Evaluation of the antibacterial effect of chitosan, cold atmospheric plasma and sodium hypochlorite on Enterococcus faecalis in mandibular second primary molars: An in vitro study

Evaluation of the antibacterial effect of chitosan, cold atmospheric plasma and sodium hypochlorite on Enterococcus faecalis in mandibular second primary molars: An in vitro study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of the antibacterial effect of chitosan, cold atmospheric plasma and sodium hypochlorite on Enterococcus faecalis in mandibular second primary molars: An in vitro study Arshia Mansouri, Maryam Pourhossein, Camellia Kianbakht This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8436881/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Persistent microbial infection is the primary cause of pulpectomy failure in primary teeth. Enterococcus faecalis is among the most resistant microorganisms associated with endodontic treatment failure. Conventional irrigants, such as sodium hypochlorite, are effective; however, their clinical use is associated with several limitations in pediatric dentistry, prompting the investigation of alternative agents such as chitosan and cold atmospheric plasma (CAP). This study aimed to evaluate and compare the antibacterial effects of chitosan, cold atmospheric plasma, and sodium hypochlorite against Enterococcus faecalis in mandibular second primary molars. Methods In this in vitro study 33 deciduous second molars with at least two-thirds of their root length were prepared by chemomechanical method. Enterococcus faecalis inoculum suspension was inoculated into the canals of each tooth and sampling was done to ensure biofilm formation. For disinfection of the canals, the teeth were divided into 3 groups: sodium hypochlorite, cold atmospheric plasma and chitosan. After applying disinfection methods, secondary sampling of the contents inside the canal was performed and the secondary CFU was counted, and finally the primary and secondary CFU levels were compared. The data were analyzed using one-way ANOVA statistical test, considering α = 0.05. Results The values ​​(mean ± standard deviation) of the primary and secondary CFU logarithms in the sodium hypochlorite (Hypo) group were (7.45 ± 0.08) and (5.89 ± 0.02), respectively, in the chitosan group (Chitosan) were (7.58 ± 0.04) and (5.89 ± 0.03), respectively, and in the cold atmospheric plasma (CAP) group were (7.56 ± 0.05) and (5.9 ± 0.03), respectively. The CFU level in the Hypo group was significantly lower than that of the Chitosan and CAP groups (p = 0.001 and p = 0.003, respectively). However, there was no statistically significant difference between the Chitosan and CAP groups (p = 0.641). Although statistically significant differences were observed among the groups, the mean percentage bacterial reduction did not differ significantly and was not considered clinically relevant. Conclusion There is no clinical difference in the antibacterial activity of chitosan, cold atmospheric plasma, and sodium hypochlorite on Enterococcus faecalis in mandibular second primary molars, These findings suggest that chitosan and cold atmospheric plasma may serve as potential alternatives to sodium hypochlorite for root canal disinfection in primary teeth. Keywords : Chitosan; cold atmospheric plasma; Enterococcus faecalis; sodium hypochlorite; primary teeth. Chitosan cold atmospheric plasma Enterococcus faecalis sodium hypochlorite primary teeth Figures Figure 1 Figure 2 Introduction Successful endodontic treatment in primary teeth depends primarily on the effective elimination of microorganisms from the root canal system. Despite advances in instrumentation techniques and irrigation protocols, Persistent microbial infection is widely recognized as a major cause of failure in pulpectomy procedures. The unique anatomical characteristics of primary molars—including ribbon-shaped canals, accessory canals, apical ramifications, and physiological root resorption—significantly limit the effectiveness of mechanical instrumentation and facilitate bacterial persistence within the canal system [ 1 , 2 , 3 ]. Microorganisms associated with endodontic infections are diverse; however, Enterococcus faecalis has been repeatedly identified as one of the most resistant and clinically significant species in persistent and secondary root canal infections. This bacterium is frequently isolated from failed endodontic treatments and exhibits several virulence factors that contribute to its survival, including the ability to tolerate extreme pH levels, survive prolonged nutrient deprivation, penetrate deep into dentinal tubules, and form structured biofilms [ 4 , 5 , 6 , 7 , 8 ]. Biofilm formation is particularly relevant, as biofilm-associated bacteria demonstrate markedly increased resistance to antimicrobial agents compared with planktonic cells [ 3 ]. Sodium hypochlorite has long been regarded as the gold standard root canal irrigant due to its broad-spectrum antimicrobial activity and unique ability to dissolve organic tissue. Its mechanism of action involves the release of hypochlorous acid and chlorine ions, which lead to irreversible oxidation of bacterial enzymes, degradation of cell membranes, and disruption of essential metabolic pathways [ 9 ]. Numerous in vitro and clinical studies have demonstrated the effectiveness of sodium hypochlorite against Enterococcus faecalis and other endodontic pathogens, even in the presence of organic debris and biofilm structures [ 10 , 11 ]. Despite its well-documented antimicrobial efficacy, the use of sodium hypochlorite is associated with several limitations. Its cytotoxic effects on periapical tissues, unpleasant taste and odor, potential for allergic reactions, and risk of accidental extrusion beyond the apex raise significant clinical concerns [ 10 , 12 ]. In primary teeth, these concerns are amplified due to the presence of physiologic root resorption, thinner dentinal walls, and the close proximity of developing permanent tooth buds. Additionally, sodium hypochlorite has been reported to alter the mechanical properties of dentin by reducing microhardness and elastic modulus, which may compromise the structural integrity of primary teeth [ 12 ]. These limitations highlight the need to investigate alternative disinfection strategies with improved safety profiles, particularly in pediatric dentistry. Chitosan is a naturally occurring polysaccharide obtained through the deacetylation of chitin and has attracted increasing attention in dentistry due to its antimicrobial, antifungal, antibiofilm, and biocompatible properties [ 13 , 14 , 15 , 16 , 17 ]. The antimicrobial activity of chitosan is primarily attributed to its polycationic nature, which enables electrostatic interactions with the negatively charged components of bacterial cell walls. This interaction results in increased membrane permeability, leakage of intracellular contents, and inhibition of bacterial enzyme activity [ 17 , 18 , 19 ]. Several studies have demonstrated the effectiveness of chitosan against Enterococcus faecalis in both planktonic and biofilm forms, suggesting its potential application as an intracanal disinfectant [ 11 , 15 ]. In addition to its direct antibacterial effects, chitosan has been shown to interfere with biofilm formation and maturation by disrupting extracellular polymeric substances and inhibiting bacterial adhesion to dentinal surfaces [ 20 ]. Moreover, chitosan has been reported to exhibit chelating properties and favorable interactions with dentin, which may enhance its penetration into dentinal tubules and contribute to sustained antimicrobial activity [ 18 ]. Cold atmospheric plasma (CAP) has emerged as a novel non-thermal antimicrobial technology with growing applications in dentistry and medicine. Plasma is an ionized gas composed of reactive oxygen and nitrogen species, electrons, ions, and ultraviolet photons, all of which contribute to its antimicrobial effects [ 21 , 22 ]. Unlike thermal plasma, cold atmospheric plasma operates at near-room temperature, allowing its application to biological tissues without causing thermal damage. The antimicrobial mechanism of cold atmospheric plasma involves oxidative damage to bacterial cell walls, lipid peroxidation, protein denaturation, and DNA fragmentation, ultimately leading to microbial cell death [ 23 , 24 , 25 ]. Previous studies have demonstrated the effectiveness of cold atmospheric plasma against a wide range of oral microorganisms, including Enterococcus faecalis, even within biofilm structures [ 26 , 27 ]. One of the advantages of cold atmospheric plasma is its ability to penetrate complex anatomical structures, such as dentinal tubules and irregular canal spaces, which are difficult to access using conventional irrigation methods. Additionally, plasma treatment does not rely on chemical diffusion, reducing the risk of cytotoxic effects on surrounding tissues. These properties suggest that cold atmospheric plasma may represent a valuable adjunct or alternative approach for root canal disinfection, particularly in primary teeth with complex morphology. Therefore, the aim of this in vitro study was to evaluate and compare the antibacterial effects of chitosan, cold atmospheric plasma, and sodium hypochlorite against Enterococcus faecalis in mandibular second primary molars. Methods This in vitro experimental study was conducted using extracted mandibular second primary molars at the Faculty of Dentistry, Islamic Azad University, Tehran, Iran. Based on the results reported by Armand et al. [ 1 ], sample size calculation was performed using one-way ANOVA power analysis in PASS 11 software, considering a significance level of α = 0.05 and a statistical power of 80% (β = 0.2). Assuming a mean standard deviation of the logarithmic colony count equal to 1.00 and an effect size of 0.55, the minimum required sample size was calculated as 10 samples per group for each of the three study groups. In addition, one extra sample per group (three samples) was allocated for determining the baseline bacterial count inside the root canals. A total of 33 extracted mandibular second primary molars, meeting the inclusion and exclusion criteria, were selected for the study (Table 1 ). The teeth were randomly assigned to the experimental groups using simple randomization, with the aid of a random number table, by an independent operator. Table 1 Inclusion and exclusion criteria Inclusion criteria Exclusion criteria Extracted mandibular second primary molars with at least two-thirds of root length remaining Presence of internal or external physiological or pathological root resorption (based on radiographic evaluation) History of previous pulpotomy or pulpectomy Perforation of the pulpal floor (based on radiographic evaluation) Debris present on the tooth surfaces was removed using a No. 15 surgical scalpel blade (Scalpel blade, ATP, Trinon Co., Germany). The teeth were then cleaned using a brush attached to a low-speed handpiece under water irrigation. Following cleaning, the samples were rinsed with normal saline (Normal saline, Shiraz Serum Co., Iran) and stored in 0.5% thymol solution (Thymol, Sigma, Iran) for one week. Subsequently, until the time of experimentation, the teeth were kept in distilled water at 4°C. All roots were sectioned at the level of the cemento-enamel junction (CEJ) using a diamond disc mounted on a high-speed handpiece. The root canals were initially prepared using size 15 and 20 K-files (Mani Co., Japan) and subsequently instrumented with size 25 and 30 rotary files of the Denco system (Denco, Shenzhen, China) with a 4% taper, to a working length 1 mm short of the radiographic apex. During canal preparation, irrigation was performed using 5 mL of normal saline (Normal saline, Shiraz Serum Co., Iran). A 30-gauge irrigation syringe with a side-vented, closed-end needle was used for canal irrigation. To remove the smear layer and maintain the patency of the dentinal tubules for bacterial penetration, the canals were irrigated sequentially with 2 mL of 17% EDTA (Asia Chemi Teb Co., Tehran, Iran) for 1 minute, followed by 2 mL of 2.5% sodium hypochlorite (Nik Darman Co., Iran) for 1 minute, and finally flushed with 5 mL of normal saline (Shiraz Serum Co., Iran). To prevent apical leakage, the apical foramen of each tooth was sealed using flowable composite resin A2 shade (Denfil, Vericom Co., South Korea). The samples were then placed in autoclavable microtubes containing Brain Heart Infusion (BHI) broth and sterilized in an autoclave at 121°C and 15 psi for 30 minutes. To confirm sterility, the samples were incubated at 37°C for 48 hours. In cases where turbidity was observed in the culture medium, the sterilization process was repeated (Fig. 1 ) . iii II: Teeth sectioned adjacent to CEJ, III: a = Hand filing b = Rotary filing c = Irrigation d = Apical sealing using flowable composite An inoculum suspension of Enterococcus faecalis was obtained from the Iranian Research Organization for Science and Technology and prepared at a concentration of 1.5 × 10⁸ CFU/mL, equivalent to 0.5 McFarland standard. Enterococcus faecalis was cultured on the appropriate culture medium and incubated at 37°C with CO₂ for 48 hours. Fifteen microliters of the E. faecalis inoculum suspension were introduced into the root canal of each tooth using a sampler for 30 seconds. The samples were then incubated in BHI culture medium at 37°C, with continuous agitation at 150 rpm, for three weeks. To ensure bacterial viability and biofilm formation, 15 µL of fresh BHI broth (BHI, Merck Co., Germany) was injected daily into each canal. To confirm biofilm formation, baseline sampling was performed. In each sample, biofilm disruption was achieved using a vortex mixer (Vortex Mixer, KST, Iran) at 2500 rpm for 1 minute. The canal contents were transferred into vials containing 0.9 mL phosphate-buffered saline (PBS). The suspension was then cultured on agar plates and incubated at 37°C for 96 hours, after which the baseline colony-forming units (CFU) were counted. These values served as the reference for comparison with post-treatment CFU values. The samples were randomly assigned using a random number table into three groups, and all disinfection procedures were performed by a single trained operator. Group 1: Sodium Hypochlorite Root canals were irrigated with 2 mL of 2.5% sodium hypochlorite (Nik Darman Co., Iran) for 1 minute. To neutralize the residual sodium hypochlorite prior to bacterial sampling, 3 mL of 5% sodium thiosulfate (Sodium Thiosulfate, Tamadkala, Iran) was applied into the canals for 1 minute. Final irrigation was performed using normal saline (Shiraz Serum Co., Iran). Group 2: Cold Atmospheric Plasma Cold atmospheric plasma was applied using a helium-based cold plasma device (Nariatech Plasmart Co., Iran) with an average frequency of 50 Hz, input power of 55 W, gas flow rate of 3 L/min, and intensity level 4. Cold atmospheric plasma was applied for 60 seconds, with the nozzle positioned at a distance of 5 mm from the canal orifice. Group 3: Chitosan A 2% chitosan solution was used as an intracanal dressing. The solution was injected into the canal using a syringe with a 30-gauge needle, positioned 2 mm short of the apex, and left in the canal for 3 minutes prior to final irrigation. Chitosan was supplied in powder form. To prepare a 2% chitosan solution, 2 g of chitosan powder (Aprinatd, Tehran, Iran) was dissolved in 100 mL of 1% (0.1 M) acetic acid. The mixture was placed on a magnetic stirrer (Domel, Domel Co., Slovenia) at 37°C and stirred at 400 rpm for 24 hours until a homogeneous 2% chitosan solution was obtained. After application of the disinfection protocols, all samples were irrigated with 1 mL of normal saline (Shiraz Serum Co., Iran). Sterile paper points were then used to remove excess saline and planktonic bacteria, while minimizing disruption of the adherent biofilm. For secondary sampling, the samples were subjected to vibration using a vortex mixer for 30 seconds to release any remaining biofilm within the canals. Secondary samples were collected from the canal contents, cultured on agar plates, and incubated at 37°C for 48 hours. The secondary CFU values were counted and compared with the baseline CFU values. All collected data were coded and entered into SPSS software version 24. Data analysis was performed using one-way ANOVA, and the level of statistical significance was set at P < 0.05. Results The antibacterial effects of sodium hypochlorite, chitosan, and cold atmospheric plasma against Enterococcus faecalis in mandibular second primary molars are presented below. The number of viable bacteria was quantified by counting colony-forming units (CFU/mL). For statistical analysis, CFU values were log-transformed, and all analyses were performed using the logarithmic data. According to the results presented in Table 2 , the mean ± standard deviation of the logarithmic primary and secondary CFU values in the sodium hypochlorite group were 7.45 ± 0.08 and 5.89 ± 0.02, respectively. In the chitosan group, these values were 7.58 ± 0.04 and 5.89 ± 0.03, respectively, while in the cold atmospheric plasma group they were 7.56 ± 0.05 and 5.90 ± 0.03, respectively. All three disinfection methods resulted in a marked reduction in bacterial counts following intervention. Table 2 Log-transformed colony-forming unit (CFU) counts of Enterococcus faecalis before and after intervention Repeated-measures ANOVA demonstrated a statistically significant reduction in bacterial counts after treatment in all groups (P < 0.001). Intergroup comparisons showed that the reduction in bacterial load achieved with sodium hypochlorite was significantly greater than that observed with chitosan (P = 0.001) and cold atmospheric plasma (P = 0.003). However, no statistically significant difference was found between the chitosan and cold atmospheric plasma groups (P = 0.641). The results of pairwise comparisons are summarized in Table 3 . Group (n = 10) Before intervention (Mean ± SD) After intervention (Mean ± SD) Sodium hypochlorite (2.5%) 7.45 ± 0.08 5.89 ± 0.02 Chitosan (2%) 7.58 ± 0.04 5.89 ± 0.03 Cold atmospheric plasma (CAP) 7.56 ± 0.05 5.90 ± 0.03 Table 3 Pairwise comparison of antibacterial efficacy among the study groups based on log-transformed CFU reduction. The clinical relevance of antibacterial efficacy among the study groups is presented in Table 4 and Fig. 2 . Comparison Mean difference (log CFU) Standard error P value Sodium hypochlorite vs Chitosan -0.064 0.017 0.001 Sodium hypochlorite vs CAP -0.056 0.017 0.003 Chitosan vs CAP 0.008 0.017 0.641 Table 4 Clinical relevance of antibacterial efficacy among study groups Group Mean percentage bacterial reduction Sodium hypochlorite 97.20 Cold atmospheric plasma 97.79 Chitosan 97.93 P value > 0.05 Discussion The present in vitro study evaluated the antibacterial efficacy of sodium hypochlorite, chitosan, and cold atmospheric plasma against Enterococcus faecalis in primary molar root canals. The findings reported by Hee-Eun Kim et al. (2024) are consistent with the results of the present study, which also demonstrated a substantial antibacterial effect of cold atmospheric plasma against Enterococcus faecalis. Kim et al. showed that the antibacterial efficacy of cold atmospheric plasma was maintained regardless of biofilm maturity, underscoring its ability to penetrate and disrupt complex biofilm structures. Similarly, in the present study, cold atmospheric plasma produced a significant reduction in bacterial counts, comparable to that achieved with conventional chemical disinfectants. Although sodium hypochlorite exhibited a statistically greater antibacterial effect, cold atmospheric plasma demonstrated a level of bacterial reduction that was not clinically different from the other tested modalities. Taken together, these findings support the potential role of cold atmospheric plasma as an effective adjunctive disinfection method, particularly in clinical situations involving resistant or mature biofilms where conventional irrigants may have limited penetration [ 23 ]. The results of Ibrahim et al. (2023) further support the antibacterial potential of cold atmospheric plasma observed in the present study. In their in vitro investigation, argon-based cold atmospheric plasma significantly reduced colony-forming units of both Streptococcus mutans and Candida albicans at all tested exposure times, demonstrating broad-spectrum antimicrobial activity. These findings align with the results of the present study, in which cold atmospheric plasma produced a substantial reduction in Enterococcus faecalis counts within infected root canals. Although Ibrahim et al. reported a greater susceptibility of Candida albicans compared with Streptococcus mutans, both studies collectively highlight the effectiveness of cold atmospheric plasma against microorganisms known for their resistance within oral biofilms. The consistency between these results suggests that the antimicrobial action of cold atmospheric plasma is not limited to a single species or experimental setup, supporting its potential application as an adjunctive disinfection method in endodontic treatment, particularly in cases involving resistant or biofilm-associated microorganisms [ 25 ]. The results reported by Kumar et al. (2023) partially contrast with the findings of the present study. In their investigation, 5.25% sodium hypochlorite demonstrated significantly greater antibacterial efficacy against Enterococcus faecalis than cold atmospheric plasma across all tested time intervals, leading the authors to recommend sodium hypochlorite as the most effective disinfection method. In the present study, although sodium hypochlorite also exhibited a statistically greater reduction in bacterial counts compared with cold atmospheric plasma, this difference was not considered clinically significant. This discrepancy may be attributed to differences in experimental design, including variations in plasma device parameters, exposure time, canal anatomy, and evaluation methods. Notably, Kumar et al. reported that cold atmospheric plasma achieved a significant reduction in bacterial load after a minimum exposure time of 5 minutes, which is consistent with the substantial antibacterial effect observed in the present study. Therefore, despite differences in the magnitude of antibacterial efficacy reported, both studies support the potential role of cold atmospheric plasma as an effective adjunctive disinfection method, particularly in situations where conventional irrigants may be limited by penetration depth or cytotoxicity concerns [ 24 ]. The findings of Bushra M et al. (2022) are largely consistent with the results of the present study, while also highlighting important differences related to formulation and contact time. Bushra et al. demonstrated that a combination of chitosan and propolis exhibited superior antibacterial activity against Enterococcus faecalis during the early phase of treatment; however, this advantage was not maintained at later time points, where its efficacy became comparable to that of calcium hydroxide. Similarly, in the present study, chitosan showed a significant antibacterial effect against Enterococcus faecalis but did not demonstrate a clinically superior outcome when compared with other disinfection modalities. The observed differences between the studies may be attributed to the use of a chitosan–propolis combination rather than chitosan alone, as well as differences in exposure duration and evaluation time points. Collectively, these findings suggest that while chitosan possesses notable antibacterial properties, its clinical effectiveness may be influenced by formulation, adjunctive agents, and duration of application [ 28 ]. The findings reported by Asnaashari et al. (2022) are generally in agreement with the results of the present study, while also revealing a relative difference in the magnitude of antibacterial efficacy among treatment modalities. In their laboratory investigation, cold atmospheric plasma significantly reduced Enterococcus faecalis biofilm compared with control groups, confirming its notable antimicrobial potential. This observation is consistent with the present study, in which cold atmospheric plasma produced a substantial reduction in bacterial counts within infected root canals. However, Asnaashari et al. reported that triple antibiotic paste achieved the greatest reduction in CFU values, surpassing the antibacterial effect of cold atmospheric plasma. In contrast, the present study demonstrated that although sodium hypochlorite showed a statistically greater antibacterial effect, the reduction achieved by cold atmospheric plasma was not clinically inferior to that of the other tested disinfection methods. The discrepancy between these findings may be attributed to differences in experimental conditions, including the use of a prolonged plasma exposure time, the presence of a mature 21-day biofilm model, and comparison with intracanal medicaments rather than irrigants. Collectively, both studies support the effectiveness of cold atmospheric plasma in reducing E. faecalis biofilms, while suggesting that its relative performance may vary depending on the comparator material and experimental design [ 26 ]. The findings reported by Wang et al. (2020) are largely in agreement with the results of the present study regarding the antibacterial efficacy of chitosan against Enterococcus faecalis. Wang et al. demonstrated that chitosan, particularly when dissolved in double-distilled water, exhibited strong bactericidal activity against both planktonic and biofilm forms of E. faecalis, with a low minimum bactericidal concentration and no detectable cytotoxicity to MC3T3-E1 pre-osteoblast cells. These observations are consistent with the present study, in which chitosan produced a significant reduction in E. faecalis counts within infected root canals. However, while Wang et al. emphasized the pronounced antibacterial activity of chitosan at specific concentrations under controlled laboratory conditions, the present study found that chitosan did not demonstrate a clinically superior antibacterial effect when compared with sodium hypochlorite or cold atmospheric plasma. This difference may be attributed to variations in experimental models, including concentration-dependent testing, solvent effects, and the use of isolated planktonic and biofilm systems versus a standardized root canal model. Nevertheless, both studies support the conclusion that chitosan possesses notable antibacterial properties against E. faecalis with favorable biocompatibility, reinforcing its potential role as a safe and effective alternative or adjunctive agent for root canal disinfection [ 17 ]. This study has several limitations. As an in vitro investigation, the experimental conditions do not fully replicate the complex biological environment of the oral cavity. In addition, the antibacterial efficacy was evaluated against a single microbial species, which may not reflect the polymicrobial nature of endodontic infections. Therefore, the results should be interpreted with caution. Conclusion Based on the findings of the present study and their comparison with similar investigations, no clinically significant difference was observed in the antibacterial efficacy of chitosan, cold atmospheric plasma, and sodium hypochlorite against Enterococcus faecalis in mandibular second primary molars. Accordingly, chitosan and cold atmospheric plasma may be considered potential alternatives to sodium hypochlorite for root canal disinfection. Future studies with larger sample sizes and longer evaluation periods are recommended. In addition, investigations assessing different concentrations of chitosan and sodium hypochlorite, as well as studies involving different bacterial species or multiple microorganisms simultaneously, should be conducted. Furthermore, the design of clinical trial studies is recommended to allow better generalization of the findings. Declarations Human Ethics and Consent to Participate This in vitro study was conducted on extracted human teeth and was approved by the Ethics Committee of the Faculty of Dentistry, Islamic Azad University, Tehran, Iran (Approval No. IR.IAU.DENTAL.REC.1403.167). All procedures were conducted in accordance with the Declaration of Helsinki. Informed consent to participate was waived by the ethics committee as the study used anonymized extracted teeth for laboratory research only. Consent to Publish Not applicable. Competing Interests The authors declare that they have no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution Camellia Kianbakht and Maryam Pourhossein conceived and designed the study and performed the experiments. Arshia Mansouri analyzed the data, and drafted the manuscript. All authors critically revised the manuscript and approved the final version. Acknowledgements Not applicable. Availability of Data and Materials Data sharing is not applicable to this article as no datasets were generated or analysed during the current study. References Armand A, Khani M, Asnaashari M, AliAhmadi A, Shokri B. Comparison study of root canal disinfection by cold plasma jet and photodynamic therapy. Photodiagnosis Photodyn Ther. 2019;26:327–33. Cancio V, Carvalho Ferreira D, Cavalcante FS, Rosado AS, Teixeira LM, Braga Oliveira Q, et al. Can the Enterococcus faecalis identified in the root canals of primary teeth be a cause of failure of endodontic treatment? Acta Odontol Scand. 2017;75(6):423–8. Sakko M, Tjäderhane L, Rautemaa-Richardson R. Microbiology of Root Canal Infections. Prim Dent J. 2016;5(2):84–9. Alghamdi F, Shakir M. The Influence of Enterococcus faecalis as a Dental Root Canal Pathogen on Endodontic Treatment: A Systematic Review. Cureus. 2020;12(3):e7257. Guguloth S, Jampanapalli SR, Patloth T, Suhasini K, Ingua HC, Shaik H. Evaluation of Chitosan and Ferric Sulphate as Pulpotomy Agents in Primary Teeth: A Randomized Controlled Trial. Int J Clin Pediatr Dent. 2023;16(2):223–6. Pourhajibagher M, Ghorbanzadeh R, Bahador A. Culture-dependent approaches to explore the prevalence of root canal pathogens from endodontic infections. Braz Oral Res. 2017;31:e108. Radeva E, Karayasheva D. Importance of Enterococci (Enterococcus faecalis) for Dental Medicine - Microbiological Characterization, Prevalence and Resistance. Int J Sci Res (IJSR). 2017;6. Singh H. Microbiology of Endodontic Infections. 2016. Estrela C, Estrela CR, Barbin EL, Spanó JC, Marchesan MA, Pécora JD. Mechanism of action of sodium hypochlorite. Braz Dent J. 2002;13(2):113–7. Berber VB, Gomes BP, Sena NT, Vianna ME, Ferraz CC, Zaia AA, Souza-Filho FJ. Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubules. Int Endod J. 2006;39(1):10–7. Goel P, Galhotra V, Makkar S, Mohan J, Bala N, Kaur T. An In Vitro Study Comparing the Antimicrobial Efficacy of 0.2% Chitosan, 3% Sodium Hypochlorite, 2% Chlorhexidine against Enterococcus faecalis, Alone and in Conjunction with Diode Laser. Int J Clin Pediatr Dent. 2022;15(1):109–14. Zhang K, Kim YK, Cadenaro M, Bryan TE, Sidow SJ, Loushine RJ, et al. Effects of different exposure times and concentrations of sodium hypochlorite/ethylenediaminetetraacetic acid on the structural integrity of mineralized dentin. J Endod. 2010;36(1):105–9. Fan W, Yan W, Xu Z, Ni H. Erythrocytes load of low molecular weight chitosan nanoparticles as a potential vascular drug delivery system. Colloids Surf B Biointerfaces. 2012;95:258–65. Pu Y, Liu A, Zheng Y, Ye B. In vitro damage of Candida albicans biofilms by chitosan. Exp Ther Med. 2014;8(3):929–34. Supotngarmkul A, Panichuttra A, Ratisoontorn C, Nawachinda M, Matangkasombut O. Antibacterial property of chitosan against E. faecalis standard strain and clinical isolates. Dent Mater J. 2020;39(3):456–63. Thienngern P, Panichuttra A, Ratisoontorn C, Aumnate C, Matangkasombut O. Efficacy of chitosan paste as intracanal medication against Enterococcus faecalis and Candida albicans biofilm compared with calcium hydroxide in an in vitro root canal infection model. BMC Oral Health. 2022;22(1):354. Wang N, Ji Y, Zhu Y, Wu X, Mei L, Zhang H, et al. Antibacterial effect of chitosan and its derivative on Enterococcus faecalis associated with endodontic infection. Exp Ther Med. 2020;19(6):3805–13. Silva PV, Guedes DF, Pécora JD, da Cruz-Filho AM. Time-dependent effects of chitosan on dentin structures. Braz Dent J. 2012;23(4):357–61. Tanikonda R, Ravi RK, Sirisha K, Divella S. Chitosan: Applications in dentistry. Trends Biomaterials Artif Organs. 2014;28:74–8. Husain S, Al-Samadani KH, Najeeb S, Zafar MS, Khurshid Z, Zohaib S, Qasim SB. Chitosan Biomaterials for Current and Potential Dental Applications. Mater (Basel). 2017;10(6). Asnaashari M, Motamedi S, Asnaashari N, Azari-Marhabi S. Antimicrobial Activity of Cold Plasma Treatment on Acrylic Denture Bases: An In Vitro Evaluation. J Lasers Med Sci. 2019;10(Suppl 1):S13–7. Hoffmann C, Berganza C, Zhang J. Cold Atmospheric Plasma: methods of production and application in dentistry and oncology. Med Gas Res. 2013;3(1):21. Kim HE. Influence of Biofilm Maturity on the Antibacterial Efficacy of Cold Atmospheric Plasma in Oral Microcosm Biofilms. Biomedicines. 2024;12(5). Kumar P, Soundharrajan P, Prakash R, Kombade SP, Yadav P, Chugh A, Patnana AK. An in-vitro analysis to evaluate the disinfection effectiveness of Cold Atmospheric Pressure (CAP) plasma jet in Enterococcus faecalis infected root canals. Biomater Investig Dent. 2023;10(1):2193214. Salam G, Maha I, Abbas M, Ali E, Al-Rubaee E. The antimicrobial effects of Cold Atmospheric Plasma jet on microorganisms causing dental caries (in vitro study). Mustansiria Dent J. 2023;19:6–2023. Asnaashari M, Mehrabinia P, Yadegari Z, Hoseini H, Sadafi M, Shojaeian S. Evaluation of Antibacterial Effects of Cold Atmospheric Plasma, Calcium Hydroxide, and Triple Antibiotic Paste on Enterococcus faecalis Biofilm in the Root Canal System: An In Vitro Study. J Lasers Med Sci. 2022;13:e50. Theinkom F, Singer L, Cieplik F, Cantzler S, Weilemann H, Cantzler M, et al. Antibacterial efficacy of cold atmospheric plasma against Enterococcus faecalis planktonic cultures and biofilms in vitro. PLoS ONE. 2019;14(11):e0223925. Shamma B, Abo-Arraj E, Rajab A, Al Kurdi S. Anti-bActeriAl Activity of Applying chitosAn And propolis dressing AgAinst EntErococcus faEcalis in primAry teeth: in vitro study. J Stomatology. 2022;75:36–43. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 02 Feb, 2026 Reviewers agreed at journal 29 Jan, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviewers agreed at journal 23 Jan, 2026 Reviewers invited by journal 23 Jan, 2026 Editor assigned by journal 22 Jan, 2026 Editor invited by journal 02 Jan, 2026 Submission checks completed at journal 01 Jan, 2026 First submitted to journal 01 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8436881","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":580778443,"identity":"7fb11794-f799-4bd5-98c2-ace1a46b7fc5","order_by":0,"name":"Arshia Mansouri","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Arshia","middleName":"","lastName":"Mansouri","suffix":""},{"id":580778446,"identity":"7c474083-d2cf-4c14-bdc7-6a0cfe524057","order_by":1,"name":"Maryam Pourhossein","email":"","orcid":"","institution":"Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Pourhossein","suffix":""},{"id":580778448,"identity":"2b6ae693-036d-4380-b8b9-578e5c34ad0b","order_by":2,"name":"Camellia Kianbakht","email":"data:image/png;base64,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","orcid":"","institution":"Islamic Azad University","correspondingAuthor":true,"prefix":"","firstName":"Camellia","middleName":"","lastName":"Kianbakht","suffix":""}],"badges":[],"createdAt":"2025-12-23 20:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8436881/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8436881/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101362836,"identity":"65acf961-2d8b-4505-ae80-e1516c0b387a","added_by":"auto","created_at":"2026-01-29 00:32:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114895,"visible":true,"origin":"","legend":"\u003cp\u003eSample preparation I: A schematic illustration of sample preparation protocol\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8436881/v1/306d5876b0738f51a2c602f0.jpg"},{"id":101362837,"identity":"525c3c4e-7753-4cfd-a96d-a8d164e9fd3e","added_by":"auto","created_at":"2026-01-29 00:32:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9970,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the mean percentage reduction of Enterococcus faecalis among sodium hypochlorite, chitosan, and cold atmospheric plasma groups following intervention.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8436881/v1/31b0113bb3efaceb2d59492b.jpg"},{"id":101362839,"identity":"40e9eed5-2e8f-4bdf-a497-654bd7e09454","added_by":"auto","created_at":"2026-01-29 00:32:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":674691,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8436881/v1/aec5ca90-d0ec-4528-b12f-9e32f7f663df.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of the antibacterial effect of chitosan, cold atmospheric plasma and sodium hypochlorite on Enterococcus faecalis in mandibular second primary molars: An in vitro study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSuccessful endodontic treatment in primary teeth depends primarily on the effective elimination of microorganisms from the root canal system. Despite advances in instrumentation techniques and irrigation protocols, Persistent microbial infection is widely recognized as a major cause of failure in pulpectomy procedures. The unique anatomical characteristics of primary molars\u0026mdash;including ribbon-shaped canals, accessory canals, apical ramifications, and physiological root resorption\u0026mdash;significantly limit the effectiveness of mechanical instrumentation and facilitate bacterial persistence within the canal system [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicroorganisms associated with endodontic infections are diverse; however, Enterococcus faecalis has been repeatedly identified as one of the most resistant and clinically significant species in persistent and secondary root canal infections. This bacterium is frequently isolated from failed endodontic treatments and exhibits several virulence factors that contribute to its survival, including the ability to tolerate extreme pH levels, survive prolonged nutrient deprivation, penetrate deep into dentinal tubules, and form structured biofilms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Biofilm formation is particularly relevant, as biofilm-associated bacteria demonstrate markedly increased resistance to antimicrobial agents compared with planktonic cells [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSodium hypochlorite has long been regarded as the gold standard root canal irrigant due to its broad-spectrum antimicrobial activity and unique ability to dissolve organic tissue. Its mechanism of action involves the release of hypochlorous acid and chlorine ions, which lead to irreversible oxidation of bacterial enzymes, degradation of cell membranes, and disruption of essential metabolic pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Numerous in vitro and clinical studies have demonstrated the effectiveness of sodium hypochlorite against Enterococcus faecalis and other endodontic pathogens, even in the presence of organic debris and biofilm structures [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Despite its well-documented antimicrobial efficacy, the use of sodium hypochlorite is associated with several limitations. Its cytotoxic effects on periapical tissues, unpleasant taste and odor, potential for allergic reactions, and risk of accidental extrusion beyond the apex raise significant clinical concerns [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In primary teeth, these concerns are amplified due to the presence of physiologic root resorption, thinner dentinal walls, and the close proximity of developing permanent tooth buds. Additionally, sodium hypochlorite has been reported to alter the mechanical properties of dentin by reducing microhardness and elastic modulus, which may compromise the structural integrity of primary teeth [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These limitations highlight the need to investigate alternative disinfection strategies with improved safety profiles, particularly in pediatric dentistry.\u003c/p\u003e \u003cp\u003eChitosan is a naturally occurring polysaccharide obtained through the deacetylation of chitin and has attracted increasing attention in dentistry due to its antimicrobial, antifungal, antibiofilm, and biocompatible properties [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The antimicrobial activity of chitosan is primarily attributed to its polycationic nature, which enables electrostatic interactions with the negatively charged components of bacterial cell walls. This interaction results in increased membrane permeability, leakage of intracellular contents, and inhibition of bacterial enzyme activity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Several studies have demonstrated the effectiveness of chitosan against Enterococcus faecalis in both planktonic and biofilm forms, suggesting its potential application as an intracanal disinfectant [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition to its direct antibacterial effects, chitosan has been shown to interfere with biofilm formation and maturation by disrupting extracellular polymeric substances and inhibiting bacterial adhesion to dentinal surfaces [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, chitosan has been reported to exhibit chelating properties and favorable interactions with dentin, which may enhance its penetration into dentinal tubules and contribute to sustained antimicrobial activity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCold atmospheric plasma (CAP) has emerged as a novel non-thermal antimicrobial technology with growing applications in dentistry and medicine. Plasma is an ionized gas composed of reactive oxygen and nitrogen species, electrons, ions, and ultraviolet photons, all of which contribute to its antimicrobial effects [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Unlike thermal plasma, cold atmospheric plasma operates at near-room temperature, allowing its application to biological tissues without causing thermal damage. The antimicrobial mechanism of cold atmospheric plasma involves oxidative damage to bacterial cell walls, lipid peroxidation, protein denaturation, and DNA fragmentation, ultimately leading to microbial cell death [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previous studies have demonstrated the effectiveness of cold atmospheric plasma against a wide range of oral microorganisms, including Enterococcus faecalis, even within biofilm structures [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. One of the advantages of cold atmospheric plasma is its ability to penetrate complex anatomical structures, such as dentinal tubules and irregular canal spaces, which are difficult to access using conventional irrigation methods. Additionally, plasma treatment does not rely on chemical diffusion, reducing the risk of cytotoxic effects on surrounding tissues. These properties suggest that cold atmospheric plasma may represent a valuable adjunct or alternative approach for root canal disinfection, particularly in primary teeth with complex morphology.\u003c/p\u003e \u003cp\u003eTherefore, the aim of this in vitro study was to evaluate and compare the antibacterial effects of chitosan, cold atmospheric plasma, and sodium hypochlorite against Enterococcus faecalis in mandibular second primary molars.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis in vitro experimental study was conducted using extracted mandibular second primary molars at the Faculty of Dentistry, Islamic Azad University, Tehran, Iran.\u003c/p\u003e \u003cp\u003eBased on the results reported by Armand et al. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], sample size calculation was performed using one-way ANOVA power analysis in PASS 11 software, considering a significance level of α\u0026thinsp;=\u0026thinsp;0.05 and a statistical power of 80% (β\u0026thinsp;=\u0026thinsp;0.2). Assuming a mean standard deviation of the logarithmic colony count equal to 1.00 and an effect size of 0.55, the minimum required sample size was calculated as 10 samples per group for each of the three study groups. In addition, one extra sample per group (three samples) was allocated for determining the baseline bacterial count inside the root canals.\u003c/p\u003e \u003cp\u003eA total of 33 extracted mandibular second primary molars, meeting the inclusion and exclusion criteria, were selected for the study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The teeth were randomly assigned to the experimental groups using simple randomization, with the aid of a random number table, by an independent operator.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInclusion and exclusion criteria\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInclusion criteria\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eExclusion criteria\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eExtracted mandibular second primary molars with at least \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003etwo-thirds of root length remaining\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresence of internal or external physiological or pathological root resorption (based on radiographic evaluation)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHistory of previous pulpotomy or pulpectomy\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePerforation of the pulpal floor (based on radiographic evaluation)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eDebris present on the tooth surfaces was removed using a No. 15 surgical scalpel blade (Scalpel blade, ATP, Trinon Co., Germany). The teeth were then cleaned using a brush attached to a low-speed handpiece under water irrigation. Following cleaning, the samples were rinsed with normal saline (Normal saline, Shiraz Serum Co., Iran) and stored in 0.5% thymol solution (Thymol, Sigma, Iran) for one week. Subsequently, until the time of experimentation, the teeth were kept in distilled water at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003eAll roots were sectioned at the level of the cemento-enamel junction (CEJ) using a diamond disc mounted on a high-speed handpiece. The root canals were initially prepared using size 15 and 20 K-files (Mani Co., Japan) and subsequently instrumented with size 25 and 30 rotary files of the Denco system (Denco, Shenzhen, China) with a 4% taper, to a working length 1 mm short of the radiographic apex. During canal preparation, irrigation was performed using 5 mL of normal saline (Normal saline, Shiraz Serum Co., Iran). A 30-gauge irrigation syringe with a side-vented, closed-end needle was used for canal irrigation.\u003c/p\u003e \u003cp\u003eTo remove the smear layer and maintain the patency of the dentinal tubules for bacterial penetration, the canals were irrigated sequentially with 2 mL of 17% EDTA (Asia Chemi Teb Co., Tehran, Iran) for 1 minute, followed by 2 mL of 2.5% sodium hypochlorite (Nik Darman Co., Iran) for 1 minute, and finally flushed with 5 mL of normal saline (Shiraz Serum Co., Iran). To prevent apical leakage, the apical foramen of each tooth was sealed using flowable composite resin A2 shade (Denfil, Vericom Co., South Korea).\u003c/p\u003e \u003cp\u003eThe samples were then placed in autoclavable microtubes containing Brain Heart Infusion (BHI) broth and sterilized in an autoclave at 121\u0026deg;C and 15 psi for 30 minutes. To confirm sterility, the samples were incubated at 37\u0026deg;C for 48 hours. In cases where turbidity was observed in the culture medium, the sterilization process was repeated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eiii\u003c/p\u003e \u003cp\u003eII: Teeth sectioned adjacent to CEJ, III: a\u0026thinsp;=\u0026thinsp;Hand filing b\u0026thinsp;=\u0026thinsp;Rotary filing c\u0026thinsp;=\u0026thinsp;Irrigation d\u0026thinsp;=\u0026thinsp;Apical sealing using flowable composite\u003c/p\u003e \u003cp\u003eAn inoculum suspension of Enterococcus faecalis was obtained from the Iranian Research Organization for Science and Technology and prepared at a concentration of 1.5 \u0026times; 10⁸ CFU/mL, equivalent to 0.5 McFarland standard. Enterococcus faecalis was cultured on the appropriate culture medium and incubated at 37\u0026deg;C with CO₂ for 48 hours. Fifteen microliters of the E. faecalis inoculum suspension were introduced into the root canal of each tooth using a sampler for 30 seconds. The samples were then incubated in BHI culture medium at 37\u0026deg;C, with continuous agitation at 150 rpm, for three weeks. To ensure bacterial viability and biofilm formation, 15 \u0026micro;L of fresh BHI broth (BHI, Merck Co., Germany) was injected daily into each canal. To confirm biofilm formation, baseline sampling was performed. In each sample, biofilm disruption was achieved using a vortex mixer (Vortex Mixer, KST, Iran) at 2500 rpm for 1 minute. The canal contents were transferred into vials containing 0.9 mL phosphate-buffered saline (PBS). The suspension was then cultured on agar plates and incubated at 37\u0026deg;C for 96 hours, after which the baseline colony-forming units (CFU) were counted. These values served as the reference for comparison with post-treatment CFU values.\u003c/p\u003e \u003cp\u003eThe samples were randomly assigned using a random number table into three groups, and all disinfection procedures were performed by a single trained operator.\u003c/p\u003e \u003cp\u003eGroup 1: Sodium Hypochlorite\u003c/p\u003e \u003cp\u003eRoot canals were irrigated with 2 mL of 2.5% sodium hypochlorite (Nik Darman Co., Iran) for 1 minute. To neutralize the residual sodium hypochlorite prior to bacterial sampling, 3 mL of 5% sodium thiosulfate (Sodium Thiosulfate, Tamadkala, Iran) was applied into the canals for 1 minute. Final irrigation was performed using normal saline (Shiraz Serum Co., Iran).\u003c/p\u003e \u003cp\u003eGroup 2: Cold Atmospheric Plasma\u003c/p\u003e \u003cp\u003eCold atmospheric plasma was applied using a helium-based cold plasma device (Nariatech Plasmart Co., Iran) with an average frequency of 50 Hz, input power of 55 W, gas flow rate of 3 L/min, and intensity level 4. Cold atmospheric plasma was applied for 60 seconds, with the nozzle positioned at a distance of 5 mm from the canal orifice.\u003c/p\u003e \u003cp\u003eGroup 3: Chitosan\u003c/p\u003e \u003cp\u003eA 2% chitosan solution was used as an intracanal dressing. The solution was injected into the canal using a syringe with a 30-gauge needle, positioned 2 mm short of the apex, and left in the canal for 3 minutes prior to final irrigation. Chitosan was supplied in powder form. To prepare a 2% chitosan solution, 2 g of chitosan powder (Aprinatd, Tehran, Iran) was dissolved in 100 mL of 1% (0.1 M) acetic acid. The mixture was placed on a magnetic stirrer (Domel, Domel Co., Slovenia) at 37\u0026deg;C and stirred at 400 rpm for 24 hours until a homogeneous 2% chitosan solution was obtained. After application of the disinfection protocols, all samples were irrigated with 1 mL of normal saline (Shiraz Serum Co., Iran). Sterile paper points were then used to remove excess saline and planktonic bacteria, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ewhile minimizing disruption of the adherent biofilm.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eFor secondary sampling, the samples were subjected to vibration using a vortex mixer for 30 seconds to release any remaining biofilm within the canals. Secondary samples were collected from the canal contents, cultured on agar plates, and incubated at 37\u0026deg;C for 48 hours. The secondary CFU values were counted and compared with the baseline CFU values. All collected data were coded and entered into SPSS software version 24. Data analysis was performed using one-way ANOVA, and the level of statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe antibacterial effects of sodium hypochlorite, chitosan, and cold atmospheric plasma against Enterococcus faecalis in mandibular second primary molars are presented below. The number of viable bacteria was quantified by counting colony-forming units (CFU/mL). For statistical analysis, CFU values were log-transformed, and all analyses were performed using the logarithmic data. According to the results presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation of the logarithmic primary and secondary CFU values in the sodium hypochlorite group were 7.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 and 5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, respectively. In the chitosan group, these values were 7.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and 5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, respectively, while in the cold atmospheric plasma group they were 7.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 and 5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, respectively. All three disinfection methods resulted in a marked reduction in bacterial counts following intervention.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLog-transformed colony-forming unit (CFU) counts of Enterococcus faecalis before and after intervention Repeated-measures ANOVA demonstrated a statistically significant reduction in bacterial counts after treatment in all groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Intergroup comparisons showed that the reduction in bacterial load achieved with sodium hypochlorite was significantly greater than that observed with chitosan (P\u0026thinsp;=\u0026thinsp;0.001) and cold atmospheric plasma (P\u0026thinsp;=\u0026thinsp;0.003). However, no statistically significant difference was found between the chitosan and cold atmospheric plasma groups (P\u0026thinsp;=\u0026thinsp;0.641). The results of pairwise comparisons are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBefore intervention (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAfter intervention (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSodium hypochlorite (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitosan (2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCold atmospheric plasma (CAP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePairwise comparison of antibacterial efficacy among the study groups based on log-transformed CFU reduction. The clinical relevance of antibacterial efficacy among the study groups is presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComparison\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean difference (log CFU)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStandard error\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSodium hypochlorite vs Chitosan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.064\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSodium hypochlorite vs CAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.056\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.003\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitosan vs CAP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.641\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical relevance of antibacterial efficacy among study groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean percentage bacterial reduction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSodium hypochlorite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e97.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCold atmospheric plasma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e97.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChitosan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e97.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present in vitro study evaluated the antibacterial efficacy of sodium hypochlorite, chitosan, and cold atmospheric plasma against Enterococcus faecalis in primary molar root canals. The findings reported by Hee-Eun Kim et al. (2024) are consistent with the results of the present study, which also demonstrated a substantial antibacterial effect of cold atmospheric plasma against Enterococcus faecalis. Kim et al. showed that the antibacterial efficacy of cold atmospheric plasma was maintained regardless of biofilm maturity, underscoring its ability to penetrate and disrupt complex biofilm structures. Similarly, in the present study, cold atmospheric plasma produced a significant reduction in bacterial counts, comparable to that achieved with conventional chemical disinfectants. Although sodium hypochlorite exhibited a statistically greater antibacterial effect, cold atmospheric plasma demonstrated a level of bacterial reduction that was not clinically different from the other tested modalities. Taken together, these findings support the potential role of cold atmospheric plasma as an effective adjunctive disinfection method, particularly in clinical situations involving resistant or mature biofilms where conventional irrigants may have limited penetration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results of Ibrahim et al. (2023) further support the antibacterial potential of cold atmospheric plasma observed in the present study. In their in vitro investigation, argon-based cold atmospheric plasma significantly reduced colony-forming units of both Streptococcus mutans and Candida albicans at all tested exposure times, demonstrating broad-spectrum antimicrobial activity. These findings align with the results of the present study, in which cold atmospheric plasma produced a substantial reduction in Enterococcus faecalis counts within infected root canals. Although Ibrahim et al. reported a greater susceptibility of Candida albicans compared with Streptococcus mutans, both studies collectively highlight the effectiveness of cold atmospheric plasma against microorganisms known for their resistance within oral biofilms. The consistency between these results suggests that the antimicrobial action of cold atmospheric plasma is not limited to a single species or experimental setup, supporting its potential application as an adjunctive disinfection method in endodontic treatment, particularly in cases involving resistant or biofilm-associated microorganisms [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results reported by Kumar et al. (2023) partially contrast with the findings of the present study. In their investigation, 5.25% sodium hypochlorite demonstrated significantly greater antibacterial efficacy against Enterococcus faecalis than cold atmospheric plasma across all tested time intervals, leading the authors to recommend sodium hypochlorite as the most effective disinfection method. In the present study, although sodium hypochlorite also exhibited a statistically greater reduction in bacterial counts compared with cold atmospheric plasma, this difference was not considered clinically significant. This discrepancy may be attributed to differences in experimental design, including variations in plasma device parameters, exposure time, canal anatomy, and evaluation methods. Notably, Kumar et al. reported that cold atmospheric plasma achieved a significant reduction in bacterial load after a minimum exposure time of 5 minutes, which is consistent with the substantial antibacterial effect observed in the present study. Therefore, despite differences in the magnitude of antibacterial efficacy reported, both studies support the potential role of cold atmospheric plasma as an effective adjunctive disinfection method, particularly in situations where conventional irrigants may be limited by penetration depth or cytotoxicity concerns [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings of Bushra M et al. (2022) are largely consistent with the results of the present study, while also highlighting important differences related to formulation and contact time. Bushra et al. demonstrated that a combination of chitosan and propolis exhibited superior antibacterial activity against Enterococcus faecalis during the early phase of treatment; however, this advantage was not maintained at later time points, where its efficacy became comparable to that of calcium hydroxide. Similarly, in the present study, chitosan showed a significant antibacterial effect against Enterococcus faecalis but did not demonstrate a clinically superior outcome when compared with other disinfection modalities. The observed differences between the studies may be attributed to the use of a chitosan\u0026ndash;propolis combination rather than chitosan alone, as well as differences in exposure duration and evaluation time points. Collectively, these findings suggest that while chitosan possesses notable antibacterial properties, its clinical effectiveness may be influenced by formulation, adjunctive agents, and duration of application [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings reported by Asnaashari et al. (2022) are generally in agreement with the results of the present study, while also revealing a relative difference in the magnitude of antibacterial efficacy among treatment modalities. In their laboratory investigation, cold atmospheric plasma significantly reduced Enterococcus faecalis biofilm compared with control groups, confirming its notable antimicrobial potential. This observation is consistent with the present study, in which cold atmospheric plasma produced a substantial reduction in bacterial counts within infected root canals. However, Asnaashari et al. reported that triple antibiotic paste achieved the greatest reduction in CFU values, surpassing the antibacterial effect of cold atmospheric plasma. In contrast, the present study demonstrated that although sodium hypochlorite showed a statistically greater antibacterial effect, the reduction achieved by cold atmospheric plasma was not clinically inferior to that of the other tested disinfection methods. The discrepancy between these findings may be attributed to differences in experimental conditions, including the use of a prolonged plasma exposure time, the presence of a mature 21-day biofilm model, and comparison with intracanal medicaments rather than irrigants. Collectively, both studies support the effectiveness of cold atmospheric plasma in reducing E. faecalis biofilms, while suggesting that its relative performance may vary depending on the comparator material and experimental design [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings reported by Wang et al. (2020) are largely in agreement with the results of the present study regarding the antibacterial efficacy of chitosan against Enterococcus faecalis. Wang et al. demonstrated that chitosan, particularly when dissolved in double-distilled water, exhibited strong bactericidal activity against both planktonic and biofilm forms of E. faecalis, with a low minimum bactericidal concentration and no detectable cytotoxicity to MC3T3-E1 pre-osteoblast cells. These observations are consistent with the present study, in which chitosan produced a significant reduction in E. faecalis counts within infected root canals. However, while Wang et al. emphasized the pronounced antibacterial activity of chitosan at specific concentrations under controlled laboratory conditions, the present study found that chitosan did not demonstrate a clinically superior antibacterial effect when compared with sodium hypochlorite or cold atmospheric plasma. This difference may be attributed to variations in experimental models, including concentration-dependent testing, solvent effects, and the use of isolated planktonic and biofilm systems versus a standardized root canal model. Nevertheless, both studies support the conclusion that chitosan possesses notable antibacterial properties against E. faecalis with favorable biocompatibility, reinforcing its potential role as a safe and effective alternative or adjunctive agent for root canal disinfection [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study has several limitations. As an in vitro investigation, the experimental conditions do not fully replicate the complex biological environment of the oral cavity. In addition, the antibacterial efficacy was evaluated against a single microbial species, which may not reflect the polymicrobial nature of endodontic infections. Therefore, the results should be interpreted with caution.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on the findings of the present study and their comparison with similar investigations, no clinically significant difference was observed in the antibacterial efficacy of chitosan, cold atmospheric plasma, and sodium hypochlorite against Enterococcus faecalis in mandibular second primary molars. Accordingly, chitosan and cold atmospheric plasma may be considered potential alternatives to sodium hypochlorite for root canal disinfection. Future studies with larger sample sizes and longer evaluation periods are recommended. In addition, investigations assessing different concentrations of chitosan and sodium hypochlorite, as well as studies involving different bacterial species or multiple microorganisms simultaneously, should be conducted. Furthermore, the design of clinical trial studies is recommended to allow better generalization of the findings.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eHuman Ethics and Consent to Participate\u003c/h2\u003e \u003cp\u003e This in vitro study was conducted on extracted human teeth and was approved by the Ethics Committee of the Faculty of Dentistry, Islamic Azad University, Tehran, Iran (Approval No. IR.IAU.DENTAL.REC.1403.167). All procedures were conducted in accordance with the Declaration of Helsinki. Informed consent to participate was waived by the ethics committee as the study used anonymized extracted teeth for laboratory research only.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent to Publish\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eCamellia Kianbakht and Maryam Pourhossein conceived and designed the study and performed the experiments. Arshia Mansouri analyzed the data, and drafted the manuscript. All authors critically revised the manuscript and approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e \u003cp\u003eData sharing is not applicable to this article as no datasets were generated or analysed during the current study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArmand A, Khani M, Asnaashari M, AliAhmadi A, Shokri B. Comparison study of root canal disinfection by cold plasma jet and photodynamic therapy. Photodiagnosis Photodyn Ther. 2019;26:327\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCancio V, Carvalho Ferreira D, Cavalcante FS, Rosado AS, Teixeira LM, Braga Oliveira Q, et al. Can the Enterococcus faecalis identified in the root canals of primary teeth be a cause of failure of endodontic treatment? Acta Odontol Scand. 2017;75(6):423\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakko M, Tj\u0026auml;derhane L, Rautemaa-Richardson R. Microbiology of Root Canal Infections. Prim Dent J. 2016;5(2):84\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlghamdi F, Shakir M. The Influence of Enterococcus faecalis as a Dental Root Canal Pathogen on Endodontic Treatment: A Systematic Review. Cureus. 2020;12(3):e7257.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuguloth S, Jampanapalli SR, Patloth T, Suhasini K, Ingua HC, Shaik H. Evaluation of Chitosan and Ferric Sulphate as Pulpotomy Agents in Primary Teeth: A Randomized Controlled Trial. Int J Clin Pediatr Dent. 2023;16(2):223\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePourhajibagher M, Ghorbanzadeh R, Bahador A. Culture-dependent approaches to explore the prevalence of root canal pathogens from endodontic infections. Braz Oral Res. 2017;31:e108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRadeva E, Karayasheva D. Importance of Enterococci (Enterococcus faecalis) for Dental Medicine - Microbiological Characterization, Prevalence and Resistance. Int J Sci Res (IJSR). 2017;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh H. Microbiology of Endodontic Infections. 2016.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstrela C, Estrela CR, Barbin EL, Span\u0026oacute; JC, Marchesan MA, P\u0026eacute;cora JD. Mechanism of action of sodium hypochlorite. Braz Dent J. 2002;13(2):113\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerber VB, Gomes BP, Sena NT, Vianna ME, Ferraz CC, Zaia AA, Souza-Filho FJ. Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubules. Int Endod J. 2006;39(1):10\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoel P, Galhotra V, Makkar S, Mohan J, Bala N, Kaur T. An In Vitro Study Comparing the Antimicrobial Efficacy of 0.2% Chitosan, 3% Sodium Hypochlorite, 2% Chlorhexidine against Enterococcus faecalis, Alone and in Conjunction with Diode Laser. Int J Clin Pediatr Dent. 2022;15(1):109\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang K, Kim YK, Cadenaro M, Bryan TE, Sidow SJ, Loushine RJ, et al. Effects of different exposure times and concentrations of sodium hypochlorite/ethylenediaminetetraacetic acid on the structural integrity of mineralized dentin. J Endod. 2010;36(1):105\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan W, Yan W, Xu Z, Ni H. Erythrocytes load of low molecular weight chitosan nanoparticles as a potential vascular drug delivery system. Colloids Surf B Biointerfaces. 2012;95:258\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePu Y, Liu A, Zheng Y, Ye B. In vitro damage of Candida albicans biofilms by chitosan. Exp Ther Med. 2014;8(3):929\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSupotngarmkul A, Panichuttra A, Ratisoontorn C, Nawachinda M, Matangkasombut O. Antibacterial property of chitosan against E. faecalis standard strain and clinical isolates. Dent Mater J. 2020;39(3):456\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThienngern P, Panichuttra A, Ratisoontorn C, Aumnate C, Matangkasombut O. Efficacy of chitosan paste as intracanal medication against Enterococcus faecalis and Candida albicans biofilm compared with calcium hydroxide in an in vitro root canal infection model. BMC Oral Health. 2022;22(1):354.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang N, Ji Y, Zhu Y, Wu X, Mei L, Zhang H, et al. Antibacterial effect of chitosan and its derivative on Enterococcus faecalis associated with endodontic infection. Exp Ther Med. 2020;19(6):3805\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilva PV, Guedes DF, P\u0026eacute;cora JD, da Cruz-Filho AM. Time-dependent effects of chitosan on dentin structures. Braz Dent J. 2012;23(4):357\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanikonda R, Ravi RK, Sirisha K, Divella S. Chitosan: Applications in dentistry. Trends Biomaterials Artif Organs. 2014;28:74\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHusain S, Al-Samadani KH, Najeeb S, Zafar MS, Khurshid Z, Zohaib S, Qasim SB. Chitosan Biomaterials for Current and Potential Dental Applications. Mater (Basel). 2017;10(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsnaashari M, Motamedi S, Asnaashari N, Azari-Marhabi S. Antimicrobial Activity of Cold Plasma Treatment on Acrylic Denture Bases: An In Vitro Evaluation. J Lasers Med Sci. 2019;10(Suppl 1):S13\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffmann C, Berganza C, Zhang J. Cold Atmospheric Plasma: methods of production and application in dentistry and oncology. Med Gas Res. 2013;3(1):21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim HE. Influence of Biofilm Maturity on the Antibacterial Efficacy of Cold Atmospheric Plasma in Oral Microcosm Biofilms. Biomedicines. 2024;12(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar P, Soundharrajan P, Prakash R, Kombade SP, Yadav P, Chugh A, Patnana AK. An in-vitro analysis to evaluate the disinfection effectiveness of Cold Atmospheric Pressure (CAP) plasma jet in Enterococcus faecalis infected root canals. Biomater Investig Dent. 2023;10(1):2193214.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalam G, Maha I, Abbas M, Ali E, Al-Rubaee E. The antimicrobial effects of Cold Atmospheric Plasma jet on microorganisms causing dental caries (in vitro study). Mustansiria Dent J. 2023;19:6\u0026ndash;2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsnaashari M, Mehrabinia P, Yadegari Z, Hoseini H, Sadafi M, Shojaeian S. Evaluation of Antibacterial Effects of Cold Atmospheric Plasma, Calcium Hydroxide, and Triple Antibiotic Paste on Enterococcus faecalis Biofilm in the Root Canal System: An In Vitro Study. J Lasers Med Sci. 2022;13:e50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTheinkom F, Singer L, Cieplik F, Cantzler S, Weilemann H, Cantzler M, et al. Antibacterial efficacy of cold atmospheric plasma against Enterococcus faecalis planktonic cultures and biofilms in vitro. PLoS ONE. 2019;14(11):e0223925.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShamma B, Abo-Arraj E, Rajab A, Al Kurdi S. Anti-bActeriAl Activity of Applying chitosAn And propolis dressing AgAinst EntErococcus faEcalis in primAry teeth: in vitro study. J Stomatology. 2022;75:36\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chitosan, cold atmospheric plasma, Enterococcus faecalis, sodium hypochlorite, primary teeth","lastPublishedDoi":"10.21203/rs.3.rs-8436881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8436881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePersistent microbial infection is the primary cause of pulpectomy failure in primary teeth. Enterococcus faecalis is among the most resistant microorganisms associated with endodontic treatment failure. Conventional irrigants, such as sodium hypochlorite, are effective; however, their clinical use is associated with several limitations in pediatric dentistry, prompting the investigation of alternative agents such as chitosan and cold atmospheric plasma (CAP). This study aimed to evaluate and compare the antibacterial effects of chitosan, cold atmospheric plasma, and sodium hypochlorite against Enterococcus faecalis in mandibular second primary molars.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this in vitro study 33 deciduous second molars with at least two-thirds of their root length were prepared by chemomechanical method. Enterococcus faecalis inoculum suspension was inoculated into the canals of each tooth and sampling was done to ensure biofilm formation. For disinfection of the canals, the teeth were divided into 3 groups: sodium hypochlorite, cold atmospheric plasma and chitosan. After applying disinfection methods, secondary sampling of the contents inside the canal was performed and the secondary CFU was counted, and finally the primary and secondary CFU levels were compared. The data were analyzed using one-way ANOVA statistical test, considering α\u0026thinsp;=\u0026thinsp;0.05.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe values ​​(mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation) of the primary and secondary CFU logarithms in the sodium hypochlorite (Hypo) group were (7.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08) and (5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02), respectively, in the chitosan group (Chitosan) were (7.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04) and (5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), respectively, and in the cold atmospheric plasma (CAP) group were (7.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05) and (5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), respectively. The CFU level in the Hypo group was significantly lower than that of the Chitosan and CAP groups (p\u0026thinsp;=\u0026thinsp;0.001 and p\u0026thinsp;=\u0026thinsp;0.003, respectively). However, there was no statistically significant difference between the Chitosan and CAP groups (p\u0026thinsp;=\u0026thinsp;0.641). Although statistically significant differences were observed among the groups, the mean percentage bacterial reduction did not differ significantly and was not considered clinically relevant.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThere is no clinical difference in the antibacterial activity of chitosan, cold atmospheric plasma, and sodium hypochlorite on Enterococcus faecalis in mandibular second primary molars, These findings suggest that chitosan and cold atmospheric plasma may serve as potential alternatives to sodium hypochlorite for root canal disinfection in primary teeth. \u003cb\u003eKeywords\u003c/b\u003e: Chitosan; cold atmospheric plasma; Enterococcus faecalis; sodium hypochlorite; primary teeth.\u003c/p\u003e","manuscriptTitle":"Evaluation of the antibacterial effect of chitosan, cold atmospheric plasma and sodium hypochlorite on Enterococcus faecalis in mandibular second primary molars: An in vitro study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-29 00:32:24","doi":"10.21203/rs.3.rs-8436881/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-02T09:08:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151635174525342561976026545768372206581","date":"2026-01-29T09:25:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66412669890216584653650158805816476627","date":"2026-01-28T19:27:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296249973320781908528592736711956200248","date":"2026-01-23T07:34:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-23T06:51:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T11:24:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-02T13:25:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-01T10:12:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2026-01-01T10:04:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cf5f76ec-19d1-419f-a9e0-92ec56c5c930","owner":[],"postedDate":"January 29th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-29T00:32:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-29 00:32:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8436881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8436881","identity":"rs-8436881","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}