Evaluation of the sealing ability of different root canal sealers: a combined SEM and micro-CT study

Abstract Objective The purpose of this study was to analyze the ability of multiple compounds to seal the dental tubules using scanning electron microscopy (SEM) and micro-computed tomogra-phy (micro-CT). Material and Methods Twenty-four single-root human mandibular premolars were selected and instrumented with nickel-titanium rotary file and the final file size was #40/06. They were then randomly allocated into 2 groups, and all samples were filled with single cone gutta-percha (#40/06) and one of the tested sealers (AH Plus and EndoSequence BC sealers). All specimens were scanned using micro-CT and then three from each group were randomly selected for SEM analysis. Results According to SEM, both root canal sealers showed sufficient adaptation to dentin along the whole length of the root canal, though the coronal sections presented superior sealing than the apical sections. Micro porosity analyses revealed that the volume of closed pores and the surface of closed pores had the largest values in the coronal sections, followed by the middle and the apical sections for both sealants (p<0.05). However, no significant difference was observed for those two parameters between AH Plus and EndoSequence BC sealers in any of the three sections (p>0.05), whereas they were larger in the apical section when the AH Plus sealer was used. Conclusions By using the single cone technique, neither EndoSequence or AH Plus pro-vides a porosity-free root canal filling. The EndoSequence BC sealer may have similar sealing abilities regarding the whole root canal as the AH Plus sealer. A better sealing effect could be obtained in the coronal and middle sections of a root canal than the apical part by using the tested sealers.


Introduction
The long-term success of endodontic therapies relies on complete filling after root canal obturation 12 .
Microleakage is one of the significant causes for endodontic failure, which occurs due to poor contacts between the gutta-percha and the sealer, the sealer and the dentin, or through voids within the sealer 16 It is not easy to achieve a complete filling with the current root-filling materials used in the clinic, due to the dimensional changes and lack of adhesion from gutta-percha, which is also the reason to use endodontic sealers in combination of gutta-percha.
Thus, the adaptability of a sealer to the dentin is the Inc, Vancover, Brtish Columbia, Canada) has been introduced as an ideal premixed and injectable biomaterial in the clinical, exhibiting excellent radiopaque, zero-shrinkage, insoluble, and hydrophilic (using moisture from the dentinal tubules to initiate and complete its setting reaction) characteristics 9 .
The adaptation of a sealant to the dentin has generally been evaluated using stereo-microscopy, confocal laser microscopy, scanning electron microscopy (SEM), leakage tests, and digital imaging 13,20,25 . Compared to other two-dimensional (2D), time-consuming, and destructive evaluation ways, micro-computed tomography (micro-CT) is one kind of advanced imaging modality used to scan filled roots and reconstruct them three-dimensionally (3D) for the assessment of the sealant's adaptation to the root canal walls 13 . To the best of our knowledge, however, no research has been performed on 3D micro porosity by using micro-CT to assess the sealing ability of BC sealer in the whole root canal system and its circumferential dentin area. Moreover, there is no study yet using SEM and micro-CT for this purpose.
Therefore, the aim of this study was to quantitatively evaluate and compare the sealing ability of BC sealer and AH Plus at the apical, middle, and coronal dental tubules using SEM and micro-CT. The study was performed under the null hypothesis that no differences in the ability of sealing dental tubules would be observed between the two tested root canal sealants.

Material and methods
In this study, similarly sized, single-rooted, human mandibular premolars, extracted by orthodontic reasons, were collected from patients in the clinic, after their verbal informal consent for the use of these teeth in the lab with the ethical approval from the Ethics Committee of Mustafa Kemal University (No. 20012017/4919). All teeth were decontaminated in 5.25% sodium hypochlorite for 2 hours. They were then stored in distilled water until further testing.
Teeth were examined using an operating microscope (Carl Zeiss Meditec AG, Oberkochen, Germany) at 20× magnification, and those with immature apices, caries, restorations, fractures, or cracks were excluded from the study, and only teeth with oval canals were included in the standardization procedure for the experiment. Preoperative radiographs were obtained in the mesiodistal and buccolingual directions to confirm the presence of a single unmanipulated root canal without root caries, resorption, or calcification.
In total 24 teeth were included and decoronated at the cemento-enamel junction, and each root was adjusted to about 12 mm in length. Subsequently, a #10 K-File

Micro-CT imaging acquisition
Scanning was performed using high-resolution micro-CT Skyscan 1172 (Brüker, Kontich, Belgium) at 100 kVp, 100 mA beam current, 0.5 mm Al/Cu filter, 13.67 µm pixel size, 0.5 step rotation, and 30% beam hardening. To minimize ring artefacts, air calibration of the detector was performed prior to each scan. Each sample was rotated 360° with an integration time of 5 min. Additional settings were also implemented, including a beam-hardening correction as previously described, and optimal contrast limits (0-0.06) based on prior scanning and reconstruction of the teeth. For visualization and quantification of 1,000 × 1,000 pixel two-dimensional (2D) axial images, NRecon software (ver. 1.6.7.2; Brüker, Kontich, Belgium) was used with an algorithm described by Bouxsein, et al. 5

(2010).
For the reconstruction, the smoothing was initially set to zero, followed by a setting of 40% when the ring artefact correction (flat field correction) was applied.
The contrast limits were set according to the Skyscan instructions. was selected, binary images were obtained and an appropriate greyscale threshold was manually selected to distinguish the gutta-percha, root canal sealant, and dentin. Despeckling was performed to remove white speckles in the 3D images that were fewer than 10 voxels. Following this, 3D imaging analyses were performed to calculate the porosity of the sealant within the ROI volume.

Scanning electron microscopy evaluation
After micro-CT scanning, three teeth from each group were randomly selected for SEM analysis.
Roots were sectioned longitudinally in the labiolingual direction and divided into apical (0-5 mm) and coronal (7-12 mm) sections. Sections were vacuum dried, coated with gold, and then examined by SEM (Carl Zeiss NTS GmbH, Oberkochen, Germany). The penetration of sealants into the dentinal tubules and adaptation of each sealant to the dentin were examined from the coronal to apical ends at 1,500× magnification, and finally, the microphotographs were taken.

3D micro porosity analysis
In this study, the total ROI volume (mm 3      the open porosity, the differences between two sealers vanished, suggesting that BC sealer may have more penetration into the dentin tubules. This may be due to the small particle size of the EndoSequence BC sealer (about 2 μm in diameter) or the viscosity of the calcium phosphate silicate ceramic-based materials that facilitate the flow of the sealant into the dentinal tubules.
Bioceramic materials contain alumina, zirconia, bioactive glass, glass ceramics, hydroxyapatite, and calcium phosphates 17 . The alkaline nature of bioceramic by-products has been reported to denature collagen fibers, which facilitates the penetration of sealers into the dentin tubules 2 . However, AH Plus is naturally acidic, which may limit its bonding to dentin. Moreover, AH Plus contains a polymer that contracts upon polymerization, which may result in sealant cracking and deterioration. Thus, it is likely for these reasons that the EndoSequence BC sealer shows superior sealing ability than AH Plus, but has yet to be confirmed by further in-vivo follow-up studies.
In the current study, a similar volume of closed pores was observed between the EndoSequence BC sealer and the AH Plus, which indicated that tested sealers adapted or penetrated equally to the dentin in the coronal, middle, and apical sections. Similarly, The degree of adhesion of the sealer to the dentin wall depends largely on the intermolecular surface energy and cleanliness of the dentin, as well as the surface tension and wetting ability of the sealant.
Dentin at the coronal, middle, and apical sections has different surface energies and cleanliness. Cleanliness is an important factor for sealer adaption, which could be difficult to achieve in the apical region due to difficulties in removing the smear layer. The smear layer often blocks sealer entry to the dentin tubules, and it was suggested that differences between the apical and coronal regions may be due to the lower density and diameter of dentin tubules in the apical regions 10 . This suggestion may also explain the In summary, within the limitation of the study, the null hypothesis that no differences would be observed between the abilities of EndoSequence and AH Plus to seal dentin tubules is accepted while using the single cone technique, which suggests that EndoSequence BC sealer has a similar sealing ability in the entire root canal as the AH Plus sealer does. A better sealing effect could be obtained in the coronal and middle sections than the apical part by using any of those tested sealers.