In vitro Cytotoxicity of Silver Nanoparticles Incorporated in a Soft Silicone Liner

Introduction

Soft liners are commonly used to improve the quality of injured tissue, support denture structures, and enhance the retention and stability of dentures in patients with thin and sharp alveolar ridges, extensive ridge resorption, severe bony undercuts, genetic or congenital defects of the palate, and ill-fitting dentures (1). However, these materials easily degrade and are susceptible to colonization by microorganisms (2, 3). About 65% of patients using dentures develop denture stomatitis in which Candida Albicans is the main responsible (1). It adheres to both denture base and soft liner and causes serious complications, especially in elderly patients (2, 3). The regular replacement of the liner, along with proper oral hygiene and use of antimicrobial agents may be efficient in controlling the colonization of microorganisms. Sodium hypochlorite, chlorhexidine, glutaraldehyde, triclosan, peroxides, and microwave radiation have also shown to be effective for this purpose (4). Silver and silver-containing compounds have long been used as antimicrobial agents with optimal tissue response and low toxicity (5, 6). Silver nitrate is used for the treatment of visceral diseases, salivary gland fistula, and bone abscess, and silver sulfadiazine is also used as a broad-spectrum antibiotic in burns (7). Silver-zeolite has shown antiviral and antifungal properties when added to soft liners (3, 8). Following the introduction of nanotechnology, silver nanoparticles (SNPs) were introduced as the new generation of antimicrobial agents (7). Due to their excellent antimicrobial properties, SNPs are suitable for wound healing; therefore; they are extensively used in medicine (9,10). The AgNPs have been incorporated into dental biomaterials to prevent or reduce biofilm formation (11). A greater surface to volume ratio and small particle size results in excellent antimicrobial action without affecting the mechanical properties, and this unique property makes these materials the filler of choice in different biomaterials (11, 12). The SNPs added to soft liners may serve as hidden antifungal agents, decreasing microorganism adherence, which is optimal in the oral environment (13). This fungicidal activity of SNPs has been documented to be equal to other antifungal agents, such as amphotericin B and fluconazole (14). Although recent studies have proved the antimicrobial effect of SNPs on denture hard and soft liners (13, 14), the hazards associated with human exposure to these materials have not been investigated to facilitate the risk assessment process. Some authors even posed the possibility of the cytotoxic effect of soft denture lining materials themselves, whereas others considered them quite biocompatible (16-19). Silver seems to be distributed in all organs, especially the intestine and stomach. In the skin, silver induces a blue-grey discoloration termed argyria (20). Another potential route of exposure to SNPs is through the respiratory system. Cytotoxicity and genotoxicity of SNPs in the human lung cancer cell line have been evaluated, and the levels of cytotoxicity were reported following the production of reactive oxygen species and increased release of lactate, causing mitochondrial and DNA damage (21, 22). Therefore, a Tolerable Daily Intake value of 2.5 µg/kg bw/day has been reported for silver (20). The size of nanoparticles is a critical factor in determining inflammatory immune response even when noncytotoxic concentrations are employed (22). In vitro tests for the assessment of cytotoxicity of SNPs have shown that these nanoparticles can pass through the nuclear membrane. This enhances the action of medications containing SNPs as they can target the function of hydrophilic surfactants, such as Tween 80 (23). The interactions between cells and nanoparticles have also been shown to result in DNA damage, cancers, developmental toxicities associated with future growth retardation, deformity, or even fetal death (24). Therefore, the necessity of designing new studies aimed at overcoming the toxicity of SNPs concerning their antimicrobial activities is emphasized. This study aimed to evaluate the cytotoxicity of SNPs added to Mucopren soft silicone liner material (Mucopren; Kettenbach, Germany).

Materials and Methods

The soft liner selected in this study was Mucopren cold cure silicone long-term liner (Mucopren; Kettenbach, Germany) supplied as two pastes.

Specimens preparation

In a sterile condition, under a cell culture laminar hood, SNPs (Nanoshel; 80-100 nm in size; 99.9% purity) were poured into a ceramic mortar, and the particles were milled to make a homogenous mass. The soft liner base and catalyst were injected into two mixing pads in equal volumes. Each mixing pad, base, and catalyst was separately weighed by a digital scale (0.000 gr, Kern, Germany). Considering the required 0.5, 1, 2, and 3 weight percentages (wt.%) of SNPs, they were added to the base paste under the hood and mixed for 2 min. The mixture was then mixed with the catalyst for another 1 min. To ensure uniform distribution and homogenous dispersion of SNPs, transverse sections were made from each wt.% samples and evaluated under a scanning electron microscope (KYKY-EM3200, KYKY Technology, Shanghai, China) (Figure 1). Then,the specimens were cut in 10 mg pieces (weighted by a digital scale, 0.000 gr, Kern, Germany), exposed to ultraviolet light for half an hour, and placed in sterile tubes to prevent microorganism contamination.

Figure 1. Electron microscopic micrograph of a sample containing silver nanoparticles

Methylthiazol Tetrazolium Test

 Cytotoxicity assay was performed according to the International Organization of Standardization (ISO) 10993-5. In this standard,  6 samples were required for each group.The L929 standard fibroblast cell line was obtained from the National Cell Bank of Iran and grown on a 100-mm diameter Petri dish. Fourth passage cells were counted, and 30,000 cells were seeded in wells of a 96-well plate and incubated at 37°C in 5% CO₂. After 24 h, the pieces of each concentration of Mucopren incorporated SNPs specimens were placed in each well together with 100 µl of Dulbecco's Modified Eagle Medium, fetal bovine serum, and antibiotics (i.e., penicillin/streptomycin) (Figure 2). Table I demonstrates the study groups and subgroups. Therefore, the cells were exposed to Mucopren incorporated into different concentrations of SNPs or SNPs alone. It should be mentioned that the amount of SNPs in the SNPs alone group was measured to be the same as the amount of SNPs incorporated in corresponding Mucopren groups. Culture medium without the mixture served as the negative control, and distilled water, which is fatal for the cells, was used as a positive control. Cell viability was assessed by the conversion of 3-(4,5-dimethylthiazol–2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to insoluble formazan (25). The MTT assay was performed at 24, 48, and 72 h following the incubation of cells. Continuing the test does not seem logical due to a reduction in cell metabolism after being confluent, and making subculture with changing the media will return the condition to the first step. The overlaying solution was removed, and the cells were rinsed with phosphate-buffered saline (PBS) (Biowest, France). The MTT solution (Atocel, Austria)

Table I. Study groups and subgroups

SNPs: Silver nanoparticles

with 5 mg/mL concentration in PBS was prepared and diluted 1:10 with a culture medium and added to each well. The plates were incubated at 37°C for 3 hours in 98% humidity and 5% CO₂. During this time, viable cells with active metabolism converted the yellow MTT salt to insoluble formazan crystals using the mitochondrial succinate dehydrogenase enzyme (Figure 2). These crystals were seen under a light microscope. After ensuring the formation of formazan crystals under an inverted microscope, the overlaying medium was gently removed and dimethyl sulfoxide was added to wells to dissolve the formazan crystals to form a purple solution. Optical density, which had a direct association with the metabolic activity of cells, was read by an ELISA reader (Metertech M965 Microplate reader, Korea) at 570 nm wavelength. Cell viability (compared to the positive and negative controls and expressed in percentage) was calculated and reported at different time points.

Figure 2. 96-well plate containing cells and samples with (C-G)₂ to (C-G)₁₁ wells assigned to test groups and other wells assigned to negative and positive control groups

Statistical Analysis

The SPSS software (version 22, SPSS Inc., IL, USA) was used to assess the data of the MTT test with the analysis of variance (ANOVA) and Tukey’s HSD paired comparison test. The results were expressed as the mean ±standard deviation of viable cells for different experimental groups. P-value less than 0.05 was considered statistically significant.

Results

The highest percentage of cell viability on the first, second, and third days of incubation was noted in groups 1, 3C, and 3D, respectively. Table II and Figure 3 describe the percentage of cell viability at different experiment days. One-way ANOVA revealed a  significant difference at 1, 2, and 3 days in different groups (P=0.04, P<0.0001, P0.05). Cell viability values were also higher in SNP concentrations without Mucopren. According to two-way ANOVA, the effect of material (i.e., the combination of different concentrations of Mucopren incorporated SNPs) and storage time on the incubator and their interaction effect on the percentage of cell viability were also statistically significant (P

Figure 3. Results of cell viability in different groups in comparison to those in control group after 1, 2, and 3 days of incubation

Table II. Percentage of cell viability in different groups in comparison to those for positive and negative controls after 1, 2, and 3 days of incubation (n=6)

SNPs: Silver nanoparticles