Influence of Continuous Chelation Protocols and Activation Techniques on Root Canal Sealer Penetration: An In Vitro Study

Influence of Continuous Chelation Protocols and Activation Techniques on Root Canal Sealer Penetration: An In Vitro Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Influence of Continuous Chelation Protocols and Activation Techniques on Root Canal Sealer Penetration: An In Vitro Study Emine Odabaşı Tezer, Dilan Kırmızı, Dilara Koruk Yürün, Abdullah Sebai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9213015/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Smear layer removal is essential for sealer penetration and obturation quality. This study aimed to compare different final irrigation solutions and activation methods on sealer penetration using confocal laser scanning microscopy (CLSM). Methods Sixty extracted single-rooted mandibular premolars were instrumented and randomly allocated into six groups (n = 10) based on the final irrigation protocol: ethylenediaminetetraacetic acid (EDTA), 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) or clodronate (CLO) combined with conventional syringe irrigation (CI) or EndoActivator (EA) system. Root canals were obturated using a single-cone technique with Rhodamine B–labeled BioRoot Flow sealer. Apical and middle sections were evaluated under CLSM, and penetration parameters were analyzed using ImageJ with non-parametric statistics (p < 0.05). Results Sealer penetration depth was significantly higher in the apical than in the middle third (p < 0.05), with sonic activation producing greater depths than conventional irrigation. The EA+HEDP group showed the highest apical penetration depth (499.41 µm), whereas the largest penetration areas were observed in the EA + CLO group and the lowest in the CI+EDTA group. Conclusions Sonic activation enhanced sealer penetration with chelating agents. Activated HEDP mainly increased penetration depth in the apical region, while activated clodronate produced wider penetration areas in the middle third. Chelating agents Clodronate HEDP Sealer penetration Smear layer removal Sonic activation Figures Figure 1 Background Root canal therapy aims to effectively debride and shape the root canal system before obturating it with an inert filling material to prevent reinfection [ 1 ]. Mechanical preparation of the root canal inevitably generates a smear layer that attaches to the walls of the canal [ 2 ]. Smear layer may restrict the dentinal tubules' penetration by sealers and can facilitate bacterial infiltration into the dentinal tubules [ 3 ]. Consequently, various irrigation solutions and activation systems are being developed to eliminate the smear layer and improve canal cleanliness and obturation quality [ 4 , 5 ]. Sodium hypochlorite (NaOCl), utilized at 0.5% to 5.25% concentrations, is the main irrigant used in root canal therapy. Nonetheless, when used alone, it is insufficient to efficiently eliminate the smear layer [ 6 ]. Therefore, using a chelating agent for irrigation of the root canal system is suggested to facilitate smear layer removal and to allow deeper penetration of the root canal sealer [ 7 ]. Ethylenediaminetetraacetic acid (EDTA) usage following NaOCl constitutes the most commonly employed irrigation protocol in root canal therapy [ 8 ]. In recent years, the use of alternative chelating agents in endodontic therapy has been explored, among which 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) has attracted attention [ 8 , 9 ]. Unlike EDTA, HEDP does not react with sodium hypochlorite (NaOCl), which permits their combined use during final irrigation. This characteristic allows organic and inorganic components of the smear layer to be removed simultaneously within a single irrigation step [ 10 ]. Lately, a further chelating agent clodronate (CLO) used at an alkaline pH has been introduced for use in continuous chelation. NaOCl-clodronate combinations have been shown to remove the smear layer [ 11 ] and to preserve free available chlorine levels at root canal temperature for durations of time that are clinically relevant [ 12 ]. These properties suggest that clodronate-NaOCl mixtures may demonstrate notable antimicrobial activity [ 13 ]. Conventional syringe irrigation (CI) alone does not consistently achieve adequate penetration of irrigants into the dentinal tubules, which can limit smear layer removal and sealer adaptation [ 14 ]. For this reason, various activation techniques have been introduced to improve the performance of irrigating solutions during root canal treatment [ 15 ]. Sonic activation is based on low frequency vibrations (1–6 kHz) produced by elastic polymer tips and applied with short, controlled pecking motions inside the root canal. These movements generate fluid motion within the canal, which can increase irrigant penetration and support deeper sealer penetration into the dentinal tubules [ 16 , 17 ]. The EndoActivator system (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA) is a sonic irrigation device designed to agitate the irrigant within the root canal system and has shown better irrigation performance than conventional syringe irrigation [ 18 , 19 ]. Nevertheless, available studies provide limited and inconsistent data on the comparative performance of different chelating agents used in continuous chelation, particularly when these protocols are combined with sonic activation. In addition, the extent to which such irrigation strategies influence dentin surface conditioning and subsequent sealer penetration has not been clearly established. The purpose of this study was to investigate how different final irrigation solutions, used with or without sonic activation, affect dentin surface conditioning and root canal sealer penetration into dentinal tubules. These outcomes were evaluated using confocal laser scanning microscopy (CLSM) to allow a comparative assessment of irrigation protocols applied during the final stage of root canal treatment. Materials and Methods The study was conducted in accordance with the Declaration of Helsinki and approved by the Non-Clinical Scientific Research Ethics Committee of *XXX* University (Approval No: 65/2023) on 12-10-2025. The study was conducted as an ex vivo laboratory investigation using extracted human teeth. The sample size was calculated a priori using G*Power software (version 3.1.9.7; Heinrich Heine University, Düsseldorf, Germany) for a fixed-effects one-way ANOVA (omnibus F test). The study design included six independent groups. The effect size was set at Cohen’s f = 0.50, based on comparable studies evaluating sealer penetration using confocal laser scanning microscopy. The Type I error probability (α) was set at 0.05 and the desired statistical power (1 − β) at 0.80. Based on these parameters, the required total sample size was calculated as 60 specimens, corresponding to 10 specimens per group. The achieved power was 0.8247, indicating that the study was adequately powered to detect statistically significant differences among groups at the 5 percent significance level. Selection of Samples: The current study utilized 60 extracted permanent mandibular premolars collected from patients who had provided written informed consent for the use of their teeth for research purposes at the Department of Maxillofacial Surgery. All teeth exhibited a single root and a single canal. Verification of canal configuration was performed using radiographs from both buccal and proximal views. Teeth displaying signs of fracture, caries, root resorption, prior restorations, pulp canal obliteration, or incomplete root development were excluded following radiographic screening. Specimens were kept in distilled water at 4°C until experimentation. Surface residues were thoroughly removed using an ultrasonic scaler. The crowns were removed at the cementoenamel junction with a diamond disc under water cooling, resulting in a standardized root length of 18 mm. A size 10 K-file (Dentsply Maillefer, Switzerland) was utilized to assess the canal length. Once the file tip appeared flush with the apical foramen, the working length was set 1 mm short of this measurement. Root Canal Preparation of Samples: Canal instrumentation was performed by the ProTaper Ultimate rotary file system (Dentsply Sirona, Switzerland) up to instrument size F3. Following each instrumentation step, the canals were irrigated with 2 mL of 2.5% NaOCl using a 27-gauge endodontic irrigation needle positioned at a level 1 mm coronal to the working length. Prior to the final irrigation protocol, the apical foramina were sealed utilizing pink wax in order to avoid apical extrusion of the irrigant. Final Stage of Root Canal Irrigation Regimen: Following canal preparation, the teeth were randomly allocated into six experimental groups (n = 10) based on two different irrigation approaches (conventional syringe irrigation or sonic activation) and three distinct final irrigation solutions. Specimen preparation and irrigation procedures were performed according to the predefined group sequence, as detailed below. Conventional Syringe Irrigation (CI) Groups CI+EDTA Group: This group received a 2.5% NaOCl solution (5 mL), and after that, 17% EDTA (5 mL) was delivered with the syringe. Finally, 5 mL of distilled water was applied to rinse the canals. CI+HEDP Group: The irrigation protocol consisted of 2.5% NaOCl solution (5 mL), followed by 5 mL of a freshly mixed solution containing 2.5% NaOCl with 18% HEDP. The procedure ended with 5 mL of distilled water as the final rinse. CI + CLO Group: In this group, 2.5% NaOCl solution (5 mL) was used first, and then 5 mL of clodronate solution was applied. The protocol was completed with 5 mL of distilled water. The clodronate solution (0.26 mol·L⁻¹) was prepared by dissolving disodium clodronate tetrahydrate (CAS 88416-50-6; Tokyo Chemical Industry, Japan) in deionized water. The pH was adjusted to 10.7 through titration with sodium hydroxide. Sonic Activation (EndoActivator, EA) Groups EA+EDTA Group: The canals were irrigated using 2.5% NaOCl solution (5 mL) followed by 17% EDTA (5 mL). Sonic agitation of both irrigants was performed using the EndoActivator system rather than being used passively. EA+HEDP Group: This group received 2.5% NaOCl solution (5 mL), and then a mixture prepared from 2.5% NaOCl and 18% HEDP was used as the second irrigant. Activation with the EndoActivator was applied for both solutions. EA + CLO Group: For this protocol, the canals were first rinsed with 5 mL of 2.5% NaOCl, and afterwards 5 mL of the clodronate solution was delivered. Both irrigants in this group were activated using the EndoActivator system. In all groups, irrigation was performed by positioning a 27-gauge side-vented needle to a point 2 mm short of the predetermined working length. In the conventional syringe groups (CI), the needle was moved in an in-and-out manual motion, and each solution was delivered over 30 seconds. In the EndoActivator (EA) groups, the activation was performed with a medium tip (size 25, 0.04 taper) operating at a rate of 10,000 cpm for 30 seconds. After the main irrigation sequence, all canals were flushed with 5 mL of distilled water for 30 seconds. Obturation Protocol: Following root canal shaping and completion of the final irrigation procedures, including activation where applicable, sterile paper points (size F3) were used to dry all canals (ProTaper Ultimate Paper Point, Dentsply Sirona, Switzerland). F3 gutta-percha cones were adjusted to the established working length, and their apical conformity was verified through the assessment of definitive apical tug-back. The BioRoot Flow (Septodont, Saint-Maur-des-Fosses, France), a calcium silicate–based bioceramic root canal sealer was supplemented with 0.1% Rhodamine B to provide fluorescence for confocal laser scanning microscopy. A small amount of sealer was initially introduced into the canals, after which the corresponding master cone (size F3) was coated with the sealer and inserted to the working length. Root canal obturation was performed using the single-cone technique. A heated ball burnisher was used to trim the gutta-percha to 1 mm below the coronal reference surface. Vertical compaction of the filling material was achieved using a plugger. Any residual sealer in the access cavity was cleaned with a moistened cotton pellet, and the cavities were sealed with a temporary coronal sealing material (Orafil G, Prevest DenPro, Jammu, India). Subsequently, all samples were incubated at 37°C in a fully humid environment for one week to ensure complete sealer setting. Sample Sectioning and CLSM Analysis: All specimens (n = 10 / per group) were embedded vertically in acrylic resin blocks, and two horizontal sections (1 mm thick) were obtained 3 mm apically and 5 mm in the middle third from the apical foramen. Overall, 120 sections were prepared, corresponding to 20 sections derived from 10 teeth in each group. Sectioning was performed using a low-speed, water-cooled precision diamond saw (Micracut 201). After sectioning, each specimen was polished with silicon carbide abrasive papers under continuous water irrigation to remove surface irregularities. The polished slices were then placed on microscope slides by fixing their apical surfaces to double-sided adhesive tape and afterwards prepared for confocal laser scanning microscopy (CLSM). Confocal images were captured using a CLSM system set to a 543 nm excitation wavelength, with a spatial resolution of 512 × 512 pixels and a ×10 objective. When a single frame did not cover the full canal circumference, several overlapping images were taken and later combined into a single view using Adobe Photoshop. All images were calibrated with a 100 µm scale bar and analyzed in ImageJ to measure maximum penetration depth, the percentage of tubules showing penetration, and the overall penetration area. Statistical Analysis: The data obtained in current study were analyzed using IBM SPSS Statistics 22.0 (IBM Corp., Armonk, NY, USA). Analysis of each variable’s distribution using the Shapiro–Wilk test showed non-normal behavior, prompting the use of non-parametric methods for all subsequent statistical procedures. Differences between anatomical regions (apical and middle) within each experimental group were evaluated using the Mann–Whitney U test, a non-parametric method appropriate for comparing two independent samples. For examining differences across the six experimental groups at the same anatomical level, the Kruskal–Wallis H test was applied. Whenever statistically significant differences were detected by the Kruskal–Wallis H test, a post-hoc analysis with the Dunn test and Bonferroni correction was performed to determine the source of the variance. The level of statistical significance was set at p < 0.05 for all comparisons. Results Table 1 summarizes the descriptive statistics of maximum sealer penetration depth (µm) for different chelators and irrigation approaches in the middle and apical root canal sections. In all groups, penetration depth values were significantly higher in the apical third than in the middle third (p < 0.05). Sonic activation consistently increased apical penetration compared with conventional irrigation. The EA+HEDP protocol yielded the highest mean penetration depth in the apical region (499.41 µm), whereas the CI+HEDP group showed significantly lower apical values (310.30 µm) than all activated groups (p < 0.01). In the middle third, conventional EDTA resulted in the lowest penetration depth (136.94 µm). Representative CLSM images are presented in Fig. 1 . Penetration area measurements (µm²) are given in Table 2 . In all groups, the middle third displayed a larger penetration area than the apical part (p < 0.05). Among the protocols, the EA + CLO group showed the widest penetration areas (middle: 8077.74 µm²; apical: 4637.68 µm²). Conversely, conventional irrigation resulted in reduced penetration areas, with the lowest values observed in the CI+EDTA group (middle: 2844.45 µm²; apical: 371.84 µm²). Taken together, these findings indicate that activation improves irrigant distribution across all chelators, with the most pronounced effect observed in the middle third of the canal. Table 1 Descriptive statistics (mean ± SD) of maximum sealer penetration depth (µm) in the middle and apical regions of root canals according to different chelators and final irrigation approaches. Different superscript letters indicate statistically significant differences among groups (p < 0.05). Final Irrigation Approaches (n = 10 per group) Middle Region Apical Region Mean ± SD Mean ± SD CI+EDTA Group 136.94 ± 12.38 a 338.32 ± 45.37 a CI+HEDP Group 225.32 ± 45.19 a 310.30 ± 58.11 a CI + CLO Group 151.90 ± 97.14 a 324.83 ± 143.17 a EA+EDTA Group 300.01 ± 32.35 b 369.11 ± 50.40 b EA+HEDP Group 431.78 ± 31.75 b 499.41 ± 31.69 b EA + CLO Group 304.63 ± 130.96 b 463.38 ± 141.69 b Table 2 Descriptive statistics (mean ± SD) of sealer penetration area (µm²) in the middle and apical regions of root canals according to different chelators and final irrigation approaches. Different superscript letters indicate statistically significant differences among groups (p < 0.05). Final Irrigation Approaches (n = 10 per group) Middle Region Apical Region Mean ± SD Mean ± SD CI+EDTA Group 2844.45 ± 400.44 a 371.84 ± 125.93 a CI+HEDP Group 2312.53 ± 353.29 a 1718.31 ± 387.25 a CI + CLO Group 5910.45 ± 3835.79 b 2885.07 ± 2515.04 a EA+EDTA Group 3123.32 ± 972.69 a 2134.53 ± 429.09 b EA+HEDP Group 5024.49 ± 961.51 b 2851.20 ± 363.37 b EA + CLO Group 8077.74 ± 3711.76 b 4637.68 ± 2539.83 b Discussion During root canal preparation, a smear layer is always produced as a result of instrument contact with dentin and soft tissues. This mixture closes the dentinal tubules and reduces how well the sealer can adapt to the canal wall. For this reason, many studies have associated the presence of the smear layer with leakage problems and bacterial persistence in the long term [ 20 , 21 ]. Because bioceramic and other bioactive sealers need direct contact with dentin to perform properly, removing this layer becomes especially important [ 22 ]. In the present study, different chelating agents were evaluated in combination with different irrigation approaches to evaluate their effect on sealer penetration. In this study, activation was included because it is thought to improve the way the irrigant moves inside the canal. By increasing the internal flow, the irrigant is expected to reach parts of the canal that may otherwise remain untouched. Similar ideas have been discussed in endodontic literature and earlier studies have also described comparable findings [ 23 , 24 ]. More recent research reports that combining activation with chelators may help the sealer penetrate more deeply into the dentinal tubules [ 25 , 26 ]. In the present study, sealer penetration depth was higher in the apical third, whereas penetration area was greater in the middle third. This finding reflects the structural characteristics of different root canal levels. The apical region is characterized by fewer dentinal tubules, increased sclerosis, and restricted canal space, which limits irrigant movement and may reduce sealer–dentin interaction. In addition, the restricted canal space in this region limits irrigant movement and makes smear layer removal less predictable. When the smear layer is not adequately removed, the interaction between the sealer and dentin is reduced, which may negatively influence penetration. Similar differences between apical and middle regions have been reported in previous studies evaluating sealer penetration at different root canal levels [ 27 , 28 ]. In the present study, the apical preparation size was extended to F3, which was intended to improve irrigant access in the apical region. A larger apical preparation has been associated with better irrigant flow and more effective cleaning by allowing greater fluid exchange within the canal [ 25 ]. This factor may have played a role in the penetration patterns observed in the apical third. Similar observation has been made in study where ProTaper Next X4 was used together with activation, reporting better sealer penetration under those conditions [ 26 ]. Compared with EDTA and HEDP, clodronate has been examined in a more limited number of studies. Nevertheless, the existing literature suggests that clodronate can interact with calcium-containing components of the smear layer and supports its removal at a level like EDTA [ 13 , 29 ]. In the present study, EDTA and HEDP showed comparable sealer penetration values under passive irrigation conditions, which agrees with previous reports indicating similar behavior of these agents when activation is not applied [ 30 ]. Once activation was introduced, both HEDP and Clodronate showed a noticeable increase in penetration. Activation improved penetration depth mainly in the apical third, while its effect on penetration area was more pronounced in the middle third. This outcome reflects the trend described in recent literature, where sonic activation and other newly developed irrigation systems have been found to improve irrigant distribution compared with standard syringe irrigation [ 31 , 32 ]. Overall, these findings suggest that while EDTA, HEDP, and clodronate show comparable performance under passive irrigation, the application of sonic activation plays a decisive role in enhancing the effectiveness of chelating agents by improving irrigant distribution and dentin surface conditioning, thereby facilitating deeper sealer penetration. Across all groups, the penetration was higher in the middle third than in the apical third. Anatomical factors explain this trend: the apical region has fewer tubules, more sclerosis and limited space for irrigant movement. These characteristics naturally restrict how deep the sealer can penetrate, and similar results have been reported by earlier studies [ 22 , 33 , 34 ]. Improved penetration of the sealer into dentinal tubules has been linked with better sealing behaviour and long-term success of root canal treatment [ 20 , 23 ]. Bioactive sealers benefit from deeper penetration because they interact chemically with dentin and may form hydroxyapatite at the interface [ 22 , 25 ]. In this study, irrigation protocols incorporating activation resulted in higher penetration values than the conventional irrigation protocol, which is compatible with earlier findings using sonic or laser activation [ 35 , 36 ]. However, some studies have shown that activation does not always lead to better penetration compared with conventional irrigation [ 37 ]. Differences in sealer type, irrigant protocol, or canal morphology may contribute to such inconsistent findings. In current study, CLSM was selected mainly because it allows samples to be examined without the dehydration or coating procedures required for SEM, which can interfere with the sealer–dentin interface. The method also makes it possible to evaluate both penetration depth and the overall penetration area with reasonable accuracy [ 38 , 39 ]. For visualization, a small amount of Rhodamine B was mixed into the sealer. Although the concentration was low, dyes may still have some minor influence on the material, and this should be kept in mind when interpreting the results [ 40 ]. Since the current study is carried out under ex vivo conditions, it was not possible to reproduce certain clinical factors, such as the natural behaviour of periapical tissues or internal fluid pressure. The dentin slices taken at 3 mm and 5 mm permitted a comparison between the apical and middle thirds. As expected, the apical region showed more limited penetration, which can be explained by its structural features particularly increased sclerosis and reduced tubule permeability [ 33 , 34 ]. Further research is still required, especially in curved canals and minimally prepared systems, and in understanding the behaviour of alternative chelators such as Clodronate under activated conditions [ 13 , 32 , 35 ]. Conclusions Sonic activation during final irrigation enhanced sealer penetration by improving the effectiveness of chelating agents and their interaction with dentin surfaces. These findings highlight the clinical relevance of irrigation dynamics in achieving effective dentin conditioning and more consistent sealer adaptation under activated conditions. Declarations Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki and approved by the Non-Clinical Scientific Research Ethics Committee of Near East University (Approval No: 65/2023, 12 October 2025). Informed consent was obtained from all participants involved in the study. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Clinical trial number Not applicable. Funding This research received no external funding. Author Contribution Conceptualization: F.B.Methodology: D.K., D.K.Y.Software: A.S.Validation: E.O.T., D.K., A.S.Formal analysis: E.O.T., D.K.Y., A.S.Investigation: D.K., D.K.Y.Resources: E.O.T.Data curation: E.O.T., D.K.Y., D.K.Writing – original draft: D.K., D.K.Y.Writing – review & editing: F.B., M.D.Ö.Visualization: A.S.Supervision: F.B., M.D.Ö.Project administration: F.B., M.D.Ö.All authors read and approved the final manuscript. 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BMC Oral Health. 2025;25:991. Bolles JA, He J, Svoboda KK, Schneiderman E, Glickman GN. Comparison of Vibringe, EndoActivator, and needle irrigation on sealer penetration in extracted human teeth. J Endod. 2013;39(5):708–11. Urban K, Donnermeyer D, Schafer E, Burklein S. Canal cleanliness using different irrigation activation systems: a SEM evaluation. Clin Oral Investig. 2017;21(9):2681–7. Aguiar BA, Frota LM, Taguatinga DT, Vivan RR, Camilleri J, Duarte MA, et al. Influence of ultrasonic agitation on bond strength, marginal adaptation, and tooth discoloration provided by three coronary barrier endodontic materials. Clin Oral Investig. 2019;23(11):4113–22. Donnermeyer D, Schmidt S, Rohrbach A, Berlandi J, Burklein S, Schafer E. Debunking the concept of dentinal tubule penetration of endodontic sealers: sealer staining with rhodamine B fluorescent dye is an inadequate method. Mater (Basel). 2021;14(12):3211. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9213015","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":631558243,"identity":"6c7613d8-ba4a-4882-bd7b-4fbb1b7b3c95","order_by":0,"name":"Emine Odabaşı Tezer","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Emine","middleName":"Odabaşı","lastName":"Tezer","suffix":""},{"id":631558246,"identity":"61b88522-b988-4ef6-b7fe-344e3fba548e","order_by":1,"name":"Dilan Kırmızı","email":"","orcid":"","institution":"Near East University","correspondingAuthor":false,"prefix":"","firstName":"Dilan","middleName":"","lastName":"Kırmızı","suffix":""},{"id":631558250,"identity":"e07ed15d-5839-4a17-b64b-5440427a1b3b","order_by":2,"name":"Dilara Koruk Yürün","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDCCw8wNIMoAiJkZPkDEDAhoYURoYZwB15KAR8sBJC3MPMRo4TvO2Pi5oobBmF/6jLGx7Q6bxAb25m0SjD/u4dQieZixWfLMMQYzyb4c4+TcM2mJDTzHyiQYEopxajEA+kWygY3BxuAMj/Hh3LbDiQ0SOWZALbhdBtTS/LPhH1SLZdv/xAb5NwS1tEk2tjGYgbQkM7YdANrCg18L0C9tlo19EsaSPWzFhr1tycZtPGnFFglpuLXwnT98+GbDNxvDfh7mzRI/2+xk+9kPb7zxwQa3FiiQQDDZQARBDaNgFIyCUTAK8AIAxa9MG1GjwnsAAAAASUVORK5CYII=","orcid":"","institution":"Near East University","correspondingAuthor":true,"prefix":"","firstName":"Dilara","middleName":"Koruk","lastName":"Yürün","suffix":""},{"id":631558251,"identity":"080bc228-92ff-4944-bb15-aaa0bab87eaf","order_by":3,"name":"Abdullah Sebai","email":"","orcid":"","institution":"Near East University","correspondingAuthor":false,"prefix":"","firstName":"Abdullah","middleName":"","lastName":"Sebai","suffix":""},{"id":631558263,"identity":"34fede66-5af6-4a67-b776-2270d0996376","order_by":4,"name":"Fatma Basmacı","email":"","orcid":"","institution":"Near East University","correspondingAuthor":false,"prefix":"","firstName":"Fatma","middleName":"","lastName":"Basmacı","suffix":""},{"id":631558266,"identity":"b99827bb-2799-426e-985d-68881228a587","order_by":5,"name":"Meltem Dartar Öztan","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Meltem","middleName":"Dartar","lastName":"Öztan","suffix":""}],"badges":[],"createdAt":"2026-03-24 14:09:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9213015/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9213015/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108215544,"identity":"771703b7-f573-4347-8f2d-217eed2c7fa1","added_by":"auto","created_at":"2026-04-30 14:25:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5431124,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative CLSM images of sealer penetration in the middle and apical thirds of root canals following different irrigation protocols.\u003c/p\u003e","description":"","filename":"FIGURES1rev2.1.png","url":"https://assets-eu.researchsquare.com/files/rs-9213015/v1/7984fe2a36b7726357eea500.png"},{"id":108627467,"identity":"a680fac0-711f-49f7-8f87-c00f06e8e31a","added_by":"auto","created_at":"2026-05-06 15:56:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6298060,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9213015/v1/7e033a65-16b3-4a1a-923e-2bd8a0b5d877.pdf"},{"id":108215545,"identity":"7c08f649-4e55-42c7-afe8-60674c0c4e3e","added_by":"auto","created_at":"2026-04-30 14:25:51","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":378420,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-9213015/v1/75146126cde121eed29e2948.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Influence of Continuous Chelation Protocols and Activation Techniques on Root Canal Sealer Penetration: An In Vitro Study","fulltext":[{"header":"Background","content":"\u003cp\u003eRoot canal therapy aims to effectively debride and shape the root canal system before obturating it with an inert filling material to prevent reinfection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Mechanical preparation of the root canal inevitably generates a smear layer that attaches to the walls of the canal [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Smear layer may restrict the dentinal tubules' penetration by sealers and can facilitate bacterial infiltration into the dentinal tubules [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Consequently, various irrigation solutions and activation systems are being developed to eliminate the smear layer and improve canal cleanliness and obturation quality [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSodium hypochlorite (NaOCl), utilized at 0.5% to 5.25% concentrations, is the main irrigant used in root canal therapy. Nonetheless, when used alone, it is insufficient to efficiently eliminate the smear layer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, using a chelating agent for irrigation of the root canal system is suggested to facilitate smear layer removal and to allow deeper penetration of the root canal sealer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Ethylenediaminetetraacetic acid (EDTA) usage following NaOCl constitutes the most commonly employed irrigation protocol in root canal therapy [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In recent years, the use of alternative chelating agents in endodontic therapy has been explored, among which 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) has attracted attention [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Unlike EDTA, HEDP does not react with sodium hypochlorite (NaOCl), which permits their combined use during final irrigation. This characteristic allows organic and inorganic components of the smear layer to be removed simultaneously within a single irrigation step [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLately, a further chelating agent clodronate (CLO) used at an alkaline pH has been introduced for use in continuous chelation. NaOCl-clodronate combinations have been shown to remove the smear layer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and to preserve free available chlorine levels at root canal temperature for durations of time that are clinically relevant [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These properties suggest that clodronate-NaOCl mixtures may demonstrate notable antimicrobial activity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConventional syringe irrigation (CI) alone does not consistently achieve adequate penetration of irrigants into the dentinal tubules, which can limit smear layer removal and sealer adaptation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For this reason, various activation techniques have been introduced to improve the performance of irrigating solutions during root canal treatment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSonic activation is based on low frequency vibrations (1\u0026ndash;6 kHz) produced by elastic polymer tips and applied with short, controlled pecking motions inside the root canal. These movements generate fluid motion within the canal, which can increase irrigant penetration and support deeper sealer penetration into the dentinal tubules [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The EndoActivator system (Dentsply Tulsa Dental Specialties, Tulsa, OK, USA) is a sonic irrigation device designed to agitate the irrigant within the root canal system and has shown better irrigation performance than conventional syringe irrigation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nevertheless, available studies provide limited and inconsistent data on the comparative performance of different chelating agents used in continuous chelation, particularly when these protocols are combined with sonic activation. In addition, the extent to which such irrigation strategies influence dentin surface conditioning and subsequent sealer penetration has not been clearly established.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to investigate how different final irrigation solutions, used with or without sonic activation, affect dentin surface conditioning and root canal sealer penetration into dentinal tubules. These outcomes were evaluated using confocal laser scanning microscopy (CLSM) to allow a comparative assessment of irrigation protocols applied during the final stage of root canal treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki and approved by the Non-Clinical Scientific Research Ethics Committee of *XXX* University (Approval No: 65/2023) on 12-10-2025. The study was conducted as an ex vivo laboratory investigation using extracted human teeth. The sample size was calculated a priori using G*Power software (version 3.1.9.7; Heinrich Heine University, D\u0026uuml;sseldorf, Germany) for a fixed-effects one-way ANOVA (omnibus F test). The study design included six independent groups. The effect size was set at Cohen\u0026rsquo;s f\u0026thinsp;=\u0026thinsp;0.50, based on comparable studies evaluating sealer penetration using confocal laser scanning microscopy. The Type I error probability (α) was set at 0.05 and the desired statistical power (1\u0026thinsp;\u0026minus;\u0026thinsp;β) at 0.80. Based on these parameters, the required total sample size was calculated as 60 specimens, corresponding to 10 specimens per group. The achieved power was 0.8247, indicating that the study was adequately powered to detect statistically significant differences among groups at the 5 percent significance level.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSelection of Samples:\u003c/h2\u003e \u003cp\u003eThe current study utilized 60 extracted permanent mandibular premolars collected from patients who had provided written informed consent for the use of their teeth for research purposes at the Department of Maxillofacial Surgery. All teeth exhibited a single root and a single canal. Verification of canal configuration was performed using radiographs from both buccal and proximal views. Teeth displaying signs of fracture, caries, root resorption, prior restorations, pulp canal obliteration, or incomplete root development were excluded following radiographic screening. Specimens were kept in distilled water at 4\u0026deg;C until experimentation. Surface residues were thoroughly removed using an ultrasonic scaler. The crowns were removed at the cementoenamel junction with a diamond disc under water cooling, resulting in a standardized root length of 18 mm. A size 10 K-file (Dentsply Maillefer, Switzerland) was utilized to assess the canal length. Once the file tip appeared flush with the apical foramen, the working length was set 1 mm short of this measurement.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRoot Canal Preparation of Samples:\u003c/h3\u003e\n\u003cp\u003eCanal instrumentation was performed by the ProTaper Ultimate rotary file system (Dentsply Sirona, Switzerland) up to instrument size F3. Following each instrumentation step, the canals were irrigated with 2 mL of 2.5% NaOCl using a 27-gauge endodontic irrigation needle positioned at a level 1 mm coronal to the working length. Prior to the final irrigation protocol, the apical foramina were sealed utilizing pink wax in order to avoid apical extrusion of the irrigant.\u003c/p\u003e\n\u003ch3\u003eFinal Stage of Root Canal Irrigation Regimen:\u003c/h3\u003e\n\u003cp\u003eFollowing canal preparation, the teeth were randomly allocated into six experimental groups (n\u0026thinsp;=\u0026thinsp;10) based on two different irrigation approaches (conventional syringe irrigation or sonic activation) and three distinct final irrigation solutions. Specimen preparation and irrigation procedures were performed according to the predefined group sequence, as detailed below.\u003c/p\u003e\n\u003ch3\u003eConventional Syringe Irrigation (CI) Groups\u003c/h3\u003e\n\u003cp\u003eCI+EDTA Group:\u003c/p\u003e \u003cp\u003eThis group received a 2.5% NaOCl solution (5 mL), and after that, 17% EDTA (5 mL) was delivered with the syringe. Finally, 5 mL of distilled water was applied to rinse the canals.\u003c/p\u003e \u003cp\u003eCI+HEDP Group:\u003c/p\u003e \u003cp\u003eThe irrigation protocol consisted of 2.5% NaOCl solution (5 mL), followed by 5 mL of a freshly mixed solution containing 2.5% NaOCl with 18% HEDP. The procedure ended with 5 mL of distilled water as the final rinse.\u003c/p\u003e \u003cp\u003eCI\u0026thinsp;+\u0026thinsp;CLO Group:\u003c/p\u003e \u003cp\u003eIn this group, 2.5% NaOCl solution (5 mL) was used first, and then 5 mL of clodronate solution was applied. The protocol was completed with 5 mL of distilled water.\u003c/p\u003e \u003cp\u003eThe clodronate solution (0.26 mol\u0026middot;L⁻\u0026sup1;) was prepared by dissolving disodium clodronate tetrahydrate (CAS 88416-50-6; Tokyo Chemical Industry, Japan) in deionized water. The pH was adjusted to 10.7 through titration with sodium hydroxide.\u003c/p\u003e\n\u003ch3\u003eSonic Activation (EndoActivator, EA) Groups\u003c/h3\u003e\n\u003cp\u003eEA+EDTA Group:\u003c/p\u003e \u003cp\u003eThe canals were irrigated using 2.5% NaOCl solution (5 mL) followed by 17% EDTA (5 mL). Sonic agitation of both irrigants was performed using the EndoActivator system rather than being used passively.\u003c/p\u003e \u003cp\u003eEA+HEDP Group:\u003c/p\u003e \u003cp\u003eThis group received 2.5% NaOCl solution (5 mL), and then a mixture prepared from 2.5% NaOCl and 18% HEDP was used as the second irrigant. Activation with the EndoActivator was applied for both solutions.\u003c/p\u003e \u003cp\u003eEA\u0026thinsp;+\u0026thinsp;CLO Group:\u003c/p\u003e \u003cp\u003eFor this protocol, the canals were first rinsed with 5 mL of 2.5% NaOCl, and afterwards 5 mL of the clodronate solution was delivered. Both irrigants in this group were activated using the EndoActivator system.\u003c/p\u003e \u003cp\u003eIn all groups, irrigation was performed by positioning a 27-gauge side-vented needle to a point 2 mm short of the predetermined working length. In the conventional syringe groups (CI), the needle was moved in an in-and-out manual motion, and each solution was delivered over 30 seconds. In the EndoActivator (EA) groups, the activation was performed with a medium tip (size 25, 0.04 taper) operating at a rate of 10,000 cpm for 30 seconds. After the main irrigation sequence, all canals were flushed with 5 mL of distilled water for 30 seconds.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eObturation Protocol:\u003c/h2\u003e \u003cp\u003eFollowing root canal shaping and completion of the final irrigation procedures, including activation where applicable, sterile paper points (size F3) were used to dry all canals (ProTaper Ultimate Paper Point, Dentsply Sirona, Switzerland). F3 gutta-percha cones were adjusted to the established working length, and their apical conformity was verified through the assessment of definitive apical tug-back. The BioRoot Flow (Septodont, Saint-Maur-des-Fosses, France), a calcium silicate\u0026ndash;based bioceramic root canal sealer was supplemented with 0.1% Rhodamine B to provide fluorescence for confocal laser scanning microscopy.\u003c/p\u003e \u003cp\u003eA small amount of sealer was initially introduced into the canals, after which the corresponding master cone (size F3) was coated with the sealer and inserted to the working length. Root canal obturation was performed using the single-cone technique. A heated ball burnisher was used to trim the gutta-percha to 1 mm below the coronal reference surface. Vertical compaction of the filling material was achieved using a plugger. Any residual sealer in the access cavity was cleaned with a moistened cotton pellet, and the cavities were sealed with a temporary coronal sealing material (Orafil G, Prevest DenPro, Jammu, India). Subsequently, all samples were incubated at 37\u0026deg;C in a fully humid environment for one week to ensure complete sealer setting.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample Sectioning and CLSM Analysis:\u003c/h3\u003e\n\u003cp\u003eAll specimens (n\u0026thinsp;=\u0026thinsp;10 / per group) were embedded vertically in acrylic resin blocks, and two horizontal sections (1 mm thick) were obtained 3 mm apically and 5 mm in the middle third from the apical foramen. Overall, 120 sections were prepared, corresponding to 20 sections derived from 10 teeth in each group. Sectioning was performed using a low-speed, water-cooled precision diamond saw (Micracut 201). After sectioning, each specimen was polished with silicon carbide abrasive papers under continuous water irrigation to remove surface irregularities. The polished slices were then placed on microscope slides by fixing their apical surfaces to double-sided adhesive tape and afterwards prepared for confocal laser scanning microscopy (CLSM).\u003c/p\u003e \u003cp\u003eConfocal images were captured using a CLSM system set to a 543 nm excitation wavelength, with a spatial resolution of 512 \u0026times; 512 pixels and a \u0026times;10 objective. When a single frame did not cover the full canal circumference, several overlapping images were taken and later combined into a single view using Adobe Photoshop. All images were calibrated with a 100 \u0026micro;m scale bar and analyzed in ImageJ to measure maximum penetration depth, the percentage of tubules showing penetration, and the overall penetration area.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis:\u003c/h2\u003e \u003cp\u003eThe data obtained in current study were analyzed using IBM SPSS Statistics 22.0 (IBM Corp., Armonk, NY, USA). Analysis of each variable\u0026rsquo;s distribution using the Shapiro\u0026ndash;Wilk test showed non-normal behavior, prompting the use of non-parametric methods for all subsequent statistical procedures. Differences between anatomical regions (apical and middle) within each experimental group were evaluated using the Mann\u0026ndash;Whitney U test, a non-parametric method appropriate for comparing two independent samples. For examining differences across the six experimental groups at the same anatomical level, the Kruskal\u0026ndash;Wallis H test was applied. Whenever statistically significant differences were detected by the Kruskal\u0026ndash;Wallis H test, a post-hoc analysis with the Dunn test and Bonferroni correction was performed to determine the source of the variance. The level of statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all comparisons.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summarizes the descriptive statistics of maximum sealer penetration depth (\u0026micro;m) for different chelators and irrigation approaches in the middle and apical root canal sections. In all groups, penetration depth values were significantly higher in the apical third than in the middle third (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Sonic activation consistently increased apical penetration compared with conventional irrigation. The EA+HEDP protocol yielded the highest mean penetration depth in the apical region (499.41 \u0026micro;m), whereas the CI+HEDP group showed significantly lower apical values (310.30 \u0026micro;m) than all activated groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the middle third, conventional EDTA resulted in the lowest penetration depth (136.94 \u0026micro;m). Representative CLSM images are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003ePenetration area measurements (\u0026micro;m\u0026sup2;) are given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In all groups, the middle third displayed a larger penetration area than the apical part (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Among the protocols, the EA\u0026thinsp;+\u0026thinsp;CLO group showed the widest penetration areas (middle: 8077.74 \u0026micro;m\u0026sup2;; apical: 4637.68 \u0026micro;m\u0026sup2;). Conversely, conventional irrigation resulted in reduced penetration areas, with the lowest values observed in the CI+EDTA group (middle: 2844.45 \u0026micro;m\u0026sup2;; apical: 371.84 \u0026micro;m\u0026sup2;). Taken together, these findings indicate that activation improves irrigant distribution across all chelators, with the most pronounced effect observed in the middle third of the canal.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescriptive statistics (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of maximum sealer penetration depth (\u0026micro;m) in the middle and apical regions of root canals according to different chelators and final irrigation approaches. Different superscript letters indicate statistically significant differences among groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFinal Irrigation Approaches\u003c/p\u003e \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;10 per group)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMiddle Region\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eApical Region\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCI+EDTA Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e136.94\u0026thinsp;\u0026plusmn;\u0026thinsp;12.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e338.32\u0026thinsp;\u0026plusmn;\u0026thinsp;45.37 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCI+HEDP Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e225.32\u0026thinsp;\u0026plusmn;\u0026thinsp;45.19 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e310.30\u0026thinsp;\u0026plusmn;\u0026thinsp;58.11 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCI\u0026thinsp;+\u0026thinsp;CLO Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e151.90\u0026thinsp;\u0026plusmn;\u0026thinsp;97.14 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e324.83\u0026thinsp;\u0026plusmn;\u0026thinsp;143.17 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEA+EDTA Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e300.01\u0026thinsp;\u0026plusmn;\u0026thinsp;32.35 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e369.11\u0026thinsp;\u0026plusmn;\u0026thinsp;50.40 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEA+HEDP Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e431.78\u0026thinsp;\u0026plusmn;\u0026thinsp;31.75 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e499.41\u0026thinsp;\u0026plusmn;\u0026thinsp;31.69 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEA\u0026thinsp;+\u0026thinsp;CLO Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e304.63\u0026thinsp;\u0026plusmn;\u0026thinsp;130.96 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e463.38\u0026thinsp;\u0026plusmn;\u0026thinsp;141.69 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescriptive statistics (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of sealer penetration area (\u0026micro;m\u0026sup2;) in the middle and apical regions of root canals according to different chelators and final irrigation approaches. Different superscript letters indicate statistically significant differences among groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFinal Irrigation Approaches\u003c/p\u003e \u003cp\u003e\u003cem\u003e(n\u0026thinsp;=\u0026thinsp;10 per group)\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMiddle Region\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eApical Region\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCI+EDTA Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2844.45\u0026thinsp;\u0026plusmn;\u0026thinsp;400.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e371.84\u0026thinsp;\u0026plusmn;\u0026thinsp;125.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCI+HEDP Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2312.53\u0026thinsp;\u0026plusmn;\u0026thinsp;353.29 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1718.31\u0026thinsp;\u0026plusmn;\u0026thinsp;387.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCI\u0026thinsp;+\u0026thinsp;CLO Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5910.45\u0026thinsp;\u0026plusmn;\u0026thinsp;3835.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2885.07\u0026thinsp;\u0026plusmn;\u0026thinsp;2515.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEA+EDTA Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3123.32\u0026thinsp;\u0026plusmn;\u0026thinsp;972.69 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2134.53\u0026thinsp;\u0026plusmn;\u0026thinsp;429.09 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEA+HEDP Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5024.49\u0026thinsp;\u0026plusmn;\u0026thinsp;961.51\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2851.20\u0026thinsp;\u0026plusmn;\u0026thinsp;363.37 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEA\u0026thinsp;+\u0026thinsp;CLO Group\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8077.74\u0026thinsp;\u0026plusmn;\u0026thinsp;3711.76\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4637.68\u0026thinsp;\u0026plusmn;\u0026thinsp;2539.83 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring root canal preparation, a smear layer is always produced as a result of instrument contact with dentin and soft tissues. This mixture closes the dentinal tubules and reduces how well the sealer can adapt to the canal wall. For this reason, many studies have associated the presence of the smear layer with leakage problems and bacterial persistence in the long term [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Because bioceramic and other bioactive sealers need direct contact with dentin to perform properly, removing this layer becomes especially important [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, different chelating agents were evaluated in combination with different irrigation approaches to evaluate their effect on sealer penetration. In this study, activation was included because it is thought to improve the way the irrigant moves inside the canal. By increasing the internal flow, the irrigant is expected to reach parts of the canal that may otherwise remain untouched. Similar ideas have been discussed in endodontic literature and earlier studies have also described comparable findings [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. More recent research reports that combining activation with chelators may help the sealer penetrate more deeply into the dentinal tubules [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, sealer penetration depth was higher in the apical third, whereas penetration area was greater in the middle third. This finding reflects the structural characteristics of different root canal levels. The apical region is characterized by fewer dentinal tubules, increased sclerosis, and restricted canal space, which limits irrigant movement and may reduce sealer\u0026ndash;dentin interaction. In addition, the restricted canal space in this region limits irrigant movement and makes smear layer removal less predictable. When the smear layer is not adequately removed, the interaction between the sealer and dentin is reduced, which may negatively influence penetration. Similar differences between apical and middle regions have been reported in previous studies evaluating sealer penetration at different root canal levels [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, the apical preparation size was extended to F3, which was intended to improve irrigant access in the apical region. A larger apical preparation has been associated with better irrigant flow and more effective cleaning by allowing greater fluid exchange within the canal [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This factor may have played a role in the penetration patterns observed in the apical third. Similar observation has been made in study where ProTaper Next X4 was used together with activation, reporting better sealer penetration under those conditions [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCompared with EDTA and HEDP, clodronate has been examined in a more limited number of studies. Nevertheless, the existing literature suggests that clodronate can interact with calcium-containing components of the smear layer and supports its removal at a level like EDTA [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the present study, EDTA and HEDP showed comparable sealer penetration values under passive irrigation conditions, which agrees with previous reports indicating similar behavior of these agents when activation is not applied [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Once activation was introduced, both HEDP and Clodronate showed a noticeable increase in penetration. Activation improved penetration depth mainly in the apical third, while its effect on penetration area was more pronounced in the middle third. This outcome reflects the trend described in recent literature, where sonic activation and other newly developed irrigation systems have been found to improve irrigant distribution compared with standard syringe irrigation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Overall, these findings suggest that while EDTA, HEDP, and clodronate show comparable performance under passive irrigation, the application of sonic activation plays a decisive role in enhancing the effectiveness of chelating agents by improving irrigant distribution and dentin surface conditioning, thereby facilitating deeper sealer penetration.\u003c/p\u003e \u003cp\u003eAcross all groups, the penetration was higher in the middle third than in the apical third. Anatomical factors explain this trend: the apical region has fewer tubules, more sclerosis and limited space for irrigant movement. These characteristics naturally restrict how deep the sealer can penetrate, and similar results have been reported by earlier studies [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Improved penetration of the sealer into dentinal tubules has been linked with better sealing behaviour and long-term success of root canal treatment [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Bioactive sealers benefit from deeper penetration because they interact chemically with dentin and may form hydroxyapatite at the interface [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, irrigation protocols incorporating activation resulted in higher penetration values than the conventional irrigation protocol, which is compatible with earlier findings using sonic or laser activation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, some studies have shown that activation does not always lead to better penetration compared with conventional irrigation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Differences in sealer type, irrigant protocol, or canal morphology may contribute to such inconsistent findings. In current study, CLSM was selected mainly because it allows samples to be examined without the dehydration or coating procedures required for SEM, which can interfere with the sealer\u0026ndash;dentin interface. The method also makes it possible to evaluate both penetration depth and the overall penetration area with reasonable accuracy [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For visualization, a small amount of Rhodamine B was mixed into the sealer. Although the concentration was low, dyes may still have some minor influence on the material, and this should be kept in mind when interpreting the results [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince the current study is carried out under ex vivo conditions, it was not possible to reproduce certain clinical factors, such as the natural behaviour of periapical tissues or internal fluid pressure. The dentin slices taken at 3 mm and 5 mm permitted a comparison between the apical and middle thirds. As expected, the apical region showed more limited penetration, which can be explained by its structural features particularly increased sclerosis and reduced tubule permeability [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Further research is still required, especially in curved canals and minimally prepared systems, and in understanding the behaviour of alternative chelators such as Clodronate under activated conditions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eSonic activation during final irrigation enhanced sealer penetration by improving the effectiveness of chelating agents and their interaction with dentin surfaces. These findings highlight the clinical relevance of irrigation dynamics in achieving effective dentin conditioning and more consistent sealer adaptation under activated conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki and approved by the Non-Clinical Scientific Research Ethics Committee of Near East University (Approval No: 65/2023, 12 October 2025). Informed consent was obtained from all participants involved in the study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no external funding.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: F.B.Methodology: D.K., D.K.Y.Software: A.S.Validation: E.O.T., D.K., A.S.Formal analysis: E.O.T., D.K.Y., A.S.Investigation: D.K., D.K.Y.Resources: E.O.T.Data curation: E.O.T., D.K.Y., D.K.Writing \u0026ndash; original draft: D.K., D.K.Y.Writing \u0026ndash; review \u0026amp; editing: F.B., M.D.\u0026Ouml;.Visualization: A.S.Supervision: F.B., M.D.\u0026Ouml;.Project administration: F.B., M.D.\u0026Ouml;.All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eAll data generated or analysed during this study are available and included as 3rd dataset of the published repository.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBatinić M, Ročan M, Budimir A, Anić I, Bago I. 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Effectiveness of different activated irrigation techniques on debris and smear layer removal from curved root canals: a SEM evaluation. Aust Endod J. 2020;46(1):40\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMancini M, Cerroni L, Palopoli P, Olivi G, Olivi M, Buoni C, Cianconi L. FESEM evaluation of smear layer removal from conservatively shaped canals: laser activated irrigation (PIPS and SWEEPS) compared to sonic and passive ultrasonic activation\u0026mdash;an ex vivo study. BMC Oral Health. 2021;21(1):81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNassar M, Hiraishi N, Tamura Y, Otsuki M, Aoki K, Tagami J. Phytic acid, an alternative root canal chelating agent. J Endod. 2015;41(2):242\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatos FDS, da Silva FR, Paranhos LR, Moura CCG, Bresciani E, Valera MC. The effect of 17% EDTA and QMiX ultrasonic activation on smear layer removal and sealer penetration: ex vivo study. Sci Rep. 2020;10(1):10311.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUlusoy OI, Savur IG, Alacam T, Celik B. The effectiveness of various irrigation protocols on organic tissue removal from simulated internal resorption defects. Int Endod J. 2018;51:1030\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiardino L, Savadori P, Generali L, Mohammadi Z, Del Fabbro M, De Vecchi E, Bidossi A. Antimicrobial effectiveness of etidronate powder (Dual Rinse HEDP) and two EDTA preparations against Enterococcus faecalis: a preliminary laboratory study. Odontology. 2020;108:396\u0026ndash;405.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoruk D, Basmacı F, Kırmızı D, Aksoy U. The impact of laser-activated and conventional irrigation techniques on sealer penetration into dentinal tubules. Photobiomodul Photomed Laser Surg. 2020;40(8):565\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWright PP, Cooper C, Kahler B, Walsh LJ. From an assessment of multiple chelators, clodronate shows potential for use in continuous chelation. Int Endod J. 2020;53(1):122\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWright PP, Kahler B, Walsh LJ. The effect of temperature on the stability of sodium hypochlorite in a continuous chelation mixture containing the chelator clodronate. Aust Endod J. 2020;46(2):244\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWright PP, Cooper C, Kahler B, Walsh LJ. Multiple assessment methodologies in determining the antibiofilm actions of sodium hypochlorite mixed with clodronate or etidronate in endodontic irrigation. J Microbiol Methods. 2021;180:106107.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGaller KM, Grubm\u0026uuml;ller V, Schlichting R, Widbiller M, Eidt A, Schuller C, Buchalla W. Penetration depth of irrigants into root dentine after sonic, ultrasonic and photoacoustic activation. Int Endod J. 2019;52:1210\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYilmaz A, Yalcin TY, Helvacioglu-Yigit D. Effectiveness of various final irrigation techniques on sealer penetration in curved roots: a confocal laser scanning microscopy study. Biomed Res Int. 2020;2020:8060489.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaudhry S, Yadav S, Talwar S, Verma M. Effect of EndoActivator and Er,Cr:YSGG laser activation of QMix as final endodontic irrigant on sealer penetration: a confocal microscopic study. J Clin Exp Dent. 2017;9:e218\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026eacute;rez-Alfayate R, Algar-Pinilla J, Mercade M, Foschi F. Sonic activation improves bioceramic sealer penetration into the tubular dentin of curved root canals: a confocal laser scanning microscopy investigation. Appl Sci. 2021;11(9):3902.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGomes B, Aveiro E, Kishen A. Irrigants and irrigation activation systems in endodontics. Braz Dent J. 2023;34(4):1\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachado R, da Silva I, Comparin D, de Mattos BAM, Alberton LR, da Silva Neto UX. Smear layer removal by passive ultrasonic irrigation and two new mechanical methods for activation of the chelating solution. Restor Dent Endod. 2021;46(1):e11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Souza Matos F, Rosatto CMPD, Cunha TC, Vidigal MTC, Blumenberg C, Paranhos LR, Moura CCG. Influence of chelating solutions on tubular dentin sealer penetration: a systematic review with network meta-analysis. Aust Endod J. 2021;47(3):715\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGawdat SI, Bedier MM. Influence of dual rinse irrigation on dentinal penetration of a bioceramic root canal sealer: a confocal microscopic analysis. Aust Endod J. 2022;48(3):481\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFurtado TC, de Bem IA, Machado LS, Pereira JR, S\u0026oacute; MVR, da Rosa RA. Intratubular penetration of endodontic sealers depends on the fluorophore used for CLSM assessment. Microsc Res Tech. 2021;84(2):305\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZehnder M. Root canal irrigants. J Endod. 2006;32(5):389\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Sluis LWM, Wu MK, Wesselink PR. The evaluation of removal of calcium hydroxide paste from an artificial standardized groove in the apical root canal using different irrigation methodologies. Int Endod J. 2007;40(1):52\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang SH, Gao ZR, Zhou YH, Tan L, Feng Y, Ye Q, et al. Comparison of Easydo Activator, ultrasonic and needle irrigation techniques on sealer penetration and smear layer removal in vitro. BMC Oral Health. 2024;24(1):56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGunes B, Yeter KY, Altay Y. Impact of different activation procedures on sodium hypochlorite penetration into dentinal tubules after endodontic retreatment via confocal laser scanning microscopy. BMC Oral Health. 2024;24(1):1103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzasir T, Eren B, Gulsahi K, Ungor M. The effect of different final irrigation regimens on the dentinal tubule penetration of three different root canal sealers: a confocal laser scanning microscopy study in vitro. Scanning. 2021;2021:8726388.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoskun Basoglu E, Kocak S, Ozdemir O, Kocak MM, Saglam BC. Efficacy of various activation techniques on tubule penetration of resin-based and bioceramic root canal sealers: an in vitro confocal microscopy study. Aust Endod J. 2023;49:381\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWright PP, Scott S, Kahler B, Walsh LJ. Organic tissue dissolution in clodronate and etidronate mixtures with sodium hypochlorite. J Endod. 2020;46(2):289\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlim Uysal BA, Kotan G, Guneser MB, Dincer AN, Senturk H, Rafiqi AM. Investigation of the effect of different chelation solutions on penetration of resin-based and bioceramic sealers with a novel method. Microsc Res Tech. 2021;84(7):1571\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Feng B, Shi S, Sun D, Wu D. The effect of different activation irrigations on intracanal smear layer removal: an in vitro study. Front Bioeng Biotechnol. 2024;12:1507525.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIandolo A, Armogida NG, Mancino D, Spagnuolo G, Cernera M, Abdellatif D. Evaluation of root canal cleaning and irrigant penetration using different irrigation protocols: a combined SEM and single-tooth micro-CT study. Clin Exp Dent Res. 2025;11(4):e70175.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarrigan PJ, Morse DR, Furst ML, Sinai IH. A scanning electron microscopic evaluation of human dentinal tubules according to age and location. J Endod. 1984;10(8):359\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeters OA, Laib A, Gohring TN, Barbakow F. Changes in root canal geometry after preparation assessed by high-resolution computed tomography. J Endod. 2001;27(1):1\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMochizuki S, Watanabe S, Liu J, Okiji T. Smear layer removal efficacy of different irrigation techniques in conservatively instrumented root canals. J Dent Sci. 2024;19(3):1546\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKilic Y, Tulgar MM, Karataslioglu E, Turk T. Comparative analysis of dentinal tubule penetration: effects of irrigation activation methods and root canal sealers: an in vitro study. BMC Oral Health. 2025;25:991.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolles JA, He J, Svoboda KK, Schneiderman E, Glickman GN. Comparison of Vibringe, EndoActivator, and needle irrigation on sealer penetration in extracted human teeth. J Endod. 2013;39(5):708\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrban K, Donnermeyer D, Schafer E, Burklein S. Canal cleanliness using different irrigation activation systems: a SEM evaluation. Clin Oral Investig. 2017;21(9):2681\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAguiar BA, Frota LM, Taguatinga DT, Vivan RR, Camilleri J, Duarte MA, et al. Influence of ultrasonic agitation on bond strength, marginal adaptation, and tooth discoloration provided by three coronary barrier endodontic materials. Clin Oral Investig. 2019;23(11):4113\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDonnermeyer D, Schmidt S, Rohrbach A, Berlandi J, Burklein S, Schafer E. Debunking the concept of dentinal tubule penetration of endodontic sealers: sealer staining with rhodamine B fluorescent dye is an inadequate method. Mater (Basel). 2021;14(12):3211.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chelating agents, Clodronate, HEDP, Sealer penetration, Smear layer removal, Sonic activation","lastPublishedDoi":"10.21203/rs.3.rs-9213015/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9213015/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSmear layer removal is essential for sealer penetration and obturation quality. This study aimed to compare different final irrigation solutions and activation methods on sealer penetration using confocal laser scanning microscopy (CLSM).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSixty extracted single-rooted mandibular premolars were instrumented and randomly allocated into six groups (n\u0026thinsp;=\u0026thinsp;10) based on the final irrigation protocol: ethylenediaminetetraacetic acid (EDTA), 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) or clodronate (CLO) combined with conventional syringe irrigation (CI) or EndoActivator (EA) system. Root canals were obturated using a single-cone technique with Rhodamine B\u0026ndash;labeled BioRoot Flow sealer. Apical and middle sections were evaluated under CLSM, and penetration parameters were analyzed using ImageJ with non-parametric statistics (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSealer penetration depth was significantly higher in the apical than in the middle third (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with sonic activation producing greater depths than conventional irrigation. The EA+HEDP group showed the highest apical penetration depth (499.41 \u0026micro;m), whereas the largest penetration areas were observed in the EA\u0026thinsp;+\u0026thinsp;CLO group and the lowest in the CI+EDTA group.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eSonic activation enhanced sealer penetration with chelating agents. Activated HEDP mainly increased penetration depth in the apical region, while activated clodronate produced wider penetration areas in the middle third.\u003c/p\u003e","manuscriptTitle":"Influence of Continuous Chelation Protocols and Activation Techniques on Root Canal Sealer Penetration: An In Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-30 14:25:36","doi":"10.21203/rs.3.rs-9213015/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09aae02d-4216-45f1-8bc4-b033f076fd2a","owner":[],"postedDate":"April 30th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Rejected","date":"2026-05-06T15:43:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T15:55:48+00:00","index":50,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T15:55:54+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-30 14:25:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9213015","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9213015","identity":"rs-9213015","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}