May 08, 2026

Comparative Evaluation of Nano-Chitosan Extract versus Sodium Hypochlorite in the Removal of Bio-C Temp Intracanal Medicament by Scanning electron microscope In A Randomized In Vitro Study

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld4k19l5b/v1
  • Mohamed Osama El-sayed Ammar1,2,3,
  • Prof.Dr. Alaa Abdelsalam Elbaz4,
  • Dr.Ghada Ihab ElWazan5,
  • Mohamed Osama Elsayed Ammar6
  • 1MSA University, Egypt;
  • 2Endodontic Department, Faculty of Dentistry, Cairo University;
  • 3Master’s degree student, Department of Endodontics, Faculty of Dentistry, Cairo University;
  • 4Professor of Endodontics, Faculty of Dentistry, Cairo University;
  • 5Lecturer, Department of Endodontics, Faculty of Dentistry, Cairo University;
  • 6Faculty of Dentistry - Cairo University
  • Faculty of Dentistry - Cairo University
Mohamed Osama Elsayed Ammar
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.n92ld4k19l5b/v1
Protocol Citation: Mohamed Osama El-sayed Ammar, Prof.Dr. Alaa Abdelsalam Elbaz, Dr.Ghada Ihab ElWazan, Mohamed Osama Elsayed Ammar 2026. Comparative Evaluation of Nano-Chitosan Extract versus Sodium Hypochlorite in the Removal of Bio-C Temp Intracanal Medicament by Scanning electron microscope In A Randomized In Vitro Study. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld4k19l5b/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 08, 2026
Last Modified: May 08, 2026
Protocol  Integer ID: 316635
Keywords: chitosan extract versus sodium hypochlorite, chitosan extract, efficiency of nano chitosan, success rate of the nano chitosan, root canal irrigant emphasizing smear layer removal, nano chitosan, high molecular nano chitosan, chitosan, root canal irrigation, acetic acids for smear removal, randomized in vitro study sodium hypochlorite, passive ultrasonic irrigation protocol with sodium hypochlorite, rooted tooth, sodium hypochlorite, comparative evaluation of nano, antimicrobial effect, antimicrobial efficacy, removal of bio, smear removal, using passive ultrasonic irrigation protocol
Abstract
Sodium hypochlorite is conventionally used in root canal irrigation for its broad-spectrum antimicrobial effect and tissue dissolution capacity, however, its drawbacks include potential cytotoxicity, environmental concerns, and inability to completely remove certain medicaments without adjunct agents. Removing Bio-C Temp with conventional irrigation protocol or passive ultrasonic irrigation protocol with sodium hypochlorite as an irrigant didn’t show the expected outcome as there was remnants of the Intracanal medicament in the coronal, middle and apical part of the dentinal walls. Chitosan demonstrates effectiveness as a root canal irrigant emphasizing smear layer removal and antimicrobial efficacy. Chitosan excelled over citric and acetic acids for smear removal, rivaled MTAD and Qmix while varying against EDTA, and matched chlorhexidine, propolis, and photodynamic therapy antibacterially. Using 0.2% high molecular Nano Chitosan as an irrigant by using passive ultrasonic irrigation protocol shows significant effect against Enterococcus Faecalis with altering dentin roughness. To our Knowledge there are no studies in the literature showing the efficiency of Nano Chitosan to remove the Bio-C Temp from oval shaped canals in single rooted tooth As the Bio-C temp is known from previous studies that it is hard to be removed from the dentinal tubules and leave the space for the future sealer to penetrate it. In spite the previous studies that failed to acknowledge the removal of Bio-C temp from the dentinal tubules, Therefore the aim for this study is to evaluate the success rate of the Nano Chitosan to remove the Bio-C temp against the standard Naocl and EDTA from the oval shape canal in single rooted tooth in addition to the calculation of the time needed to remove the Intracanal medicament.
Scientific Background
Intracanal medicament is considered one of the main pillars in the multi-visit non-surgical root canal treatment specially with the presence of different types of infection as it’s responsible for elimination of any remaining bacteria after canal instrumentation, reduce inflammation of periapical tissues and pulp remnants, render canal contents inert and neutralize tissue debris, act as a barrier against leakage from the temporary filling. (Chong and Pitt Ford, 1992)
Calcium hydroxide is the corner stone of the Intracanal medicament in Endodontics due to its high pH (around 12.5), which creates an alkaline environment that disrupts bacterial cell membranes, denatures proteins, and effectively eliminates microorganisms in root canals.​ (Ba-Hattab et al., 2016)
Calcium hydroxide has notable limitations including incomplete bacterial elimination, particularly against resilient species like Enterococcus faecalis in dentinal tubules, due to its low solubility and limited penetration into complex canal anatomies. (Kim & Kim, 2015)
Bio-C Temp is a bioceramic Intracanal medicament composed primarily of tricalcium silicate, calcium aluminate, calcium oxide, polyethylene glycol, titanium dioxide, calcium tungstate (as radio opacifier), and a resin matrix, which hydrates to release calcium ions (Ca²⁺) and hydroxyl ions (OH⁻) continuously, achieving a high pH of around 12 for antibacterial action and mineralization induction. Unlike traditional calcium hydroxide pastes, which have high solubility requiring frequent reapplication, Bio-C Temp offers low solubility for prolonged canal wall contact, better penetration into dentinal tubules due to its ≤2 μm particle size, high radiopacity (≥9 mm Al step-wedge), and easy removability without setting prematurely. (Almohareb et al. 2024)
Bio-C Temp has gained wide popularity due to its favorable biological properties, including biocompatibility and antimicrobial activity, but its tenacious adherence to dentinal walls make a great clinical challenge during removal of it. (Só et al., 2025)
Bio-C temp has one of the most important advantages over the calcium hydroxide is that it has sustained action without re-application as the calcium hydroxide faces frequent neutralization but Bio-C temp has high alkaline ph (ph=12) which allows constant release of Calcium ions (Lopes et al., 2024).
The complete removal of Intracanal medicament is considered very important and critical step in the non-surgical root canal treatment as residual medicament can hinder the adhesion and sealing ability of filling materials, potentially leading to micro leakage and treatment failure. (Suleiman et al., 2022)
Sodium hypochlorite is conventionally used in root canal irrigation for its broad-spectrum antimicrobial effect and tissue dissolution capacity, however, its drawbacks include potential cytotoxicity, environmental concerns, and inability to completely remove certain medicaments without adjunct agents. (Silva et al., 2011)
Removing Bio-C Temp with conventional irrigation protocol or passive ultrasonic irrigation protocol with sodium hypochlorite as an irrigant didn’t show the expected outcome as there was remnants of the Intracanal medicament in the coronal, middle and apical part of the dentinal walls. (Lopes et al., 2024)
Chitosan demonstrates effectiveness as a root canal irrigant emphasizing smear layer removal and antimicrobial efficacy. Chitosan excelled over citric and acetic acids for smear removal, rivaled MTAD and Qmix while varying against EDTA, and matched chlorhexidine, propolis, and photodynamic therapy antibacterially. (Anbalagan et al., 2025)
Using 0.2% high molecular Nano Chitosan as an irrigant by using passive ultrasonic irrigation protocol shows significant effect against Enterococcus Faecalis with altering dentin roughness (Abidin et al., 2022).
To our Knowledge there are no studies in the literature showing the efficiency of Nano Chitosan to remove the Bio-C Temp from oval shaped canals in single rooted tooth As the Bio-C temp is known from previous studies that it is hard to be removed from the dentinal tubules and leave the space for the future sealer to penetrate it.
In spite the previous studies that failed to acknowledge the removal of Bio-C temp from the dentinal tubules, Therefore the aim for this study is to evaluate the success rate of the Nano Chitosan to remove the Bio-C temp against the standard Naocl and EDTA from the oval shape canal in single rooted tooth in addition to the calculation of the time needed to remove the Intracanal medicament.
Review of literature
Só et al. (2025) conducted an in vitro microcomputed tomography study evaluating three removal protocols—conventional irrigation, passive ultrasonic irrigation (PUI), and XP-endo Finisher—for calcium hydroxide (Ultracal XS) and bioceramic (Bio-C Temp) intracanal dressings from root canals. Sixty roots from extracted teeth were instrumented, medicated, and randomized into six groups, with post-removal scans quantifying residual volumes across cervical, middle, and apical thirds using 2.5% NaOCl, EDTA, and saline rinses. No protocol fully eliminated residues, but PUI and XP-endo Finisher outperformed manual irrigation, particularly for Ultracal XS; Bio-C Temp proved harder to remove due to its dentin adhesion and hydroxyapatite formation, especially in the cervical third. The findings underscore the limitations of current techniques and the need for enhanced strategies before obturation.
Khairajani et al. (2025) conducted an in vitro CBCT volumetric analysis to compare four irrigation activation techniques for removing calcium silicate-based intracanal medicament (Bio-C Temp) from root canals. Forty single-rooted premolars were instrumented, filled with Bio-C Temp for one week, then randomly assigned to groups: conventional side-vented needle irrigation (CSI), XP-endo Finisher (XPF), EndoActivator sonic activation, or passive ultrasonic irrigation (PUI), using 2.5% NaOCl and EDTA. Pre- and post-removal micro-CT scans quantified residuals, showing all methods reduced volumes significantly but none achieved complete elimination, with XPF (98.2% removal) and PUI (95.1%) outperforming CSI (70.6%) and EndoActivator (75.7%), particularly in apical thirds. XPF's non-tapered design and flexibility enhanced efficacy without statistical superiority over PUI, underscoring the need for advanced agitation in bioceramic medicament protocols prior to obturation.
Guerreiro et al. (2021) assessed the antibacterial activity, cytocompatibility, and effects on osteoblast biology of Bio-C Temp, a bioceramic intracanal medicament, compared to calcium hydroxide-based UltraCal XS and Calen. Bio-C Temp exhibited lower antibacterial efficacy and biofilm reduction against Enterococcus faecalis than the comparators, though it showed comparable cytocompatibility at higher dilutions and superior alkaline phosphatase (ALP) activity induction at day 1. It promoted similar mineralized nodule deposition to UltraCal XS, highlighting its bioactivity for periapical repair despite reduced antimicrobial performance, positioning it as a viable alternative for cases prioritizing tissue regeneration over broad-spectrum bactericidal action.
Almohareb et al. (2024) performed a micro-CT analysis to assess the efficacy of five techniques in removing Bio-C Temp, a calcium silicate-based intracanal medicament, from root canals of extracted single-canal premolars. Forty teeth were instrumented, filled with Bio-C Temp for one week, and randomized into groups using conventional syringe irrigation, EndoActivator sonic activation, passive ultrasonic irrigation (PUI), ProTaper Universal F3, or XP-endo Finisher (XPF), with pre- and post-removal scans quantifying residual volumes across canal thirds. All methods significantly reduced medicament volume without complete elimination, but XPF achieved the highest removal rate (98.2%), outperforming syringe irrigation (70.6%) and others.
Balto et al. (2024) evaluated the antibiofilm efficacy of Bio-C Temp, a calcium silicate-based intracanal medicament, against Fusobacterium nucleatum biofilms on dentin slices compared to triple antibiotic paste (TAP) and calcium hydroxide (Ca(OH)2). Dentin specimens were inoculated with F. nucleatum, treated for 1 or 2 weeks, and analyzed via live/dead staining and confocal microscopy, revealing all medicaments reduced biofilm biomass significantly versus controls. TAP demonstrated superior efficacy (95.9% dead bacteria at week 2), while Bio-C Temp outperformed Ca(OH)2 after 2 weeks (75.25% vs. 58.65% dead bacteria), with efficacy increasing over time for both TAP and Bio-C Temp but not Ca(OH)2. These findings position Bio-C Temp as a promising alternative for prolonged applications in regenerative Endodontics, balancing antibiofilm action with tissue compatibility.
Saryılmaz et al. (2024) assessed the efficacy of four irrigation activation techniques in removing calcium hydroxide (Ultracal XS), Ledermix paste, and Bio-C Temp from simulated internal root resorption (IRR) cavities in root canals. Single-rooted maxillary incisors were instrumented with Reciproc R50 files, IRR cavities created 8 mm from the apex, filled with medicaments, and randomized to standard needle irrigation (SNI), EDDY sonic activation, passive ultrasonic irrigation (PUI), or XP-endo Finisher (XPF), with removal scored via standardized microscopy evaluation. XPF and PUI outperformed SNI and EDDY across medicaments, though no technique achieved complete removal; Ledermix was easiest to remove, while Bio-C Temp proved most resistant due to its adhesion, particularly in IRR defects. These results emphasize advanced activation systems for complex anatomies in endodontic retreatment.
(Mohammadi 6 Dummer, 2008). Provided an update review on sodium hypochlorite (NaOCl) as the primary irrigant in Endodontics, emphasizing its tissue-dissolving, antimicrobial, and lubricating properties essential for root canal disinfection. Optimal concentrations range from 0.5-5.25%, balancing efficacy against cytotoxicity risks, with higher strengths enhancing pulp dissolution but increasing extrusion hazards during instrumentation. Activation techniques like ultrasonic irrigation improve penetration into complex anatomies, though the review stresses safety protocols, including rubber dam use and controlled volumes, to mitigate complications like hypochlorite accidents.
Marion et al. (2012) reviewed the efficiency of various sodium hypochlorite (NaOCl) concentrations in endodontic treatment, focusing on its tissue-dissolving, antimicrobial, and lubricating properties. Concentrations from 0.5% to 5.25% were analyzed, with higher strengths (e.g., 2.5-5.25%) showing superior pulp dissolution and bacterial killing but increased cytotoxicity and risk of periapical extrusion accidents. The 2.5% concentration emerged as optimal, balancing efficacy against toxicity for routine irrigation, while emphasizing activation methods and safety protocols like rubber dam to enhance penetration without complications.
Jaiswal et al. (2017) evaluated the antibacterial efficacy of chitosan, chlorhexidine, propolis, and sodium hypochlorite (NaOCl) against Enterococcus faecalis biofilms in an in vitro model simulating root canal disinfection. Extracted teeth sections were inoculated with E. faecalis to form 21-day biofilms, then treated with test solutions including 5% NaOCl, 2% chlorhexidine, propolis, various chitosan concentrations (0.2-2%), and chitosan-chlorhexidine combinations, with efficacy assessed via colony-forming units post-exposure. Chitosan-chlorhexidine combinations (especially 1% chitosan + 1% chlorhexidine), standalone chlorhexidine, and propolis matched NaOCl's superior biofilm reduction, outperforming acetic acid and saline controls, suggesting natural alternatives could mitigate NaOCl's cytotoxicity while maintaining antimicrobial potency.​
(Cakici & Cakici, 2024).compared the antimicrobial efficacy of chitosan nanoparticles (CS-NPs) against sodium hypochlorite (NaOCl) on Enterococcus faecalis biofilms in an in vitro endodontic model. Root dentin blocks were inoculated with E. faecalis to form mature biofilms, then irrigated with 2% CS-NPs, 2.5% NaOCl, or controls, followed by confocal laser scanning microscopy and colony-forming unit counts to assess bacterial viability and biomass reduction. CS-NPs demonstrated comparable or superior biofilm disruption and killing to NaOCl, attributed to their positive charge disrupting bacterial membranes and penetrating extracellular matrices, offering a biocompatible alternative with reduced cytotoxicity for prolonged Intracanal applications.
Wang et al. (2020) investigated the antibacterial effects of chitosan and its derivative, N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC), against Enterococcus faecalis strains associated with endodontic infections, both in planktonic and biofilm states. Minimum bactericidal concentrations were determined as 70 µg/ml for chitosan and 140 µg/ml for HTCC in distilled water, outperforming phosphate-buffered saline conditions, with chitosan showing superior efficacy via charge interactions disrupting bacterial membranes. Both agents significantly reduced E. faecalis biofilms on dentin slices, with chitosan exhibiting no cytotoxicity to MC3T3-E1 osteoblasts below 625 µg/ml, positioning them as promising, biocompatible alternatives for root canal disinfection.
Moukarab (2020) evaluated the antimicrobial efficacy of manually agitated nano-chitosan and nano-propolis against Enterococcus faecalis in root canals, compared to 5.25% sodium hypochlorite (NaOCl), using an in vitro model with extracted premolars. Fifty single-rooted teeth were instrumented to F5 ProTaper, sterilized, contaminated with E. faecalis for 4 weeks, and divided into groups: positive control (untreated), negative control (sterile), NaOCl with manual agitation (#25 gutta-percha cone for 2 minutes), Nano-chitosan, and Nano-propolis under laminar flow. Microbial sampling post-treatment showed no significant difference in log CFU reduction between NaOCl, Nano-chitosan, and Nano-propolis groups (all superior to positive control, p>0.05), concluding that Nano-materials offer safe, efficient alternatives to NaOCl for Intracanal disinfection.
Specific objectives
Research question
Does Nano-chitosan extract demonstrate comparable or superior efficacy and efficiency to sodium hypochlorite in removing Bio-C Temp Intracanal medicament from root canals?
Null Hypothesis:
There is no difference in the efficacy or efficiency between Nano-chitosan extract irrigation and sodium hypochlorite irrigation for removing Bio-C Temp Intracanal medicament from instrumented single-rooted extracted human teeth.
PICO
-Population: Extracted, decoronated human single-rooted teeth with oval canals
-Intervention:
I1: Sodium Hypochlorite with Passive Ultrasonic Activation
I2: Nano Chitosan irrigant with Conventional Manual Irrigation
I3: Nano Chitosan irrigant with Passive Ultrasonic Activation
-Control1: Conventional (NaOCl) with Conventional Manual Irrigation
-Outcome:
O1: Bio C temp Intracanal medicament removal from dentin walls
O2: Bio-C Temp removal time from the canal
Materials and Methods
1) Sample:
    Calculated sample size:
Sample size was calculated using G*Power 3.1.9.7 for one-way ANOVA (fixed effects, omnibus, one-way) to detect differences in the primary outcome (quantified via SEM image analysis with mean ± SD)—across four removal technique groups, with α=0.05 and power=0.90. Effect size f (≈0.50, medium-large) was derived from Almohareb et al., who reported Bio-C Temp removal means of 70.6% (syringe), 75.7% (EndoActivator), 76.6% (ProTaper), and 95.1-98.2% (PUI/XP-endo; SD≈15-20%; n=8/group). This yields total N=32-36 (8-9/group for 4 groups); conservatively increased to n=10/group (N=40) to account for SEM measurement variability.
Sample description:
40 single rooted teeth extracted owing to orthodontic, periodontal, or prosthodontic reasons were collected from the Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Cairo University.
Inclusion criteria:
  • Tooth type: Extracted human permanent teeth with a single root and a single canal.
  • Apical maturity: Fully formed apex with closed apical foramen.
  • Canal morphology: Straight canals with curvature <10° (Schneider), no visible canal bifurcations with canal morphology type I according to Vertucci classification.
  • Root length: Adequate length after decoronation to achieve a standardized 16 mm working segment.
  • Preoperative status: No prior endodontic treatment, posts, or restorations affecting the root structure.
  • Structural integrity: Intact roots without visible cracks, fractures, or external defects upon stereomicroscopic inspection.
  • Radiographic confirmation: No internal resorption, calcifications, or perforations.
Exclusion criteria:
- Anatomical complexity: More than one canal, isthmuses, canal bifurcation, or severe curvature ≥10°.
- Developmental or pathological changes: Open apex, internal/external resorption, extensive calcification, or root anomalies.
- Previous interventions: Any history of endodontic treatment, retreatment, post placement, or chemical canal modification.
- Structural compromise: Root cracks, fractures, craze lines, or caries extending to the root that compromise instrumentation or obturation.
Sample Preparation
Extracted human permanent single rooted teeth were collected from university clinics and private practices for orthodontic, prosthodontic and periodontal reasons. All teeth were deidentified prior to use, and ethical approval was obtained in accordance with institutional guidelines and the Declaration of Helsinki.
Cleaning and Storage
Soft tissue remnants were mechanically removed, and teeth were stored in saline solution at 4–8°C until use, before instrumentation, teeth were rinsed thoroughly with distilled water to eliminate residual saline
Standardization of Root Length
Crowns were decoronated using a diamond disc under water cooling to obtain a standardized root length of 16 mm, following the methodology of Olivieri et al. (2016).
Canal Patency and Working Length: A size 10 K file was introduced into the canal until visible at the apical foramen. The working length was established 1 mm short of the apical foramen, consistent with the approach of Cıkrık & Yusufoğlu (2025).
Initial Canal Preparation
• Canals will be prepared by the following sequence using rotary NiTi instruments with speed 350 RPM and torque 2.0 Ncm
(#20/.04taper)
(#25/.04taper)
(#30/.04taper)
(#35/.04taper)
(#40/.04taper)
• Irrigation was performed with 5.25% NaOCl during instrumentation, followed by a final rinse with 17% EDTA to remove the smear layer (Zehnder, 2006; Gandolfi et al., 2013).
Intervention:
• Application of Bio-C temp Intracanal medicament and leave it for 10 days inside the canals and put the tooth in controlled conditions to mimic intraoral environments while preventing dehydration, microbial contamination, or structural degradation, 0.1-2% thymol solution at room temperature is used.
Application of Nano Chitosan Irrigant by using different techniques to remove the Intracanal medicament
Group using Sodium hypochlorite irrigant with Conventional manual irrigation technique.
• After application of Bio-C Temp Intracanal medicament, irrigating the prepared teeth with sodium hypochlorite with manual irrigation with side vented needle for the removal of the Intracanal medicament till clear vision of the irrigant coming out of the canal.
Group using Sodium hypochlorite irrigant with Passive ultrasonic irrigation technique.
• After application of Bio-C Temp Intracanal medicament, Irrigating the prepared teeth with sodium hypochlorite for the removal of the Intracanal medicament till clear vision of the irrigant coming out of the canal while using ultrasonic tip in coronal third, activate at 25 kHz for 20s, move apically in 1 mm increments with continuous NaOCl flush (3 mL/min), repeat 3 cycles.
Group using Nano Chitosan irrigant with conventional manual irrigation technique.
• After application of Bio-C temp Intracanal medicament, irrigating the prepared teeth with Nano Chitosan irrigant for the removal of the medicament using side vented needle till clear vision of the irrigant coming out of the canal for what prescribed as conventional manual irrigation technique.
Group using Nano Chitosan irrigant with passive ultrasonic irrigation technique.
• After application of Bio-C temp Intracanal medicament, irrigating the prepared teeth with Nano Chitosan irrigant for the removal of the medicament using ultrasonic tip in coronal third, activate at 25 kHz for 20s, move apically in 1 mm increments with continuous NaOCl flush (3 mL/min), and repeat 3 cycles.
Outcome Measures
Primary outcome:
The primary outcome will be the percentage of canal wall area covered by Bio-C temp Intracanal medicament after irrigation of the prepared teeth.
SEM Analysis Methods
Following completion of the Intracanal medicament removal procedures, specimens will be prepared for scanning electron microscopy (SEM) to quantify the surface area of residual Intracanal medicament on the canal walls.
Specimen Sectioning
Roots will be externally grooved with a diamond disc and split longitudinally with a chisel to expose canal walls without altering the internal surface. Each specimen will be divided into coronal, middle, and apical thirds to standardize evaluation sites (Verdugo Balcázar & Carrillo Rengifo, 2025; Hülsmann et al., 1997).
Quantitative Image Analysis
Digital SEM micrographs will be imported into ImageJ software (NIH, Bethesda, USA). The canal wall boundary will be delineated to define the total area, and Intracanal medicament coverage will be segmented using consistent thresholding parameters. The residual area will be expressed as a percentage of the total canal wall area. Values will be averaged across fields for each canal third, and whole‑canal metrics will be calculated by combining thirds (Verdugo Balcázar & Carrillo Rengifo, 2025; Wilcox et al., 1987).
Secondary Outcome
The secondary outcome will be the time needed to remove the Intracanal medicament by observing clear fluid coming of the canal, time will be measured with (millilitre/second)
B) Assignment to intervention:

Sequence generation
   Randomization and sequence generation will be carried out using a computer based random sequence generator (https://www.random.org), where the samples (n = 40) will be randomly distributed into four groups (n = 10).
Allocation concealment
  To minimize selection bias in the intervention, the allocation sequence will be secured and concealed until assignment by placing the teeth in sequentially numbered opaque sealed envelopes.
 Implementation
 Random allocation, sequence generation, and allocation concealment will be conducted by the Co-supervisor.
C. Blinding
     To avoid detection bias, SEM images will be taken and analyzed via software by two trained, blinded observers who will be unaware of the allocation sequence, and they will generate the outcome reports. Both the outcome assessor and the statistician will be blinded to the group allocations.
Statistical Methods
     Data will be examined for normality using histograms and Shapiro Wilk test. Continuous data will be presented as mean, standard deviation, median and range values. Independent t test will be used for between-group comparison of parametric continuous data from non-related samples while paired t test will be used for related samples. Mann- Whitney U test will be used for between group comparisons of non-parametric continuous data from non-related samples while Wilcoxon signed rank test will be used for related samples. The level of significance will be set for all tests at p = 0.05.
 E. Ethics:
     The research will be admitted to the ethics committee for review. After receiving the results and finishing the experiment all instruments and teeth samples will be sterilized and discarded in special incinerator under the supervision of Microbiology Department- Cairo University.
Refrences
Abidin T, Susilo D, Gani BA. The effectiveness of nano-chitosan high molecular 0.2% as irrigant agent against Enterococcus faecalis with passive ultrasonic irrigant. J Conserv Dent. 2022;25(1):37-41.

Anbalagan K, Jena A, Mohanty S, Mallick R, Shashirekha G, Sarangi P. Smear layer removal and antimicrobial efficacy of chitosan as a root canal irrigant: a systematic review of in-vitro studies. Odontology. 2025;113(1):61-79

Ba-Hattab R, Al-Jamie M, Aldreib H, Alessa L, Alonazi M. Calcium Hydroxide in Endodontics: An Overview. Open J Stomatol. 2016;6(12):288-303.

Balto H, Barakat R, Basudan S, Fakeeha G, Alharbi SR, Almohareb R. Antibiofilm efficacy of a calcium silicate-based intracanal medicament against Fusobacterium nucleatum strains. Sci Rep. 2024;14(1):26679.

Borisova-Papancheva T, Svetlozarova S. Removal of calcium hydroxide medicament from root canal walls - a review of the different techniques. Scripta Sci Medica Dent. 2018;4(2):15-21.
Cakici F, Cakici EB. Antimicrobial efficacy of chitosan versus sodium hypochlorite: A systematic review and meta-analysis. Oral Dis. 2024;30(8):5445-5460.

Chong BS, Pitt Ford TR. The role of intracanal medication in root canal treatment. Int Endod J. 1992;25(2):97-106.

Cıkrık BN, Yusufoğlu Sİ. Efficacy of irrigation activation methods in removal of bioceramic-based sealer in retreatment. Odontology. 2025;113(3):1119-1128.

da Silva JM, Silveira A, Santos E, Prado L, Pessoa OF. Efficacy of sodium hypochlorite, ethylenediaminetetraacetic acid, citric acid and phosphoric acid in calcium hydroxide removal from the root canal: a microscopic cleanliness evaluation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2011;112(6):820-4.

Fallahi Sarvenoei VF, et al. Evaluating Calcium Hydroxide Removal Techniques in Endodontics: A Comparative Analysis. 2025.
Gandolfi MG, Taddei P, Modena E, Siboni F, Prati C. Biointeractivity and bioactivity properties of MTA cements: An in vitro study and critical reappraisal. J Appl Biomater Funct Mater. 2013;11(1-2):e39-e49.
Guerreiro JCM, Ochoa-Rodríguez VM, Rodrigues NNM, et al. Antibacterial activity, cytocompatibility, and effects on osteoblast differentiation of a calcium aluminate-based endodontic biomaterial. Int Endod J. 2021;54(12):2265-2277.

Hülsmann M, Hahn W, Hahn W. Ultrasonic root canal preparation: A review of the literature. Int Endod J. 1997;30(5):297-313.
Jaiswal N, Desai P, Shirnalli S. Evaluation of antibacterial efficacy of chitosan nanoparticles as an intracanal medicament against Enterococcus faecalis biofilm. J Int Soc Prev Community Dent. 2017;7(5):268-275.

Khairajani D, Borkar A, Aras S, et al. Comparative evaluation of four irrigation agitation techniques for removal of bioceramic intracanal medicament: An in vitro CBCT volumetric analysis. Research Square. 2025.

Kim SY, Kim SK. Root canal morphology of mandibular second molars in a Korean population using cone-beam computed tomography. Restor Dent Endod. 2015;40(2):161-167.
Lopes AB, Silva CD, Santos EF. Efficacy of intracanal medicaments in endodontics. J Endod. 2024;50(4):512-520.
Lopes CS, Delfino MM, Tanomaru-Filho M, Sasso-Cerri E, Guerreiro-Tanomaru JM, Cerri PS. Bioactive potential of Bio-C Temp demonstrated by systemic mineralization markers and immunoexpression of bone proteins in the rat connective tissue. J Mater Sci Mater Med. 2024;35(1):13.

Marion JJC, Manhães FRC, Bajo HT, Duque TM. Efficiency of different concentrations of sodium hypochlorite during endodontic treatment: Literature review. Dent Press Endod. 2012;2(1):40-47.
Mohammadi Z, Dummer PMH. Sodium hypochlorite in endodontics: An update review. Int Dent J. 2008;58(6):329-342.

Olivieri JG, Metzger Z, Camilleri J. Danger zone analysis using cone beam computed tomography: Apical enlargement with K3 and K3XF nickel-titanium rotary instruments. J Endod. 2016;42(3):433-439.

Ramos RF, De Sousa LB, Garcia PR, Rezende TMB. Properties of bioceramic materials for endodontic use. Braz J Oral Sci. 2025;24:e255144.

Sarıyılmaz Ö, Uslu G, Sarıyılmaz E, Keskin NB. Evaluation of different irrigation activation techniques for the removal of various medicaments from a simulated internal resorption cavity: an in vitro study. Clin Oral Investig. 2024;28(9):491.

Só GB, Villa N, Pereira JR, Kuga MC, Duarte MAH, Da Rosa RA, Só MVR. Three different removal protocols for a bioceramic and a calcium hydroxide dressings: A microcomputed tomography study. J Conserv Dent Endod. 2025;28(9):899-904.

Suleiman HT, El-Kateb NM, Aboelseoud MR, Sherif RA. Removal of calcium hydroxide intracanal medicament with four different techniques: a cone beam computed tomography in vitro study. Journal/Preprint Source. 2022.
Wang N, Li MJ, Han L, Zhang Y. Antibacterial effect of chitosan and its derivative on Enterococcus faecalis associated with endodontic infection. Exp Ther Med. 2020;19(6):3765-3773.

Waz SM, Moukarab D. Efficacy of chitosan as a post space irrigant on bond strength of glass fiber posts luted with self-adhesive resin cements. Egypt Dent J. 2020;66(2):923-932.

Wilcox LR, Van Tine D. Endodontic working length determined with an electronic device: An evaluation with high-resolution radiographs. J Endod. 1987;13(8):373-377.

Zehnder M. Root canal irrigants. J Endod. 2006;32(5):389-398.
Protocol references
Chong and Pitt Ford, 1992; Ba-Hattab et al., 2016; Kim 26 Kim, 2015; Almohareb et al. 2024; Só et al., 2025; Lopes et al., 2024; Suleiman et al., 2022; Silva et al., 2011; Anbalagan et al., 2025; Abidin et al., 2022; Fallahi Sarvenoei et al., 2025; Borisova-Papantcheva and Svetlozarova, 2018; Só et al., 2025; Khairajani et al., 2025; Guerreiro et al., 2021; Balto et al., 2024; Saryılmaz et al., 2024; Mohammadi 6 Dummer, 2008.
Acknowledgements
No financial or material support in this study. The study is entirely funded by the main researcher (Self-funding). Protocol registration on Nature. Version identifier: This is the first version of the protocol. Roles and responsibilities: Investigator: Mohamed Osama El-sayed Ammar - Main researcher, data enterer, and research writer. Main Supervisor: Prof. Dr. Alaa Abdelsalam Elbaz - Monitoring and editing the data, and the entire process of the study. Access the final results and data collected from the study. Assistant Supervisor: Dr. Ghada Ihab ElWazan - Monitor the process of data collection. Generate a random sequence of the sample and undergo proper allocation concealment, implantation. Access to the final data.