Model resin composites incorporating ZnO-NP: activity against S. mutans and physicochemical properties characterization

Abstract Although resin composites are widely used in the clinical practice, the development of recurrent caries at composite-tooth interface still remains as one of the principal shortcomings to be overcome in this field. Objectives To evaluate the activity against S. mutans biofilm of model resin composites incorporating different concentrations of ZnO-nanoparticles (ZnO-NP) and characterize their physicochemical properties. Materials and Methods Different concentrations of ZnO-NP (wt.%): E1=0, E2=0.5, E3=1, E4=2, E5=5 and E6=10 were incorporated into a model resin composite consisting of Bis-GMA-TEGDMA and barium borosilicate particles. The activity against S. mutans biofilm was evaluated by metabolic activity and lactic acid production. The following physicochemical properties were characterized: degree of conversion (DC%), flexural strength (FS), elastic modulus (EM), hardness (KHN), water sorption (Wsp), water solubility (Wsl) and translucency (TP). Results E3, E4, E5 and E6 decreased the biofilm metabolic activity and E5 and E6 decreased the lactic acid production (p<0.05). E6 presented the lowest DC% (p<0.05). No significant difference in FS and EM was found for all resin composites (p>0.05). E5 and E6 presented the lowest values of KHN (p<0.05). E6 presented a higher Wsp than E1 (p<0.05) and the highest Wsl (p<0.05). The translucency significantly decreased as the ZnO- NP concentration increased (p<0.05). Conclusions The incorporation of 2 – 5 wt.% of ZnO-NP could endow antibacterial activity to resin composites, without jeopardizing their physicochemical properties.


Introduction
Since their development, the use of resin composites in the dental clinical practice has increased exponentially. However, irrespective of advantages such as the capacity to mimic the optical properties of the dental tissues and the on demand Methacrylate quaternary ammonium monomers (MQAM) chemically immobilized into the polymeric matrix have been used as a promising method to inhibit bacterial growth at resin composite surfaces. It is claimed that the great advantage of this approach is that the bioactive agent is not released from the matrix, which could contribute to the maintenance of the composites' properties 8 . However, previous reports have shown that depending on the functionality and the concentration in which they are immobilized into the organic matrix, MQAMs can also reduce the mechanical properties 19 , and increase the water sorption of the composites 4 . Different bacteriostatic and bactericide chemicals, e.g., chlorhexidine, Ag salts and particles, oxides, and others, have also been tested to confer antibacterial activity to resin composites 7,25 .
Unfortunately, these chemicals also jeopardized the composites' physicochemical properties.
Recent studies have shown that resin composites incorporating ZnO presented antibacterial activity against S. mutans and S. sobrinus 9,31 . However, these works have used commercially available resin composites to test this possibility. Since the exact composition of commercial resin composites is not known, it is plausible to claim that any chemical present in them could mask or interfere with the action mechanism of ZnO. Therefore, the goal of the present study was to evaluate the activity of a model resin

Lactic acid production
After 72 h of biofilm formation, the medium growth was removed and the composite disks were rinsed with 1 ml of PBS. Then, the specimens were transferred to a new 24-well plate and rinsed with 1 ml of buffered peptone water supplemented with 0.2 % sucrose (BPW). This medium was replaced with a fresh one and the plate was incubated at 37°C in an anaerobic condition for 3 h 6 . After the incubation period, the BPW solutions were used for lactic acid analysis, which was determined using a lactate dehydrogenase (LDH) reaction. Afterwards, the increments were light-cured and the spectra were recorded again (n=3). The DC% was calculated from the equation (1): Where R is the ratio between the integrated area of absorption bands of the aliphatic C=C bond (1638 cm -1 ) to that of aromatic C=C bond (1608 cm -1 ).

Flexural strength and elastic modulus
Ten bar-shaped specimens were prepared for each experimental composite. The composite was bulk inserted into a stainless-steel mold with 10 mm  (2) and (3), respectively: Where R is the ratio between the integrated area of absorption bands of the aliphatic C=C bond (1638 cm -1 ) to that of aromatic C=C bond (1608 cm -1 ).

ZnO-NP size distribution
The result of the size distribution analysis is presented in Figure 2. The ZnO-NP ranged from 7 to 810 nm, with a mean size of 12±19 nm.

Activity against S. mutans biofilm
The results of the MTT assay metabolic activity and the production of lactic acid by the S. mutans biofilm are depicted in Figure 3.      Caries starts with the progressive damage of the mineral structure of the dental hard tissues by organic acids produced by the cariogenic biofilm after fermentable carbohydrate intake 11 . Thus, quantifying the amount of lactic acid produced by S. mutans biofilm is also of great relevance in terms of antibacterial activity 6,35 . Only the composites with 5 and 10 wt.% of ZnO-NP significantly reduced the production of lactic acid by S. mutans biofilm ( Figure   4), with the total amount of acid being three-fold less than that produced by the control composite.
According to He, et al. 18 (2002), this reduction in lactic acid production is due to the ability of Zn 2+ to interfere  ZnO-NP wt.% and DC% (Figure 5a). This result is in accordance with a previous study 3  Microhardness suffered a significant decrease with the increase of ZnO-NP concentration (Table 1). This result is in agreement with that of Garcia,et al. 15 (2016), who showed that the incorporation of ZnO nanoparticles significantly decreased the hardness of a experimental adhesive, and could be linked to the ZnO-NP hardness itself being lower than that of barium borosilicate glass particles 22 . Additionally, the decrease of DC% could also explain this result. This possibility corroborates the results of previous studies that showed a positive relationship between DC% and the microhardness of unfilled resins 12 and resin composites 16 .
Although ISO4049 standard establishes specimens with 15 mm in diameter and 1 mm thick to evaluate W sp and W sl , in the current study, we used specimens with 6 mm in diameter and 1 mm thick. This was done in order to allow the specimen to be light-cured in one step, which is more in conformity with clinical practice 17 . The water sorption (24.4 to 26.1 µg/mm 3 ) and the water solubility (2.4 to 3.9 µg/mm 3 ) obtained in the current study (Table 1) are in agreement with the maximum values established by the ISO 4049 standard: W sp ≤ 40 µg/mm 3 and W sl ≤ 7.5 µg/mm 3 20 , suggesting that the composites formulated here may be resistant to hydrolytic degradation. E6 presented the highest solubility (3.9 µg/mm 3 ), while the other composites did not differ from each other ( Table 1).
As ZnO is an amphoteric oxide insoluble in water and alcohols, this behaviour can be only explained through the lowest DC% developed by E6 (Table 1). This thought is supported by previous studies that showed good relationships between the degree of conversion and solubility of different resin-based materials 17,24 .
Because the degree of conversion, hardness, water sorption, water solubility, and translucency were influenced by the different amounts of ZnO-NP, the second research hypothesis of the current study was rejected.

Conclusion
Although the translucency was negatively affected by ZnO-NP, it is reasonable to conclude that the incorporation of 2 to 5 wt.% of ZnO-NP may endow antibacterial activity to resin composites, without jeopardizing their physicochemical properties.
Irrespective of these findings, aspects such as the effect of pH-cycling, in situ simulations, and long-term evaluation must be addressed in future investigations.