Inhibitory effect of reduced graphene oxide-silver nanocomposite on progression of artificial enamel caries

Abstract The use of antimicrobial agents is an efficient method to prevent dental caries. Also, nanometric antibacterial agents with wide antibacterial spectrum and strong antibacterial effects can be applied for prevention of dental caries. Objectives: The aim of this study was to evaluate the inhibitory effect of reduced graphene oxide-silver nanoparticles (rGO/Ag) composite on the progression of artificial enamel caries in a Streptococcus mutans biofilm model. Material and Methods: Enamel specimens from bovine incisors were divided into eight treatment groups (n = 13), as follows: group 1 was inoculated with S. mutans grown in Brain Heart Infusion containing 1% sucrose (1% BHIS), as negative control; groups 2-4 were inoculated with S. mutans grown in the presence of different rGO/Ag concentrations (0.08, 0.12, 0.16 mg/mL) + 1% BHIS; group 5-7 were inoculated with S. mutans grown in the presence of different agents (0.16 mg/mL reduced graphene oxide, 0.16 mg/mL silver nanoparticles, 10 ppm NaF) + 1% BHIS; group 8 was mixed with 1% BHIS, without inoculation. Artificial enamel carious lesions were produced by S. mutans biofilm model for 7 days. Confocal laser scanning microscopy and atomic force microscopy were used to analyze roughness and morphology of the enamel surface. Polarized light microscopy and confocal laser scanning microscopy were employed to measure the lesion depth and the relative optical density (ROD) of the demineralized layer. Results: Compared with the control groups, the rGO/Ag groups showed: (a) reduced enamel surface roughness; (b) much smoother and less eroded surfaces; (c) shallower lesion depth and less mineral loss. Conclusion: As a novel composite material, rGO/Ag can be a promising antibacterial agent for caries prevention.


Introduction
Dental caries is a common chronic infectious oral disease affecting both adults and children. Extensive efforts in preventing caries through reduction of sucrose intake, enhancement of public awareness, and administration of fluoride have led to a decline in caries prevalence, 1 but it is still a major public health problem worldwide. Cariogenic bacteria are the known risk factor for dental caries. Consequently, application of antibacterial agents to inhibit biofilm accumulation over tooth surface is an innovative method for dental caries prevention. 2 However, the problem of antibiotic resistance hinders the application of such drugs.
In recent years, metallic nanomaterials with wide antibacterial spectrum and strong antibacterial effects have been drawing great attention and have gradually been applied in the field of dentistry. 3 Graphene, as one atom-thick sheet of sp 2 -bonded carbon atoms packed in a dense two-dimensional honeycomb crystal, 4 is of the utmost interest due to its unique structure and valuable properties. Rich in oxygen-containing functional groups, graphene oxide nanosheets have good water dispersion and high cell compatibility. 5 Due to their advantageous properties, graphene oxide sheets have already been developed as substrates to carry inorganic antibacterial nanoparticles, such as silver (Ag), gold (Au), and titanium oxide (TiO 2 ). 6,7 Silver nanoparticles (AgNPs), as broad-spectrum antimicrobial agents, have been used in many fields, such as infection treatment and production of dental materials. 8 However, the antibacterial properties of AgNPs could be diminished or even totally lost due to their strong tendency to self-aggregation. 9 Graphene oxide can effectively disperse AgNPs in water by acting as a substrate to anchor nanoparticles, thus improving the antibacterial property of AgNPs. As novel antibacterial systems, AgNPs assembled on graphene oxide nanosheets are increasingly attracting researchers' interests. 10 Cai,  13 (2017) compared the antibacterial activity of reduced graphene-silver nanoparticles (R-GNs/Ag), reduced graphene, and AgNPs; the results indicated that R-GNs/Ag composite exhibited the highest antibacterial action. Therefore, applying rGO/Ag as novel antibacterial agents in dental caries is promising, and the anticariogenic potential of this novel agent in combatting microbial biofilm-induced artificial caries remains as a matter to be investigated.
In this study, the Streptococcus mutans biofilminduced caries model was used to evaluate the inhibitory effect of rGO/Ag composite on the progression of artificial enamel caries. The null hypothesis is that rGO/Ag composite has no effect on the progression of artificial enamel caries.

Material and methods
Preparation and characterization of rGO/Ag GO  Antimicrobial effects of rGO/Ag on S. mutans

Characterization of rGO/Ag
Imaging analysis of the rGO/Ag composite by scanning electron microscope and transmission electron microscopy showed that rGO sheets effectively dispersed AgNPs by acting as substrates to anchor AgNPs nanoparticles, and that AgNPs attached to rGO sheets had no observed self-aggregation ( Figure 1A-C).
The curve of the thermogravimetric analysis of the rGO/Ag composite is shown in Figure 1D

Enamel surface roughness
Confocal laser scanning microscopy (CLSM) was used to obtain three-dimensional topography images.
Apparent differences were observed on the surface roughness between the treatment and the control groups ( Figure 2). Based on data for the average surface roughness (Figure 2A      ROD was calculated as ODR=ODl/ODs × 100%; ODl represents the OD of the enamel demineralized layer, ODs represents the OD of the sound enamel tissue (blank control) measured at the corresponding tooth specimen level. Values annotated with different superscripts (a-d) within the same row indicate statistically significant differences (p<0.05) between different groups Table 1-Relative optical density (ROD) of artificial enamel lesions treated with rGO/AG, GO and AgNPs at different depths of tooth specimens (n=10)  Therefore, the intensity and width of the fluorescent layer represents the depth of the demineralized layer. 27 In the groups treated with the rGO/Ag composite, the Based on the results of this study, the rGO/Ag composite has a promising anticariogenic activity, and it can potentially be incorporated into the existing dental materials, as glass ionomer cements, dental resin composites, adhesive and denture resin bases, aiming at preventing secondary caries. Meanwhile, rGO/Ag composite with excellent antibacterial activity may also be used in root canal irrigation and disinfection, as well as surface modification of dental implant to reduce the chance of peri-implantitis. However, most of these promising results were achieved under mono-species biofilm model. To apply this novel composite material for caries prevention, the preventive effect of rGO/Ag composite on progression of caries in multi-species biofilm model needs to be confirmed. The long-term antibacterial effect of the use of rGO/Ag as additive to modify dental materials remains to be investigated.