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ORIGINAL ARTICLE
Year : 2017  |  Volume : 7  |  Issue : 2  |  Page : 87-91

Evaluation of efficacy of chitosan-silver nanocomposite on Candida albicans when compared to three different antifungal agents in combination with standard irrigation protocol: An ex vivo study


Department of Conservative Dentistry and Endodontics, Sree Mookambika Institute of Dental Sciences, Kanyakumari, Tamil Nadu, India

Date of Web Publication25-Apr-2017

Correspondence Address:
Betty Babu
Sree Mookambika Institute of Dental Sciences, Kulasekharam, Kanyakumari - 629 161, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1658-5984.205124

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  Abstract 

Aims: This study aims to evaluate the efficacy of chitosan-silver nanocomposite on Candida albicans when compared to three different antifungal agents in combination with standard irrigation protocol.
Materials and Methods: Fifty experimental teeth were biomechanically prepared and inoculated with suspension of C. albicans. At the end of 96 h, teeth were divided into five experimental groups. The groups were treated with respective irrigating solutions for 1 min and compared to the antifungal. An inoculation loop was used to remove aliquots from the fluid and was plated on 4% Sabouraud Dextrose Agar and incubated for 48 h. After incubation, the growth of C. albicans was assessed with light microscopy at ×400.
Statistical Analysis Used: The data were statistically analyzed using one-way ANOVA. Post hoc followed by Dunnet t-test.
Results: Colony forming unit (CFU) was determined for all five groups. One percent clotrimazole and chitosan-silver nanocomposite showed complete inhibition in all the samples. Control group (5.25% sodium hypochlorite, 17% ethylenediaminetetraacetic acid, and 0.9% saline), 0.2% fluconazole and 0.2% amphotericin B showed complete inhibition in 8 samples and reduced CFU in two samples. 0.2% fluconazole showed better inhibition of C. albicans compared to control group and 0.2% amphotericin B.
Conclusions: Chitosan-silver nanocomposite as an endodontic irrigant can inhibit the growth of C. albicans in combination with standard irrigation protocol.

Keywords: Antifungal agents, Candida albicans, chitosan-silver nanocomposite, irrigants, nanoparticle


How to cite this article:
Babu B, Nair RS, Angelo JM, Mathai V, Vineet R V. Evaluation of efficacy of chitosan-silver nanocomposite on Candida albicans when compared to three different antifungal agents in combination with standard irrigation protocol: An ex vivo study. Saudi Endod J 2017;7:87-91

How to cite this URL:
Babu B, Nair RS, Angelo JM, Mathai V, Vineet R V. Evaluation of efficacy of chitosan-silver nanocomposite on Candida albicans when compared to three different antifungal agents in combination with standard irrigation protocol: An ex vivo study. Saudi Endod J [serial online] 2017 [cited 2017 Aug 17];7:87-91. Available from: http://www.saudiendodj.com/text.asp?2017/7/2/87/205124


  Introduction Top


Endodontic infections are polymicrobial in nature with predominance toward anaerobic species. Among the microorganisms, fungi may play an important role in the failed endodontic treatment.[1] The most important oral fungi belong to genus Candida with Candida albicans being the most predominant.[2] The incidence of C. albicans in the oral cavity is 30% to 45% in healthy adults and 95% of patients infected with human immunodeficiency virus. C. albicans is a dimorphic fungus that exists in many morphologic forms such as germ tubes, blastospores, pseudohyphae, true hyphae, and chlamydospores. Each form of growth depends on environmental conditions such as the pH level, temperature, and nutritional source. C. albicans can grow on the dentinal surfaces in the absence of oral tissue fluids and penetrate into the dentinal tubules by its various growth patterns.[3] The presence of C. albicans in the contents of infected root canals and aspirates of cellulitis/abscesses of endodontic origin is 21%.[4] The transition of C. albicans from a harmless commensal to a pathogenic organism appears to be dependent on minor changes in predisposing conditions that cause the expression of a variety of virulence.[5]

The goal of endodontic treatment is to achieve the disinfection of root canals by the eradication of microbes and prevention of reinfection. Although mechanical instrumentation can reduce bacterial population, effective elimination of bacteria cannot be achieved without the use of antimicrobial root canal irrigation and medication.[6] Endodontic irrigants are used for the removal of inflamed and necrotic tissue, microbes/biofilms from the root canal space.[7]

Sodium hypochlorite (NaOCl) is one of the most popular and widely used endodontic irrigants. Studies prove that 5.25% NaOCl has been widely used for many years, and it prevails as the golden standard today.[8],[9] Ethylenediaminetetraacetic acid (EDTA) is a chelating agent, removes smear layer by chelating the inorganic component of the dentin.[10] Normal saline accomplishes gross debridement and lubrication.

Chitosan, a natural polysaccharide, is a derivative of chitin, which is commonly found in shells and exoskeletons of some crustacean and is the second most abundant biopolymer.[11] Chitosan exhibits a broad spectrum of antimicrobial activity. Silver nanoparticles have a wide range of antimicrobial activities and exhibit high performance even at a very low concentration.[12] In chitosan-silver nanocomposite, chitosan stabilizes and prolongs the action of silver.[13]

In this study, four antifungal agents have been used to assess the antifungal efficacy against C. albicans. There is limited evidence of researches regarding chitosan-silver nanocomposite as an antifungal irrigant. Hence, the study hypothesis was that chitosan-silver nanocomposite has better antifungal efficacy against C. albicans.


  Materials and Methods Top


Tooth preparation

Freshly extracted fifty single-rooted mandibular premolars extracted for orthodontic purpose and which were free of cracks, caries, restorations, resorptive defects, and open apices were chosen using a dental operating microscope (Pico, Carl Zeiss, Germany) at ×20 magnification. The ethical clearance for the study protocol was obtained from Institutional Ethical Committee. The teeth were stored in 0.2% sodium azide (Choice Organochem, Mumbai, India) and used within 3 months of extraction.

The teeth were decoronated at the cementoenamel junction using the diamond disc (SS White, NJ, USA) and pulpal remnants was extirpated using barbed broaches (Dentsply, Maillefer, Switzerland). A 25 mm size 15 stainless steel K-file (Mani Inc., Utsunomiya, Japan) was inserted into the root canal until it was seen at the apical foramen. Working length was obtained by subtracting 1 mm from this length. Gates Glidden Drills 1–4 (Mani Inc., Utsunomiya, Japan) was used for coronal flaring, and apical preparation was done using step back technique until International Organization for Standardization size 50. Between each filing 3 mL of 5.25% NaOCl (Novo Dental Products, Mumbai, India) was used. After instrumentation, the removal of smear layer was accomplished with a final rinse of 1 mL of 17% EDTA (Desmear, Anabond Stedman Pharma Research, Tamil Nadu, India) for 1 min followed by 3 mL of 5.25% NaOCl. Finally, the canals were flushed with 5 mL of saline (0.9% w/v, Nirlife, Nirma Ltd., Ahmedabad, India) to remove any debris and residual irrigants. The roots were coated with two coats of nail varnish and apical foramen sealed with Type II GIC (GC). Subsequently, the roots were sterilized in an autoclave for 15 min, at 121°C and 15 lb pressure.

Inoculation of fungi into the samples

A suspension of ATCC 10231 strains of C. albicans was adjusted to 0.5% turbidity on the McFarland scale. The canals of the experimental teeth were cautiously inoculated with 0.5 mL of the freshly prepared suspension. The samples were stored and incubated at 37°C and 91% humidity for 96 h. Every 24 h, the vials containing the experimental teeth were replenished with the freshly prepared suspension of C. albicans (ATCC, Himedia Laboratories, Mumbai, India). At the end of 96 h, teeth were removed and then subjected to the experimental groups of 10 teeth each.

Synthesis of chitosan-silver nanocomposite

The chitosan suspension was prepared by solubilizing chitosan (1.0 g) (Sigma-Aldrich, Karnataka, India) in acetic acid (50 mL, 1.0 wt %) (High Purity laboratory chemicals, Mumbai, India) solution. Then, AgNO3 (50 mL, 0.01 M) (High Purity laboratory chemicals Pvt. Ltd., Mumbai, India) was added immediately into the suspension under constant stirring for 2.0 h for the preparation of the AgNO3 in chitosan suspension. NaBH4(20 mL, 0.04 M) (Sisco research laboratories, Mumbai, India) was added to the suspension of AgNO3/Cts, and an immediate color change from pale yellow to brown indicating the formation of silver nanoparticles was noted.

  • Group I: 5.25% NaOCl + 17% EDTA + 0.9% saline
  • Group II: 5.25% NaOCl + 17% EDTA + chitosan-silver nanocomposite (20% Ag) +0.9% saline
  • Group III: 5.25% NaOCl + 17% EDTA + 0.2% fluconazole (Cipla Pvt Ltd., Kashipur, India) +0.9% saline
  • Group IV: 5.25% NaOCl + 17% EDTA + 1% clotrimazole (Glenmark Pharmaceuticals Ltd., Mumbai, India) +0.9% saline
  • Group V: 5.25% NaOCl + 17% EDTA + 0.2% amphotericin B (United Biotech Pvt. Ltd., Solan, Himachal Pradesh, India) +0.9% saline.


The time of contact of each irrigant was 1 min and final flush with 5 mL of distilled water. The antifungal agents were injected into the root canal and the time of contact was 5 min; after which, the sample was flushed with 5 mL of distilled water to prevent carryover of irrigants. All experimental teeth were flushed with 15 mL of sterile saline and canals were dried with sterile absorbent paper points (Dentsply, Maillefer, Switzerland). A small amount of saline solution was introduced into the canal, and then an endodontic hand file was used in a filing motion. A 1 μm inoculation loop was used to remove aliquots from the fluid and was plated on 4% Sabouraud Dextrose Agar (Himedia Laboratories, Mumbai, India). The plates were incubated at 36°C and 91% humidity for 48 h. After the incubation period, the growth of C. albicans was assessed with light microscopy at ×400. The number of colony forming units (CFUs) of Candida serves as a measure of the antifungal activity.


  Results Top


Group II (chitosan-silver nanocomposite) and Group IV (1% clotrimazole) showed better antifungal efficacy when compared to other antifungal agents. Complete inhibition of growth of C. albicans was shown by Group II and Group IV. CFUs was shown by Group I (control), Group III (0.2% fluconazole), and Group V (0.2% amphotericin B) proved their lesser efficacies in inhibition of growth of Candida as shown in [Table 1].
Table 1: Multiple comparison of antifungal activity of different groups

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  Discussion Top


Knowledge regarding the nature of endodontic microbiota depends on the recognition of those microorganisms present in the root canal system of teeth with necrotic pulp and failed endodontic treatment. Previous studies proved that bacteria was the root cause of endodontic disease and therefore advocated to sterilize the root canal by thorough instrumentation, irrigation, intracanal medications, and finally obturation.[1]

A single irrigant cannot successfully fulfill all the requirements, a minimum of two irrigation solutions are required to optimize the effect of irrigation. In this study, 5.25% NaOCl and 17% EDTA followed by 0.9% normal saline have been used as routine root canal irrigants. In this study, a contact time of 5 min was taken as the standard time for all the irrigants. According to current literature, NaOCl is the most commonly used irrigation solution in the concentration range of 0.5–6%. It has an excellent antimicrobial and tissue dissolution properties.[14]

Due to the presence of the C. albicans in 7–18% cases of persistent apical periodontitis and used extensively in endodontic research because it was detected in 30–45% of posttreatment diseases and due to high level of resistance to a wide range of antimicrobial agents.[15],[16] ATCC 10231 strains of C. albicans which have been the standard strain used in the previous in vitro studies was selected for present study to determine the efficacy of endodontic irrigants.

In this study, Group II (chitosan-silver nanocomposite) and Group IV (1% clotrimazole) showed better antifungal efficacy when compared to other antifungal agents. Complete inhibition of growth of C. albicans was shown by Group II and Group IV. Chitosan-silver nanocomposite (Group II) showed complete inhibition of growth of C. albicans and did not show significant difference with and Group IV (1% clotrimazole). Chitosan is a derivative of chitin, works by binding to the negatively charged bacterial cell wall followed by attachment to the DNA, inhibiting its replication. The introduction of silver into bacterial cells induces a high degree of structural and morphological changes, which can lead to cell death.[13] Akmaz et al. evaluated the antibacterial properties and characterization of chitosan-silver nanocomposite materials. They concluded that antibacterial effect of chitosan-silver nanoparticle materials was increased by increasing Ag amount of the composite materials. The presence of small amount of metal nanoparticles in the composite was enough to significantly enhance antibacterial activity as compared with pure chitosan.[17] Vimala et al. evaluated the antimicrobial and antifungal activity of the chitosan-polyvinyl alcohol silver nanoparticle films and had demonstrated significant effects against Escherichia coli, Pseudomonas, Staphylococcus, Micrococcus, C. albicans, and Pseudomonas aeruginosa. To improve further their therapeutic efficacy as antimicrobial agents, curcumin encapsulated chitosan-polyvinyl alcohol silver nanocomposite films were developed which showed enormous growth inhibition of E. coli compared to curcumin and chitosan-polyvinyl alcohol silver nanoparticles film alone.[18]

One percent clotrimazole (Group IV) irrigated specimens showed complete inhibition of growth of C. albicans and showed similar antifungal effect with Group II. Clotrimazole, an imidazole derivative works to kill individual Candida or fungal cells by altering the permeability of fungal cell wall. Wadhwani et al. evaluated the antifungal efficacy of 5.25% NaOCl, 2% chlorhexidine gluconate, and 17% EDTA as final irrigant with and without the inclusion of an antifungal agent that is 1% clotrimazole on C. albicans. They concluded that 5.25% NaOCl exhibited superior antifungal efficacy compared to 2% chlorhexidine gluconate and 17% EDTA. On inclusion of 1% clotrimazole, there was a significant decrease in CFUs. 5.25% NaOCl and 2% chlorhexidine gluconate with clotrimazole showed significantly greater antifungal properties than 17% EDTA with clotrimazole.[19]

Group I (control), 0.2% fluconazole (Group III) and 0.2% amphotericin B (Group V) irrigated specimens did not show a significant difference in inhibition of growth of C. albicans and these groups showed less efficacy compared to Group II and Group IV. However, when comparing Group III with Group I and Group V, 0.2% fluconazole showed more antifungal activity than 0.2% amphotericin B and Group I (control). Fluconazole inhibits the fungal cytochrome P450 enzyme 14α-demethylase. Amphotericin B exerts its antifungal effect by disruption of fungal cell wall synthesis. Ignatius and Pradeep evaluated the efficiency of NaOCl and four other intracanal medicaments in eliminating the C. albicans in the root canal system. They concluded that irrigation with 2.5% NaOCl was effective in 90% of the samples, followed in decreasing order of effectiveness by amphotericin B powder and distilled water (80% effectiveness), ZnO powder and 2% CHX (70% effectiveness), Ca (OH)2 powder and 2% CHX (60% effectiveness), Ca (OH)2 powder and saline (50% effectiveness), and saline + no intracanal medication.[20] Dumani et al. antimicrobial activity of 4 antibiotic agents (for Enterococcus faecalis) and 4 antifungal agents (for C. albicans) by agar dilution method. They concluded that for C. albicans, 18 isolates were susceptible to amphotericin B, nystatin, fluconazole but showed resistance to Ketoconazole.[21]

The constant increase in antibiotic resistant strains and side effects caused by synthetic drugs has prompted researchers to look for nanoformulations. The use of chitosan-silver nanocomposite as a root canal irrigant might prove to be advantageous considering the several undesirable characteristics of standard irrigants and other frequently used antimicrobials. Further research is warranted to conclusively recommend nanoformulations as a root canal irrigant because of the staining property of silver nanoparticles used in this nanocomposite.


  Conclusions Top


Within the limitations of this study, we concluded that chitosan-silver nanocomposite (20% Ag) and 1% clotrimazole showed complete inhibition when compared to control (5.25% NaOCl + 17% EDTA + 0.9% Saline), 0.2% fluconazole and 0.2% amphotericin B which showed lesser efficacy in the inhibition of growth of C. albicans when used in conjunction with standard irrigation protocol. With ever increasing resistance, adverse drug reactions, application difficulties of synthetic drugs and typical features of C. albicans, chitosan-silver nanocomposite can be an alternative option provided all the ideal properties of an irrigant are satisfied.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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