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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 10  |  Issue : 2  |  Page : 51-56

Elevation of the resistance of heat-cured acrylic denture base resin against biofilm-forming Candida albicans by incorporating Amphotericin B or Clotrimazole


1 Department of Microbiology, College of Medicine, University of Karbala, Karbala, Iraq
2 Department of Prosthodontics, College of Dentistry, University of Karbala, Karbala, Iraq

Date of Web Publication10-Sep-2018

Correspondence Address:
Ali Abdul Hussein S. AL-Janabi
Department of Microbiology, College of Medicine, University of Karbala, Karbala
Iraq
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jorr.jorr_36_17

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  Abstract 


Aim: The ability of antifungal agents to reduce or eliminate biofilm formation by Candida albicans after incorporating heat-cured acrylic denture base materials was investigated.
Materials and Methods: Different concentrations of Amphotericin B (AmB) and Clotrimazole (CT) were incorporated into polymethylmethacrylate (PMMA) specimens (10 mm × 10 mm × 2 mm). C. albicans was stimulated to produce biofilms on the surface of specimens. Biofilm count was determined by crystal violet and transmittance percentage assays. Porosity percentage of PMMA specimens was measured.
Results: AmB and CT showed more effective action against C. albicans biofilms at low concentrations (1, 5, and 10 μg/ml). Meanwhile, high concentrations (25 and 50 μg/ml) showed less activity. Porosity percentage was decreased in PMMA containing low concentrations of both antifungal agents.
Conclusion: Incorporation of AmB and CT into denture materials has a significant inhibitory effect on the biofilm produced by C. albicans, especially at low concentrations. Decrease in porosity level is another advantage evidenced by incorporating low concentrations of AmB and CT within denture.

Keywords: Amphotericin B, candidiasis, Clotrimazole, denture


How to cite this article:
S. AL-Janabi AA, Abdulkareem MM. Elevation of the resistance of heat-cured acrylic denture base resin against biofilm-forming Candida albicans by incorporating Amphotericin B or Clotrimazole. J Oral Res Rev 2018;10:51-6

How to cite this URL:
S. AL-Janabi AA, Abdulkareem MM. Elevation of the resistance of heat-cured acrylic denture base resin against biofilm-forming Candida albicans by incorporating Amphotericin B or Clotrimazole. J Oral Res Rev [serial online] 2018 [cited 2018 Nov 18];10:51-6. Available from: http://www.jorr.org/text.asp?2018/10/2/51/240923




  Introduction Top


Denture is a prosthesis structure used by most elderly individuals to fill the gap resulting from teeth loss. It is considered a suitable solid surface for the yeast of oral normal flora to produce a biofilm. This type of growth can stimulate an inflammatory reaction in the denture-bearing mucosa called denture stomatitis, which is found in about 11%–67% of denture wearers.[1],[2],[3] Although denture stomatitis is caused by a multifactorial etiology, Candida albicans is recorded as the most common causative factor.[1],[2],[4],[5] Other factors can increase the development of the disease such as the poor oral cavity or denture hygiene, trauma, low pH under prosthesis, or the presence of a sufficient concentration of sugar and iron.[1],[2],[3],[6] C. albicans has greater ability to produce biofilms on the denture surface in plaque form than it produces on the tooth surface.[6] Biofilm production on the denture is mainly dependent on several factors, including adherence ability, interaction with oral commensal bacteria, redox potential of the site, and surface properties of acrylic resin.[4]

Prevention or elimination of C. albicans to form biofilm on denture surface is the first priority to limit the development of denture stomatitis or dental caries. Several compounds have been evaluated against C. albicans growth, including chemical materials such as with silver nanoparticles,[7] chlorhexidine,[8],[9],[10] natural products such as with alpha-Mangostin,[11] or standard antifungal agents such as polyene group and some of azole group.[2],[3],[12],[13] Amphotericin B (AmB), as one of the polyene group elements, and clotrimazole (CT), as one of the azole group elements, are usually used in a different pharmaceutical formula such as solution, cream, ointment, jelly, tissue conditioner, or lozenge for the treatment of oral candidiasis or denture stomatitis.[2],[3],[12],[14] The clinical effectiveness of these forms has several unsuitable characteristics that limit their appeal as a treatment drug such as unpleasant taste, chances of getting washed off by saliva, gastrointestinal side effects, and neurological toxicity by CT.[3],[10],[15] Thus, incorporation of antifungal agents into denture materials could be the best solution to reduce the previous unfavorable characteristics.

AmB and CT were incorporated into polymethylmethacrylate (PMMA) denture materials to evaluate their inhibitory effects of biofilm formation by C. albicans on the surface of a prosthetic denture.


  Materials and Methods Top


Chemicals

AmB and CT were purchased from Sigma-Aldrich chem., (Germany). Polymer powder and monomer solution were purchased from Vertex-Dental, prosthetic dental products, (Netherlands). Sabouraud's dextrose agar (SDA) and Sabouraud's dextrose broth (SDB) were purchased from (Mast Group, Ltd., UK).

Specimen preparation

Ninety-nine square PMMA specimens (10 mm × 10 mm × 2 mm) were prepared from a heat-cured acrylic resin denture base with a polymer and monomer ratio of 2.2 g: 1 ml. The specimens were divided into 11 groups with 3 specimens in each group. The first 10 groups were prepared by adding different concentrations (1, 5, 10, 25, and 50 μg) of either of AmB or CT to each milliliter of monomer before mixing with polymer powder. The second group was processed without any drug to consider it as a control. The mixing ratio and conditions for processing and polymerization recommended by the manufacturers were strictly followed. The required specimen size of heat-cured acrylic denture base resin was first prepared by cutting the elastic foil of 2-mm thickness into plastic specimens of 10 mm × 10 mm × 2 mm. Flasking and packing were done by the conventional methods. Packing was accomplished according to the manufacturer's instructions. According to the American National Standard/American Dental Association,[16] curing of the heat-cured acrylic resin specimens was carried out by placing the clamped flask in a thermostatically controlled water bath for 90 min at 74°C, and then 60 min at 100°C. Flasks were allowed to cool at room temperature after curing. The acrylic specimens were removed from their stone molds. Any flashes of excess resin material were removed from the specimens using acrylic bur, finished with stone bur, and polished with pumice. The specimens were stored in distilled water (DW) at 37°C for 48 h for conditioning. All the prepared specimens were sterilized by autoclaving at 121°C with 15 bars for 15 min.

Biofilm formation

C. albicans was obtained from the microbial bank of the Central Public Health Laboratory of Karbala. Biofilm was induced on the PMMA specimens as mentioned by Dhale et al.[17] with some modifications including denture use. Briefly, C. albicans grown on SDA were activated by inoculating in SDB supplied with 8% glucose and incubated at 37°C for 24 h. Growth density of approximately 1.5 × 108 cells of C. albicans in each milliliter of sterilized normal saline was obtained after matching with the 0.5 McFarland standard. The PMMA specimens were singly distributed in a sterile screw vial, with three replicates for each type of specimen. Each vial received 2 ml of SDB and 0.1 ml of standardized yeast solution. Mixtures were agitated at 130 rpm for 1 h to stimulate adhesion, followed by incubation at 37°C for 24 h. After incubation, the inoculated PMMA specimens were washed three times with 1 ml of phosphate-buffered saline (PBS) to remove nonbiofilm-forming cells. Determination of biofilm formation was performed by two assays: crystal violet staining and transmission assays.

Crystal violet staining

Total count of C. albicans in the form of biofilm was determined by the processing of crystal violet staining as described by Melo et al.[18] with some modifications including denture use. Briefly, after washing of specimens with PBS solution, they were left at room temperature to dry for 45 min. Dry specimens were stained by submerging in 2 ml of 0.4% aqueous crystal violet solution for 45 min. Then, specimens were washed three times with 1 ml of sterilized DW and destained with 2 ml of 95% ethanol for 45 min. Absorption of destaining solution was measured with a spectrophotometer (APEL, PD-303 UV, Japan) at 595 nm.

Transmission assay

After the incubation period, each washed PMMA specimen was immediately added to a vial with 2 ml of sterilized normal saline for measurement of percent transmittance (%T) at a wavelength of 405 nm as mentioned by Tumbarello et al.[19] The %T value for each specimen was subtracted from the %T value for the reagent blank to obtain a measure of the amount of light blocked when passing through destaining solution (%T block).

Porosity analysis

Porosity percentage of PMMA specimens was calculated according to the measurement method used by Kasina et al.[20] Briefly, specimen weight in air and specimen weight in water (in a glass beaker with 1000 ml) were measured. Several equations were applied to obtain the final results depending on the known values as follows: dr = 1.1986 ± 0.01 g/ml, da = 1.23 kg/m3, dw = 1000 kg/m3, and g = 9.8066 m/s2. Percentage of porosity was calculated according to the following equations:

Wa = g (drda)(vspvip) (1)

Ww = g (drdw)(vspvip) + g (dadw) vip (2)

% of porosity = vip/vsp × 100 (3)

where

Wa: Specimen weight in air

Ww: Specimen weight in water

g: Gravitational constant

dr: Density of acrylic resin

da: Density of the air

dw: Density of the water

vip: Specimen volume

vsp: Internal porosity volume.

Statistical analysis

All the experiments were triplicated. The data were analyzed statistically with paired t-test. The minimum level of P < 0.05 is considered a statistically significant level.


  Results Top


Investigation for antifungal activity of AmB and CT against C. albicans after incorporation into acrylic denture base material showed promising results. Low concentrations (1, 5, and 10 μg/ml) of both antifungal agents revealed a significant ability to reduce the biofilm formation by C. albicans as indicated by an absorption value of stained specimens with crystal violet [Figure 1]. On comparison of absorption values, less biofilm formed as indicated by the high percentage of transmission was noted under the effect of high concentrations (25 and 50 μg/ml) of the two antifungal agents [Figure 2]. However, both assays showed that AmB and CT in different concentrations have inhibitory activity against the development of C. albicans biofilms in comparison to PMMA specimens without any agents. At the first three concentrations (1, 5, and 10 μg/ml), AmB showed more inhibitory effects on C. albicans than CT, while this condition was reflected in other two higher concentrations (25 and 50 μg/ml) [Figure 1] and [Figure 2].
Figure 1: Absorption value of stained denture base with Candida albicans biofilms

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Figure 2: Transmission percentage of stained denture base with Candida albicans biofilms

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Antifungal agents played an effective role in the porosity value of PMMA. Incorporation of the first three low concentrations of AmB and CT within PMMA materials decreased the porosity percentages. Meanwhile, elevation of antifungal concentrations had significant increasing effects on the porous nature of the PMMA structure [Figure 3]. After comparing with control, it was found that AmB was a more effective factor in reducing the porosity value of PMMA than CT, but with no statistically significant differences (P < 0.05).
Figure 3: Porosity percentage of denture base with Candida albicans biofilms

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  Discussion Top


C. albicans, as a member of the normal flora, is normally found on the epithelial surface of the skin and oral cavity. It can cause some serious diseases under specific conditions. In the oral cavity, denture stomatitis and dental caries are the most common types of infection resulting from an overgrowth of C. albicans on the mucosal surface, especially in denture-wearing individuals.[2],[3],[21] Adhesion is an important virulence factor of C. albicans that gives the yeast the ability to attach to the surface of normal tissues or of synthetic PMMA denture materials by a lectin-like component that is assisted by hydrophobicity of C. albicans' cell wall.[1],[4],[22] Denture may play a role in the development of denture stomatitis by C. albicans in the presence of sugar, low level of oxygen, and saliva.[2] Denture type also affects the adhesion of C. albicans on its surface. A flexible denture base material (Valplast®) was found to have a lower count of C. albicans than of acrylic resin.[23]

Several types of antifungal agents that are prepared in different pharmaceutical formulations are recommended for use in the treatment of oral candidiasis or denture stomatitis. Topical administration of AmB and nystatin related to the polyene group is the most common type. Azole group elements such as ketoconazole, fluconazole, and CT in systemic administration could also be useful in the treatment.[2],[8],[10],[13] AmB as well as other polyene agents has many undesirable characteristics that affect its suitability for the treatment of oral candidiasis, including unpleasant bitter taste, poor absorption through the gut, problems in maintenance of sufficient levels of drug at the site of infection due to it being washed off by saliva, and recurrence of the infection signs.[3],[8],[10],[15],[24] CT that is related to azole group has less of those unfavorable characteristics.[8] However, all the unwanted characteristics mentioned above can be eliminated if the contact of antifungal drug with oral tissues is limited. Incorporation of suitable antifungal drugs into denture materials is proposed to be the successful management to reduce such characteristics without imposing effect on the antifungal ability against C. albicans.

Polyene group represented by AmB and azole group represented by CT were chosen for incorporation with PMMA structure to test their antifungal activity against biofilm formation by C. albicans. They revealed a significant ability to reduce C. albicans biofilms. Thus, the methodology used in the study and the results can be considered a step ahead in limiting unsuitable characteristics of such antifungal agents, especially AmB, without a concern about their unpleasant taste, elimination by saliva, or even poor absorption by gastrointestinal tissues. Incorporation strategy of antifungal agents with PMMA was evaluated by some previous studies. Fluconazole, chlorhexidine, and a combination of them that were incorporated with PMMA showed continuous release into distilled water at mouth temperature throughout 28 days of the test period without loss of their antifungal activity against C. albicans.[9] Nystatin and miconazole also revealed in vitro and in vivo antifungal activities against C. albicans biofilms after incorporation with soft-liner denture materials.[25],[26],[27]

C. albicans is usually sensitive to AmB at minimum inhibitory concentration (MIC) ≤1 μg/ml and to CT at MIC ≤0.1 μg/ml,[12],[28],[29] while non-C. albicans needs higher concentrations (MIC ≤2 μg/ml).[30] Biofilm formation usually gives the candida the resistant ability to antifungal action up to 1000 fold higher than those needed to inhibit nonbiofilm cells.[31] Protective value of C. albicans biofilm against AmB was found to be 50% compared to nonbiofilm cells.[32] Moreover, C. albicans growing on denture surface was recorded to need a higher MIC value of AmB than when it was found on oral tissues[29] and this resistance increased with progressive development of biofilms.[33] Thus, persistence of antifungal activity close to denture surface should be effective to reduce biofilm formation by C. albicans in the underlying tissue of denture-wearing individuals. Although some studies had a fear about the possible emergence of antifungal resistance,[6],[8] incorporation of antifungal agents with denture materials is still the best choice to treat this condition. Our results showed that AmB and CT even at standard MIC concentrations have effective antifungal activity against biofilm formation more than at higher concentrations, while activity of these agents remains within the standard range that is mentioned in the M27-A3 document of the Clinical and Laboratory Standards Institute (CLSI or NCCLS).[34]

Porosity feature of denture is considered an important factor for encouraging of C. albicans' adherence. It mainly originates from an acrylic resin polymer that is used to prepare denture structure due to water sorption development in acrylic resin by a primary diffusion mechanism.[5],[25] Other factors that may be responsible for creating the porosity in denture structure include air entrapped during mixing, insufficient mixing of monomer and polymer, contraction of monomer during polymerization, monomer vaporization during exothermic reaction and in the presence of residual monomer, processing temperature higher than 74°C, or packing the mold and inadequate compression on the flask.[35] However, the method of denture processing has an important effect on the porosity of the final product. Denture specimens prepared by conventional heat polymerization technique have the least percent porosity when compared to the same thickness of microwave polymerization method.[23],[35]

From the present results, porosity percentage was increased in association with elevated antifungal concentration. This can explain the increase of biofilm production on the surface of denture containing high concentrations of antifungal agents instead of inhibition of C. albicans' growth. Therefore, low porosity level in denture that is incorporated with MICs of antifungal agents can be a beneficial factor for the synthesis of the denture. Some of other antifungal agents such as chlorhexidine, nystatin, and ketoconazole showed no effect on the porous nature of denture as recorded with soft liners.[25]


  Conclusion Top


Incorporation of AmB and CT into denture base materials has a significant inhibitory effect on the biofilm produced by C. albicans, especially at low concentrations. Decreasing porosity level is another advantage evidenced by incorporating low concentrations of AmB and CT within denture. Higher concentrations of antifungal agent will raise porosity percentage and limited antifungal effects against biofilm formation. Therefore, synthesis of denture base containing effective antifungal agents is the most important recommendation that can be made from our results to prevent the development of denture stomatitis or other undesirable characteristics resulting from fungal overgrowth in the mouth of denture wearers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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