The Effect of Silver Nano Particles on Candida Albicans and Streptococcus Mutans in Denture Acrylic Resins

Introduction

Silver nanoparticles are particles of silver ranging in size from 1 to 100 nm. The peculiar characteristic of silver ions and compounds is their toxic impact on microorganisms. This fact persuades researchers to use them for medical purposes (1). Nitrate form of silver is generally used for inducing antimicrobial effects; however silver nanoparticles increase the available surface area to microbes (1, 2). In Kim’s study (3), nanosilver were used to inhibit yeast, E.coli and S.aureous proliferation and they were successful in preventing yeast and E.coli growth in low concentrations. Although silver nanoparticles are used in various antibacterial applications, the mechanism of action is not well recognized. It has been hypothesized that silver nanoparticles can cause cell lysis or inhibit cell transduction (1). Kim indicated that nanoparticles of silver could affect cell membrane and cause an antifungal effect, a mechanism similar to amphotericin B, without similar levels of hemolytic effect (4). In another study, silver nanoparticles presented more antifungal effects on C. albicans compared with Amphotericin B and fluconazole (5). Regardless of the details of silver nanoparticles used and the targeted microorganisms, it has been used in previous studies to control the colonization of certain microorganisms around medical or dental appliances such as denture bases, resin composites, etc. (1, 6, 7).

Polymethyl Methacrylate acrylic resin is a choice for applying Candida the antimicrobial purposes of silver nanoparticles. Growth of the microorganisms under acrylic denture bases, the main material to replace the lost tissue in treatment of edentulous patients, is common. Denture related oral ulcers like denture stomatitis can be the most prevalent lesion in these patients (73% of prosthodontic patients) in which Candida albicans is responsible for about 70% of cases, Common treatment plans for these infections such as Nystatin, fluconazole, and Amphotericin B therapy are short-term, so we tried to produce a denture with self-antimicrobial features (8(.

In several studies such as Kassaee’s experiment (9), different concentrations of Nano silver particles in acrylic resin have been examined. In this experiment, strong antibacterial effect of 0.5% silver nanoparticles on E.coli has been demonstrated. Furthermore, Casemiro (8), showed that addition of 5% and 2.5% silver nanoparticles to acrylic resin brings about a high antimicrobial effect on C.albicans. Another study illustrated that the addition of 10% β-AgVO3 (β- Silver Vanadium Oxide) to acrylic resin promotes antimicrobial activity against P. aeruginosa, S. aureus, C. albicans, and S. mutans (6). Torres et al. found that the antifungal effect of PMMA-silver nanoparticle disk is related to the reduction of C. albicans adherence with no effect on metabolism or proliferation of cells (10). Moreover, Zhe Li’s study demonstrated an antifungal activity, resultant inhibitory effect on adhesion and biofilm formation on denture base resin containing nano-silver ,especially at higher concentrations (11.(

The aim of this study is to examine whether acrylic resins containing silver nanoparticles, release silver ions and generate long-term antimicrobial effect.

Materials and Methods

Silver nanoparticle solution preparation: Colloidal silver nanoparticles (fig 1) were prepared via chemical process of reduction. 0.001 M of silver nitrate (Ghataran Shimi T. Co. Iran) was dissolved in water and heated up to 80 ºC. Next, 5 ml of 1% trisodium citrate was added to the solution drop by drop while it was being shaken severely. Warming process continued until the color of the solution changed to brown. Finally, the solution was cooled in room temperature, while being shaken.

The shape of nanoparticles shown under an electronic microscope was spherical (fig 2). A nanosizer (nano- sz, Malvern Instruments Ltd, England) device was also used to measure the size. The average size of the particles was about 22 nm. In the end, a potential zeta device was utilized to specify the ionic charge of the silver nanoparticles, the result of which was a -2 surface charge.

Preparing acrylic samples without silver nanoparticles (control samples).

Cylindrical pieces with 10 mm diameter and 4 mm thickness were used to prepare the samples (fig 3).

Plaster and water were mixed using manufacturer instructions and poured in the inferior part of the flask leaving 10 mm free space. After plaster setting was completed, 10 mm of silicon (putty) was placed in it. Then, 10 metallic cylinders were submerged completely into the unset putty.

Next, the superior part of the flask was placed on the inferior part, plaster poured in it and pressed under a hydraulic pressure device. Metallic cylinders were extracted after three hours and the acrylic paste was prepared considering manufacturer liquid/powder ratio. While the mixture was going through its doughy phase, we placed it in the empty spaces. Finally, the flask was pressed under a hydraulic pressure device andthen placed into a hot water tub (72 degrees C) for six hours. After 24 hours, the flask was opened and samples were extracted from the plaster. After the finishing procedures were carried out using acrylic burs, they were placed in normal saline solution. (Fig. 4).

Figure 1. Colloidal silver nanoparticles

Figure 2. Electronic microscopic image of silver nanoparticles (×8000)

Figure 3. Cylindrical pieces

Figure 4. Acrylic samples (without nanosilver)

Preparing acrylic samples containing different concentrations of silver nanoparticles:

We poured about 25cc of colloid into a special bulb and set the device temperature at 85^oC and speed at 200 rpm until colloids were concentrated to a volume of 2 cc. The weight of 1.5 cc silver nanoparticle colloid was 2 gr after concentration.

Then, the volume of silver nanoparticle colloids for sample preparation was evaluated considering weight percentage of silver nanoparticles in the acrylic samples.

Fifty samples were prepared without silver nanoparticles to estimate the weight of each sample. The average weight of each sample was 0.4gr.

The required weight of silver nanoparticle colloid for preparation of an acrylic sample containing 2.5% silver nanoparticles: Weight of each acrylic sample (0.4 gr) × 2.5% (weight of silver nanoparticles (gr))/ (weight of each acrylic sample (gr)) = 0.01gr

The required weight of silver nanoparticle colloid for preparation of 40 acrylic samples containing 2.5% silver nanoparticles: 0.01gr × 40 = 0.4 gr

The required volume of colloid for preparation of 40 acrylic samples containing 2.5% silver nanoparticles:

0.4 gr silver nanoparticles × (1.5 cc of silver nanoparticle colloids)/ (2 gr of silver nanoparticle colloids) = 0.3cc

regarding the above-mentioned equations, the required volume of colloid for preparing 40 blocks of 0.01 gr acrylic samples in 2.5, 5 and 10 w/w % (mass fraction) is 0.3 cc, 0.6 cc and 1.2 cc, respectively.

We mixed 10 cc of acrylic liquid and 20 cc of acrylic powder. Then, the whole paste was cured and its weight was measured after it was cooled down. The resulting 6 gr was equivalent to 15 samples.

The required volume of powder for preparing 40 acrylic samples:

(Number of samples (15))/ (number of samples (40)) = (required volume (10 cc))/ (required volume (x cc))                x = 26.6 cc

We had to mix .3 cc, 0.6 cc and 1.2 cc of silver nanoparticle colloids with 53.3 cc of acrylic powder and 26.6 cc of acrylic liquid to prepare 40 acrylic samples containing silver nanoparticles concentrated at 2.5, 5 and 10 w/w%, respectively.

The prepared mixture was mixed up properly until it became completely homogenous, then it was poured into a flask designed empty space and other procedures were followed just like samples without silver nanoparticles.

Antimicrobial effects

Studied Microorganisms comprised:

1). Candida albicans ATCC 10231

2). Streptococcus mutans ATCC 35668

3). Streptococcus mutans hospital isolated

4). Candida albicans hospital isolated

Sample 1 and 2 were taken from Industrial and Scientific Research Organization of Iran and 3 and 4 were taken from a hospital (Ghaem Hospital, Mashhad, Iran).

Acrylic samples were prepared in four different concentrations:

1). Acrylic specimens without silver nanoparticles as control group (n=40)

2). 2.5% silver nanoparticles (n=40)

3). 5% silver nanoparticles (n=40)

4). 10% silver nanoparticles (n=40)

Each group samples were divided into 4 subgroups depending on the microorganisms.

Samples were kept in physiologic serum after preparation until they were placed in separate isolated laboratory tubes and were sterilized in 120 degrees centigrade for 15 minutes.

Brain Heart Broth culture environment was prepared and sterilized according to the manufacturer’s instructions.

Blood Agar culture environment was prepared as usual. In this process, Muller Hinton Broth environment was prepared and after being sterilized in autoclave and cooling in room temperature to 10 percent of the volume, Blood sheep was added to the solution and the solution was poured in 8centimeter sterilized plates. Bacterial strains were solved in Brain Heart Broth and incubated for 24 hours. Then, linear culture was applied on the sterilized plates to grow the bacteria. After the growth of the bacteria, bacterial suspension was provided with concentration of 0.5 Mc Farland (equals 1.5×108cfu) in comparison with Mc Farland standard. In the next stage, this suspension was diluted to 1.5×105cfu.

To prepare 1.5×105 CFU bacterial suspension, 1 cc of suspension with 1.5×108 CFU was mixed with 9 cc of the culture environment and a suspension with 1.5×107 CFU was obtained. The same process was repeated twice unitl a suspension with the concentration of 1.5×105 was provided.

One milliliter of the suspension was poured in laboratory tubes with sampler, and an acrylic sample was placed in each tube. Then, 10 µl of every suspension was isolated via sampler, which was cultured linearly, during the primary time of exposure to the bacteria.

In the next stage, laboratory tube was placed in the incubator shaker for 24 hours in 37 ºC. 10 µl of the suspension was taken after 1, 6, and 24 hours and cultured.Each plate was incubated for 24 hours post cultureand bacterial colonies were counted afterward.

Statistical Analysis

Mean and standard deviation were employed to describe the data. One-way ANOVA was used for comparing the amount of bacteria in silver nanoparticle.

Results

Obtained results after analysis of the gathered data were shown in the tables 1,2 and 3.

Mean and standard deviation of the reduction of bacterial suspension in different concentrations of silver nanoparticles were shown in table 1. The results indicated a significant difference among all groups in 2.5% concentration after 0, 1, and 24 hours of exposure (P<0.001). The maximum effect occurred after 24hours against standard S. mutans. 

Significant difference was observed among all groups of 5% concentration (P<0.001). The maximum effect on Standard Candida occurred at 6 hours after exposure Candida.

There was a significant difference among all groups in concentration of 10% (P<0.001). The maximum effect occurred on Standard Candida at 24 hours post exposure Candida. The anti-microbial property after 6h of exposure was approximately equal to 24h post exposure.

Analyses illustrated that the maximum effect occurred at 24 hours post exposure in concentration of 2.5% in standard S.mutans. However, in standard Candida, the maximum effect occurred at 24 hours post exposure in concentration of 10%. In hospital Candida and hospital S.mutans, there was a maximum effect at 24 hours post exposure in concentration of 10%.

Table 1.  Mean and standard deviation of the reduction of bacterial suspension in different concentrations of silver nanoparticles considering type of bacteria, concentration, and exposure time

C = average bacteria in 10 test tubes of the initial control sample (C0), an hour later (C1), six hours later (C6), and 24 hours later (C24)

T = average bacteria in 10 test tubes of silver nanoparticles in initial time (T0), an hour later (T1), six hours later (T6), and 24 hours later (T24)

Table 2.  The results of one way variation ANOVA categorized by a bacterium and separated by concentration and time

Table 3.  Results of one way variation analysis categorized by concentration and separated by bacterium and time