ANTIMIKROBNA SVOJSTVA AKRILATNIH SMOLA ZA STOMATOLOŠKE PROTEZE IMPREGNIRANIH NANOČESTICAMA SREBRA ANTIMICROBIAL PROPERTIES OF ACRYLIC RESINS FOR DENTURES IMPREGNATED WITH SILVER NANOPARTICLES

Uvod. Poroznost i površinska adherentnost akrilata čine ih kolektorima infektivnog sadržaja iz usne duplje. To se posebno odnosi na hladno polimerizujuće akrilate, čija je struktura manje kompaktna. Cilj rada bio je ispitivanje antimikrobnog dejstva hladno polimerizujućeg akrilata nakon njegove impregnacije nanočesticama srebra. Materijal i metode. Polimernoj komponenti (prahu) hladno polimerizovanog akrilata dodate su različite koncentracije (2%, 5% i 10%) nanočestica srebra, nakon čega su napravljeni uzorci oblika diska promera 10 mm. Kao kontrola poslužio je disk od nanočestica srebra. Antimikrobna aktivnost ispitivana je disk difuzionom metodom na dva česta izazivača infekcija usne duplje – Gram pozitivnoj bakteriji, Staphylococcus aureus ATCC 25923, i gljivici, Candidi albicans ATCC2091. Rezultati su pokazali da uzorak čistog srebra, kao i uzorci polimera sa srebrom, pokazuju antibakterijsku aktivnost. Zona inhibicije rasta Staphylococcus aureusa na hranjivoj podlozi upravno je srazmerna koncentraciji nanočestica srebra u akrilatu. Sa druge strane, ispitivani uzorci nisu ihhibirali rast Candide albicans na hranjivoj podlozi. Zaključak. Nanočestice srebra u akrilatu pokazale su antibakterijsku aktivnost. Proširenje njihovog spektra delovanja, kao i mogućnost eventualne kliničke primene biće predmet budućih istraživanja.


Introduction
The oral cavity provides habitats for a wide diversity of microorganisms, including bacteria, yeasts and viruses, with members of all groups being associated with oral infections.Bacteria are the predominant components of this resident microflora that colonizes surfaces on the teeth, mucosa and tongue, forming an oral biofilm 1,2 .
Most bacterial infections within the oral cavity are polymicrobial in nature, and it is quite unusual to find any that is clearly due to a single species.The relative contribution of different bacterial components in such infections is thus difficult to determine.
Plaque-related diseases are probably the most common bacterial diseases occurring in men.Dental caries (dental decay) is a destructive condition of the dental hard tissues that, if uncontrolled, can progress to inflammation and death of the vital pulp tissue, with eventual spread of the infection to the periapical area of the tooth and beyond.The disease process involves acidogenic plaque bacteria, including Streptococcus mutans, Streptococcus sobrinus and Lactobacillus spp. 1 .
The use of prosthetic devices within the oral cavity changes the oral conditions and promotes the deposit of biofilms on dental surfaces and on the prosthetic device 3 .Dentures are made of poly(methyl methacrylate) (PMMA) acrylic resin 4 .The porous surface and irregularities of acrylic resins favor the accumulation of microorganisms, which are determining agents in the vast majority of oral problems, such as candidiasis, caries, gingivitis, and periodontitis 5,6 .
The antimicrobial properties of silver (Ag) dates to 3000 years ago, and the mechanism is based on the interaction of silver with thiol groups of enzymes involved in bacterial cell metabolism thus causing cell death 7 .
Silver nanoparticles can kill all pathogenic microorganisms, and no report as yet has shown that any organism can readily build up resistance to them.Silver nanoparticles are also reported to be nontoxic to humans and very effective against bacteria, viruses, and other eukaryotic micro-organisms at very low concentrations and without side effects 8 .Due to this property, silver ions and silver nanoparticles (AgNPs) have also been introduced in dental materials 9,10 .srebra, Sigma-Aldrich) impregniranog u stomatološke akrilne smole za izradu zubnih proteza (poli (metil metakrilat), PMMA) u odnosu na Candidu albicans i Streptococcus aureus, najčešće patogene mikroorganizme koji se javljaju kod osoba koje nose zubne proteze.

Material and Methods
Silver nanoparticles (576832 Colloidal silver Nano powder, Sigma-Aldrich) of 2%, 5% and 10%concentrations were added to the powder component of the cold curing acrylic resin (Triplex Cold, Ivoclar Vivadent).Samples were made according to the manufacturer's instructions and poured into a disk shapemold 10mmin diameter.The obtained samples were then shredded into powder for further usage in the experiment.A disk of silver nanoparticles was used as a control for our study.
Antimicrobial activity was investigated using the disc diffusion method 11 .Antimicrobial activity of all four samples was tested on Gram positive bacterium-Staphylococcus aureus ATCC 25923 and fungus Candida albicans ATCC 2091.
The substrates used for the growth of microorganisms were nutrient agar for bacteria and Sabouraud maltose agar (Torlak, Belgrade) for fungi.Substrates were sterilized for 15 minutes by autoclaving at 121°C under a pressure of 110 kPa.A 0.1cm3 of the proper inoculum culture was added to 10cm 3 of substrate and poured into Petri dishes.The samples were placed on the inoculated surface of the substrate.The incubation was carried out for 24 hours at 37°C for the bacteria and for 48 hours at 25°C for the fungi.After incubation, the growth inhibition zones were measured and values were expressed in mm.The presence of inhibition zone indicates the antimicrobial activity of the samples.

Results
The results have shown that the control sample (Sample 23) as well as the samples of silver impregnated acrylics resins (Samples 20-22) exhibit antibacterial activity.The growth zone inhibition of Staphylococcus aureus (Figure 1) in the culture medium is proportional to the concentration of silver nanoparticles in the cold curing acrylic resin (Table 1).Test samples did not inhibit the growth of Candida albicans in the medium (Figure 2).

Discussion
Compared with other metals that are a part of nanoparticles, silver in low concentrations shows weak toxic effects on human cells [12][13][14][15] .Studies have shown that the positively charged Ag+ ion is responsible for its antimicrobial activity, which allows the binding of the negatively charged electrostatic bacterial cell membrane and the positively charged nanoparticles 16,17 .Kwakye-Awauach et al. concluded that the mechanism of antimicrobial activity is based on the interaction of Ag and the thiol enzyme group in the metabolism of the bacterial cell membrane, thus causing cell death 18 .
The oral cavity is populated by a large number of microorganisms which under certain conditions can lead to a wide variety of oral infections.Wearing dentures, and therefore removable dentures, changes the conditions of the oral cavity, and undoubtedly affects the oral flora.The acrylic materials, such as cold polymerized acrylates, are suitable materials for colonization of bacteria and fungi and as such their good hygiene is an imperative.Bearing in mind the age and social structure of denture holders, the improvement of the biological properties of acrylates would significantly reduce the possible complications in the form of infection and inflammatory reactions.
The research objective was refining cold polymerized acrylic material AgNPs to improve its biological quality.It was presumed that the resin material impregnated with silver acts in the mouth as fungicidal and antimicrobial, and that the purely mechanical compensation provides prophylactic and possibly therapeutic agent.Different concentrations of the tested AgNPs were added to the powder component of the cold polymerized acrylates, and such acrylates were in contact with Staphylococcus aureus and Candida albicans.The results showed a positive effect of antimicrobial activity of the impregnated material on Staphylococcus aureus because the inhibitory effect was greater with the increasing concentration AgNPs.The results are consistent with the findings of Castro et al. who have shown that the addition of 5% and 10% β-AgVO3 significantly reduces the metabolic activity of the Pseudomonas aeruginosa and Staphylococcus aureus in both types of polymerization of the acrylate denture base [13][14][15][16][17][18][19] .Khurana i sar.ukazuju na značaj smanjenja veličine partikle AgNPs u inhibiciji rasta Staphylococcus aureusa 21 .Sa druge strane, istraživanja Morrison i sar.nisu pokazala uticaj kombinacije AgNPs i praha akrilata na formiranje biofilma Staphilococcus pseudointermedius 22 .
The results did not prove the fungicidal effect of AgNPs.Literary available data clearly describe the effect AgNPs on the colonization of Candida in the mouth, dictating the necessity for the repetition of the same methodological research process, as well as the introduction of new research methods 23,24 .
Given the limitations of this study, we cannot make a clear judgment on the antimicrobial properties of resin impregnated materials.Their proven inhibitory effect on the growth of Staphylococcus aureus is incentive for future research on the impact of impregnation of hot and cold polymerized acrylates with AgNPs on different types of microorganisms that inhabit the oral cavity.

Conclusion
Silver nanoparticles in cold curing acrylic resin demonstrated antibacterial activity.Expanding their antimicrobial spectrum of activity and their potential clinical application will be the subject of the future research.

Figure 2 .
Candida albicans ATCC 2091 growth inhibition zone for samples 20-22 (a) and for the control sample 23 (b).

Table 1 .
AgNPs Antimicrobial analysis of acrylic resin samples impregnated with AgNPs Slane et al. concluded that the AgNPs added to the PMMA bone cement have a strong antimicrobial activity with respect to Staphylococcus aureus 20 .Khurana et al. point to the importance of reducing the size of AgNPs particles to inhibit the growth of Staphylococcus aureus 21 .On the other hand, the research of Morrison et al. did not confirm the effect of the combination of AgNPs and powder acrylate component on the biofilm formation by Staphylococcus pseudo-intermedius