Assessment of the penetration of an endodontic sealer into dentinal tubules with three different compaction techniques using confocal laser scanning microscopy

Citation: Barbero-Navarro, I.; Velázquez-González, D.; Irigoyen-Camacho, M.E.; Zepeda-Zepeda, M.A.; Mauricio, P.; Ribas-Perez, D.; Castano-Seiquer, A. Assessment of the Penetration of an Endodontic Sealer into Dentinal Tubules with Three Different Compaction Techniques Using Confocal Laser Scanning Microscopy. J. Funct. Biomater. 2023,14, 542. https://doi.org/10.3390/jfb14110542 Academic Editors: Lavinia Cosmina Ardelean and Laura-Cristina Rusu Received: 11 October 2023 Revised: 31 October 2023 Accepted: 3 November 2023 Published: 7 November 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Journal of Functional Biomaterials Article Assessment of the Penetration of an Endodontic Sealer into Dentinal Tubules with Three Different Compaction Techniques Using Confocal Laser Scanning Microscopy Ignacio Barbero-Navarro 1,2, Diego Velázquez-González 1, María Esther Irigoyen-Camacho 3,*, Marco Antonio Zepeda-Zepeda 3, Paulo Mauricio 4, David Ribas-Perez 2,* and Antonio Castano-Seiquer 2 1 Dental School, University Institute Egas Moniz (IUEM), 2800-064 Almada, Portugal; [email protected] (I.B.-N.) 2Dental School, University of Seville, 41009 Seville, Spain; [email protected] 3Health Care Department, Autonomous Metropolitan University-Xochimilco, Mexico City 04960, Mexico; [email protected] 4Interdisciplinary Research Centre, University Institute Egas Moniz (IUEM), 2800-064 Almada, Portugal *Correspondence: [email protected] (M.E.I.-C.); [email protected] (D.R.-P.) Abstract: Adequate root canal sealing is essential for the success of endodontic treatment. There are numerous techniques available; identifying simple and efficient techniques is important to provide good patient care. The purpose of the study was to compare the maximum penetration depth and the percentage of sealant penetration of an endodontic sealer into dentine tubules using cold lateral condensation, continuous wave, and hybrid techniques, and to contrast the effectiveness of two different tapered gutta-percha master cones (0.02 and 0.04). A sample of sixty single root teeth was used. Six experimental groups were formed from the three filling techniques and the two tapered master cones. Images were acquired using a confocal laser scanning microscope. In the apical root third, the penetration percentage was higher in the hybrid compared with the continuous wave technique. The results indicated a higher penetration depth of hybrid compared with cold lateral condensation in the middle and coronal thirds, and in the apical third, a higher penetration was identified in the hybrid group compared with the continuous wave group. No significant differences in penetration were found comparing 0.02 with 0.04 taper gutta-percha groups. The coronal cross-sections presented a higher penetration than the apical third sections. In conclusion, the hybrid technique a had higher maximum sealer penetration than the continuous wave in the apical third, and the coronal third hybrid and continuous wave had a higher penetration than cold lateral condensation. Keywords: endodontic obturation; obturation technique; sealers’ penetration depth; dentin tubule penetration; confocal laser scanning microscope 1. Introduction Successful root canal treatment requires an impermeable tridimensional seal along the root canal’s length, thereby ensuring the healing of periapical tissues and preventing intracanal recontamination from coronal leakage [ 1 ]. The techniques for root canal filling have improved; however, there are still challenges, such as inadequately prepared areas affecting the adaptation of obturation material [ 2 ], apical extrusion of gutta-percha and sealer, difficulty in performing the technique, the lack of hermetic sealing leading to favorable environments for bacterial colonization, and the challenge of addressing obturations in the face of different anatomical variations within endodontic spaces, ranging from oval and curved canals to lateral canals, isthmuses, and other issues, resulting in obturation failure. The cold lateral condensation technique remains the most accepted approach for root canal filling, being a reliable technique that may be used in most cases [ 3 ]. An important advantage of cold lateral condensation is the controlled placement of the gutta-percha in J. Funct. Biomater. 2023,14, 542. https://doi.org/10.3390/jfb14110542 https://www.mdpi.com/journal/jfb J. Funct. Biomater. 2023,14, 542 2 of 18 the canal [ 4 ]. Additionally, it is frequently used for comparison with other compaction techniques [ 5 ]. However, cold lateral condensation has some limitations, such as potential deficiencies in gutta-percha mass homogeneity, partial filling in certain hard-to-reach areas of the root canal system, and, in the case of fragile teeth, there is a risk of root fractures [ 6 – 8 ]. Techniques based on gutta-percha heating were introduced to improve the three-dimensional filling of root canal systems. Schilder [ 9 ] developed a technique using heat and vertical condensation. This was modified, and the continuous wave condensation technique was introduced [ 10 ]. Root canal obturation with injected thermoplasticized gutta-percha was presented by Yee et al. [ 11 ] Subsequently, Tagger [ 12 ] developed a hybrid technique combining mechanical and thermal obturation with cold lateral condensation. Thermoplastic methods offer the benefit of achieving good adaptation to the root canal walls and require less treatment time when compared to cold lateral condensation. Nevertheless, there is a possibility of gutta-percha undergoing physicochemical changes with the use of this technique. Thermomechanical compaction has demonstrated commendable adaptation, especially within the middle and coronal thirds of the root. Although there is limited supporting evidence regarding the effectiveness of a hybrid approach combining cold lateral condensation in the apical third and thermoplastic techniques in the middle and coronal thirds of the root canal, it has the potential to harness the advantages of both techniques. Yet, the assessment of filling quality in hybrid condensation techniques has yielded inconclusive findings [13,14]. The introduction of the nickel-titanium (Ni-Ti) rotary instruments in root canal preparation can improve the results and reduce the time required during the canal’s instrumentation. Rotatory instruments may help maintain the root canals’ original curvature, shape, and patency [ 15 , 16 ]. To match up with the various tapers of Ni-Ti instruments, firms have produced gutta-percha with a large taper (0.04 and 0.06). Gordon [ 17 ] found that higher taper gutta-percha points had better adaptation in a study of filling efficacy and reduced the time required for filling the canal compared with the standard tapered cones, using the cold lateral condensation. Nevertheless, some studies found different results [18,19]. A high percentage of endodontic sealer penetration may be an indirect indicator of potential resistance to microbial and fluid filtration between the canalicular system and the periapex, favoring three-dimensional obturations. Several factors may influence sealer penetration, including the effectiveness of the removal of the smear layer, [ 20 ] the anatomy of the root canal system, [ 21 , 22 ] the obturation techniques, [ 23 ] the physical and chemical properties of the sealer, [ 24 ] and the gutta-percha characteristics [ 17 ]. Confocal laser scanning microscopy (CLSM) is considered an appropriate technique for evaluating the amount of sealer that has penetrated the dentinal tubules [17,25]. The root filling efficacy of various compaction techniques employing gutta-percha points has yielded inconclusive findings, particularly in the context of hybrid techniques and the influence of different tapered gutta-percha master cones. This study postulates two key hypotheses. Firstly, it is hypothesized that the hybrid compaction technique will yield superior results in terms of root canal sealing when compared to the cold lateral compaction and continuous wave techniques. Secondly, the second hypothesis posits that the utilization of more tapered gutta-percha master cones (0.04) will result in improved sealing proficiency compared to their less tapered counterparts (0.02), leading to greater sealant penetration within the root canal dental tubules. Accordingly, this study aims to compare the maximum sealer penetration depth and the percentage of sealer penetration into dentinal tubules using three condensation techniques: cold lateral condensation, continuous wave, and a hybrid, while employing two different tapered gutta-percha master cones (0.02 and 0.04). 2. Materials and Methods The Ethics Committee of the Egas Moniz University Instituteo (CIIEM), Portugal, approved the study protocol (CEPI/0893). The sample size was calculated for the percent of sealer penetration using cold lateral condensation as a reference technique, and information J. Funct. Biomater. 2023,14, 542 3 of 18 from a confocal study [ 26 ], with a mean of 73 and a standard deviation of 8.7; we assumed that the experimental technique (hybrid) had a higher standard deviation (sd 14) than the reference technique. The effect size established was 15 percent points. The type I error alpha was set at 0.05 and the power (1 −β ) was 0.80. The sample size obtained was n = 10. The STATA V17 (StataCorp LLC, College Station, TX, USA) program used a sample size calculation. The root specimens for laboratory tests were obtained from Egas Moniz Instituto Universitrio CIIEM. Sixty extracted fully developed human teeth with single, straight canals were selected and radiographically examined (Kodak 2100; Kodak Dental Systems, Atlanta, GA, USA) with a Trophy RVG Ultimate 6100 digital sensor (Kodak Dental Systems, Atlanta, GA, USA) from facial and proximal views to ensure the presence of a single canal and to evaluate the root canal morphology. The study applied inclusion criteria, which required teeth to be erupted with fully developed apices and straight root canals. Exclusion criteria included teeth with visible root cracks, fractures, calcified root canals, or dental caries affecting the root portion of the tooth. Furthermore, teeth with dental development anomalies were also excluded. The teeth were stored in a 0.2% sodium azide solution. 2.1. Root Canal Preparation and Filling The root length was standardized as 12 mm from the apex and cut with a 910P diamond disc (Drewdel Zweilinf, Berlin, Germany). The roots were debrided with ultrasonic scalers and washed with distilled water. Subsequently, they were immersed for 15 min at room temperature in a 6% NaOCl solution to remove the remaining organic debris, following Gharib protocol [27]. The working length of each canal was established by measuring the penetration of a 10 K-file (Flexofile; Dentsply Maillefer, Ballaigues, Switzerland) until it reached the apical foramen and then subtracting 0.5 mm. The canal spaces were cleaned and mechanically shaped with Mtwo rotary system files (VDW, Munich, Germany) in conjunction with Slick- Gel ES (SybronEndo Company, Orange, CA, USA) and 1.8 mL 5.25% sodium hypochlorite (NaOCl) between each file size. A handpiece was used with an electric engine (X-Smart; Dentsply Maillefer, Ballaigues, Switzerland) at 280 rpm. The manufacturer’s recommendations were followed during the root canal preparation. All the canals were enlarged to an ISO size 35, 0.04 taper, to working length. A dentist instrumented all the teeth. Apical patency was maintained throughout the instrumentation using a #10 K-file (Flexofile; Dentsply Maillefer, Ballaigues, Switzerland). After preparation, the canals were irrigated for 3 min with 17% ethylenediaminetetraacetic acid (EDTA) to remove the smear layer, followed by 5 mL NaOCl and final irrigation with 10 mL of distilled water to avoid the prolonged effect of the EDTA and NaOCl solutions. The canals were subsequently dried with paper points. AH plus (DeTrey Dentsply, Konstanz, Germany) was used as a sealer in all the cases. This sealer was labelled with rhodamine B (0.1%) (Sigma-Aldrich, St. Louis, MO, USA) to allow analysis under confocal laser scanning microscopy (CLSM). The sealer was applied using a #35 Lentulo Spiral (Zipperer-VDW, Munich, Germany). 2.2. Root Canal Preparation and Filling Technique Sixty roots were randomly divided into six groups of 10 teeth each. According to the condensation technique and the taper of the gutta-percha master cone, we formed the following groups: Group 1 (cold lateral condensation 0.02). Ten root canals were filled with 0.02 tapered gutta-percha (VDW, Munich, Germany) using the cold lateral condensation technique. A gutta-percha cone of 0.02 taper was fitted into the root canal at the working length, making firm pressure in an apical direction. A finger spreader (Dentsply Maillefer, Ballaigues, Switzerland) number 20 was introduced at 1 mm short of the working length to laterally compact the gutta-percha to create space and be able to insert auxiliary 0.02 taper gutta-percha cones. This process was repeated until the digital spreader only J. Funct. Biomater. 2023,14, 542 4 of 18 entered the coronal third of the root canal. A hot metal instrument was used for trimming excess coronary gutta-percha and was subsequently compacted vertically with a Buchanan instrument. Group 2 (cold lateral condensation 0.04). Ten roots were filled with gutta-percha with a 0.04 taper, using the cold lateral condensation as described in Group 1. Group 3 (continuous wave 0.02). Ten root canals were sealed with gutta-percha of standardized 0.02 taper using a continuous vertical wave condensation technique with thermoplastic injection. After inserting a standard gutta-percha point adapted to 1 mm short of the working length, the corresponding tip of the Elements Obturation Unit (EOU) sealing system was heated to 200 ◦ C, and a depth of 3 mm above the canal’s length was used. Once the apical third was sealed, the other two-thirds of the root canal was sealed using the thermoplastic gutta-percha injection technique, elements obturation unit EOU system (SybronEndo Company, Glendora, CA, USA) by applying manual vertical condensation, and the corresponding Buchanan Pluggers instrument (Sybron Endodontics, Orange, CA, USA). Group 4 (continuous wave 0.04). Ten roots were filled with gutta-percha with 0.04 taper, using continuous wave as described in Group 3. Group 5 (hybrid 0.02). Ten root canals were sealed with a gutta-percha 0.02 taper using a hybrid filling technique. The cold lateral condensation and thermoplastic filling condensation techniques were combined. After filling the root canal using the cold lateral condensation as described for Group I, a hot instrument was used to remove the guttapercha from the middle and coronal thirds. Subsequently, vertical pressure was applied using the corresponding Buchanan Pluggers (Sybron Endodontics, Orange, CA, USA) within 5 to 7 mm of working lengths to improve the gutta-percha adaptation. The coronal and middle third portions of the root canal were sealed with thermoplastic gutta-percha injected by the corresponding terminal of the elements gutta-percha cartridges (SybronEndo, Glendora, CA, USA) using the elements obturation unit (SybronEndo Company, Glendora, CA, USA). Subsequently, gutta-percha was vertically compacted with a Buchanan Pluggers instrument (Sybron Endodontics, Orange, CA, USA). Group 6 (hybrid 0.04). Ten roots were filled with gutta-percha with 0.04 taper, using the hybrid technique described in Group 5. In all the filled root canals, vestibule-lingual and mesiodistal radiographs were taken of each root to verify its correct condensation. Facial and proximal views of digital radiographs of all teeth roots were taken to verify the canal obturations. Figure 1illustrates radiographic images depicting root canals filled using the three techniques examined in the current study. The teeth were placed in a Memmert BE 500 heater (Memmert, Heilbronn, Germany) and incubated at 37 ◦ C for three days to set the sealer completely. Each root was embedded in an epofix hardener resin block (Struers, Ballerup, Denmark). J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 5 of 19 Figure 1. Illustrates radiographic images of teeth filled with each of the three compaction techniques evaluated. Cold lateral condensation ((1a) vestibule-lingual and (1b) mesiodistal radiograph); continuous wave ((2a) vestibule-lingual and (2b) mesiodistal radiograph) and hybrid technique ((3a) vestibule-lingual and (3b) mesiodistal radiograph). 2.3. Sectioning and Image Analysis One-millimeter transversal sections were made in the 3, 7, and 10 mm levels from the apex of sixty roots using a 0.3 mm Isomet saw (Buehler IsoMet, Lake Bluff, IL, USA) at 200 rpm and continuous water cooling. In this manner, three slices per root were created (coronal, middle, apical), resulting in 180 pieces. The samples were then mounted onto glass slides. Only the apically facing surface of each slice was examined. All the sections were sequentially polished with Sof-Lex discs (3M ESPE, Seefeld, Germany) using running tap water as a lubricant to smooth the surfaces. Images of the filled areas were acquired using the epifluorescence mode of an inverted Leica TCS-SP2 confocal laser scanning microscopy (CLSM) (Leica, Mannheim, Germany). An argon-mixed gas laser was used (Ar/HeNe) as the light source. Excitation light had a wavelength maximum of 543 nm. The respective absorption and emission wavelengths for rhodamine B were 554 and 649 nm. The images were recorded at 40×, with a 1024 × 1024 pixels resolution. The images were acquired and analyzed using the Leica Confocal software (Leica Microsystems, Heidelberg, Germany). All the pictures were taken by using 10 sections with a 4 μm step size. The images were evaluated according to the method used by Gharib et al. [27] for measuring the depth of penetration; each image was imported into the Leica Confocal Figure 1. Cont. J. Funct. Biomater. 2023,14, 542 5 of 18 J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 5 of 19 Figure 1. Illustrates radiographic images of teeth filled with each of the three compaction techniques evaluated. Cold lateral condensation ((1a) vestibule-lingual and (1b) mesiodistal radiograph); continuous wave ((2a) vestibule-lingual and (2b) mesiodistal radiograph) and hybrid technique ((3a) vestibule-lingual and (3b) mesiodistal radiograph). 2.3. Sectioning and Image Analysis One-millimeter transversal sections were made in the 3, 7, and 10 mm levels from the apex of sixty roots using a 0.3 mm Isomet saw (Buehler IsoMet, Lake Bluff, IL, USA) at 200 rpm and continuous water cooling. In this manner, three slices per root were created (coronal, middle, apical), resulting in 180 pieces. The samples were then mounted onto glass slides. Only the apically facing surface of each slice was examined. All the sections were sequentially polished with Sof-Lex discs (3M ESPE, Seefeld, Germany) using running tap water as a lubricant to smooth the surfaces. Images of the filled areas were acquired using the epifluorescence mode of an inverted Leica TCS-SP2 confocal laser scanning microscopy (CLSM) (Leica, Mannheim, Germany). An argon-mixed gas laser was used (Ar/HeNe) as the light source. Excitation light had a wavelength maximum of 543 nm. The respective absorption and emission wavelengths for rhodamine B were 554 and 649 nm. The images were recorded at 40×, with a 1024 × 1024 pixels resolution. The images were acquired and analyzed using the Leica Confocal software (Leica Microsystems, Heidelberg, Germany). All the pictures were taken by using 10 sections with a 4 μm step size. The images were evaluated according to the method used by Gharib et al. [27] for measuring the depth of penetration; each image was imported into the Leica Confocal Figure 1. Illustrates radiographic images of teeth filled with each of the three compaction techniques evaluated. Cold lateral condensation (( 1a ) vestibule-lingual and ( 1b ) mesiodistal radiograph); continuous wave (( 2a ) vestibule-lingual and ( 2b ) mesiodistal radiograph) and hybrid technique ((3a) vestibule-lingual and (3b) mesiodistal radiograph). 2.3. Sectioning and Image Analysis One-millimeter transversal sections were made in the 3, 7, and 10 mm levels from the apex of sixty roots using a 0.3 mm Isomet saw (Buehler IsoMet, Lake Bluff, IL, USA) at 200 rpm and continuous water cooling. In this manner, three slices per root were created (coronal, middle, apical), resulting in 180 pieces. The samples were then mounted onto glass slides. Only the apically facing surface of each slice was examined. All the sections were sequentially polished with Sof-Lex discs (3M ESPE, Seefeld, Germany) using running tap water as a lubricant to smooth the surfaces. Images of the filled areas were acquired using the epifluorescence mode of an inverted Leica TCS-SP2 confocal laser scanning microscopy (CLSM) (Leica, Mannheim, Germany). An argon-mixed gas laser was used (Ar/HeNe) as the light source. Excitation light had a wavelength maximum of 543 nm. The respective absorption and emission wavelengths for rhodamine B were 554 and 649 nm. The images were recorded at 40 × , with a 1024 × 1024 pixels resolution. The images were acquired and analyzed using the Leica Confocal software Version 2.1E (Leica Microsystems, Heidelberg, Germany). All the pictures were taken by using 10 sections with a 4 µm step size. The images were evaluated according to the method used by Gharib et al. [ 27 ] for measuring the depth of penetration; each image was imported into the Leica Confocal Software Version 2.1E and, using the distance tool software, the point of deepest penetration was measured from the canal wall to the area of maximum sealer penetration (Figure 2). Each image was imported into the Image J software (Rasband WS, ImageJ, 1.54; US National Institute of Health, Bethesda, MD, USA) to calculate the percentage of sealer penetration, and the root canal’s circumference wall was measured. Next, the areas along the canal walls in which the sealer penetrated dentinal tubules (sealer tags) were outlined and measured using the same method. The percentage of the canal walls where the J. Funct. Biomater. 2023,14, 542 6 of 18 sealer had penetrated was calculated by dividing the canal circumference’s outline by the entire circumference (Figure 2). J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 6 of 19 Software and, using the distance tool software, the point of deepest penetration was measured from the canal wall to the area of maximum sealer penetration (Figure 2). Each image was imported into the Image J software (Rasband WS, ImageJ, 1.54; US National Institute of Health, Bethesda, MD, USA) to calculate the percentage of sealer penetration, and the root canal’s circumference wall was measured. Next, the areas along the canal walls in which the sealer penetrated dentinal tubules (sealer tags) were outlined and measured using the same method. The percentage of the canal walls where the sealer had penetrated was calculated by dividing the canal circumference’s outline by the entire circumference (Figure 2). Figure 2. Representative confocal laser microscopic image showing the maximum depth of sealer penetration illustrated by a green line. The root canal wall with sealer penetration is illustrated using a violet curved line, and a yellow line depicts the perimeter. 2.4. Statistical Analysis The average percentage of penetration and the maximum depth of sealer penetration were calculated in the three root levels analysed: 3, 7, and 10 mm. These indicators were compared among the three condensation techniques used in the levels of the root canal examined using mixed linear regression models, considering that the observations were nested within teeth. Models with and without interaction terms were tested (interactions between the root level and the compaction technique, and between the technique and the gutta-percha taper master cones). Unstandardized β coefficients, and 95% confidence intervals (95% CI) were obtained. Akaike’s information criterion was used for the model selection. After fitting the models and obtaining estimates for coefficients (βo,β1,...,βk) linear combinations of these estimators were calculated in order to obtain the interaction terms effects as well as their 95% confidence intervals and p-values. The statistical significance value was set at α = 0.05. Data analysis was performed using Stata V15 (StataCorp LLC., College Station, TX, USA). 3. Results The results of the percentage sealer penetration using the cold lateral condensation technique presented a mean of 57.49 (±28.9), for continuous wave, 56.8 (±33.1), and, for the hybrid technique, 64.5 (±29.8). Table 1 presents the mean sealer penetration by technique and gutta-percha taper across the root level. In the coronal sections, the lowest percentage of penetration corresponded to the cold lateral condensation technique (74.3%) and values above 80% were observed in the continuous wave and hybrid techniques. In the middle Figure 2. Representative confocal laser microscopic image showing the maximum depth of sealer penetration illustrated by a green line. The root canal wall with sealer penetration is illustrated using a violet curved line, and a yellow line depicts the perimeter. 2.4. Statistical Analysis The average percentage of penetration and the maximum depth of sealer penetration were calculated in the three root levels analysed: 3, 7, and 10 mm. These indicators were compared among the three condensation techniques used in the levels of the root canal examined using mixed linear regression models, considering that the observations were nested within teeth. Models with and without interaction terms were tested (interactions between the root level and the compaction technique, and between the technique and the gutta-percha taper master cones). Unstandardized β coefficients, and 95% confidence intervals (95% CI) were obtained. Akaike’s information criterion was used for the model selection. After fitting the models and obtaining estimates for coefficients ( β o, β1 , . . . , βk ) linear combinations of these estimators were calculated in order to obtain the interaction terms effects as well as their 95% confidence intervals and p-values. The statistical significance value was set at α = 0.05. Data analysis was performed using Stata V15 (StataCorp LLC., College Station, TX, USA). 3. Results The results of the percentage sealer penetration using the cold lateral condensation technique presented a mean of 57.49 ( ± 28.9), for continuous wave, 56.8 ( ± 33.1), and, for the hybrid technique, 64.5 ( ± 29.8). Table 1presents the mean sealer penetration by technique and gutta-percha taper across the root level. In the coronal sections, the lowest percentage of penetration corresponded to the cold lateral condensation technique (74.3%) and values above 80% were observed in the continuous wave and hybrid techniques. In the middle section, a similar pattern was observed, and in the apical third, continuous wave had the lowest percentage of penetration. None of the groups studied achieved a full adaptation of sealer material on the dentin walls. Figure 3a–c illustrates the root sealer penetration into the coronal (Figure 3a), middle (Figure 3b), and apical (Figure 3c) sections, using the hybrid technique. The higher depth of sealer penetration was observed in the coronal third. J. Funct. Biomater. 2023,14, 542 7 of 18 Table 1. Percentage of sealer penetration in µ m into dentinal tubules in the coronal (10 mm), the middle (7 mm), and the apical (3 mm) thirds by groups according with technique and gutta-percha point taper size. Groups Condensation Technique and Gutta-Percha Taper Root Level Coronal (10 mm) Mean (±SD) Cold Lateral Condensation 74.3 (19.1) Cold Lateral Condensation 0.02 175.4 (20.5) Cold Lateral Condensation 0.04 273.2 (18.6) Continuous Wave 82.7 (21.0) Continuous Wave 0.02 174.3 (26.1) Continuous Wave 0.04 291.3 (9.6) Hybrid 81.3 (20.9) Hybrid 0.02 179.6 (22.3) Hybrid 0.04 283.0 (20.4) Middle (7 mm) Mean (±SD) Cold Lateral Condensation 63.9 (25.2) Cold Lateral Condensation 0.02 162.9 (24.1) Cold Lateral Condensation 0.04 264.8 (27.5) Continuous Wave 64.8 (19.5) Continuous Wave 0.02 163.5 (23.1) Continuous Wave 0.04 266.0 (16.4) Hybrid 69.0 (27.9) Hybrid 0.02 175.4 (22.9) Hybrid 0.04 262.6 (22.1) Apical (3 mm) Mean (±SD) Cold Lateral Condensation 34.3 (26.4) Cold Lateral Condensation 0.02 137.5 (28.8) Cold Lateral Condensation 0.04 231.3 (24.9) Continuous Wave 22.8 (23.9) Continuous Wave 0.02 120.1 (31.6) Continuous Wave 0.04 225.4 (14.0) Hybrid 43.1 (31.5) Hybrid 0.02 133.5 (28.2) Hybrid 0.04 252.8 (33.1) 1Gutta-percha master cone taper 0.02, 2Gutta-percha master cone taper 0.04. J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 8 of 19 Figure 3. Representative confocal laser microscopic image of root sealer penetration. (a) corresponds to the coronal third, (b) depicts the sealer penetration in the middle third. And (c) depicts sealer penetration in the apical third, using the hybrid technique. The results of the maximum penetration of sealer using the cold lateral condensation technique showed a mean of 918.2 (±383.2), for continuous wave, 960.1 (±623.4), and for hybrid, 1144.2 (±571.1). Table 2 presents the results of the maximum penetration depth of condensation techniques by root level and taper of the gutta-percha master cones. In the coronal section, the hybrid technique presented a high penetration mean (1401.6 μm), similarly, it was high in the middle section (1423.4 μm). In the coronal and middle root thirds, the lowest penetration depth was observed in the cold lateral condensation groups. Figure 3. Cont. J. Funct. Biomater. 2023,14, 542 8 of 18 J. Funct. Biomater. 2023, 14, x FOR PEER REVIEW 8 of 19 Figure 3. Representative confocal laser microscopic image of root sealer penetration. (a) corresponds to the coronal third, (b) depicts the sealer penetration in the middle third. And (c) depicts sealer penetration in the apical third, using the hybrid technique. The results of the maximum penetration of sealer using the cold lateral condensation technique showed a mean of 918.2 (±383.2), for continuous wave, 960.1 (±623.4), and for hybrid, 1144.2 (±571.1). Table 2 presents the results of the maximum penetration depth of condensation techniques by root level and taper of the gutta-percha master cones. In the coronal section, the hybrid technique presented a high penetration mean (1401.6 μm), similarly, it was high in the middle section (1423.4 μm). In the coronal and middle root thirds, the lowest penetration depth was observed in the cold lateral condensation groups. Figure 3. Representative confocal laser microscopic image of root sealer penetration. ( a ) corresponds to the coronal third, ( b ) depicts the sealer penetration in the middle third. And ( c ) depicts sealer penetration in the apical third, using the hybrid technique. The results of the maximum penetration of sealer using the cold lateral condensation technique showed a mean of 918.2 ( ± 383.2), for continuous wave, 960.1 ( ± 623.4), and for hybrid, 1144.2 ( ± 571.1). Table 2presents the results of the maximum penetration depth of condensation techniques by root level and taper of the gutta-percha master cones. In the coronal section, the hybrid technique presented a high penetration mean (1401.6 µ m), similarly, it was high in the middle section (1423.4 µ m). In the coronal and middle root thirds, the lowest penetration depth was observed in the cold lateral condensation groups. Table 2. Depth of sealer penetration in µ m into dentinal tubules in the coronal (10 mm), the middle (7 mm), and the apical (3 mm) thirds, by groups according with condensation technique and guttapercha taper sizes. Group by Condensation Technique and Gutta-Percha Taper Root Level Coronal (10 mm) Mean (±SD) Cold Lateral Condensation 1084.9 (197.9) Cold Lateral Condensation 0.02 11294.5 (450.1) Cold Lateral Condensation 0.04 2875.5 (461.5) Continuous Wave 1337.1 (291.1) Continuous Wave 0.02 1271.9 (678.4) Continuous Wave 0.04 1402.8 (514.0) Hybrid 1401.6 (477.3) Hybrid 0.02 1170.7 (796.1) Hybrid 0.04 1632.9 (585.8) J. Funct. Biomater. 2023,14, 542 9 of 18 Table 2. Cont. Group by Condensation Technique and Gutta-Percha Taper Root Level Middle (7 mm) Mean (±SD) Cold Lateral Condensation 982.8 (467.4) Cold Lateral Condensation 0.02 11187.7 (552.7) Cold Lateral Condensation 0.04 2778.0 (449.5) Continuous Wave 1177.3 (694.0) Continuous Wave 0.02 1152.1 (666.0) Continuous Wave 0.04 1202.5 (756.1) Hybrid 1423.4 (442.8) Hybrid 0.02 1394.7 (506.2) Hybrid 0.04 1452.0 (394.8) Apical (3 mm) Mean (±SD) Cold Lateral Condensation 686.8 (329.1) Cold Lateral Condensation 0.02 1711.4 (412.9) Cold Lateral Condensation 0.04 2662.3 (565.2) Continuous Wave 366.0 (263.8) Continuous Wave 0.02 244.7 (280.5) Continuous Wave 0.04 487.4 (422.5) Hybrid 607.7 (365.9) Hybrid 0.02 745.5 (586.9) Hybrid 0.04 470.0 (501.1) 1Gutta-percha master cone taper 0.02, 2Gutta-percha master cone taper 0.04. Table 3presents the results of the mixed linear regression models fitted for percentage of penetration as the outcome variable, using as predictors the root level, technique, and gutta-percha taper size master cone (model 1 without interaction terms). In this model, the taper size was not significant (p= 0.803). Table 3. Regression coefficients of percentage of sealer penetration and root level and type of condensation technique, and gutta-percha cone tapper (model 1), and regression coefficients of maximum penetration depth, technique and interaction between root level and technique (model 2). Model 1 1 Variable Coefficient 95% (CI) p Root level Coronal ref - - Middle −13.6 (−20.2, −7.0) <0.001 Apical −46.1 (−52.7, −39.5) <0.001 Technique Cold lateral condensation ref - - Continuous wave −0.8 (−11.9, 10.4) 0.895 Hybrid 7.0 (−4.2, 18.1) 0.219 Tapper 0.02 ref - - 0.04 −1.2 (−10.3, 7.9) 0.803 Model 2 1 Variable Coefficient 95% (CI) p Root level Coronal ref - - Middle −10.4 (−21.4, 0.6) 0.063 Apical −40.0 (−50.9, −29.0) <0.001 Technique Cold lateral condensation ref - - J. Funct. Biomater. 2023,14, 542 16 of 18 Author Contributions: Conceptualization, I.B.-N. and D.V.-G.; methodology, I.B.-N.; software, P.M.; validation, I.B.-N. and P.M.; formal analysis, M.A.Z.-Z.; investigation, I.B.-N. and D.V.-G.; data curation, M.E.I.-C. and M.A.Z.-Z.; writing—original draft preparation, I.B.-N.; writing—review and editing, M.E.I.-C., M.A.Z.-Z. and D.R.-P.; supervision, A.C.-S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and aproved by The Ethics Committee of the Egas Moniz University Institute (CIIEM), Portugal, with the register number CEPI/0893. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data will be available from corresponding authors. Conflicts of Interest: The authors declare no conflict of interest. References 1. Suguro, H.; Takeichi, O.; Hayashi, M.; Okamura, T.; Hira, A.; Hirano, Y.; Ogiso, B. Microcomputed tomographic evaluation of techniques for warm gutta-percha obturation. J. Oral Sci. 2018,60, 165–169. [CrossRef] [PubMed] 2. Campello, A.F.; Marceliano-Alves, M.F.; Siqueira, J.F.; Fonseca, S.C.; Lopes, R.T.; Alves, F.R.F. Unprepared surface areas, accumulated hard tissue debris, and dentinal crack formation after preparation using reciprocating or rotary instruments: A study in human cadavers. Clin. Oral Investig. 2021,25, 6239–6248. [CrossRef] [PubMed] 3. Qualtrough, A.J.E.; Whitworth, J.M.; Dummer, P.M.H. Preclinical endodontology: An international comparison. Int. Endod. J. 1999,32, 406–414. [CrossRef] [PubMed] 4. Bhandi, S.; Mashyakhy, M.; Abumelha, A.S.; Alkahtany, M.F.; Jamal, M.; Chohan, H.; Raj, A.T.; Testarelli, L.; Reda, R.; Patil, S. Complete Obturation—Cold Lateral Condensation vs. Thermoplastic Techniques: A Systematic Review of Micro-CT Studies. Materials 2021,14, 4013. [CrossRef] 5. Dummer, P.M.H. Comparison of undergraduate endodontic teaching programmes in the United Kingdom and in some dental schools in Europe and the United States. Int. Endod. J. 1991,24, 169–177. [CrossRef] 6. Bhagat, K.; Jasrotia, A.; Bhagat, R.K. A comparison of cold lateral compaction and warm vertical compaction using continuous wave of compaction technique. Int. J. Appl. Dent. Sci. 2021,7, 244–246. [CrossRef] 7. Llena-Puy, M.C.; Forner-Navarro, L.; Barbero-Navarro, I. Vertical root fracture in endodontically treated teeth: A review of 25 cases. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 2001,92, 553–555. [CrossRef] 8. Lertchirakarn, V.; Palamara, J.E.; Messer, H.H. Load and strain during lateral condensation and vertical root fracture. J. Endod. 1999,25, 99–104. [CrossRef] 9. Schilder, H. Filling root canals in three dimensions. Dent. Clin. N. Am. 1967,11, 723–744. [CrossRef] 10. Buchanan, L. The continuous wave of obturation technique: ‘centered’ condensation of warm gutta percha in 12 seconds. Dent. Today 1996,15, 60–67. 11. Yee, F.S.; Marlin, J.; Krakow, A.A.; Gron, P. Three-dimensional obturation of the root canal using injection-molded, thermoplasticized dental gutta-percha. J. Endod. 1977,3, 168–174. [CrossRef] 12. Tagger, M. Use of thermo-mechanical compactors as an adjunct to lateral condensation. Quintessence Int. Dent. Dig. 1984 ,15, 27–30. 13. Marciano, M.A.; Bramante, C.M.; Duarte, M.A.H.; Delgado, R.J.R.; Ordinola-Zapata, R.; Garcia, R.B. Evaluation of single root canals filled using the lateral compaction, tagger’s hybrid, microseal and guttaflow techniques. Braz. Dent. J. 2010 ,21, 411–415. [CrossRef] [PubMed] 14. Robberecht, L.; Colard, T.; Claisse-Crinquette, A. Qualitative evaluation of two endodontic obturation techniques: Tapered single-cone method versus warm vertical condensation and injection system An in vitro study. J. Oral Sci. 2012,54, 99–104. [CrossRef] [PubMed] 15. Haupt, F.; Pult, J.R.W.; Hülsmann, M. Micro–computed Tomographic Evaluation of the Shaping Ability of 3 Reciprocating Single-File Nickel-Titanium Systems on Single- and Double-Curved Root Canals. J. Endod. 2020 ,46, 1130–1135. [CrossRef] [PubMed] 16. Schafer, E.; Schulzbongert, U.; Tulus, G. Comparison of Hand Stainless Steel and Nickel Titanium Rotary Instrumentation: A Clinical Study. J. Endod. 2004,30, 432–435. [CrossRef] [PubMed] 17. Gordon, M.P.J.; Love, R.M.; Chandler, N.P. An evaluation of 0.06 tapered gutta-percha cones for filling of 0.06 taper prepared curved root canals. Int. Endod. J. 2005,38, 87–96. [CrossRef] [PubMed] 18. Motamedi, M.R.K.; Mortaheb, A.; Jahromi, M.Z.; Gilbert, B.E. Micro-CT Evaluation of Four Root Canal Obturation Techniques. Scanning 2021,2021, 1–7. [CrossRef] J. Funct. Biomater. 2023,14, 542 17 of 18 19. Romania, C.; Beltes, P.; Boutsioukis, C.; Dandakis, C. Ex-vivo area-metric analysis of root canal obturation using gutta-percha cones of different taper. Int. Endod. J. 2009,42, 491–498. [CrossRef] 20. Haupt, F.; Meinel, M.; Gunawardana, A.; Hülsmann, M. Effectiveness of different activated irrigation techniques on debris and smear layer removal from curved root canals: A SEM evaluation. Aust. Endod. J. 2020,46, 40–46. [CrossRef] 21. Gok, T.; Capar, I.D.; Akcay, I.; Keles, A. Evaluation of Different Techniques for Filling Simulated C-shaped Canals of 3-dimensional Printed Resin Teeth. J. Endod. 2017,43, 1559–1564. [CrossRef] [PubMed] 22. Du, Y.; Soo, I.; Zhang, C.F. A case report of six canals in a maxillary first molar. Chin. J. Dent. Res. 2011 ,14, 151–153. Available online: http://www.ncbi.nlm.nih.gov/pubmed/22319758 (accessed on 2 February 2022). [PubMed] 23. Karatekin, A.O.; Kele¸s, A.; Genço˘glu, N. Comparison of continuous wave and cold lateral condensation filling techniques in 3D printed simulated C-shape canals instrumented with Reciproc Blue or Hyflex EDM. PLoS ONE 2019 ,14, e0224793. [CrossRef] [PubMed] 24. Komabayashi, T.; Colmenar, D.; Cvach, N.; Bhat, A.; Primus, C.; Imai, Y. Comprehensive review of current endodontic sealers. Dent. Mater. J. 2020,39, 703–720. [CrossRef] [PubMed] 25. Akcay, M.; Arslan, H.; Durmus, N.; Mese, M.; Capar, I.D. Dentinal tubule penetration of AH Plus, iRoot SP, MTA fillapex, and guttaflow bioseal root canal sealers after different final irrigation procedures: A confocal microscopic study. Lasers Surg. Med. 2016,48, 70–76. [CrossRef] [PubMed] 26. Furtado, T.C.; de Bem, I.A.; Machado, L.S.; Pereira, J.R.; Só, M.V.R.; da Rosa, R.A. Intratubular penetration of endodontic sealers depends on the fluorophore used for CLSM assessment. Microsc. Res. Tech. 2020,84, 305–312. [CrossRef] 27. Gharib, S.R.; Tordik, P.A.; Imamura, G.M.; Baginski, T.A.; Goodell, G.G. A Confocal Laser Scanning Microscope Investigation of the Epiphany Obturation System. J. Endod. 2007,33, 957–961. [CrossRef] 28. Kierklo, A.; Tabor, Z.; Pawi´nska, M.; Jaworska, M. A microcomputed tomography-based comparison of root canal filling quality following different instrumentation and obturation techniques. Med. Princ. Pract. 2015,24, 84–91. [CrossRef] 29. De Macedo, L.M.D.; Silva-Sousa, Y.; Da Silva, S.R.C.; Baratto, S.S.P.; Baratto-Filho, F.; Rached-Júnior, F.J.A. Influence of Root Canal Filling Techniques on Sealer Penetration and Bond Strength to Dentin. Braz. Dent. J. 2017,28, 380–384. [CrossRef] 30. Yücel, A.Ç.; Çiftçi, A. Effects of different root canal obturation techniques on bacterial penetration. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 2006,102, e88–e92. [CrossRef] 31. Holmes, S.; Gibson, R.; Butler, J.; Pacheco, R.; Askar, M.; Paurazas, S. Volumetric Evaluation of 5 Root Canal Obturation Methods in TrueTooth 3-dimensional–Printed Tooth Replicas Using Nano–computed Tomography. J. Endod. 2021 ,47, 485–491.e4. [CrossRef] [PubMed] 32. Saberi, E.; Akbari, N.; Ebrahimipour, S.; Jalilpour, H. In-vitro evaluation of coronal microbial leakage after post space tooth preparation. Minerva Stomatol. 2016 ,65, 127–133. Available online: http://www.ncbi.nlm.nih.gov/pubmed/27075369 (accessed on 2 March 2022). [PubMed] 33. Peters, O.A.; Paqué, F. Root Canal Preparation of Maxillary Molars With the Self-adjusting File: A Micro-computed Tomography Study. J. Endod. 2011,37, 53–57. [CrossRef] [PubMed] 34. Haji, T.H.; Selivany, B.J.; Suliman, A.A. Sealing ability in vitro study and biocompatibility in vivo animal study of different bioceramic based sealers. Clin. Exp. Dent. Res. 2022,8, 1582–1590. [CrossRef] 35. Ordinola-Zapata, R.; Bramante, C.M.; Graeff, M.S.; Perochena, A.d.C.; Vivan, R.R.; Camargo, E.J.; Garcia, R.B.; Bernardineli, N.; Gutmann, J.L.; de Moraes, I.G. Depth and percentage of penetration of endodontic sealers into dentinal tubules after root canal obturation using a lateral compaction technique: A confocal laser scanning microscopy study. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endodontol. 2009,108, 450–457. [CrossRef] 36. Mjör, I.A.; Smith, M.R.; Ferrari, M.; Mannocci, F. The structure of dentine in the apical region of human teeth. Int. Endod. J. 2001,34, 346–353. [CrossRef] 37. Yan, W.; Chen, H.; Fernandez-Arteaga, J.; Paranjpe, A.; Zhang, H.; Arola, D. Root fractures in seniors: Consequences of acute embrittlement of dentin. Dent. Mater. 2020,36, 1464–1473. [CrossRef] 38. Allan, N.A.; Walton, R.E.; Schaffer, M. Setting Times for Endodontic Sealers Under Clinical Usage and In Vitro Conditions. J. Endod. 2001,27, 421–423. [CrossRef] 39. Donfrancesco, O.; Del Giudice, A.; Zanza, A.; Relucenti, M.; Petracchiola, S.; Gambarini, G.; Testarelli, L.; Seracchiani, M. SEM Evaluation of Endosequence BC Sealer Hiflow in Different Environmental Conditions. J. Compos. Sci. 2021,5, 99. [CrossRef] 40. Balguerie, E.; van der Sluis, L.; Vallaeys, K.; Gurgel-Georgelin, M.; Diemer, F. Sealer penetration and adaptation in the dentinal tubules: A scanning electron microscopic study. J. Endod. 2011,37, 1576–1579. [CrossRef] 41. Vo, K.; Daniel, J.; Ahn, C.; Primus, C.; Komabayashi, T. Coronal and apical leakage among five endodontic sealers. J. Oral Sci. 2022,64, 95–98. [CrossRef] [PubMed] 42. Piai, G.G.; Duarte, M.A.H.; Nascimento, A.L.D.; da Rosa, R.A.; Só, M.V.R.; Vivan, R.R. Penetrability of a new endodontic sealer: A confocal laser scanning microscopy evaluation. Microsc. Res. Tech. 2018,81, 1246–1249. [CrossRef] [PubMed] 43. Donnermeyer, D.; Schmidt, S.; Rohrbach, A.; Berlandi, J.; Bürklein, S.; Schäfer, E. Debunking the concept of dentinal tubule penetration of endodontic sealers: Sealer staining with rhodamine B fluorescent dye is an inadequate method. Materials 2021,14, 3211. [CrossRef] [PubMed] 44. E˘gemen, A.; Belli, S. The Effect of Primary Root Canal Treatment on Dentinal Tubule Penetration of Calcium Silicate–based Sealers during Endodontic Retreatment. J. Endod. 2022,48, 1169–1177. [CrossRef] [PubMed] J. Funct. Biomater. 2023,14, 542 18 of 18 45. Hembrough, M.; Robertsteiman, H.; Belanger, K. Lateral condensation in canals prepared with nickel-titanium rotary instruments: An evaluation of the use of three different master cones. J. Endod. 2002,28, 516–519. [CrossRef] 46. Pérez Heredia, M.; Clavero González, J.; Ferrer Luque, C.M.; González Rodríguez, M.P. Apical seal comparison of low-temperature thermoplasticized gutta-percha technique and lateral condensation with two different master cones. Med. Oral Patol. Oral Cirugía Bucal. 2007,12, E175–E179. Available online: http://www.ncbi.nlm.nih.gov/pubmed/17322810 (accessed on 3 March 2022). 47. Schäfer, E.; Köster, M.; Bürklein, S. Percentage of Gutta-percha–filled Areas in Canals Instrumented with Nickel-Titanium Systems and Obturated with Matching Single Cones. J. Endod. 2013,39, 924–928. [CrossRef] 48. Lea, C.; Apicella, M.; Mines, P.; Yancich, P.; Parker, M. Comparison of the obturation density of cold lateral compaction versus warm vertical compaction using the continuous wave of condensation technique. J. Endod. 2005,31, 37–39. [CrossRef] 49. Ferrari, M.; Mannocci, F.; Vichi, A.; Cagidiaco, M.C.; Mjör, I.A. Bonding to root canal: Structural characteristics of the substrate. Am. J. Dent. 2000,13, 255–260. Available online: http://www.ncbi.nlm.nih.gov/pubmed/11764112 (accessed on 3 March 2022). 50. Paqué, F.; Luder, H.U.; Sener, B.; Zehnder, M. Tubular sclerosis rather than the smear layer impedes dye penetration into the dentine of endodontically instrumented root canals. Int. Endod. J. 2006,39, 18–25. [CrossRef] 51. De-Deus, G.; Souza, E.M.; Silva, E.J.N.L.; Belladonna, F.G.; Simões-Carvalho, M.; Cavalcante, D.M.; Versiani, M.A. A critical analysis of research methods and experimental models to study root canal fillings. Int. Endod. J. 2022 ,55 (Suppl. S2), 384–445. [CrossRef] [PubMed] 52. Mekhdieva, E.; Del Fabbro, M.; Alovisi, M.; Comba, A.; Scotti, N.; Tumedei, M.; Carossa, M.; Berutti, E.; Pasqualini, D. Postoperative Pain following Root Canal Filling with Bioceramic vs. Traditional Filling Techniques: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. J. Clin. Med. 2021,10, 4509. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.