubules is influenced by several factors such as physical and chemical properties of sealer cement, dentin permeability and intermolecular surface energy, sealer surface tension, and wetting ability, obturation technique, and smear layer removal. Smear layer removal of root canal walls is taken into account to be fundamental to permit sealer penetration, regardless of the kind of sealer used.[13]

Different microscopic techniques, such as light microscopy, SEM, and CLSM, are accustomed to showing root canal sealers within dentinal tubules. Within the present study, CLSM was used rather than SEM because it allows the detection of sealer penetration at various depths with high contrast points along the canal circumference of every sample using fluorescence, ability to regulate the depth of field, reduction of background information far away from the focal plane, and therefore the ability to assemble numerous optical sections, even from thick specimens.

In the current study, the sealer penetration depth within dentinal tubules was higher at the 7-mm and 5-mm level compared to the 3-mm root level which is per results from Camilleri and McMichael et al.[1516]

One of the possible reasons is due to the quantity and diameter of dentinal tubules decreasing apically within the root canal. Mjor et al. showed that the number of dentinal tubules decreased from 40,000 to 14,400 (coronal to apical).[17] Another explanation could also be the removal of the smear layer within the coronal region.[9]

Consistent with the O'Connell MS study, the significantly lesser penetration of sealer within the apical part can also result in the very fact that techniques that will not remove the smear layer were less effective when approximating the apex.[18]

GuttaFlow Bioseal sealer has a particularly low surface tension, high flow, limited dimensional change on setting (0.6%–0.2%), low water sorption, and having a particle size of (2–10) μm, which could enhance the penetration of the particles into the dentinal tubules deepest at 7-mm level.[19] GuttaFlow Bioseal sealer undergoes 0.14% expansion after storage in distilled water for 7 days, and 0.68% volume contraction after 30 days of storage, while AH Plus undergoes 0.5% expansion and 0.19% contraction, after 7 days and 30 days of storage, respectively.[20] Consistent with Mamootil and Messer study, physical and chemical properties, such as particle size, solubility, and viscosity, influence the depth and consistency of the sealer penetration.[21]

It was found that the sealer penetration depth was greater in the buccolingual direction compared with the mesiodistal direction. A butterfly-like phenomenon was observed based on root cross-sections of single-rooted teeth, as a result of increased sclerosis along the dentinal tubules located on the mesial and distal sides of the root canal lumen.[13]

According to Weis et al. study, the difference in sealer penetration depth was found to be not related to the obturation technique, concluding that sealer penetration is particularly associated with the permeability of dentinal tubules along with different properties of the sealer utilized.[22] Consistent with Mamootil and Messer study, sealer penetration of resin-based sealers does not depend on hydraulic forces formed during obturation; rather, the sealer is sucked into the tubules by capillary action.[21]

In this current study, more interfacial gaps were observed at the apical level for all the sealers than at the coronal level. This discrepancy is often accounted for by the lower density and diameter of dentinal tubules found at the apical region. Higher mean gaps width at the middle and apical root region might be attributed to the problem posed by the oval shape of premolar root canals. This might be another possible explanation for the presence of high interfacial gaps.[13]

The limitation of the present study is the preparing method of CLSM and SEM specimens. There is a risk that sectioning of the filled root may result in tearing or smearing of GP and sealer in both CLSM and SEM specimens. Moreover, the preparation of concentration 0.1% Rhodamine dye in sealer penetration and the vacuum desiccation process could cause an interfacial gap between different sealer materials.

CONCLUSION

Within the limitation of this study, it can be concluded that the depth of penetration of root canal sealers into dentinal tubules using the lateral compaction technique is influenced by the kind of sealer and by the root canal level, with sealer penetration decreasing apically. GuttaFlow Bioseal has shown higher sealer penetration and better marginal adaptation than Apexit Plus and AH Plus sealer in the apical area, while the overall penetration of the sealers was deeper at 7-mm level as compared to the 5-mm and 3-mm level. Further studies using different obturation techniques are necessary to get three-dimensional obturation in multirooted curved canals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.