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 Table of Contents  
Year : 2019  |  Volume : 31  |  Issue : 1  |  Page : 13-16

The effectiveness of calcium hydroxide paste mixed with 2% chlorhexidine solution against Candida albicans-infected human roots: An ex vivo study

Department of Restorative Dental Sciences, College of Dentistry, King Saud University, Riyadh, Saudi Arabia

Date of Web Publication19-Jun-2019

Correspondence Address:
Fahd Alsalleeh
Department of Restorative Dental Sciences, College of Dentistry, King Saud University, Riyadh
Saudi Arabia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/endo.endo_127_18

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It is known that Candida albicans is involved in the etiology of persistent periradicular lesions.
Aims: The aim of this study was to determine the antifungal activity of calcium hydroxide (Ca[OH]2), paste (Ultradent Products Inc., South Jordan, UT, USA) mixed with 2% chlorhexidine against C. albicans.
Subjects and Methods: Fifty extracted human single-rooted teeth were selected. The teeth were standardized and instrumented with K3™ Nickel–Titanium rotary files (Kerr Dental). Teeth were then infected with C. albicans, except negative controls. After 7 days, teeth were randomly divided into the following groups according to intracanal medicament protocols (n = 10): Ca(OH)2 mixed with 2% chlorhexidine, Ca(OH)2 mixed with normal saline, Ca(OH)2 alone, or 2% chlorhexidine alone. Normal saline alone was used as positive controls. All teeth were incubated at 37°C and 100% humidity for a week. C. albicans growth was recorded. Pairwise comparison and Tukey's analysis of mean differences were conducted with the significance level set at the 0.05 level.
Results: All samples of the positive controls, Ca(OH)2 alone and Ca(OH)2 mixed with the saline groups showed C. albicans growth (100%). Ca(OH)2 mixed with chlorhexidine showed 70% growth, whereas chlorhexidine alone group showed 60% growth. The negative controls had no growth.
Conclusions: A mixture of Ca(OH)2 and 2% chlorhexidine had better antifungal activity against C. albicans in human-extracted teeth compared to that of Ca(OH)2 alone.

Keywords: Candida albicans, intracanal medicaments, root canal disinfection

How to cite this article:
Alsalleeh F, Almohaimeed A, Almoqbel A, Allahem Z. The effectiveness of calcium hydroxide paste mixed with 2% chlorhexidine solution against Candida albicans-infected human roots: An ex vivo study. Endodontology 2019;31:13-6

How to cite this URL:
Alsalleeh F, Almohaimeed A, Almoqbel A, Allahem Z. The effectiveness of calcium hydroxide paste mixed with 2% chlorhexidine solution against Candida albicans-infected human roots: An ex vivo study. Endodontology [serial online] 2019 [cited 2020 Nov 28];31:13-6. Available from: https://www.endodontologyonweb.org/text.asp?2019/31/1/13/260698

  Introduction Top

Fungi are component of the normal flora of human oral cavity, becoming pathogenic when there are local or systemic factors predisposing the individual to infection. Candida albicans is considered the most frequent commensal pathogenic yeast in the oral cavity.[1] It has been reported that 21% of infected root canals contain C. albicans.[2] A pure culture of C. albicans has been found to be the causative factor of an acute apical abscess.[3] C. albicans, Enterococcus faecalis, and Actinomyces are the most prevalent microorganisms associated with persistent secondary endodontic infections that do not respond to current root canal therapy.[4],[5],[6],[7] These reports support that C. albicans are involved in the etiology of persistent periradicular lesions. There are multiple mechanisms involved in the pathogenicity of C. albicans. Adaptation and adhesion to a variety of surfaces, including dentine and root surfaces, are believed to be a crucial virulence factor.[5],[6],[8]

Many studies have evaluated the effectiveness of different intracanal medications against C. albicans, including, calcium hydroxide (Ca[OH]2) paste and chlorhexidine.[9],[10] Due to the high pH value, Ca(OH)2 is considered an effective anti-bacterial agent in addition to its ability to release hydroxyl ions to denatures proteins. Therefore, Ca(OH)2 can effectively eliminate the most endodontics pathogens when used as intracanal medicaments.[9] However, it has been shown that C. albicans were resistant to Ca(OH)2 and additional medications were required to increase the effectiveness of anti-fungal activity.[11],[12] Few investigators had tried to improve the effectiveness of Ca(OH)2 against C. albicans. It was reported that mixing Ca(OH)2 with chlorhexidine was more effective to eliminate C. albicans. In a study that used agar plates model, it was found that the antifungal activity of Ca(OH)2 was evident only during the first 24 h, while no colonies were seen in the presence of 2% chlorhexidine with the same concentration even after 3 days.[13] The chlorhexidine solution alone or mixed with Ca(OH)2 was able to completely inhibit the growth of C. albicans in an in vitro study.[14] However, these studies were not conducted in infected root canals models. It is well-known that the majority of intracanal disinfecting agents are able to kill even the resistant microbes when tested in vitro in a test tube, yet the effectiveness of the same agents is clearly weaker in the in vivo conditions.[15]

The aim of this study is to investigate the antifungal activity of Ca(OH)2 paste mixed with 2% chlorhexidine solution using human-extracted single-rooted teeth.

  Subjects and Methods Top

Candida albicans and growth conditions

The C. albicans wild-type ATCC: 10231 strain was grown in yeast nitrogen base medium from fresh Sabouraud dextrose agar plates (Difco Laboratories, Detroit, MI) and incubated for 24 h at 37°C in a shaker at 60 rpm. Cells were harvested and washed three times with phosphate buffered saline (PBS). Cells were resuspended in 10 mL PBS, counted after serial dilution, standardized, and used within 24 h.

Sample selection and preparation

Fifty extracted human single-rooted teeth, with an unknown history, were collected for this study. The study was approved by the Institutional Clinical Research Ethics Board (IRB #E-1Z-26Z6., CDRC #0249). Teeth with straight roots, fully developed apices, and sound or minimally carious were included. Radiographs were taken to confirm that each tooth has a single canal. Teeth were sectioned above the cementoenamel junction to standardize the length (21 mm), standardized access cavity was made, and all canals received a standardized cleaning and shaping protocol. The working length was established with a #10/0.02 K-file (Dentsply Maillefer, Switzerland) to the apex subtracting 1 mm from the length. A glide path was completed to a #25/0.02 K-file. Shaping the canal was accomplished using K3™ Nickel–Titanium rotary files (Kerr Dental) reaching size #35/0.06. The apical part was sealed with a flowable composite to prevent the extrusion of solutions, and root surface contamination and root surfaces were covered with nail polish except the access cavity. Teeth were autoclaved and stored into the sterile saline solution.

After that, all root canals were infected with C. albicans at 1 × 106 cells/mL for 1 week and allowed to incubate aerobically at 37°C for 1 week. Root canals rinsed with sterile 0.85% saline solution and dried with sterile paper points. The growth of C. albicans was confirmed in all samples using paper point samples on the agar plate. Furthermore, representative samples were subjected and prepared for a scanning electron microscopy as previously described.[16],[17] Five teeth were not infected with C. albicans and served as a negative control (Group A).

Calcium hydroxide preparations and dilutions

A saturated solution of UltraCal XS Ca(OH)2 (Ultradent Products Inc, South Jordan, UT, USA) paste was purchased. A single dilution was used at a ratio of (1:1) with either saline of chlorhexidine. The mixtures were stirred for 4 h at room temperature then centrifuged at 3000 rpm for 15 min to clarify the solutions, and the aqueous supernatant layers were filtered by using a sterile 25-mm syringe filter (Fisher Scientific, Newark, DE, USA) (Sabrah et al. 2013). Furthermore, the pH of each preparation will be measured using Jenway 3540 pH and conductivity meter (UK).

Intracanal medicaments protocols

Infected teeth were randomly distributed according to the following experimental groups:

  • Group B: Saline (positive control, n = 5)
  • Group C: Chlorhexidine alone (n = 10)
  • Group D: Ca(OH)2 alone (n = 10)
  • Group E: Ca(OH)2 mixed with 2% of chlorhexidine solution at a ratio of 1:1 (n = 10)
  • Group F: Ca(OH)2 mixed with saline solution as a ratio of 1:1 (n = 10).

The prepared medicaments then were introduced into each canal using the NaviTip needles (30 g; 25-mm long) placed as close as possible to the working length until the paste is seen within the canal orifice access cavity. The access was sealed with Cavit and specimens were stored in an incubator at 37°C and 100% humidity.

Sample processing and evaluation

After 1 week, canals were irrigated with 1 mL 0.5% citric acid to terminate the effect of Ca(OH)2. Then, all the canals were flushed with 1 mL sterile saline. A sterile H-file size 25 used to stroke it against the wall of the canals and agitate the fluid. All teeth were then flushed with 15 mL of sterile saline, and the canals were dried with sterile absorbent paper points. A small amount of saline solution was introduced into canals, and new sterile paper points were introduced and left inside for 30 s. Paper points were transferred and plated on fresh Sabouraud 4% dextrose agar (Difco Laboratories, Detroit, MI, USA). The plates were incubated at 37°C for 48 h and evaluated for the growth of C. albicans visually. Pairwise comparison and Tukey's analysis of mean differences were conducted with significance level set at the 0.05 level. Data were calculated using the SPSS software version 18.0 (SPSS Inc., Chicago, USA).

  Results Top

Before conducting the experiment, the scanning electron microscopy had confirmed a rich biofilm formation in the root canal system [Figure 1] and all infected canals showed C. albicans growth. The negative control group had no C. albicans growth. The chlorhexidine alone and Ca(OH)2 mixed with chlorhexidine had six and seven samples C. albicans growth, respectively. The difference was not significant between these two Groups (P = 0.687). All samples of the positive controls, Ca(OH)2 alone or mixed with saline showed C. albicans growth, and the difference was statistically significant compared to chlorhexidine alone and Ca(OH)2 mixed with chlorohexidine (P = 0.036). The number of samples in each group tested for C. albicans growth are shown in [Table 1]. The pH readings of different solutions used in the study are listed in [Table 2].
Figure 1: Standard error of mean analyses at accelerating voltage of 10 KV of Candida albicans biofilm formation covering intracanal wall before the application of materials tested. Scale bar = 100 μm

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Table 1: Number of samples tested positive for Candida albicans growth

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Table 2: The pH readings of all materials tested

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  Discussion Top

The antimicrobial effect of Ca(OH)2 has been studied extensively with different outcomes. Early studies reported high success with the use of Ca(OH)2. In general, intracanal medicaments and disinfecting agents used in endodontic therapy are very effective to inhibit bacteria and yeasts using in vitro models; agar plates and tubes. However, these agent when used in ex vivo models behaves poorly.[15] Therefore, the current study attempted to address this issue. It has been shown that C. albicans were resistant to Ca(OH)2 and additional medications were required to increase the effectiveness of anti-fungal activity.[11] Chlorhexidine has been shown to be an effective alternative agent against C. albicans.[18] The present study aimed to analyze the effectiveness of Ca(OH)2 against C. albicans when mixed with chlorhexidine using extracted human teeth. Previous reports had examined this mixture on Sabouraud's dextrose agar plates model.[14],[19]

The need for ideal intracanal medicaments in endodontics continued to by the dream and ultimate goal for many endodontic researchers. Ca(OH)2 have been shown to be an effective intracanal medicaments against primary endodontic infection. Recently, it had gain more popularity to be used during endodontic regeneration. Therefore, the current study was an attempt to test the suitability of mixing Ca(OH)2 with 2% chlorhexidine liquid against persistent C. albicans infecting root canals. Results herein indicated that the antifungal activity of Ca(OH)2 mixed with chlorhexidine was far better than Ca(OH)2 alone. This was consistent with the previous studies.[13] It was hypothesized that such combinations may lessen the antifungal activity of chlorhexidine due to the alkalizing effect of Ca(OH)2.[20] In the present study, the pH of the mixture of Ca(OH)2 and chlorhexidine was clearly altered compared to that of chlorhexidine alone. However, chlorhexidine alone or mixed with Ca(OH)2 had the same effect against C. albicans infection. Similar results were reported that a combination of Ca(OH)2 with chlorhexidine at a ratio 1:1 had shown to decrease enterococcus infected root canals.[21] However, it appeared C. albicans infecting root canals may able to sustain or resist the high pH environment of Ca(OH)2 alone. Furthermore, the negative impact of dentin on intracanal medicaments had been confirmed.[22]

Finally, the current study had utilized C. albicans biofilm model to test the antifungal activity of intracanal medicaments used. Microbial biofilms in the root canal system had been shown to reduce or inactivate intracanal medicaments.[23] Therefore, the research should continue to develop more effective intracanal medicaments against biofilms.

  Conclusions Top

Under the condition of the present study, a mixture of Ca(OH)2 and 2% chlorhexidine at ratio 1:1 improved the antifungal activity against C. Albicans in human-extracted teeth. The need to develop more potent and effective intracanal medicaments continues and must be tested in simulated root canal system and/or clinical trials.


The authors would like to thank Dr. Ahmed Albrag for providing Candida albicans strain. Also, Sara AlAmeer (Lab technician), Dr. Abdualrahman Niazi for laboratory assistances.

Financial support and sponsorship

This study was supported by Prince Naif bin Abdul Aziz Health Research Center.

Conflicts of interest

There are no conflicts of interest.

  References Top

Conti HR, Gaffen SL. Host responses to Candida albicans: Th17 cells and mucosal candidiasis. Microbes Infect 2010;12:518-27.  Back to cited text no. 1
Baumgartner JC, Watts CM, Xia T. Occurrence of Candida albicans in infections of endodontic origin. J Endod 2000;26:695-8.  Back to cited text no. 2
Matusow RJ. Acute pulpal-alveolar cellulitis syndrome. III. Endodontic therapeutic factors and the resolution of a Candida albicans infection. Oral Surg Oral Med Oral Pathol 1981;52:630-4.  Back to cited text no. 3
Waltimo TM, Sen BH, Meurman JH, Ørstavik D, Haapasalo MP. Yeasts in apical periodontitis. Crit Rev Oral Biol Med 2003;14:128-37.  Back to cited text no. 4
Siqueira JF Jr., Sen BH. Fungi in endodontic infections. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:632-41.  Back to cited text no. 5
Rôças IN, Hülsmann M, Siqueira JF Jr. Microorganisms in root canal-treated teeth from a German population. J Endod 2008;34:926-31.  Back to cited text no. 6
Miranda TT, Vianna CR, Rodrigues L, Monteiro AS, Rosa CA, Corrêa A Jr. Diversity and frequency of yeasts from the dorsum of the tongue and necrotic root canals associated with primary apical periodontitis. Int Endod J 2009;42:839-44.  Back to cited text no. 7
Gomes C, Fidel S, Fidel R, de Moura Sarquis MI. Isolation and taxonomy of filamentous fungi in endodontic infections. J Endod 2010;36:626-9.  Back to cited text no. 8
Delgado RJ, Gasparoto TH, Sipert CR, Pinheiro CR, de Moraes IG, Garcia RB, et al. Antimicrobial activity of calcium hydroxide and chlorhexidine on intratubular Candida albicans. Int J Oral Sci 2013;5:32-6.  Back to cited text no. 9
Sen BH, Safavi KE, Spångberg LS. Antifungal effects of sodium hypochlorite and chlorhexidine in root canals. J Endod 1999;25:235-8.  Back to cited text no. 10
Waltimo TM, Sirén EK, Orstavik D, Haapasalo MP. Susceptibility of oral Candida species to calcium hydroxide in vitro. Int Endod J 1999;32:94-8.  Back to cited text no. 11
Mohammadi Z, Shalavi S. Is chlorhexidine an ideal vehicle for calcium hydroxide? A microbiologic review. Iran Endod J 2012;7:115-22.  Back to cited text no. 12
Stavileci M, Hoxha V, Bajrami D, Dragidella A. Analysis of the effects of calcium hydroxide, chlorhexidine and mineral trioxide aggregate on the viability of Candida albicans. Dentistry 2013;3:3.  Back to cited text no. 13
Al-Nazhan S, Al-Obaida M. Effectiveness of a 2% chlorhexidine solution mixed with calcium hydroxide against Candida albicans. Aust Endod J 2008;34:133-5.  Back to cited text no. 14
Haapasalo M, Qian W, Portenier I, Waltimo T. Effects of dentin on the antimicrobial properties of endodontic medicaments. J Endod 2007;33:917-25.  Back to cited text no. 15
Alsalleeh F, Young A, Algarawi Z, Petro T. C. albicans biofilm formation is restricted by periodontal ligament cells and induces differential cytokines response compared to planktonic C. albicans. J Dent Res 2014;1:139-44.  Back to cited text no. 16
Alsalleeh F, Williams S, Jaber H. Interaction of Candida albicans with periodontal ligament fibroblasts limits biofilm formation over elastomer silicone disks. Arch Oral Biol 2016;63:47-52.  Back to cited text no. 17
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Mohammadi Z, Jafarzadeh H, Shalavi S, Sahebalam R, Kinoshita JI. Additive and reducing effects between calcium hydroxide and current irrigation solutions. J Contemp Dent Pract 2017;18:246-9.  Back to cited text no. 20
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  [Figure 1]

  [Table 1], [Table 2]


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