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. 2024 Jan 16;55(1):403–410. doi: 10.1007/s42770-024-01255-8

Efficacy of calcium hypochlorite in disinfection of gutta-percha cones contaminated with Candida albicans

Guilherme Pauletto 1,✉, Pedro Henrique Fortes Guerim 2, Arthur Brites Barbosa 2, Leonardo Quintana Soares Lopes 3, Carlos Alexandre Souza Bier 1, Patrícia Kolling Marquezan 4
PMCID: PMC10920541  PMID: 38225531

Abstract

This study aimed to compare the efficacy of 2.5% sodium hypochlorite (NaOCl), 2.5% calcium hypochlorite [Ca(OCl)2], and 2% chlorhexidine (CHX) in the rapid disinfection of gutta-percha cones contaminated with Candida albicans. The minimum inhibitory and minimum fungicidal concentrations of each solution for C. albicans were determined and the ability of each solution to destroy and inhibit biofilm in culture wells was tested. In addition, ninety-eight gutta-percha cones contaminated with the fungal suspension were disinfected according to the type of solution (2.5% NaOCl, 2.5% Ca(OCl)2 or 2% CHX) in its different application methods (without agitation, ultrasonic agitation or agitation with Easy Clean), and regarding the exposure time to each irrigating solution (1 or 5 min). Next, the samples were checked for turbidity and evaluation of viable colonies. The compounds that showed the best performance in biofilm destruction were NaOCl and Ca(OCl)2 at a concentration of 2xMIC (p < 0.001). Regarding inhibited biofilm, the only compound that was effective at all MIC concentrations tested was 2.5% Ca(OCl)2 (p < 0.0001). Regarding the viable colonies, all solutions were effective concerning the control group, for all application methods, in 1 and 5 min (p < 0.05). The densitometer reading showed that CHX was the only effective solution in all application methods performed (p < 0.05). The results demonstrate that all tested solutions were effective in the rapid decontamination of cones contaminated with C. albicans.

Keywords: Candida albicans, Sodium hypochlorite, Calcium hypochlorite, Chlorhexidine, Gutta-percha

Background

Endodontics has the main purpose of cleaning and shaping the root canal, providing ideal conditions for the repair of periapical tissues [1]. Root canal obturation is a fundamental step in endodontic treatment, as its primary objective is to seal the root canal using inert and antiseptic materials, preventing infection or reinfection of the root canal system [2]. In this operative step, gutta-percha cones are used together with an endodontic sealer to provide an adequate sealing of the canal [3]. Although gutta-percha cones are produced under aseptic conditions, they can be contaminated by aerosols or physical sources during storage [4]. Due to their nature, they cannot undergo a wet or dry heat sterilization process [5]. Thus, the use of disinfectant solutions is suggested to carry out a chemical disinfection [4].

According to previous studies, during endodontic treatment, instruments and materials can acquire a high contamination load, which potentially increases the risk of iatrogenic contamination of the root canal [6, 7]. In addition to bacteria, which are recognized as the main etiological factor of endodontic pathologies, among the potentially pathogenic microorganisms found when the aseptic chain breaks, fungi such as Candida albicans also stand out [6]. C. albicans is a multimorphic commensal organism and opportunistic fungal pathogen that is present in persistent endodontic infections [8, 9].

Sodium hypochlorite (NaOCl) and chlorhexidine (CHX) are the solutions most used worldwide to disinfect gutta-percha cones before obturation [10–12]. Despite the reported positive results, the vast majority of studies use bacterial species in their methodology, especially Enterococcus faecalis [10, 12]. There are scarce studies evaluating the respective solutions for the disinfection of gutta-percha cones contaminated with C. albicans [13, 14]. In addition, due to limitations of NaOCl and CHX in root canal irrigation, such as high chemical instability and lack of tissue dissolution capacity, respectively, new solutions are constantly investigated [15]. Recently, the use of calcium hypochlorite [Ca(OCl)2] has been considered in Endodontics, due to its satisfactory antimicrobial action and tissue dissolution capacity [16, 17], and greater stability chemical [18]. Despite the mentioned potential, there are no studies available reporting the effectiveness of this compound in the disinfection of gutta-percha cones contaminated with C. albicans.

Therefore, this investigation aimed to compare the efficacy of 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX, in the rapid disinfection of gutta-percha cones contaminated with C. albicans. The antifungal effect was evaluated from fungicidal inhibition in an isolated format and in biofilm. The study adopted the null hypothesis that there would be no difference in the antifungal effect of the different solutions.

Materials and methods

Fungal strain and culture conditions

The Candida specie used in this study were kindly provided by the Fundação Oswaldo Cruz (FIOCRUZ, Manguinhos, Rio de Janeiro, Brazil), and is identified as follows: Candida albicans (ATCC 2508). These microorganisms were maintained on a culture medium containing glycerol and cooled to -80 °C. The sample was unfrozen, inoculated on Sabouraud dextrose broth (SDB) (KASVI®, São José dos Pinhais, Paraná, Brazil), incubated for 24 h, and seeded on Sabouraud Agar after and incubated at 37 °C for 24 h.

Determination of minimum inhibitory and minimum fungicidal concentrations

The methodology followed the study of Amorim et al. [19], with modifications. The minimum inhibitory concentration (MIC) was conceptualized as a concentration compound that was capable of inhibiting 100% of fungicidal growth (MIC100) and following the standards established by the Clinical and Laboratory Standards Institute (CLSI) M27-S4 [20] for broth microdilution assays in 96-well plates.

For the determination, the inoculums were prepared in sterile saline solution (0.9%) to obtain an absorbance between 0.08—0.10 (representing of 1.0 × 106 to 5.0 × 106 CFU/mL, 1.0 on the McFarland scale), as measured in a spectrophotometer (Multiskan FC Microplate Photometer, Thermo Fisher, Waltham, MA, USA) set at 530 nm. Yeasts were diluted again (1:50) in saline and then (1:20) in SDB, to obtain inoculums of 5.0 × 102 to 2.5 × 103 CFU/mL.

After the preparation fungal, 100 µL of Sabouraud broth was added to all wells, 100 µL of treatments (2.5% NaOCl, 2.5% Ca(OCl)2 or 2% CHX) was added to the first row of wells, and serial dilution was performed. After dilution, the inoculum containing C. albicans was added.

The plate was incubated aerobically at 37 °C for 24 h. The well containing only the inoculum and broth was considered the positive control, while the negative control was considered the well containing only the broth with saline. The test was performed with 3 replicates. To determine the minimum fungal concentration (MFC), 1 μL was collected from each well, seeded onto an agar plate with SDB, and incubated aerobically at 37 °C for 24 h. Colonies were identified, and the lowest concentration at which no microbial growth was detected was considered the MFC.

In vitro biofilm destruction and inhibition

The ability of the 2.5% NaOCl, 2.5% Ca(OCl)2 or 2% CHX to interfere with Candida spp. biofilm formation was evaluated using the adapted protocol described by Amorim et al. [19] and Lohse et al. [21]. Biomass quantification in the formed biofilm was performed using the 1.0% crystal violet technique and the absorbance reading was performed in a microplate reader (Bio-Rad 550®) at 570 nm.

Then, to evaluate the ability of the compound to reduce the preformed biofilm, the MIC and two higher concentrations (2 × MIC and 4 × MIC) were used. After incubation, the biofilm biomass was quantified using the crystal violet 1.0% technique detailed above. All assays were performed in triplicate and repeated twice. Only the C. albicans strain in SDB was considered as a positive control, and only SDB was considered as a negative control.

Counting colony forming units (CFUs)

The methodology followed the study of Subha et al. [11] with modifications. Ninety-eight gutta-percha cones (Dentsply Maillefer, Ballaigues, Switzerland) were opened in aseptic conditions to prevent contamination in the laminar flow hood. The materials were tested for disinfection according to the type of solution (2.5% NaOCl, 2.5% Ca(OCl)2 or 2% CHX), and the time of exposure to each solution (1 and 5 min).

Experimental groups were composed as follows:

  • Group A: 18 cones of gutta-percha were contaminated with C. albicans. Each group was immersed in 2.5% NaOCl for 1 min (n = 9) or 5 min (n = 9), and was treated with different methodologies: without agitation (6 cones), ultrasonic agitation (6 cones), or agitation with Easy Clean (Easy Equipamentos Odontológicos, Belo Horizonte, MG, Brazil) (6 cones).

  • Group B: 18 cones of gutta-percha were contaminated with C. albicans. Each group was immersed in 2.5% Ca(OCl)2 for 1 min (n = 9) or 5 min (n = 9), and was treated with different methodologies: without agitation (6 cones), ultrasonic agitation (6 cones), or agitation with Easy Clean (Easy Equipamentos Odontológicos).

  • Group C: 18 cones of gutta-percha were contaminated with C. albicans. Each group was immersed in 2% CHX for 1 min (n = 9) or 5 min (n = 9), and was treated with different methodologies: without agitation (6 cones), ultrasonic agitation (6 cones), or agitation with Easy Clean (Easy Equipamentos Odontológicos) (6 cones).

The gutta-percha cones were contaminated by individually immersing each one in the microtube with fungal suspension for 48 h at 37 ºC. After immersion, the cones were transferred to sterile microtubes and allowed for disinfection by immersing in the treatments for 1 or 5 min with different forms: without agitation, with ultrasonic agitation, or agitation through the Easy Clean plastic file (Easy Equipamentos Odontológicos). To control sterility and contamination, after sterilization, 2 cones were sown in a nutrient medium to certify sterility, and every 24 h 3 cones were sown to guarantee 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. Regarding the agitation procedure, the Easy Clean plastic file nº 25.04 (Easy Equipamentos Odontológicas) was attached to a handpiece at 20.000 rpm and ultrasonic agitation was performed with the E1 Irrisonic tip (Helse Ultrasonic, Ribeirão Preto, SP, Brazil) 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 h days. All test tubes were observed at 24-h intervals and visually checked for turbidity.

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 fungi were confirmed by colony morphology. The data obtained were evaluated using the Pearson chi-square test with a p-value set at < 0.05. The number of cells was determined by counting colony forming units (CFUs) following plated [22].

Reading with densitometer

The growth of fungal was followed by measuring the optical density of fungal suspension by densitometry, according to Vági et al. [23] with modifications. The optical density of the samples was measured by densitometry (Carl Zeiss Jena spectrometer, Germany) at 570 nm wavelength. The growth of the fungal is manifested in the turbidity of the suspension and can be followed by the rise of optical density. Also, a fungal control was applied without any added material in the medium.

The optical density values of the control and treatments were compared to the pure liquid medium. The optical densities of mediums containing treatment were compared to the liquid medium containing only medium in the absence of fungal. The inhibition percent is defined as the optical density of the sample (ODX) of measurement compared to the optical density of control (ODAC).

Statistical analysis

Data were submitted to the Shapiro–Wilk test and showed normal distribution. One-way analysis of variance (ANOVA) was used, followed by Tukey’s test, using the GraphPad Prism 8.0.1 software (GraphPad Software Inc, San Diego, CA, USA). Statistical significance was considered at p < 0.05.

Results

Determination of minimum inhibitory and minimum fungicidal concentrations

The MIC and MFC of 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX against C. albicans are presented in Table 1. The CHX showed the lowest values for both indices.

Table 1.

Minimum inhibitory concentration [MIC] and minimum fungicidal concentrations [MFC] of 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX against C. albicans

Solution MIC [mg/ml] MFC [mg/ml]
2.5% NaOCl 12.5 12.5
2.5% Ca(OCl)2 0.3125 0.3125
2% CHX 0.00015 0.00015
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Biofilm inhibition and destruction assay

The assay demonstrated that the 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX significantly reduced the formed biofilm and inhibited the biofilm formation. The compounds that showed the best performance were 2.5% NaOCl and Ca(OCl)2 at a concentration of 2xMIC which reduced the biofilm by 32% and 35% (p < 0.001) (Fig. 1A). Regarding inhibited biofilm, the only compound that was effective at all MIC concentrations tested was 2.5% Ca(OCl)2 at concentrations 0.5 × MIC, 0.25 × MIC and 0.125 × MIC (0.156; 0.078; 0.039 mg/ml), with biofilm inhibited by 66.8%, 62.9% and 61.2%, respectively (p < 0.0001). The result is shown in Fig. 1B.

Fig. 1.

Fig. 1

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Antibiofilm activity [A] and biofilm inhibition [B] of C. albicans 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’s test was performed. 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

Counting colony forming units

The tubes seeded were incubated and the colonies were counted. The statistics showed that in 1 or 5 min of agitation, treatment with 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX showed a significant reduction compared to saline solution (p < 0.05). The result of counting is shown in Fig. 2A and B.

Fig. 2.

Fig. 2

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Counting of viable colonies of biofilm treated with 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX against C. albicans in different agitation processes in 1 [A] and 5 min [B]. Data expressed as mean ± standard deviation. Analysis of variance [ANOVA] followed by the Tukey’s test was performed. Values of p < 0.05 were considered statistically significant [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

Reading with densitometer

The turbidity was taken as an indicator of fungal density. In every case, the growth curves were compared with those obtained in a medium with salina. The inhibitory effects of 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX at different times are summarized in Fig. 3A. There was no statistical difference between the groups NaOCl without agitation or ultrasonic agitation and Ca(OCl)2without agitation or ultrasonic agitation when compared to saline during 1 min of treatment (p > 0.05). Concerning 5 min of treatment, all compound and agitations forms were statistically different when compared to saline solution (p < 0.05). Treatments with NaOCl and Ca(OCl)2, Easyclean agitation for 1 min was more efficient than ultrasonic agitation in reducing fungal density (p < 0.05) (Fig. 3B).

Fig. 3.

Fig. 3

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Antibacterial activity of 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX against C. albicans in different agitation processes in 1 [A] and 5 min [B] through the densitometer. Data expressed as mean ± standard deviation. Analysis of variance [ANOVA] followed by the Tukey’s test was performed. Values of p < 0.05 were considered statistically significant [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

Discussion

This study was designed to evaluate the antifungal activity of 2.5% NaOCl, 2.5% Ca(OCl)2, and 2% CHX solutions in the rapid disinfection of gutta-percha cones contaminated with C. albicans. Few studies have evaluated the effect of traditional irrigants (NaOCl and CHX) for this purpose, but it is known that C. albicans is resistant to intracanal medications [8], is present when the aseptic chain breaks in Dentistry [6], and is frequently found in endodontic failures [9], representing an important microorganism to be understood. The present study found different antifungal effects against C. albicans between the solutions investigated. Thus, the hypothesis initially formulated was rejected.

Comparative studies between the antimicrobial effect of chlorinated solutions and CHX on endodontic biofilms have been widely performed throughout history, however, conflicting results have been found [24, 25]. Despite the report of positive efficacy for both solutions, chlorinated solutions seem to have a greater effect in combating microbes, through a mechanism of irreversible oxidation of cellular components in the invading cell [24], which may explain our findings in tests in culture wells. It was observed that 2.5% NaOCl, 2.5% Ca(OCl)2 and 2% CHX solutions significantly reduced the biofilm formed in cell culture wells containing C. albicans biofilm, with effect varying according to with the amount of the MIC. On the other hand, 2.5% NaOCl and 2.5% Ca(OCl)2 showed positive results in inhibiting microbial growth, while 2% CHX was not effective. Probably aforementioned mechanism contributes to the better inhibitory effect of 2.5% NaOCl and 2.5% Ca(OCl)2 against C. albicans.

When examining the obtained results, it is noted that for CHX there is no necessity to employ any agitating method for the irrigant to enhance disinfection. In both analyses (CFUs and densitometer reading) and at both application times (1 min and 5 min), the use of CHX without agitation proved to be effective in rapid disinfecting cones contaminated with C. albicans. In contrast, for the use of NaOCl, when it was used without stirring at both times of application, worse results were observed than when the solution was stirred. In the four scenarios evaluated (two analysis techniques x two times), Easy Clean (Easy Equipamentos Odontológicos) presented better results than ultrasound in two scenarios, which in turn, did not present superiority in any. As for Ca(OCl)2, use in conjunction with Easy Clean (Easy Equipamentos Odontológicos) was the only subgroup that proved to be effective in the four scenarios evaluated. A previous study also reported significantly better results for 2% CHX compared to 2.5% NaOCl [14], corroborating the findings of the present study. Furthermore, although Ca(OCl)2 has a greater amount of available chlorine and greater chemical stability than NaOCl [18], which would possibly confer a greater antimicrobial effect on the solution, results similar to NaOCl were found, indicating that chlorinated solutions may have a reduced effect against C. albicans.

Previous studies emphasize that the antimicrobial effect of irrigating solutions can be variable depending on the type of surface and its composition [26, 27], which probably explains the divergence observed in results in culture wells or gutta-percha cones. Furthermore, this assumption may partly explain the better performance of Easy Clean (Easy Equipamentos Odontológicos) in the present study, as previous studies with extracted teeth demonstrate similar results of Easy Clean (Easy Equipamentos Odontológicos) with ultrasonic agitation [28, 29]. The Easy Clean plastic file (Easy Equipamentos Odontológicos) was used in continuous rotation coupled to a handpiece at 20.000 rpm, which may have increased irrigant turbulence and better results in some scenarios. In line with this hypothesis, the use of Easy Clean (Easy Equipamentos Odontológicos) in continuous rotation appears to be better than in reciprocating movement for cleaning the root canal, as previously discovered [30]. However, studies to better elucidate the relationship between the sampling surface and the effect of irrigants on disinfection must be conducted to better understand the mechanisms involved.

A limitation of the present study is the fact that it tested the effects of the solutions on only one microorganism, and the endodontic microbiota is vast and complex [31]. However, it should be noted that C. albicans is an important microorganism in the context of endodontic pathologies, and few studies are available that have investigated the effectiveness of irrigation solutions in disinfecting gutta-percha cones contaminated with this fungus.

Conclusion

In conclusion, within the limits of this study, all tested solutions proved to be effective in the rapid decontamination of cones contaminated with C. albicans. However, it is noteworthy that CHX was the only solution that proved to be effective without any method of agitation in both analyses carried out (CFUs and densitometer reading) and at both times of application (1 and 5 min).

Authors' contributions

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.

Leonardo Quintana Soares Lopes – Data curation; Methodology; Software; Formal analysis; Validation; Writing—review & editing.

Carlos Alexandre Souza Bier—Conceptualization; Funding acquisition; Investigation; Methodology; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing—review & editing.

Patrícia Kolling Marquezan—Conceptualization; Investigation; Methodology; Formal analysis; Project administration; Resources; Software; Supervision; Validation; Visualization; Writing—original draft; Writing—review & editing.

Funding

This study was partly funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) “Grant number 001”.

Data availability

Data available on request from the authors.

Declarations

Ethical approval

Not applicable.

Informed consent

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Data Availability Statement

Data available on request from the authors.

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