Preparation and Characterization of Antibacterial Dental Resin Cement Material

In this study, bisphenol A glycerol dimethacrylate (Bis-GMA), which is a dental resin cement, and hydroxypropyl methacrylate (HPMA), is a dilution agent, were used as the main materials. In addition, benzoyl peroxide (BPO) and camphorquinone (Cam) were used as photoinitiators, whereas butylated hydroxytoluene (BHT) was used as a photo catalyst, and triethylene glycol dimethacrylate (TEGDMA) was used as a cross-linking agent. Finally, 2-isocyanatoethyl methacrylate (IEM) was used as an additive for high strength, while 3-hydroxypyridine (3HP) was used as an additive for antibacterial property. All reagents used in this study were made by Sigma-Aldrich.

The main dental materials (Bis-GMA and HPMA) and a cross-linking agent (TEGDMA) were mixed together, according to their recommended proportions, before being stirred for approximately 24 hours by using an agitator (Vortex GENIE 2, Scientific Industries, USA) to prepare the desired mixture. In addition, BPO, BHT, and Cam were used as photoinitiators and photocatalyst, and IEM and 3-HP were used as additives to impart high strength and antibacterial properties to the mixture, respectively. The mixture was then stirred for approximately 5 hours by using an agitator. In regard to the conditions for photopolymerization, the mixture was polymerized for 40 seconds in a 460 nm wavelength range. The fabricated samples were then evaluated for various physical properties, such as flexural strength, flexural bonding strength, antibacterial activity, breaking strength, and viscosity. The surface of the samples was also evaluated via SEM analysis. The groups, to which 3DHP was added according to their respective proportions, were each named 3HP-1, 3HP-2, 3HP-3, and 3HP-4, whereas the groups, to which IEM was added according to their respective proportions, were named Iso-1, Iso-2, Iso-3, and Iso-4, respectively. Table 1 shows the mixing proportion of the dental resin cement used in the experiment.

Flexural Strength: The sample was filled into a mold measuring (25 ± 2) mm × (2 ± 0.1) mm × (2 ± 0.1) mm, and then it was polymerized by radiating light beams, so that their energy sources would be overlapping with each other at the center. After the specimen hardened at (37 ± 1) °C, it was stored in a thermostat for 15 minutes before it was removed from the mold. The specimens were stored in (37 ± 1) °C distilled water until the load test was performed after 24 hours.

The specimen was measured with a caliper before it was tested for its flexural strength, and the load was applied at a rate of (1 ± 0.3) until the specimen was fractured in the flexural strength measuring device before recording the maximum load at the time of fracture of the specimen. At this time, the diameter of the support roller and the load roller were 2 mm each, and the distance between the centers of the three-point bending support rollers was 20 mm. The flexural strength (σB) of each specimen was calculated in MPa units.

Flexural strength = 3 F l 2 b h 2

Breaking Strength: The casting/molding method was used in the experiment. The specimen, which was fabricated by irradiating light with a thickness of approximately 100 μm was subject to a loading force at a rate of 10 mm/min by using a universal testing machine (AGS-X 20N, SHIMADZU, Japan), where the maximum load, at which the specimen was broken, was measured in N units.

Viscosity measurement: After the temperature setting of the thermostat was completed, 500 μg of the sample was applied to the center of the cup and an appropriate torque was set for the sample. The viscosity of the sample was then measured at the stabilization time.

Antibacterial Property: Streptococcus mutans, Staphylococcus aureus, and Escherichia coli FMF were used as bacterial colonies. The frozen bacteria were cultured in a solid medium by using a platinum wire to form colonies of single cell clusters. Then, a single colony was inoculated to a 5 mL liquid medium and cultured at 37 °C for 16 hours. For the preparation of the solid medium, 6 g of brain heart infusion, 6 g of peptic digest of animal tissue, 5 g of sodium chloride, 3 g of dextrose, 14.5 g of pancreatic digest of gelatin, 2.5 g of disodium phosphate, and 15 g of agar, which altogether add up to 37 g, were mixed in a flask, and then added with distilled water to make a 1 L solution. The prepared solution was then heated for 1 minute until it has completely dissolved. Thereafter, it was autoclaved at 121 °C for 15 minutes and cooled down to 50 °C before it was dispensed to 90 mm petri dishes and cured. The BHI liquid medium was prepared in the same manner by extracting agar from the solid medium. Furthermore, the absorbance of the bacterial culture was measured at 600 nm by using an absorbance meter to calculate the number of bacteria (the number of bacteria is 1 × 10⁹ CFU/ml when the absorbance is 1 at OD 600). In order to measure the antibacterial activity of the specimen, 2 g of beef extract, 17.5 g of acid hydrolysate of casein starch, 1.5 g of starch, and 17 g of agar, which altogether add up to 38 g, were mixed in a flask, and then added with distilled water to make a 1 L solution. The prepared solution was then heated for 1 minute until it has completely dissolved. The mixture was then autoclaved at 121 °C for 15 minutes and cooled down to 50 °C before it was dispensed to 90 mm petri dishes and cured. A total of 3×10⁶ CFU/ml bacteria were rubbed onto Mueller Hinton (BM) agar medium by using a cotton swab until they spread uniformly across the medium. Sterile paper disks (2 mm in thickness and 6 mm in diameter) were placed on the bacteria-bearing media, and samples 1, 2, and 3 (each measuring 20 μl) were applied on each paper disc under dark condition. The sterilized water was used as a negative control and the antibiotic ampicillin (20 μg), which is currently available in the market, was used for Gram-negative bacteria as a positive control, whereas penicillin-streptomycin was used for Gram-positive bacteria. E. coli and S. aureus were cultured for 20 hours at 37 °C in the culture medium, and S. mutans with a slow growth rate for 72 hours until clear zones were formed. It is believed that the clear zone was formed due to the zero growth of bacteria in the culture medium. In addition, 0.04, 0.4, and 4 μg of ampicillin were each added to E. coli and S. aureus in order to compare the antibacterial activity of the samples to that of ampicillin. The size of the clear zone, which was formed as a result of the slow growth rate of the specimen, was measured because it implies that the larger the clear zone, the greater the antibacterial activity. The experiment was repeated over 5 times.

The specimens that were polymerized by adding Ref., 3-hydroxypyridine and 2-isocyanatoethyl methacrylate glowed with a clear yellow light, while the polymerization tendency was identified by measuring the time required for the entire polymerization process. The polymerized samples and matching polymerization times are shown in Fig. 1 and Table 2, respectively.

Figure1.

Polymerization of Samples.

Viscosity is also an important factor in the development of dental resin cements that promote adhesion between dental ceramics and damaged section of the tooth. The measurements show that the viscosity has the tendency to increase along with increasing the addition ratio of IEM and 3-hydroxypyridine, respectively, whereas such increase was more pronounced when 3-hydorxypyridine, rather than 2-isocyanatoethyl methacrylate, was added to the mixture. The viscosity measurement results of the samples are shown in Fig. 2.

Figure2.

Viscosity of samples (A: IEM group, B: 3HP group).

The measurements of the breaking strength of the samples showed that the breaking strength increased along with increasing the addition ratio of IEM, while it took less time until the polymerization was complete, thereby suggesting that the degree of polymerization has the tendency to increase. In addition, when 3-HP was added to the mixture, the breaking strength decreased, while it took longer until the polymerization was complete, thereby suggesting that the degree of polymerization has the tendency to decrease as a whole. The breaking strength of each sample is shown in Fig. 3 and Table 3, respectively.

Figure3.

The diagram of breaking strength of samples (A: Ref., B: Iso-3, C: 3HP-1).

The control group was prepared by fabricating a resin, which was prepared by adding approximately 10% of commercial resin cement and IEM. The average flexural strength of Iso 3 was 82.642 MPa, which was higher than that of the conventional resin cement. The measurements of flexural bond strength of each sample are shown in Table 4.

The specimen was absorbed in the absorbing disk and cultured for 20 to 72 hours before it was inspected for any changes. The specimen demonstrated strong antibacterial activity against all three bacteria, thereby forming the widest clear zone when treated with ampicillin or penicillin-streptomycin, which was used as a positive control. When it was treated with sterilized water, which was used as a negative control, no antibacterial activity was observed at all in the specimen. In the case of S. mutans, which is a Gram-positive bacterium, no apparent clear zone was formed, as it took a long time until the clear zone was formed due to the slow growth rate and insufficient number of germs in the vicinity. However, a relatively wider clear zone was formed in sample colonies 1, 2, and 3. In the case of S. mutans, there was no significant difference in the antibacterial activity among samples 1, 2, and 3. In the case of S. aureus, which is another Gram-positive bacterium, sample 3 showed the highest antibacterial activity, while samples 1 and 2 showed similar results. In the case of E. coli, which is a Gram-negative bacterium, a similar antibacterial activity was observed among samples 1, 2, and 3. However, samples 1, 2 and 3 all showed a higher antibacterial activity when E. coli, rather than S. aureus, was applied to them (Fig. 4). Ampicillin was treated according to different concentration levels in order to compare their antibacterial activity to that of ampicillin, which was used as a positive control. The results showed that a clear zone was formed in the case of S. aureus with 0.2, 0.4, 0.5, 1, and 2 μg of ampicillin, respectively. When compared to the size of the clear zone formed by ampicillin, samples 1 and 2 showed 0.2 μg of antibacterial activity, whereas sample 3 showed 0.4 μg of antibacterial activity. In the case of E. coli, a clear zone was formed by at least 1 μg of ampicillin, and when compared to the size of the clear zone formed by ampicillin, samples 1, 2, and 3 all showed an antibacterial activity that was triggered by ampicillin of 10 μg or more (Fig. 5).

Figure4.

Test of antibacterial activity of samples using absorbing disk. A is S. mutans, B is S. aureus, C is E. coli. (On the left side is the medium showing the antibacterial ability using the absorbing disk. The right side shows the size of the clear zone by numerical value. n = 5).

Figure5.

Test for antibacterial activity of ampicillin using absorbing disk.