Chelating Agents’ Effects on Dentin Properties and Bond Strength of Biodentine

Chelating Agents’ Effects on Dentin Properties and Bond Strength of Biodentine | 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 Article Chelating Agents’ Effects on Dentin Properties and Bond Strength of Biodentine Aslı ÖZDEMİR, Mevlüt Sinan OCAK This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7998211/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 This in vitro study evaluated the effects of different chelating agents on dentin properties influencing the adhesion of calcium silicate–based biomaterials. The effects of 9% etidronic acid (HEBP) and 0.2% chitosan nanoparticles on the microhardness, surface roughness, and push-out bond strength of Biodentine were assessed. Seventy-five extracted maxillary incisors were divided into three groups according to the final irrigation protocol: 9% HEBP, 0.2% chitosan, or 0.9% saline (control). Dentin microhardness (Vickers hardness number) and surface roughness (Ra, µm) were measured before and after irrigation. For push-out testing, standardized cavities in 60 dentin discs were filled with Biodentine and stored for 24 h at 37°C and 100% humidity. Failure modes were examined under a stereomicroscope. Data were analyzed using one-way ANOVA, Tukey HSD, Kruskal–Wallis, and paired t-tests (p < 0.05). All groups showed significant microhardness reduction (p < 0.001), greatest in HEBP. Surface roughness increased significantly, highest in HEBP (p < 0.001). Push-out bond strength was higher in HEBP than control (p 0.05). Both agents improved Biodentine adhesion; chitosan produced milder dentin alterations, indicating potential for dentin preservation. Biological sciences/Biotechnology Health sciences/Health care Physical sciences/Materials science Health sciences/Medical research Chitosan Dental Materials Etidronic acid Figures Figure 1 Figure 2 Introduction One of the primary objectives of root canal treatment is the removal of infected tissues and effective disinfection of the canal system [ 1 ]. During instrumentation, a smear layer is formed on the dentinal surface, which may hinder the penetration of irrigants and filling materials into the dentinal tubules, subsequently compromising the quality of adhesion [ 2 ]. Therefore, effective removal of the smear layer plays a critical role in the success of endodontic therapy. Ethylenediaminetetraacetic acid (EDTA), the most commonly used chelating agent, is effective in removing the inorganic component of the smear layer; however, previous studies have reported several undesirable effects, including reduced dentin microhardness, disruption of collagen structure, and decreased flexural strength [ 3 , 4 ]. Moreover, its limited antibacterial efficacy and potential cytotoxicity toward surrounding tissues have encouraged the search for more biocompatible alternatives [ 5 ]. In this context, alternative chelating agents such as chitosan and 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) have gained increasing attention. Chitosan, a natural polysaccharide, stands out due to its biocompatibility, antimicrobial activity, and gentler interaction with dentin surfaces [ 6 , 7 ], whereas HEBP has been considered a more surface-friendly alternative to EDTA because of its lower demineralization profile [ 5 ]. The physicochemical changes induced by chelating agents on dentin surfaces may influence the adhesion of restorative materials. While increased surface roughness may enhance micromechanical retention, excessive demineralization can weaken dentin integrity [ 8 , 9 ]. Therefore, an ideal chelating agent should effectively eliminate the smear layer while causing minimal alteration to the dentinal substrate. Bioceramic-based materials have been successfully used in endodontic applications and are capable of forming chemical and micromechanical bonds with dentin. Their clinical performance largely depends on their ability to adhere to the dentinal surface [ 10 ]. Among these materials, Biodentine has gained prominence due to its high biocompatibility, durability, and color stability [ 11 ]. Considering the reported adverse effects of EDTA on dentin, this study aimed to comparatively evaluate the effects of %9 HEBP and %0.2 chitosan nanoparticles on dentin microhardness, surface roughness, and the push-out bond strength of Biodentine to root canal dentin. Although numerous studies have investigated the individual effects of chelating agents on dentin microhardness, surface roughness, or bond strength, there is a scarcity of studies simultaneously evaluating all three properties within the same experimental design and using the same biomaterial. Furthermore, previous reports suggest that chitosan nanoparticles exert milder effects on dentin surfaces, whereas HEBP preserves dentin integrity through a soft chelation profile. Kadulkar et al. [ 12 ] evaluated the effects of HEBP, EDTA, and chitosan on dentin but did not include bond strength as a parameter. Similarly, Ratih et al. [ 13 ] and Pimenta et al. [ 14 ] investigated the effects of chitosan on dentin microhardness and smear layer removal, but their findings were not correlated with surface morphology or bond strength. In this regard, the present study aims to fill an important gap in the literature by systematically assessing the physicochemical alterations induced by %9 HEBP and %0.2 chitosan nanoparticles across three different parameters. Our hypothesis was that there would be no statistically significant difference in push-out bond strength between the HEBP and chitosan groups; however, due to its milder demineralizing effect, chitosan would lead to more favorable outcomes in terms of dentin microhardness and surface roughness. Materials and Methods This study was approved by the Non-Interventional Clinical Research Ethics Committee of Fırat University (decision no: 2022/11–25; date: 06.10.2022). The use of extracted human teeth was performed in accordance with relevant institutional and international guidelines and regulations. All experimental procedures were conducted in compliance with the Declaration of Helsinki. The research was conducted as a comparative in vitro experimental model among three groups. Power Analysis and Sample Size Calculation The power analysis for microhardness and surface roughness measurements was performed using GPower software (G Power 3.1; Heinrich Heine University, Düsseldorf, Germany). Based on repeated-measures ANOVA for three groups with a power of 95% (1-β err prob = 0.95), an effect size (f) of 0.47, and α = 0.05, a minimum sample size of 45 specimens was calculated to be sufficient. For the push-out bond strength test, the sample size was determined using effect sizes obtained from similar studies. Considering a power of 95% and α = 0.05, at least 20 specimens per group were required, resulting in a total of 60 samples. The specimens were randomly assigned to three main groups according to the irrigation protocols, and each group was further subdivided for microhardness, surface roughness, and push-out bond strength measurements (Fig. 1 ). Randomization was performed, and the researcher who conducted the evaluations was blinded to group assignments (single-blind design). Specimen Selection and Preparation A total of 75 extracted human maxillary central incisors from male and female patients aged 25–50 years, extracted for periodontal, prosthetic, or orthodontic reasons, were used. Teeth exhibited similar root morphology and size and had no cracks, fractures, caries, restorative or endodontic treatment, canal calcifications, or anatomical variations. Organic debris and soft tissue remnants were removed from root surfaces using a sharp periodontal curette. The teeth were stored in distilled water at 4°C until use. Preparation of Irrigating Solutions %9 HEBP : Prepared by adding 448.3 mL of distilled water to 60% HEBP solution (Sigma Aldrich, USA) to obtain 500 mL of 9% concentration. %0.2 Chitosan : Chitosan powder (Sigma Aldrich, Germany) was dissolved in 250 mL of 1% acetic acid at 0.5 mg/mL concentration and mixed using a magnetic stirrer for 2 hours. Control Group : Commercially prepared %0.9 saline solution was used. A 2.5% sodium hypochlorite (NaOCl) solution was used for standard irrigation before chemical interactions in all groups. The pH values of the irrigating solutions were obtained from manufacturer specifications: %9 HEBP (pH 10.7; Zimmer Schwartz, Germany), %0.2 chitosan (pH 3.2; Sigma Aldrich, Germany), saline (pH 4.5–7; PF %0.9 Isotonic, Turkey), and NaOCl (pH 10.6; Microvem, Turkey). Since pH determines the interaction profile of irrigants with dentin, these values were considered in the present study. Microhardness Test Following crown removal, the roots of 45 teeth were sectioned longitudinally in the buccolingual direction under water cooling, yielding 90 half-root specimens. Forty-five halves were allocated for microhardness testing. Samples were embedded in acrylic blocks with the canal lumen exposed, and surfaces were flattened using silicon carbide abrasive papers (500–1200 grit). Baseline microhardness measurements were performed at coronal, middle, and apical thirds, 0.5 mm from the canal lumen, using a DuraScan 20 microhardness tester (EMCO-TEST, Germany) with a 300 g load applied for 20 seconds. The mean baseline value was recorded. Specimens were then immersed in 100 mL of the assigned irrigating solution for 5 minutes, and post-treatment measurements were recorded from the same locations [ 15 ]. Vickers microhardness values (VHN) were automatically calculated using the formula VHN = 1.854 × force / (diagonal base)². Surface Roughness Test The remaining 45 half-root specimens underwent surface preparation using the same procedures described above. Standard dentin polishing was intentionally not performed to preserve native dentin morphology, as mechanical polishing may artificially alter baseline roughness values and reduce clinical relevance. Accordingly, measurements reflect the natural radicular dentin substrate. Surface roughness was measured using a Surftest SJ-410 profilometer (Mitutoyo, Japan) with a cut-off length of 0.25 mm, scanning speed of 0.5 mm/s, and total tracing length of 1.25 mm. Five measurements were recorded per specimen, and the mean baseline Ra value was calculated. Samples were then immersed in 100 mL of the assigned chelating solution for 5 minutes, and final surface roughness values were measured. The arithmetic mean of five readings was recorded as the surface roughness value [ 13 ]. Push-Out Bond Strength Test From the middle third of 30 root specimens, two dentin slices per root were obtained perpendicular to the long axis at 1-mm thickness using a low-speed water-cooled precision saw (Struers Accutom-50, Denmark; 100–200 rpm) [ 16 ]. A standardized cavity (diameter: 1.3 mm) was prepared in each slice using a no. 5 Gates-Glidden drill (Mani Inc., Tachigiken, Japan), yielding a total of 60 dentin discs. These were randomly divided into three groups (n = 20) according to the irrigating solutions [ 17 ]. All specimens were initially irrigated with 2.5% NaOCl for 5 minutes, followed by distilled water rinsing for 60 seconds to minimize chemical interactions. The discs were then exposed to 3 mL of the assigned irrigant (%9 HEBP, %0.2 chitosan, or saline) via immersion for 5 minutes [ 18 ]. Specimens were dried using cotton pellets and paper points (DentPlus, Diadent, Netherlands), after which Biodentine (Septodont, Saint Maur des Fossés, France) was placed into the cavities. Samples were stored at 37°C in 100% humidity for 24 hours to allow complete setting. Push-out testing was performed by applying a compressive load at a crosshead speed of 1 mm/min using a universal testing machine (Shimadzu Co., Kyoto, Japan) with a 1-mm stainless-steel plugger. The maximum force at dislodgement (N) was recorded and converted to MPa using the formula: Dislodgement stress (MPa) = Force (N) / Bonded area (mm²) Bonded interface area = 2πrh (r = cavity radius; h = slice thickness) After testing, failure modes were evaluated under a stereomicroscope (Leica MZ75, Cambridge, UK) at ×10 magnification and classified as adhesive, cohesive, or mixed [ 19 ]. Statistical Analysis Normality of the data was assessed using the Shapiro–Wilk and Kolmogorov–Smirnov tests. Descriptive statistics (mean ± standard deviation) were reported. For normally distributed pre- and post-treatment values, the paired-sample t-test was used. The Kruskal–Wallis test was used for non-normally distributed comparisons. One-way ANOVA was applied to determine statistically significant differences among groups, and the Tukey HSD post hoc test was used to identify pairwise differences. A significance level of p < 0.05 was adopted. Data analysis was conducted using SPSS version 22 (SPSS Inc., Chicago, IL, USA). Results Vickers Microhardness Test Before solution application, no statistically significant differences were observed among the groups in terms of microhardness at the coronal, middle, or apical thirds (p > 0.05; Table 1). The mean baseline microhardness values were 57.18 ± 4.39 VHN for the saline group, 57.52 ± 3.97 VHN for the chitosan group, and 56.20 ± 4.19 VHN for the HEBP group. Following solution application, statistically significant differences in microhardness values were detected among the groups in all regions (p < 0.001; Table 1). The lowest mean post-treatment microhardness value was recorded in the HEBP group (28.54 ± 6.47 VHN), followed by the chitosan group (47.40 ± 4.73 VHN), while the saline group exhibited the highest value (56.34 ± 4.53 VHN). According to Tukey post hoc analysis, the HEBP group differed significantly from both the chitosan and saline groups (p < 0.001). Additionally, the difference between the chitosan and saline groups was statistically significant (p < 0.05). Surface Roughness Test No statistically significant differences were observed among the groups in baseline surface roughness values (p = 0.469, ANOVA; Table 2). The mean baseline Ra values were 0.1595 ± 0.0213 µm for the saline group, 0.1488 ± 0.0268 µm for the chitosan group, and 0.1536 ± 0.0222 µm for the HEBP group. After solution application, a statistically significant difference in surface roughness was observed among the groups (p < 0.001; Table 2). The highest surface roughness was recorded in the HEBP group (0.5674 ± 0.0636 µm), followed by the chitosan (0.2788 ± 0.0492 µm) and saline (0.1704 ± 0.0230 µm) groups. Post hoc comparisons revealed that all groups differed significantly from each other (p < 0.001). Push-Out Bond Strength Test Push-out bond strength values differed significantly among the groups (p = 0.001; Table 3). The mean bond strength values were 15.73 ± 7.34 MPa for the HEBP group, 13.20 ± 5.92 MPa for the chitosan group, and 9.82 ± 4.70 MPa for the saline group. The HEBP group demonstrated significantly higher bond strength compared to the saline group (p 0.05). Failure Mode Analysis Evaluation of failure modes revealed that mixed failure was the most frequently observed type across all groups. In the HEBP group, 72% mixed, 16% cohesive, and 12% adhesive failures were observed; in the chitosan group, 68% mixed, 20% cohesive, and 12% adhesive failures were recorded; while in the saline group, 60% mixed, 22% cohesive, and 18% adhesive failures were identified (Figs. 2 ). Although this distribution suggests similar trends among the groups, it was not statistically analyzed. Discussion This study evaluated the effects of different chelating agents on dentin surface characteristics and the push-out bond strength of the bioceramic material Biodentine. The findings demonstrated that both HEBP and chitosan yielded similarly high bond strength values; however, HEBP caused the greatest reduction in microhardness and the highest increase in surface roughness. These results support our hypothesis regarding bond strength while confirming the secondary hypothesis favoring chitosan with respect to reduced dentin degradation. The adhesion of calcium silicate–based materials to root dentin enhances the integrity of the dentin–material interface and contributes to the long-term success of endodontic treatment through improved sealing [ 20 ]. The physical properties of dentin, including microhardness and the degree of surface roughening, directly influence the quality of this adhesion [ 21 ]. While strong chelators such as EDTA and citric acid can enhance the organic tissue-dissolving capacity and antimicrobial activity of NaOCl, they may also induce procedural complications such as ledge formation and perforation [ 22 ]. To minimize these risks while enhancing irrigation efficacy, the continuous soft-chelation technique—combining a weak acid with NaOCl—has been proposed. This approach aims to prevent smear layer accumulation, reduce procedural errors, facilitate clinical handling, and preserve dentin integrity [ 23 ]. For these reasons, the present study utilized %9 HEBP and %0.2 chitosan nanoparticles as potential alternatives to EDTA, and assessed their effects on dentin microhardness, surface roughness, and adhesion to Biodentine. Optimal chelation times reported in the literature vary between 1, 5, and 15 minutes. However, prolonged exposure to decalcifying agents is believed to induce detrimental effects on root dentin [ 24 ]. In this study, chelation was performed for 5 minutes to simulate clinical conditions and facilitate smear layer removal while avoiding excessive demineralization, consistent with previous reports [ 18 , 25 ]. Microhardness results indicated that the group treated with chitosan exhibited values closest to the control. This may be attributed to the ability of positively charged chitosan nanoparticles to interact electrostatically with dentin collagen, thereby maintaining mechanical stability [ 26 ]. However, contradictory findings regarding the influence of chitosan on dentin microhardness exist in the literature, which may be related to variations in concentration, formulation, and exposure time [ 27 – 29 ]. Compared with EDTA, HEBP has been reported to induce less reduction in the mechanical properties of dentin, whereas EDTA significantly decreases the calcium-to-phosphorus ratio and weakens dentin structure [ 30 ]. The decrease in dentin microhardness is associated with mineral loss in the intertubular dentin and hydroxyapatite dissolution. Due to its lower chelation capacity, etidronic acid may mitigate these detrimental effects. In the present study, the HEBP group demonstrated a significant increase in surface roughness. Although an increase in dentin surface area may enhance micromechanical adhesion, it has been suggested that excessive roughness can be clinically undesirable. Specifically, increased surface area may promote bacterial adhesion and potentially elevate the risk of microleakage [ 31 , 32 ]. Therefore, when selecting an irrigant, not only chelation efficacy but also its impact on dentin surface morphology should be considered. The use of centrally located perforations in single-rooted teeth for push-out testing provides a reproducible model with improved comparability. Off-center perforations may expose dentinal tubules at variable orientations, influencing the penetration and adaptation of calcium silicate–based materials. By preparing centrally positioned cavities, a uniform exposure of tubules was achieved, contributing to more standardized results [ 33 ]. Previous reports have shown that the diameter of the cylindrical plunger does not significantly influence test outcomes when properly positioned to avoid contact with canal walls [ 34 ]. Based on this information, a 1-mm plunger was selected to match the 1.3-mm cavity diameter. The highest bond strength values were observed in the HEBP group, followed by chitosan, although the difference between these two groups was not statistically significant. The control group exhibited significantly lower values. Consistent with these findings, Tuncel et al. [ 35 ] and Carvalho et al. [ 36 ] demonstrated that chelating agents increase the bond strength of calcium silicate–based materials, with no significant differences detected among different chelators. Similarly, Buldur et al. [ 37 ] reported enhanced bond strength following chelator use. Contrary to these results, Ballal et al. [ 38 ] evaluated bond strength in simulated root-end cavities and reported significantly lower values in samples treated with 17% EDTA compared to controls. Due to its erosive effect and strong chelating capacity, EDTA may reduce calcium content at the Biodentine–dentin interface or alter the calcium silicate fraction within the cement, thereby weakening adhesion [ 39 ]. Following push-out testing, Aguiar et al. [ 40 ] reported that Biodentine predominantly exhibited cohesive failures under scanning electron microscopy. Although the present study demonstrated a higher prevalence of mixed failures, both studies identified cohesive and mixed failures as the most frequent failure modes, whereas adhesive failures were the least common. This study has several limitations. First, because it was conducted under in vitro conditions, caution must be exercised when extrapolating the findings to clinical practice. Additionally, biological responses of dentin following irrigation, such as cell viability, inflammatory reactions, and regenerative potential, were not evaluated. Only a single bioceramic material (Biodentine) was tested, limiting generalizability to other biomaterials. Therefore, future studies should evaluate different material systems, incorporate in vivo experimental models, and employ advanced imaging techniques (e.g., micro-CT, SEM) to further assess dentinal tubule patency and surface topography. Conclusions Within the limitations of this in vitro study, this investigation comparatively evaluated the effects of different chelating agents on the physicomechanical properties of root dentin. The findings demonstrated that %9 HEBP significantly reduced dentin microhardness and markedly increased surface roughness, whereas %0.2 chitosan caused more limited alterations, preserving surface integrity more effectively. Push-out bond strength values were higher in both HEBP and chitosan groups compared to the control group, although no statistically significant difference was observed between the two chelating agents in this regard. Clinically, these results support the concept of continuous soft chelation, suggesting that the use of mild irrigating agents such as %0.2 chitosan and %9 HEBP may enhance long-term bonding stability and help preserve dentin integrity, particularly in structurally compromised teeth and regenerative procedures. The biocompatibility and collagen-stabilizing potential of chitosan further highlight its promise as an alternative irrigant in contemporary endodontic protocols. However, the in vitro nature of this study and the use of a single calcium silicate–based material limit the generalizability of these findings. Future investigations involving in vivo models, alternative biomaterials, and biological response assessments are recommended to further validate the translational applicability of these irrigation strategies in clinical practice. Declarations Acknowledgements: This manuscript originates from the thesis of Aslı ÖZDEMİR. The study was funded by the Fırat University Scientific Research Projects (FÜBAP) Coordination Unit, Elazığ, Turkey (Project No: DHF.22.05). Author Contributions: MSO: Conceptualization, methodology, resources, project administration, writing - review and editing; funding acquisition, supervision; AÖ: Conceptualization, data curation, methodology, formal analysis, resources, writing - original draft, writing - review and editing. All authors reviewed and approved the final version of the manuscript. Data availability: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Funding: This study was supported by F.U. Scientific Research Projects (Fübap) Coordination Unit, Elazig, Turkey with the project number DHF.22.05. Competing interests: The authors declare no competing interests. Ethical Statements: This study was conducted using extracted human teeth that were obtained with the patients’ informed consent and in accordance with ethical standards. The research protocol was reviewed and approved by the Ethics Committee of Fırat University (Elazığ, Türkiye) (Approval No: 2022/11-25). References Peters, O. A. et al. Guidelines for non-surgical root canal treatment. Aust Endod J. 50 (2), 202–214. https://doi.org/10.1111/aej.12848 (2024). Alamoudi, R. A. The smear layer in endodontics: to keep or remove—an updated overview. Saudi Endod J. 9 (2), 71–81. https://doi.org/10.4103/sej.sej_95_18 (2019). Ari, H., Erdemir, A. & Belli, S. Evaluation of the effect of endodontic irrigation solutions on the microhardness and the roughness of root canal dentin. J. Endod . 30 (11), 792–795. https://doi.org/10.1097/01.don.0000128747.89857.59 (2004). Barcellos, D. P. D. C. et al. 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Effect of the combination of several irrigants on dentine surface properties, adsorption of chlorhexidine and adhesion of microorganisms to dentine. Int. Endod J. 51 (12), 1420–1433. https://doi.org/10.1111/iej.12960 (2018). Scelza, M. Z. et al. Influence of a new push-out test method on the bond strength of three resin-based sealers. Int. Endod J. 48 (8), 801–806. https://doi.org/10.1111/iej.12378 (2015). Nagas, E., Uyanik, O., Durmaz, V. & Cehreli, Z. C. Effect of plunger diameter on the push-out bond values of different root filling materials. Int. Endod J. 44 (10), 950–955. https://doi.org/10.1111/j.1365-2591.2011.01913.x (2011). Tuncel, B. et al. Effect of endodontic chelating solutions on the bond strength of endodontic sealers. Braz Oral Res. 29, S1806-83242015000100256. https://doi.org/10.1590/1807-3107BOR-2015.vol29.0059 (2015). Carvalho, N. K. et al. Do smear-layer removal agents affect the push-out bond strength of calcium silicate-based endodontic sealers? Int. Endod J. 50 (6), 612–619. https://doi.org/10.1111/iej.12662 (2017). Buldur, B., Oznurhan, F. & Kaptan, A. The effect of different chelating agents on the push-out bond strength of proroot mta and endosequence root repair material. Eur. Oral Res. 53 (2), 88–93. https://doi.org/10.26650/eor.20191618 (2019). Ballal, N. V., Ulusoy, Ö. İ., Chhaparwal, S. & Ginjupalli, K. Effect of novel chelating agents on the push-out bond strength of calcium silicate cements to the simulated root-end cavities. Microsc Res. Tech. 81 (2), 214–219. https://doi.org/10.1002/jemt.22969 (2018). Atmeh, A. R., Chong, E. Z., Richard, G., Festy, F. & Watson, T. F. Dentin-cement interfacial interaction: calcium silicates and polyalkenoates. J. Dent. Res. 91 (5), 454–459. https://doi.org/10.1177/0022034512443068 (2012). Aguiar, B. A. et al. Influence of ultrasonic agitation on bond strength, marginal adaptation, and tooth discoloration provided by three coronary barrier endodontic materials. Clin. Oral Investig . 23 (11), 4113–4122. https://doi.org/10.1007/s00784-019-02850-y (2019). Tables Tablo 1 . Coronal, middle, apical, and mean microhardness values of root dentin before and after solution application. P values marked with * indicate statistically significant differences (paired samples t-test) (p<0,05). Groups N Mean Std. Dev. F P Coronal Initial test Salin 15 58,0267 5,80104 1,541 0,226 Chitosan 15 57,1933 4,11556 HEBP 15 54,6667 6,23179 Middle Initial test Salin 15 56,2267 4,70615 2,244 0,119 Chitosan 15 59,2000 5,34416 HEBP 15 54,9333 6,72986 Apical Initial test Salin 15 57,2933 4,91840 1,175 0,319 Chitosan 15 56,1733 5,88988 HEBP 15 59,0133 4,41634 Average Initial test Saline 15 57,1822 4,39484 0,399 0,673 Chitosan 15 57,5222 3,97821 HEBP 15 56,2044 4,19283 Coronal Final test Salin 15 57,1267 6,23647 77,983 0,001 * Chitosan 15 46,6200 5,13451 HEBP 15 27,9267 7,80837 Middle Final test Salin 15 55,2267 5,04798 64,536 0,001 * Chitosan 15 49,0667 4,93843 HEBP 15 28,4533 9,33938 Apical Final test Salin 15 56,6800 4,65912 62,640 0,001 * Chitosan 15 46,5200 7,39239 HEBP 15 29,2467 7,86374 Average Final test Salin 15 56,3444 4,53709 106,618 0,001 * Chitosan 15 47,4022 4,73777 HEBP 15 28,5422 6,47988 Tablo 2. Mean surface roughness values of root dentin before and after solution application. A paired samples t-test was used for data analysis. P values marked with * indicate statistically significant differences (p < 0.05). Groups (n=15) Mean Std. Dev. F P Surface Roughness Initial Test Salin 0,1595 0,02130 0,771 0,469 Chitosan 0,1488 0,02679 HEBP 0,1536 0,02225 Surface Roughness Final Test Salin 0,1704 0,02307 270,190 0,001 * Chitosan 0,2788 0,04926 HEBP 0,5674 0,06367 Tablo 3. Push-out bond strength test results are presented in the table. Data were analyzed using the Kruskal–Wallis test (p = 0.001). According to the Tukey HSD test, a statistically significant difference was found between the HEBP and saline groups (p 0.05). P values marked with * indicate statistically significant differences (p < 0.05). Groups N Mean Std. Dev. Median H P HEBP 20 15,73 7,34 15,40 13,555 0,001 * Chitosan 20 13,20 5,92 13,53 Salin 20 9,82 4,70 9,47 Additional Declarations No competing interests reported. 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08:48:15","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131389,"visible":true,"origin":"","legend":"","description":"","filename":"23c4dbfd346f4dffaa177077cb94ea8b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7998211/v1/bbc168857fb1b2158cb0af26.xml"},{"id":96160481,"identity":"9a3512f9-86d1-4e66-b6a8-fbc87883e3d0","added_by":"auto","created_at":"2025-11-18 08:48:15","extension":"html","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141065,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7998211/v1/4df22d631a8307cccf1fc252.html"},{"id":96250160,"identity":"30a6f415-dc76-4381-97ca-02d9da3a7f92","added_by":"auto","created_at":"2025-11-19 07:37:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65311,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental Group Assignment.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7998211/v1/b0f4cbabbfb2c8088b81abac.jpg"},{"id":96160473,"identity":"23097c0a-a706-46f3-8b89-6b6d4033bea4","added_by":"auto","created_at":"2025-11-18 08:48:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":113863,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images and distribution of failure modes observed in the push-out bond strength test: (A) Adhesive failure at the dentin–Biodentine interface, (B) Cohesive failure within Biodentine, and (C) Mixed failure involving both regions (arrows indicate the locations of the failures), along with a pie chart representation showing the percentage distribution of failure types among all samples.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7998211/v1/835f05c7f117d709ffa8ce01.jpg"},{"id":97674697,"identity":"2dafe34c-1134-473b-a41e-f3b82a1bf63e","added_by":"auto","created_at":"2025-12-08 09:43:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":932240,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7998211/v1/1b85c35e-7490-4eae-bbdf-73fe92a95486.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chelating Agents’ Effects on Dentin Properties and Bond Strength of Biodentine","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOne of the primary objectives of root canal treatment is the removal of infected tissues and effective disinfection of the canal system [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. During instrumentation, a smear layer is formed on the dentinal surface, which may hinder the penetration of irrigants and filling materials into the dentinal tubules, subsequently compromising the quality of adhesion [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Therefore, effective removal of the smear layer plays a critical role in the success of endodontic therapy.\u003c/p\u003e\u003cp\u003eEthylenediaminetetraacetic acid (EDTA), the most commonly used chelating agent, is effective in removing the inorganic component of the smear layer; however, previous studies have reported several undesirable effects, including reduced dentin microhardness, disruption of collagen structure, and decreased flexural strength [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Moreover, its limited antibacterial efficacy and potential cytotoxicity toward surrounding tissues have encouraged the search for more biocompatible alternatives [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this context, alternative chelating agents such as chitosan and 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) have gained increasing attention. Chitosan, a natural polysaccharide, stands out due to its biocompatibility, antimicrobial activity, and gentler interaction with dentin surfaces [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], whereas HEBP has been considered a more surface-friendly alternative to EDTA because of its lower demineralization profile [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe physicochemical changes induced by chelating agents on dentin surfaces may influence the adhesion of restorative materials. While increased surface roughness may enhance micromechanical retention, excessive demineralization can weaken dentin integrity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, an ideal chelating agent should effectively eliminate the smear layer while causing minimal alteration to the dentinal substrate.\u003c/p\u003e\u003cp\u003eBioceramic-based materials have been successfully used in endodontic applications and are capable of forming chemical and micromechanical bonds with dentin. Their clinical performance largely depends on their ability to adhere to the dentinal surface [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among these materials, Biodentine has gained prominence due to its high biocompatibility, durability, and color stability [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConsidering the reported adverse effects of EDTA on dentin, this study aimed to comparatively evaluate the effects of %9 HEBP and %0.2 chitosan nanoparticles on dentin microhardness, surface roughness, and the push-out bond strength of Biodentine to root canal dentin. Although numerous studies have investigated the individual effects of chelating agents on dentin microhardness, surface roughness, or bond strength, there is a scarcity of studies simultaneously evaluating all three properties within the same experimental design and using the same biomaterial. Furthermore, previous reports suggest that chitosan nanoparticles exert milder effects on dentin surfaces, whereas HEBP preserves dentin integrity through a soft chelation profile. Kadulkar et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] evaluated the effects of HEBP, EDTA, and chitosan on dentin but did not include bond strength as a parameter. Similarly, Ratih et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and Pimenta et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] investigated the effects of chitosan on dentin microhardness and smear layer removal, but their findings were not correlated with surface morphology or bond strength. In this regard, the present study aims to fill an important gap in the literature by systematically assessing the physicochemical alterations induced by %9 HEBP and %0.2 chitosan nanoparticles across three different parameters. Our hypothesis was that there would be no statistically significant difference in push-out bond strength between the HEBP and chitosan groups; however, due to its milder demineralizing effect, chitosan would lead to more favorable outcomes in terms of dentin microhardness and surface roughness.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis study was approved by the Non-Interventional Clinical Research Ethics Committee of Fırat University (decision no: 2022/11\u0026ndash;25; date: 06.10.2022). The use of extracted human teeth was performed in accordance with relevant institutional and international guidelines and regulations. All experimental procedures were conducted in compliance with the Declaration of Helsinki. The research was conducted as a comparative in vitro experimental model among three groups.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePower Analysis and Sample Size Calculation\u003c/h2\u003e\u003cp\u003eThe power analysis for microhardness and surface roughness measurements was performed using GPower software \u003cem\u003e(G\u003c/em\u003ePower 3.1; Heinrich Heine University, D\u0026uuml;sseldorf, Germany). Based on repeated-measures ANOVA for three groups with a power of 95% (1-β err prob\u0026thinsp;=\u0026thinsp;0.95), an effect size (f) of 0.47, and α\u0026thinsp;=\u0026thinsp;0.05, a minimum sample size of 45 specimens was calculated to be sufficient. For the push-out bond strength test, the sample size was determined using effect sizes obtained from similar studies. Considering a power of 95% and α\u0026thinsp;=\u0026thinsp;0.05, at least 20 specimens per group were required, resulting in a total of 60 samples.\u003c/p\u003e\u003cp\u003eThe specimens were randomly assigned to three main groups according to the irrigation protocols, and each group was further subdivided for microhardness, surface roughness, and push-out bond strength measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Randomization was performed, and the researcher who conducted the evaluations was blinded to group assignments (single-blind design).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSpecimen Selection and Preparation\u003c/h3\u003e\n\u003cp\u003eA total of 75 extracted human maxillary central incisors from male and female patients aged 25\u0026ndash;50 years, extracted for periodontal, prosthetic, or orthodontic reasons, were used. Teeth exhibited similar root morphology and size and had no cracks, fractures, caries, restorative or endodontic treatment, canal calcifications, or anatomical variations. Organic debris and soft tissue remnants were removed from root surfaces using a sharp periodontal curette. The teeth were stored in distilled water at 4\u0026deg;C until use.\u003c/p\u003e\n\u003ch3\u003ePreparation of Irrigating Solutions\u003c/h3\u003e\n\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e%9 HEBP\u003c/b\u003e: Prepared by adding 448.3 mL of distilled water to 60% HEBP solution (Sigma Aldrich, USA) to obtain 500 mL of 9% concentration.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003e%0.2 Chitosan\u003c/b\u003e: Chitosan powder (Sigma Aldrich, Germany) was dissolved in 250 mL of 1% acetic acid at 0.5 mg/mL concentration and mixed using a magnetic stirrer for 2 hours.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eControl Group\u003c/b\u003e: Commercially prepared %0.9 saline solution was used.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eA 2.5% sodium hypochlorite (NaOCl) solution was used for standard irrigation before chemical interactions in all groups. The pH values of the irrigating solutions were obtained from manufacturer specifications: %9 HEBP (pH 10.7; Zimmer Schwartz, Germany), %0.2 chitosan (pH 3.2; Sigma Aldrich, Germany), saline (pH 4.5\u0026ndash;7; PF %0.9 Isotonic, Turkey), and NaOCl (pH 10.6; Microvem, Turkey). Since pH determines the interaction profile of irrigants with dentin, these values were considered in the present study.\u003c/p\u003e\n\u003ch3\u003eMicrohardness Test\u003c/h3\u003e\n\u003cp\u003eFollowing crown removal, the roots of 45 teeth were sectioned longitudinally in the buccolingual direction under water cooling, yielding 90 half-root specimens. Forty-five halves were allocated for microhardness testing. Samples were embedded in acrylic blocks with the canal lumen exposed, and surfaces were flattened using silicon carbide abrasive papers (500\u0026ndash;1200 grit).\u003c/p\u003e\u003cp\u003eBaseline microhardness measurements were performed at coronal, middle, and apical thirds, 0.5 mm from the canal lumen, using a DuraScan 20 microhardness tester (EMCO-TEST, Germany) with a 300 g load applied for 20 seconds. The mean baseline value was recorded. Specimens were then immersed in 100 mL of the assigned irrigating solution for 5 minutes, and post-treatment measurements were recorded from the same locations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Vickers microhardness values (VHN) were automatically calculated using the formula VHN\u0026thinsp;=\u0026thinsp;1.854 \u0026times; force / (diagonal base)\u0026sup2;.\u003c/p\u003e\n\u003ch3\u003eSurface Roughness Test\u003c/h3\u003e\n\u003cp\u003eThe remaining 45 half-root specimens underwent surface preparation using the same procedures described above. Standard dentin polishing was intentionally not performed to preserve native dentin morphology, as mechanical polishing may artificially alter baseline roughness values and reduce clinical relevance. Accordingly, measurements reflect the natural radicular dentin substrate.\u003c/p\u003e\u003cp\u003eSurface roughness was measured using a Surftest SJ-410 profilometer (Mitutoyo, Japan) with a cut-off length of 0.25 mm, scanning speed of 0.5 mm/s, and total tracing length of 1.25 mm. Five measurements were recorded per specimen, and the mean baseline Ra value was calculated. Samples were then immersed in 100 mL of the assigned chelating solution for 5 minutes, and final surface roughness values were measured. The arithmetic mean of five readings was recorded as the surface roughness value [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePush-Out Bond Strength Test\u003c/h2\u003e\u003cp\u003eFrom the middle third of 30 root specimens, two dentin slices per root were obtained perpendicular to the long axis at 1-mm thickness using a low-speed water-cooled precision saw (Struers Accutom-50, Denmark; 100\u0026ndash;200 rpm) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A standardized cavity (diameter: 1.3 mm) was prepared in each slice using a no. 5 Gates-Glidden drill (Mani Inc., Tachigiken, Japan), yielding a total of 60 dentin discs. These were randomly divided into three groups (n\u0026thinsp;=\u0026thinsp;20) according to the irrigating solutions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAll specimens were initially irrigated with 2.5% NaOCl for 5 minutes, followed by distilled water rinsing for 60 seconds to minimize chemical interactions. The discs were then exposed to 3 mL of the assigned irrigant (%9 HEBP, %0.2 chitosan, or saline) via immersion for 5 minutes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Specimens were dried using cotton pellets and paper points (DentPlus, Diadent, Netherlands), after which Biodentine (Septodont, Saint Maur des Foss\u0026eacute;s, France) was placed into the cavities.\u003c/p\u003e\u003cp\u003eSamples were stored at 37\u0026deg;C in 100% humidity for 24 hours to allow complete setting. Push-out testing was performed by applying a compressive load at a crosshead speed of 1 mm/min using a universal testing machine (Shimadzu Co., Kyoto, Japan) with a 1-mm stainless-steel plugger. The maximum force at dislodgement (N) was recorded and converted to MPa using the formula:\u003c/p\u003e\u003cp\u003eDislodgement stress (MPa)\u0026thinsp;=\u0026thinsp;Force (N) / Bonded area (mm\u0026sup2;)\u003c/p\u003e\u003cp\u003eBonded interface area\u0026thinsp;=\u0026thinsp;2πrh\u003c/p\u003e\u003cp\u003e(r\u0026thinsp;=\u0026thinsp;cavity radius; h\u0026thinsp;=\u0026thinsp;slice thickness)\u003c/p\u003e\u003cp\u003eAfter testing, failure modes were evaluated under a stereomicroscope (Leica MZ75, Cambridge, UK) at \u0026times;10 magnification and classified as adhesive, cohesive, or mixed [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eNormality of the data was assessed using the Shapiro\u0026ndash;Wilk and Kolmogorov\u0026ndash;Smirnov tests. Descriptive statistics (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation) were reported. For normally distributed pre- and post-treatment values, the paired-sample t-test was used. The Kruskal\u0026ndash;Wallis test was used for non-normally distributed comparisons. One-way ANOVA was applied to determine statistically significant differences among groups, and the Tukey HSD post hoc test was used to identify pairwise differences. A significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was adopted. Data analysis was conducted using SPSS version 22 (SPSS Inc., Chicago, IL, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eVickers Microhardness Test\u003c/h2\u003e\u003cp\u003eBefore solution application, no statistically significant differences were observed among the groups in terms of microhardness at the coronal, middle, or apical thirds (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Table\u0026nbsp;1). The mean baseline microhardness values were 57.18\u0026thinsp;\u0026plusmn;\u0026thinsp;4.39 VHN for the saline group, 57.52\u0026thinsp;\u0026plusmn;\u0026thinsp;3.97 VHN for the chitosan group, and 56.20\u0026thinsp;\u0026plusmn;\u0026thinsp;4.19 VHN for the HEBP group.\u003c/p\u003e\u003cp\u003eFollowing solution application, statistically significant differences in microhardness values were detected among the groups in all regions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table\u0026nbsp;1). The lowest mean post-treatment microhardness value was recorded in the HEBP group (28.54\u0026thinsp;\u0026plusmn;\u0026thinsp;6.47 VHN), followed by the chitosan group (47.40\u0026thinsp;\u0026plusmn;\u0026thinsp;4.73 VHN), while the saline group exhibited the highest value (56.34\u0026thinsp;\u0026plusmn;\u0026thinsp;4.53 VHN). According to Tukey post hoc analysis, the HEBP group differed significantly from both the chitosan and saline groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, the difference between the chitosan and saline groups was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSurface Roughness Test\u003c/h2\u003e\u003cp\u003eNo statistically significant differences were observed among the groups in baseline surface roughness values (p\u0026thinsp;=\u0026thinsp;0.469, ANOVA; Table\u0026nbsp;2). The mean baseline Ra values were 0.1595\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0213 \u0026micro;m for the saline group, 0.1488\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0268 \u0026micro;m for the chitosan group, and 0.1536\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0222 \u0026micro;m for the HEBP group.\u003c/p\u003e\u003cp\u003eAfter solution application, a statistically significant difference in surface roughness was observed among the groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Table\u0026nbsp;2). The highest surface roughness was recorded in the HEBP group (0.5674\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0636 \u0026micro;m), followed by the chitosan (0.2788\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0492 \u0026micro;m) and saline (0.1704\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0230 \u0026micro;m) groups. Post hoc comparisons revealed that all groups differed significantly from each other (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003ePush-Out Bond Strength Test\u003c/h2\u003e\u003cp\u003ePush-out bond strength values differed significantly among the groups (p\u0026thinsp;=\u0026thinsp;0.001; Table\u0026nbsp;3). The mean bond strength values were 15.73\u0026thinsp;\u0026plusmn;\u0026thinsp;7.34 MPa for the HEBP group, 13.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.92 MPa for the chitosan group, and 9.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.70 MPa for the saline group. The HEBP group demonstrated significantly higher bond strength compared to the saline group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas no statistically significant difference was observed between the HEBP and chitosan groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFailure Mode Analysis\u003c/h2\u003e\u003cp\u003eEvaluation of failure modes revealed that mixed failure was the most frequently observed type across all groups. In the HEBP group, 72% mixed, 16% cohesive, and 12% adhesive failures were observed; in the chitosan group, 68% mixed, 20% cohesive, and 12% adhesive failures were recorded; while in the saline group, 60% mixed, 22% cohesive, and 18% adhesive failures were identified (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Although this distribution suggests similar trends among the groups, it was not statistically analyzed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study evaluated the effects of different chelating agents on dentin surface characteristics and the push-out bond strength of the bioceramic material Biodentine. The findings demonstrated that both HEBP and chitosan yielded similarly high bond strength values; however, HEBP caused the greatest reduction in microhardness and the highest increase in surface roughness. These results support our hypothesis regarding bond strength while confirming the secondary hypothesis favoring chitosan with respect to reduced dentin degradation.\u003c/p\u003e\u003cp\u003eThe adhesion of calcium silicate\u0026ndash;based materials to root dentin enhances the integrity of the dentin\u0026ndash;material interface and contributes to the long-term success of endodontic treatment through improved sealing [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The physical properties of dentin, including microhardness and the degree of surface roughening, directly influence the quality of this adhesion [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. While strong chelators such as EDTA and citric acid can enhance the organic tissue-dissolving capacity and antimicrobial activity of NaOCl, they may also induce procedural complications such as ledge formation and perforation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo minimize these risks while enhancing irrigation efficacy, the continuous soft-chelation technique\u0026mdash;combining a weak acid with NaOCl\u0026mdash;has been proposed. This approach aims to prevent smear layer accumulation, reduce procedural errors, facilitate clinical handling, and preserve dentin integrity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For these reasons, the present study utilized %9 HEBP and %0.2 chitosan nanoparticles as potential alternatives to EDTA, and assessed their effects on dentin microhardness, surface roughness, and adhesion to Biodentine.\u003c/p\u003e\u003cp\u003eOptimal chelation times reported in the literature vary between 1, 5, and 15 minutes. However, prolonged exposure to decalcifying agents is believed to induce detrimental effects on root dentin [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, chelation was performed for 5 minutes to simulate clinical conditions and facilitate smear layer removal while avoiding excessive demineralization, consistent with previous reports [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMicrohardness results indicated that the group treated with chitosan exhibited values closest to the control. This may be attributed to the ability of positively charged chitosan nanoparticles to interact electrostatically with dentin collagen, thereby maintaining mechanical stability [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, contradictory findings regarding the influence of chitosan on dentin microhardness exist in the literature, which may be related to variations in concentration, formulation, and exposure time [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCompared with EDTA, HEBP has been reported to induce less reduction in the mechanical properties of dentin, whereas EDTA significantly decreases the calcium-to-phosphorus ratio and weakens dentin structure [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The decrease in dentin microhardness is associated with mineral loss in the intertubular dentin and hydroxyapatite dissolution. Due to its lower chelation capacity, etidronic acid may mitigate these detrimental effects.\u003c/p\u003e\u003cp\u003eIn the present study, the HEBP group demonstrated a significant increase in surface roughness. Although an increase in dentin surface area may enhance micromechanical adhesion, it has been suggested that excessive roughness can be clinically undesirable. Specifically, increased surface area may promote bacterial adhesion and potentially elevate the risk of microleakage [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, when selecting an irrigant, not only chelation efficacy but also its impact on dentin surface morphology should be considered.\u003c/p\u003e\u003cp\u003eThe use of centrally located perforations in single-rooted teeth for push-out testing provides a reproducible model with improved comparability. Off-center perforations may expose dentinal tubules at variable orientations, influencing the penetration and adaptation of calcium silicate\u0026ndash;based materials. By preparing centrally positioned cavities, a uniform exposure of tubules was achieved, contributing to more standardized results [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious reports have shown that the diameter of the cylindrical plunger does not significantly influence test outcomes when properly positioned to avoid contact with canal walls [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Based on this information, a 1-mm plunger was selected to match the 1.3-mm cavity diameter. The highest bond strength values were observed in the HEBP group, followed by chitosan, although the difference between these two groups was not statistically significant. The control group exhibited significantly lower values.\u003c/p\u003e\u003cp\u003eConsistent with these findings, Tuncel et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and Carvalho et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] demonstrated that chelating agents increase the bond strength of calcium silicate\u0026ndash;based materials, with no significant differences detected among different chelators. Similarly, Buldur et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] reported enhanced bond strength following chelator use. Contrary to these results, Ballal et al. [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] evaluated bond strength in simulated root-end cavities and reported significantly lower values in samples treated with 17% EDTA compared to controls. Due to its erosive effect and strong chelating capacity, EDTA may reduce calcium content at the Biodentine\u0026ndash;dentin interface or alter the calcium silicate fraction within the cement, thereby weakening adhesion [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFollowing push-out testing, Aguiar et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] reported that Biodentine predominantly exhibited cohesive failures under scanning electron microscopy. Although the present study demonstrated a higher prevalence of mixed failures, both studies identified cohesive and mixed failures as the most frequent failure modes, whereas adhesive failures were the least common.\u003c/p\u003e\u003cp\u003eThis study has several limitations. First, because it was conducted under in vitro conditions, caution must be exercised when extrapolating the findings to clinical practice. Additionally, biological responses of dentin following irrigation, such as cell viability, inflammatory reactions, and regenerative potential, were not evaluated. Only a single bioceramic material (Biodentine) was tested, limiting generalizability to other biomaterials. Therefore, future studies should evaluate different material systems, incorporate in vivo experimental models, and employ advanced imaging techniques (e.g., micro-CT, SEM) to further assess dentinal tubule patency and surface topography.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWithin the limitations of this in vitro study, this investigation comparatively evaluated the effects of different chelating agents on the physicomechanical properties of root dentin. The findings demonstrated that %9 HEBP significantly reduced dentin microhardness and markedly increased surface roughness, whereas %0.2 chitosan caused more limited alterations, preserving surface integrity more effectively. Push-out bond strength values were higher in both HEBP and chitosan groups compared to the control group, although no statistically significant difference was observed between the two chelating agents in this regard.\u003c/p\u003e\u003cp\u003eClinically, these results support the concept of continuous soft chelation, suggesting that the use of mild irrigating agents such as %0.2 chitosan and %9 HEBP may enhance long-term bonding stability and help preserve dentin integrity, particularly in structurally compromised teeth and regenerative procedures. The biocompatibility and collagen-stabilizing potential of chitosan further highlight its promise as an alternative irrigant in contemporary endodontic protocols.\u003c/p\u003e\u003cp\u003eHowever, the in vitro nature of this study and the use of a single calcium silicate\u0026ndash;based material limit the generalizability of these findings. Future investigations involving in vivo models, alternative biomaterials, and biological response assessments are recommended to further validate the translational applicability of these irrigation strategies in clinical practice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript originates from the thesis of Aslı \u0026Ouml;ZDEMİR. The study was funded by the Fırat University Scientific Research Projects (F\u0026Uuml;BAP) Coordination Unit, Elazığ, Turkey (Project No: DHF.22.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMSO: Conceptualization, methodology, resources, project administration, writing - review and editing; funding acquisition, supervision; A\u0026Ouml;: Conceptualization, data curation, methodology, formal analysis, resources, writing - original draft, writing - review and editing. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was supported by F.U. Scientific Research Projects (F\u0026uuml;bap) Coordination Unit, Elazig, Turkey with the project number DHF.22.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Statements:\u003c/strong\u003e This study was conducted using extracted human teeth that were obtained with the patients\u0026rsquo; informed consent and in accordance with ethical standards. The research protocol was reviewed and approved by the Ethics Committee of Fırat University (Elazığ, T\u0026uuml;rkiye) (Approval No: 2022/11-25).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePeters, O. A. et al. Guidelines for non-surgical root canal treatment. \u003cem\u003eAust Endod J.\u003c/em\u003e \u003cb\u003e50\u003c/b\u003e (2), 202\u0026ndash;214. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/aej.12848\u003c/span\u003e\u003cspan address=\"10.1111/aej.12848\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlamoudi, R. A. 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V., Ulusoy, \u0026Ouml;. İ., Chhaparwal, S. \u0026amp; Ginjupalli, K. Effect of novel chelating agents on the push-out bond strength of calcium silicate cements to the simulated root-end cavities. \u003cem\u003eMicrosc Res. Tech.\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e (2), 214\u0026ndash;219. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jemt.22969\u003c/span\u003e\u003cspan address=\"10.1002/jemt.22969\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAtmeh, A. R., Chong, E. Z., Richard, G., Festy, F. \u0026amp; Watson, T. F. Dentin-cement interfacial interaction: calcium silicates and polyalkenoates. \u003cem\u003eJ. Dent. Res.\u003c/em\u003e \u003cb\u003e91\u003c/b\u003e (5), 454\u0026ndash;459. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/0022034512443068\u003c/span\u003e\u003cspan address=\"10.1177/0022034512443068\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAguiar, B. A. et al. Influence of ultrasonic agitation on bond strength, marginal adaptation, and tooth discoloration provided by three coronary barrier endodontic materials. \u003cem\u003eClin. Oral Investig\u003c/em\u003e. \u003cb\u003e23\u003c/b\u003e (11), 4113\u0026ndash;4122. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00784-019-02850-y\u003c/span\u003e\u003cspan address=\"10.1007/s00784-019-02850-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTablo 1 .\u0026nbsp;\u003c/strong\u003eCoronal, middle, apical, and mean microhardness values of root dentin before and after solution application. P values marked with * indicate statistically significant differences (paired samples t-test) (p\u0026lt;0,05). \u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"562\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 75px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eStd. Dev.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 83px;\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eCoronal Initial test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e58,0267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e5,80104\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1,541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 83px;\"\u003e\n \u003cp\u003e0,226\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e57,1933\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,11556\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e54,6667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e6,23179\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eMiddle Initial test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e56,2267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,70615\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 52px;\"\u003e\n \u003cp\u003e2,244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 83px;\"\u003e\n \u003cp\u003e0,119\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e59,2000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e5,34416\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e54,9333\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e6,72986\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eApical Initial test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e57,2933\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,91840\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 52px;\"\u003e\n \u003cp\u003e1,175\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 83px;\"\u003e\n \u003cp\u003e0,319\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e56,1733\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e5,88988\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e59,0133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,41634\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eAverage Initial test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSaline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e57,1822\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,39484\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 52px;\"\u003e\n \u003cp\u003e0,399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 83px;\"\u003e\n \u003cp\u003e0,673\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e57,5222\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e3,97821\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e56,2044\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,19283\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eCoronal Final test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e57,1267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e6,23647\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 52px;\"\u003e\n \u003cp\u003e77,983\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0,001\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e46,6200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e5,13451\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e27,9267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e7,80837\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eMiddle Final test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e55,2267\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e5,04798\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 52px;\"\u003e\n \u003cp\u003e64,536\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0,001\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e49,0667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,93843\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e28,4533\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e9,33938\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eApical Final test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e56,6800\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,65912\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 52px;\"\u003e\n \u003cp\u003e62,640\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0,001\u003c/strong\u003e\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e46,5200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e7,39239\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e29,2467\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e7,86374\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width: 105px;\"\u003e\n \u003cp\u003eAverage Final test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e56,3444\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,53709\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 52px;\"\u003e\n \u003cp\u003e106,618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0,001\u003c/strong\u003e\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e47,4022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e4,73777\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 75px;\"\u003e\n \u003cp\u003e28,5422\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 77px;\"\u003e\n \u003cp\u003e6,47988\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTablo 2.\u0026nbsp;\u003c/strong\u003eMean surface roughness values of root dentin before and after solution application. A paired samples t-test was used for data analysis. P values marked with * indicate statistically significant differences (p \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"578\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eGroups (n=15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 79px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 104px;\"\u003e\n \u003cp\u003eStd. Dev.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 71px;\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 81px;\"\u003e\n \u003cp\u003eP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eSurface Roughness Initial Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0,1595\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0,02130\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 71px;\"\u003e\n \u003cp\u003e0,771\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 81px;\"\u003e\n \u003cp\u003e0,469\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0,1488\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0,02679\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0,1536\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0,02225\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" valign=\"top\" style=\"width: 149px;\"\u003e\n \u003cp\u003eSurface Roughness Final Test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0,1704\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0,02307\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 71px;\"\u003e\n \u003cp\u003e270,190\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 81px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0,001\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0,2788\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0,04926\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e0,5674\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 104px;\"\u003e\n \u003cp\u003e0,06367\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTablo 3.\u0026nbsp;\u003c/strong\u003ePush-out bond strength test results are presented in the table. Data were analyzed using the Kruskal\u0026ndash;Wallis test (p = 0.001). According to the Tukey HSD test, a statistically significant difference was found between the HEBP and saline groups (p \u0026lt; 0.01). Differences among the other groups were not significant (p \u0026gt; 0.05). P values marked with * indicate statistically significant differences (p \u0026lt; 0.05).\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"591\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 19.0563%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.7931%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.9746%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 14.3376%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStd. Dev.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4301%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMedian\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 9.9819%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.4265%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0563%;\"\u003e\n \u003cp\u003eHEBP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7931%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.9746%;\"\u003e\n \u003cp\u003e15,73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3376%;\"\u003e\n \u003cp\u003e7,34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4301%;\"\u003e\n \u003cp\u003e15,40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 9.9819%;\"\u003e\n \u003cp\u003e13,555\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width: 15.4265%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0,001\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e*\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0563%;\"\u003e\n \u003cp\u003eChitosan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7931%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.9746%;\"\u003e\n \u003cp\u003e13,20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3376%;\"\u003e\n \u003cp\u003e5,92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4301%;\"\u003e\n \u003cp\u003e13,53\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 19.0563%;\"\u003e\n \u003cp\u003eSalin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.7931%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.9746%;\"\u003e\n \u003cp\u003e9,82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 14.3376%;\"\u003e\n \u003cp\u003e4,70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.4301%;\"\u003e\n \u003cp\u003e9,47\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":"Chitosan, Dental Materials, Etidronic acid","lastPublishedDoi":"10.21203/rs.3.rs-7998211/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7998211/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis in vitro study evaluated the effects of different chelating agents on dentin properties influencing the adhesion of calcium silicate\u0026ndash;based biomaterials. The effects of 9% etidronic acid (HEBP) and 0.2% chitosan nanoparticles on the microhardness, surface roughness, and push-out bond strength of Biodentine were assessed. Seventy-five extracted maxillary incisors were divided into three groups according to the final irrigation protocol: 9% HEBP, 0.2% chitosan, or 0.9% saline (control). Dentin microhardness (Vickers hardness number) and surface roughness (Ra, \u0026micro;m) were measured before and after irrigation. For push-out testing, standardized cavities in 60 dentin discs were filled with Biodentine and stored for 24 h at 37\u0026deg;C and 100% humidity. Failure modes were examined under a stereomicroscope. Data were analyzed using one-way ANOVA, Tukey HSD, Kruskal\u0026ndash;Wallis, and paired t-tests (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All groups showed significant microhardness reduction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), greatest in HEBP. Surface roughness increased significantly, highest in HEBP (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Push-out bond strength was higher in HEBP than control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with no difference between HEBP and chitosan (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Both agents improved Biodentine adhesion; chitosan produced milder dentin alterations, indicating potential for dentin preservation.\u003c/p\u003e","manuscriptTitle":"Chelating Agents’ Effects on Dentin Properties and Bond Strength of Biodentine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-18 08:48:10","doi":"10.21203/rs.3.rs-7998211/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":"5424fa7e-e876-4853-b471-040571a077a2","owner":[],"postedDate":"November 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58088515,"name":"Biological sciences/Biotechnology"},{"id":58088516,"name":"Health sciences/Health care"},{"id":58088517,"name":"Physical sciences/Materials science"},{"id":58088518,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-12-08T04:23:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-18 08:48:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7998211","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7998211","identity":"rs-7998211","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}