The aim of this study is evaluating the antibacterial activity of resin composites containing ZnO nanoparticles against Streptococcus mutans and examining their physical and mechanical properties.
The properties of flowable resin composites containing 0–5 wt.% nano-ZnO are investigated using different tests:
Antibacterial activity (including agar diffusion test on the cured resins, direct contact test using bacteria in a liquid medium, evaluating the effect of aging while the samples are adjacent to a liquid medium, and scanning electron microscopy (SEM)).
Mechanical behavior (including flexural and compressive strength and modulus).
Curing aspects (including depth of cure and degree of conversion).
Adhesion properties (including micro-shear bond strength).
Although the agar diffusion test reveals no significant difference between the groups, the direct contact test demonstrates that by increasing the nanoparticle content, the bacterial growth is significantly diminished ( p < 0.05). In the aging test, however, the antibacterial properties reduce significantly ( p < 0.05). The flexural strength and compressive modulus remains unchanged by incorporation of nanoparticles ( p > 0.05) while the compressive strength and flexural modulus significantly increase ( p < 0.05). The ZnO containing resins show significantly lower depth of cure ( p < 0.05), and higher bond strength ( p < 0.05). There is no significant difference between the degrees of conversion, measured by FTIR technique, of the groups ( p > 0.05).
Production of a dental resin composite with antibacterial activity without significant sacrificing effect on the mechanical properties is desirable in dental material science.
Secondary caries are defined as “positively diagnosed carious lesion occurs at the margins of an existing restoration” . This phenomenon remains the most frequent reason leading to shortening the service life of dental restorations and finally results in the need for the replacement of the restorative materials .
The main cause of enamel demineralization is adhesion of micro-organisms to the teeth and/or restorations which produces pathogenic plaque. Therefore, one of the most applicable methods for preventing enamel demineralization around the restorations is using dental materials resistant to the bacterial accumulation . On the other hand, as various laboratory and clinical researches have demonstrated, comparing to either other restorative materials or dental hard tissues, more plaque accumulation occurs on resin composites which results in higher prevalence of secondary caries around composite resin restorations . The more biofilm formation on resin composites is related to its surface roughness and free energy, that is the outcome of resin type, filler size, and percentage of filler. Moreover, not only none of the resin composite components have bacteriostatic property, but also they are metabolized or eaten away by microorganisms . Therefore, recent studies pay growing attention to the antibacterial activity of composite resins in order to reduce the risk of recurrent decay around esthetic direct restorative materials. Different approaches have been used to impart the antibacterial activity into the resin based dental composites and adhesives. The first approach is the incorporation of antibacterial agents into the resin matrix which are released over the time inhibiting the bacterial growth. Examples of the agents are fluoride and chlorhexidine . Although the agents are initially strong antibacterial agents, their release rates do not last for long periods. In addition, the dental composites containing the agents have shown a higher rate of bond failure because their mechanical properties are diversely affected . The other approach is the inclusion of quaternary ammonium functionality in the resin monomers or as an additive . It seems the approach is more promising as the researches have reported higher longevity for the antibacterial activity of the composites containing the materials. The third approach is the incorporation of metal (oxide) particles/ions into the restorative materials . From centuries ago, metals such as silver, gold, and zinc have been used as bactericidal and bacteriostatic agents . The antibacterial efficacy of metals is directly depends on their total contact surface area. The nano-scale dimensions of nanoparticles allow a considerable broader gamut of interactions with microorganisms increasing their antibacterial activities.
Streptococcus mutans is one of the major species of bacteria responsible for dental caries . Several researches argued that among metallic agents, silver nanoparticles are the most effective metal for inhibiting the growth of S. mutans . However, the major drawback of silver in restorative dental materials is the cosmetic changes of tooth colored materials . Hence, insoluble, tooth-colored, or colorless metal oxide powders such as silica, zirconia, alumina and zinc oxide (ZnO) may be more interesting in dental composites. Although the incorporation of ZnO into dental composites may impart antibacterial activity , the opacity of the particles against visible light may adversely affect the light curing and, consequently, the mechanical properties of the composites.
The purpose of this study is to evaluate the hypothesis whether the addition of minute amounts of ZnO-NPs into a flowable resin composite would affect its bond strength, physical and mechanical properties, and antimicrobial activity.
Materials and methods
Preparation of test specimens
In this study we had six experimental groups consisting of five resin composites containing ZnO-NPs in different concentrations of 1, 2, 3, 4, and 5 wt.% and one control group with no additive. The tested materials were prepared by the incorporation of the ZnO-NPs (average particle size of 20 nm with hexagonal crystal structure and 99.8% purity) (Nanopars Espadana, Isfahan, Iran) into the dental restorative resin composite Heliomolar Flow (Ivoclar Vivodent AG, FL-9494 Schaan/Liechtenstein). The nanoparticle powder was added to the resin composite and homogeneously mixed in a dark room for 15 min with a glass spatula. These modified composites were stored in completely opaque bottles until each test were performed.
Bacterial strains and growth conditions
S. mutans PTCC 1683 (Persian Type Culture Collection, IROST, Iran) were used in this study. The bacteria were cultured aerobically overnight in 5 ml of brain–heart infusion (BHI) (High Media, India), at 37 °C.
Preparation of the microtiter tubes
A microtiter tube (500 μl, Zimax K.A., Iran) was vertically positioned. Using a flat-ended dental instrument (dental spatula) the side walls of twelve dishes were coated evenly with an equal amount of the tested material (200 μl in each tube). The materials were polymerized for 280 s with an overlapping regimen by a light curing unit (LED, DEML, SDS Kerr, USA, with an intensity of circa 800 mW cm −2 ) in seven 40 s cycles from the top and the outside wall of the tubes.
Direct contact test (DCT)
The direct contact test (DCT) was performed to determine the antibacterial activity of the tested materials . A 10 μl of bacterial suspension (~10 6 bacteria) was placed on the surface of each tested material in a set of three tubes for each group. The tubes were then incubated in vertical position for 1 h under a sterile condition. During the incubation period, the suspension liquid was evaporated to obtain a thin layer of bacteria, ensuring direct contact between the bacteria and the tested surface. A 10 μl of the bacterial suspension was placed on the uncoated walls of three tubes, which served as control. Then 300 μl of BHI broth were added to each tube containing the material . After 3, 6, 12 and 24 h, aliquots of 50 μl of the mixture (bacteria + BHI broth) were spread on blood agar plates (High Media, India and defibrinated sheep blood) and incubated at 37 °C for 24 h subsequently. The bacterial colonies were then counted.
Similar microtiter tubes were prepared with the tested materials and aged for 48 h, 1 and 4 weeks. During this period each well was filled with 300 μl PBS (Phosphate Buffered Saline), which was replaced every 48 h, and the plates were incubated at 37 °C. The PBS was aspirated and the plates were dried under sterile condition before the DCT test .
Agar diffusion test
200 μl of bacterial suspension was spread on blood agar. Uniform resin composite test-disks (2 mm thick and 8 mm in diameter) were prepared by pressing the sample resins between two glass slides to obtain smooth surfaces. The disks were photopolymerized for 40 s using the light curing unit from the top side and an extra 40 s cycle was repeated from the bottom side in order to ensure complete photopolymerization. Test-disks of the resin composite Heliomolar Flow with 0, 1, 2, 3, 4 or 5 wt.% ZnO-NPs contents were placed on the surface of the mentioned blood agar plates. The plates were incubated for 24 h at 37 °C and the inhibition zone around each specimen was measured in mm scale .
Scanning electron microscopy
New resin composite disks prepared as previously described. A 10 μl of bacterial suspension (~10 6 bacteria) was placed on the surface of each specimen and incubated for 1 h at 37 °C. Evaporation of the suspension liquid resulted in a thin layer of bacteria, ensuring direct contact between the bacteria and the specimen surface. The samples were fixed with glutaraldehyde and osmium tetroxide solutions, dehydrated in a graded ethanol series, and then gold coated. An additional set of disks was processed as above and incubated for 24 h in 5 ml of BHI broth. The test specimens were then examined by scanning electron microscopy (HITACHI, S-4160, Japan, Field Emission Electron Microscopy (FE-SEM)) .
Measurement of degree-of-conversion
The degree of photopolymerization conversion of specimens containing 0, 1, 2, 3, 4 and 5 wt.% ZnO-NPs was measured by FTIR (EQUINOX 55, Bruker, Germany) spectroscopy. The specimens were placed between two polyethylene films, pressed to form a very thin film and the absorbance peaks of the un-cured samples were obtained. The specimens were then light-cured for 40 s using the light source and the peaks were collected for the cured specimens.
Degree of conversion (DC%) was determined from the ratio of absorbance intensities of aliphatic C C (peak at 1638 cm −1 ) against internal reference of the aromatic C⋯C (peak at 1608 cm −1 ) before and after curing of the specimen. The degree of conversion was then calculated as follows :
DC % = 1 − ( 1638 cm − 1 / 1608 cm − 1 ) peak area after curing ( 1638 cm − 1 / 1608 cm − 1 ) peak area before curing × 100
For each group of resin composites the measurement was repeated for three times.
Measurement of the depth of cure
The depth of cure of resin composite was determined following the ISO 4049 (2000) standardized technique. The composite resins were inserted into a stainless-steel split mold with a cylindrical cavity of 10 mm height and 4 mm diameter while the top of the mold were covered with transparent polymer strips. The specimens were then light-cured for 40 s from the top. Immediately after irradiation, uncured materials were scraped away with a plastic spatula. Subsequently, the height of the cured resin was measured in three different places using a digital micrometer (Mitotoyo, Japan). The measured values were divided by 2 and the average of three measurements was then reported as the depth of cure.
Flexural strength and modulus
Flexural strength is one of the most important mechanical tests for assessing the performance of dental resins. According to ISO 4049, the resins were inserted in a rectangular stainless-steel mold with 2 mm × 2 mm × 25 mm dimensions, which was placed on a glass slide. Then, the mold was covered with another glass slide and specimens were cured from both top and bottom sides by a light-curing unit irradiated for 40 s in each spot using an overlapping regime. The specimens were removed from the mold and stored in deionized water for 1 day at 37 °C prior to the test. Both surfaces of all specimens were polished using a 600 grit silicon carbide paper in a moist environment. At least 7 specimens were tested for each formulation. A three-point bending test was performed using a universal testing machine (Z20, Zwick Roell, Germany) at a cross-head speed of 0.5 mm min −1 . The flexural strength (FS) in MPa was calculated as:
FS = 3 P L 2 b d 2