Aim: The objective of this study was to compare the efcacy of 2.5% calcium hypochlorite [Ca(OCl)2], 2.5% sodium hypochlorite (NaOCl), and 2% chlorhexidine (CHX), applied under different agitation protocols, in the rapid disinfection of gutta-percha cones contaminated with Enterococcus faecalis. Methods: The antibioflm activity of each solution against E. faecalis was initially tested in cell culture wells. Next, ffty-four gutta-percha cones contaminated with the bacterial suspension were equally divided into three groups (n=18) and subjected to disinfection using one of the three solutions (2.5% NaOCl, 2.5% Ca(OCl)2, or 2% CHX). Each group was further subdivided based on the application method (no agitation, ultrasonic agitation, or agitation with Easy Clean) and exposure time to each solution (1 or 5 minutes) (n=6). After disinfection, the samples were examined for turbidity,assessed for viable colonies, and the data were analyzed using one-way analysis of variance (ANOVA) and Tukey’s post hoc tests (α = 5%). Results: The 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX solutions signifcantly destroyed and inhibited bioflm formation (p < 0.0001). In terms of reducing viable colonies, 2.5% Ca(OCl)2 and 2% CHX were effective across all application methods, both at 1 and 5 minutes (p < 0.05). However, 2.5% NaOCl was only effective under ultrasonic agitation for 5 minutes(p < 0.05). Densitometer readings showed no statistical difference between the groups (p > 0.05). Conclusions: Overall, the results demonstrated better performance of 2% CHX and 2.5% Ca(OCl)2 in the disinfection of gutta-percha cones under the simulated experimental conditions.
Keywords: Enterococcus faecalis. Calcium compounds. Chlorhexidine. Gutta-percha. Sodium hypochlorite.

Introduction

Gutta-percha is a filling material historically used in root canal treatments in conjunction with endodontic sealer, as it has adequate dimensional stability, satisfactory radiopacity, is biocompatible, and is thermoplastic. Due to its nature, gutta-percha cannot undergo a wet or dry heat sterilization process. Thus, chemical disinfection by immersion in antiseptic solutions is recommended.

Several chemical agents have been proposed to disinfect gutta-percha cones, including sodium hypochlorite (NaOCl) and chlorhexidine (CHX). The ideal disinfectant should ensure rapid disinfection without altering the cone structure3. NaOCl is the most widely used antimicrobial irrigant solution worldwide in Endodontics, and although it is effective, there is evidence that the deposition of residual crystals on the cones may end up making it difficult to bond with the sealer, leading to microleakage,. Previous studies also reported that CHX is effective for disinfecting gutta-percha cones. However, when NaOCl is used as the primary irrigant, any residual CHX on the cone surface can chemically interact with NaOCl residues within the root canal, which may occur due to inadequate drying or anatomical complexities,. This interaction can lead to the formation of para-chloroaniline, a toxic compound that interferes with the sealing of the treated root canal,.

Recently, the use of calcium hypochlorite [Ca(OCl)2 ] has been considered in End2 odontics, due to its satisfactory antimicrobial action and tissue dissolution capacity, greater compatibility with the properties of dentin, and less aggravation in adhesive procedures. Despite the aforementioned potential, studies evaluating the effectiveness of Ca(OCl)2 in disinfecting gutta-percha cones are scarce and must 2 be carried out to verify its applicability for this purpose. In addition, further investigations into the effect of different agitation methods to enhance disinfection in this context are also limited. Finally, E. faecalis is the primary microorganism identified in endodontic failures, making it essential to evaluate the effectiveness of solutions in its elimination. Therefore, the objective of this study was to compare the efficacy of 2.5% Ca(OCl)2 , 2.5% NaOCl, and 2% CHX, applied under different agitation proto2 cols, in disinfecting gutta-percha cones contaminated with E. faecalis. The study adopted the null hypothesis that there would be no difference in the antibacterial effect of the different solutions, regardless of the application method.

Materials and Methods

Bacterial strain and culture conditions

The strain used in the experiments was E. faecalis (ATCC 29212), obtained from the American Type Culture Collection. The microorganisms were preserved in a glycerol-containing culture medium and stored at -80°C. Before use, the sample was thawed, inoculated into Brain Heart Infusion (BHI) broth, and incubated for 24 hours.

Determination of minimum inhibitory and minimum bactericidal concentrations

The minimum inhibitory concentration (MIC) was determined using the broth microdilution method in a 96-well plate. To establish the minimum bactericidal concentra-tion (MBC), 1 μL was taken from each well, plated on Mueller-Hinton agar, and incubated anaerobically at 37°C for 24 hours. Colonies were then identified, and the lowest concentration at which no microbial growth was observed was recorded as the MBC.

Biofilm formation The biofilm was produced following previously described conditions with modifications. The treatments applied included MIC, 2X MIC and 4X MIC of 2.5% Ca(OCl)2, 2.5% NaOCl 2 or 2% CHX. For control purposes, only the E. faecalis strain in Mueller-Hinton broth was used as a positive control, and Mueller-Hinton broth alone served as the negative control.

Crystal violet assay Biomass was quantified using the crystal violet staining technique as described previously. The antibiofilm activity was assessed by measuring the difference in optical density between the positive control (Mueller-Hinton broth + E. faecalis) and the treatments.

Biofilm inhibition assay The anti-adhesion properties of 2.5% Ca(OCl)2 , 2.5% NaOCl, and 2% CHX were evaluated 2 using a microplate biofilm assay. A suspension containing E. faecalis, Mueller-Hinton broth, and the treatments was incubated anaerobically at 37°C for 24 hours. The assay was performed as described in the previous point. Mueller Hinton broth containing E. faecalis was considered as a positive control, and the percentage of inhibition was calculated by Optical density test/Optical density control×100.

Counting colony forming units (CFUs) The methodology followed the approach outlined by Pauletto et al.. Ninety-eight gutta-percha cones (Dentsply Maillefer, Ballaigues, Switzerland) were used and opened under aseptic conditions. Each gutta-percha cone was individually contaminated by immersing it in a bacterial suspension within a microtube for 48 hours at 37°C. To ensure sterility and monitor contamination, 2 cones were plated on a nutrient medium post-sterilization to confirm sterility. Additionally, 3 cones were plated every 24 hours to verify the absence of contamination.

Positive controls consisted of 18 gutta-percha cones, immersed in salina without treatment and negative controls consisted of 18 uncontaminated samples of each material. The remaining 54 cones were divided by according to the solution used for decontamination in three experimental groups: 2.5% NaOCl (n=18), 2.5% Ca(OCl)2 2 (n=18) or 2% CHX (n=18). Each group was treated with different methodologies: without agitation (3 cones during 1 minute and 3 cones during 5 minutes), ultrasonic agitation (3 cones during 1 minute and 3 cones during 5 minutes), or agitation with Easy Clean (Easy Equipamentos Odontológicos, Belo Horizonte, MG, Brazil) (3 cones during 1 minute and 3 cones during 5 minutes). As for the agitation procedure, the Easy Clean #25.04 plastic file (Easy Equipamentos Odontológicos) was attached to a handpiece at 20.000 rpm and the E1 Irrisonic tip (Helse Ultrasonic, Ribeirão Preto, SP, Brazil) was coupled to an ultrasonic device (Sonic Laxis BP LED, Schuster, Santa Maria, RS, Brazil), with a power level of 20%.

After treatment, all samples were placed in a microtube containing 2 mL Mueller-Hinton broth and incubated at 37°C for 48 hours. Samples from each experimental and control group were randomly chosen, seeded onto an agar plate, incubated at 37°C, and checked for growth at 2 days. In the presence of microbial growth, the bacteria were confirmed by colony morphology and Gram staining for E. faecalis. The number of cells was determined by CFUs following plated.

Reading with densitometer The growth of bacteria was followed by measuring the optical density of bacterial suspension by densitometry, according to Vági et al. with modifications. 3 ml of nutrient liquid medium was measured into a spectrophotometer, two flasks were prepared for each sample examined. The flask containing the medium only was measured, as a control. Also, a bacteria control was applied without any added material in the medium.

To obtain the bacteria concentration after the treatments, the optical density of the samples was measured by densitometry (Carl-Zeiss, Gottingen, Germany) at 570 nm wavelength. The growth of the bacteria is manifested in the turbidity of the suspension and can be followed by the rise of optical density. The inhibition percent is defined as the optical density of the sample (ODX) of measurement compared to the optical density of control (ODAC).

Scanning electron microscopy (SEM) Scanning electron microscopy images were performed to assess the effect on the 2.5 % Ca(OCl)2 . The biofilm formed on 35 mm polystyrene plates was analyzed by 2 SEM after the use of 2.5 % Ca(OCl)2 in different treatment forms and periods of time. 2 The biofilm on the plates was rinsed with distilled H O and fixed in an oven at 60°C 2 for 1 hour. The specimens were then sputter coated with gold (sputter coating 108A; Cressington Scientific Instruments Inc., Watford, UK). JSM-6360 scanning electron microscope (JEOL, Tokyo, Japan) was used.

Statistical analysis The Shapiro-Wilk test indicated normal distribution for the values collected. Therefore, all data were analyzed by one-way analysis of variance (ANOVA) and Tukey’s post hoc tests. In biofilm inhibition and destruction assay, values of p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) were considered statistically significant when compared with the positive control. In counting of viable colonies and antibacterial activity values of p < 0.05 were considered statistically significant comparing saline with treatments in the same agitation process (a) and comparing agitation processes of the same treatment with ultrasonic agitation (b). All analyses were performed using GraphPad Prism 8.0.1 software (GraphPad Software Inc, San Diego, CA, USA).

Results

Determination of minimum inhibitory and minimum bactericidal concentrations Table 1 shows the MIC and MBC of NaOCl 2.5%, Ca(OCl)2 2.5% and CHX 2% against 2 E. faecalis. In both indices, the CHX presented the best performance.

Table 1. Minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) of 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX against E. faecalis

Biofilm inhibition and destruction assay The NaOCl 2.5% and Ca(OCl)2 2.5% demonstrated significant biofilm destruction com2 pared to the control (p < 0.01), whereas CHX 2% did not show a satisfactory effect (p > 0.05). The compounds that showed the best performance were 2.5% NaOCl at a concentration of 4xMIC (2.6 mg/ml), which reduced the biofilm by 83.5% (p < 0.0001), and 2.5% Ca(OCl)2 at a concentration of 2xMIC (0.625 mg/ml) which destroyed the 2 biofilm by 76.7% (p < 0.0001) (Figure 1A). Concerning inhibited biofilm, the compound that showed the best performance was 2% CHX in the concentration 0.5x MIC (0.012 mg/ml), inhibiting biofilm in 87.6% (p < 0.0001) (Figure 1B). The data were considered statistically significant when compared with the control.

Figure 1. Antibioflm activity (A) and bioflm inhibition (B) of E. faecalis treated with different concentrations of 2.5% NaOCl, 2.5% Ca(OCl)2 , and 2% CHX. Data expressed as mean ± standard deviation. Analysis of variance (ANOVA) followed by the Tukey test was performed. Values of p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****) were considered statistically signifcant when compared with the Positive Control.

Figure 2. Counting of viable colonies of bioflm treated with 2.5% NaOCl, 2.5% Ca(OCl)2 , and 2% CHX against E. faecalis in different agitation processes in 1 (A) and 5 minutes (B). Antibacterial activity of 2.5% NaOCl, 2.5% Ca(OCl)2 , and 2% CHX against E. faecalis in different agitation processes in 1 (C) and 5 minutes (D) through the densitometer. Data expressed as mean ± standard deviation. Analysis of variance (ANOVA) followed by the Tukey test was performed. Values of p < 0.05 were considered statistically signifcant (a) comparing saline with treatments in the same agitation process and p < 0.05; (b) comparing agitation processes of the same treatment with ultrasonic agitation.

Counting colony forming units (CFUs) In 1 minute of exposure, treatment with 2.5% NaOCl showed no difference when compared to saline solution regardless of the application method (p > 0.05). Treatments with 2.5% Ca(OCl)2 and 2% CHX showed a significant reduction compared to saline 2 solution (p < 0.05). When it was shaken for 5 minutes, treatment with NaOCl under ultrasonic agitation showed a significant reduction when compared to the saline solution, and the other results observed at 1 minute were maintained (p < 0.05). The result of counting is shown in Figures 2A and 2B.

Reading with densitometer In every case, the growth curves were compared with those obtained with salina. All treatments demonstrated a significant reduction in optical density compared to the control with saline solution, except for 2.5% NaOCl without agitation and with Easy clean agitation, which did not demonstrate a decrease. There was no statistical difference in the density reduction comparing the different stirring processes when treated with 2.5% Ca(OCl)2 or CHX at both times tested, Figures 2C and 2D. 2

Scanning electric microscopy Figure 3 shows the destruction of the biofilm. SEM photographs emphasize the results obtained.

Figure 3. MEC of bioflm E. faecalis treated with salina in 1 (A) and 5 (B) minutes and 2.5% Ca(OCl)2  in different agitation processes – without agitation in 1 (C) and 5 (D) minutes; ultrasonic agitation in 1 (E) and 5 (F) minutes, and Easy clean agitation in 1 (G) and 5 (H) minutes.

Discussion This study evaluates the rapid disinfection of gutta-percha cones using Ca(OCl)2 solu2 tion with different agitation methods, comparing the results with NaOCl and CHX, solutions historically used for this purpose. The findings revealed varied antibacterial effects against E. faecalis across the scenarios simulated in this in vitro study, leading to the rejection of the initially formulated null hypothesis.

In front of tests in culture wells, our results demonstrated that the 2% CHX was the most effective solution to inhibit the E. faecalis biofilm formed, and the 2.5% NaOCl and 2.5% Ca(OCl)2 solutions had a greater power to eliminate the respective microbial 2 species. Although there are controversies in the literature regarding which solution has the greater antibacterial effect against endodontic biofilms (chlorinated solutions x biguanide solutions), it is likely to assume that chlorinated solutions are more efficient in this scenario, as they are potent oxidants and release large amounts of hypochlorous acid during its ionic dissociation, the agent directly responsible for the antibacterial effect of the solution. In this sense, there is an irreversible oxidation of enzymes essential to microorganisms, deactivating metabolic functions of the bacterial cell, thus generating cell death.

Regarding the CFUs on the surface of gutta-percha cones, our findings emphasize that for a 1 minute decontamination time, the use of 2.5% NaOCl, regardless of the method employed, was not effective in decontamination, with data comparable to control. The solutions of 2.5% Ca(OCl)2 and 2% CHX were effective concerning 2 the control group, for all application methods. For the 5 minutes decontamination time, the same results persisted, with the addition that the ultrasonically agitated 2.5% NaOCl group performed better than the control. Although it is controversial that NaOCl had effective results in eliminating E. faecalis in cell culture wells, and did not have good performance in decontaminating gutta-percha cones, there are previous studies that emphasize that the antibacterial effect of irrigants and root canal medicaments can be influenced by the type of surface and its components. Thus, additional investigations should be carried out to better elucidate this proposition.

In contrast to the findings of this study, a previous investigation showed similar results for the three solutions in disinfecting gutta-percha cones contaminated with Candida albicans. Hence, it is reasonable to imagine that the efficacy of the solutions may vary depending on the microorganism tested, and future research should explore this aspect and simulate mixed biofilms representative of the microorganisms commonly found in endodontic infections. Regarding the positive performance of 2% CHX, this finding is possibly justified by its MBC, as described in our results, and which is in line with previous studies3,. The effectiveness of 2.5% Ca(OCl)2 2 can be attributed to the fact that Ca(OCl)2 has a greater availability of active chlorine 2 than NaOCl and its ionic dissociation results in the release of a greater amount of hypochlorous acid, which can expect a greater antibacterial effect of the solution. Congruently, our bacterial density findings also reveal a trend towards greater bacterial reduction when 2.5% Ca(OCl)2 and 2% CHX were used. SEM images emphasize 2 the results obtained for Ca(OCl)2 . 2 Considering that NaOCl is the most widely used solution worldwide as the main irrigant of the root canal and that when NaOCl interacts with CHX, a chemical interaction occurs, with a toxic compound as a by-product (para-chloroaniline) that interferes with the quality of the root filling,, it is reasonable to recommend, based on our findings, the use of Ca(OCl)2 for the rapid disinfection of gutta-percha cones, since it demonstrated better performance than NaOCl. On the other hand, still based on our findings, for professionals who use CHX as the main irrigant of the root canal, it becomes more practical to use the same solution to disinfect the cones. As another possibility, it is emphasized that the chemical interaction between CHX and Ca(OCl)2 , although causes the formation of a precipitate, is not 2 para-chloroaniline. Therefore, further studies are needed to discover the accuracy of the compound formed and whether it interferes with root canal sealing, to confirm or not its potential applicability to disinfect cones when the main irrigant is CHX.

In general, we did not observe statistically significant differences regarding the methods of agitation of the test solutions to decontaminate the gutta-percha cones. We emphasize that the current literature lacks studies on the use of agitation methods to disinfect gutta-percha cones, which ends up making it difficult to compare findings. However, a previous systematic review demonstrated that there were no significant differences in root canal disinfection between agitating or not agitating the irrigating solution, demonstrating that the main role in decontamination is played by the irrigant and its characteristics.

Although our findings suggest that Ca(OCl)2 could be recommended for the rapid 2 disinfection of gutta-percha cones due to its superior performance compared to NaOCl, it is important to acknowledge that this recommendation may be influenced by the materials and bacterial species used. This is a limitation of this preliminary study. Since saliva contains a large number and variety of bacterial species, further research involving different bacterial species is needed to validate these conclusions. Finally, additional studies should be conducted to evaluate the impact of Ca(OCl)2 2 on the microstructure of gutta-percha cones and its possible consequences for endodontic treatment.

Despite the limitations of this in vitro study, the use of Ca(OCl)2 for the decontam2 ination of gutta-percha cones appears to be a promising alternative, supported by the solution’s chemical composition. During ionic dissociation, Ca(OCl)2 releases 2 two molecules of hypochlorous acid (HOCl), whereas NaOCl releases only one. As a result, the chlorine concentration is higher in Ca(OCl)2 , enhancing its disinfection 2 capacity. However, the use of this solution in endodontic practice requires clinical investigations to obtain more robust findings and guide its potential incorporation into endodontic procedures.

In conclusion, the results of this study confirm the efficacy of 2% CHX and 2.5% Ca(OCl)2 in the rapid disinfection of gutta-percha cones contaminated with E. faecalis, 2 with no statistical difference for the application methods without agitation, ultrasonic agitation, and agitation with Easy Clean. The 2.5% NaOCl was only effective after ultrasonic agitation for 5 minutes.

Acknowledgments We would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for their support.

Declaration of interest The authors declare no conflict of interests.

Data availability Datasets related to this article will be available upon request to the corresponding author.

Author Contribution Guilherme Pauletto: Conceptualization; data curation; investigation; methodology; software; validation; visualization; writing - original draft; writing - review & editing. Pedro Henrique Fortes Guerim: Data curation; investigation; methodology; software; validation; visualization; writing - review & editing. Arthur Brites Barbosa: Data curation; investigation; methodology; software; validation; visualization; writing review & editing. Natália de Freitas Daudt: Data curation; methodology; validation; writing - review & editing. Leonardo Quintana Soares Lopes: Data curation; methodology; software; formal analysis; validation; writing - review & editing. Patrícia Kolling Marquezan: Conceptualization; Investigation; Methodology; Formal analysis; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing - original draft; Writing - review & editing. Carlos Alexandre Souza Bier: Conceptualization; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing review & editing. All authors actively revised and approved the final version of the manuscript.

Attribution and scope

Pauletto G, Guerim PHF, Barbosa AB, Daudt NF, Lopes LQS, Marquezan PK, et al. Disinfection of gutta-percha cones contaminated with Enterococcus faecalis using calcium hypochlorite, sodium hypochlorite and chlorhexidine. Brazilian Journal of Oral Sciences. 2026;25:e267486. https://doi.org/10.20396/bjos.v25i00.8677486

Reproduced under the Creative Commons Attribution 4.0 International License (CC BY 4.0): https://creativecommons.org/licenses/by/4.0/.

This independent in-vitro study evaluates chemical disinfection protocols for the article's test materials. It does not test, evaluate, endorse, or establish the performance, antimicrobial activity, disinfection performance, or clinical outcome of Hygendo Gutta Percha Points. The Hygendo Catalog image is separate brand material and is not part of the study.

Any third-party product or device names appear only to faithfully reproduce or translate the study methodology; they are not a Hygendo comparison, recommendation, or affiliation statement.

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