One-year water-ageing of calcium phosphate composite containing nano-silver and quaternary ammonium to inhibit biofilms

Abstract

Dental composites are commonly used restorative materials; however, secondary caries due to biofilm acids remains a major problem. The objectives of this study were (1) to develop a composite containing quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP), and (2) to conduct the first investigation of the mechanical properties, biofilm response and acid production vs water-ageing time from 1 day to 12 months. A 4 × 5 design was utilized, with four composites (NACP-QADM composite, NACP-NAg composite, NACP-QADM-NAg composite, and a commercial control composite), and five water-ageing time periods (1 day, and 3, 6, 9, and 12 months). After each water-ageing period, the mechanical properties of the resins were measured in a three-point flexure, and antibacterial properties were tested via a dental plaque biofilm model using human saliva as an inoculum. After 12 months of water-ageing, NACP-QADM-NAg had a flexural strength and elastic modulus matching those of the commercial control (P>0.1). Incorporation of QADM or NAg into the NACP composite greatly reduced biofilm viability, metabolic activity and acid production. A composite containing both QADM and NAg possessed a stronger antibacterial capability than one with QADM or NAg alone (P<0.05). The anti-biofilm activity was maintained after 12 months of water-ageing and showed no significant decrease with increasing time (P>0.1). In conclusion, the NACP-QADM-NAg composite decreased biofilm viability and lactic acid production, while matching the load-bearing capability of a commercial composite. There was no decrease in its antibacterial properties after 1 year of water-ageing. The durable antibacterial and mechanical properties indicate that NACP-QADM-NAg composites may be useful in dental restorations to combat caries.

Figure 1

Mechanical properties of composites during water-ageing. (a) Flexural strength; (b) elastic modulus. Each value represents the mean of six measurements, with the error bar indicating one standard deviation (mean±standard deviation; n=6). In each plot, values with dissimilar letters are significantly different from each other (P<0.05). There was a moderate decrease in the first 3 months, with little subsequent decrease from 3 to 12 months. At 12 months, the NACP-QADM-NAg composite had mechanical properties similar to those of the commercial control composite without antibacterial or remineralizing properties (P>0.1). NACP, nanoparticles of amorphous calcium phosphate; NAg, nanoparticles of silver; QADM, quaternary ammonium dimethacrylate.

Figure 2

Live/dead staining of 2-day dental plaque microcosm biofilms on the composites. Live bacteria were stained green, and dead bacteria were stained red. Live and dead bacteria in proximity resulted in yellow/orange colours. The control composite was covered by a thick biofilm with green staining. NACP composites containing QADM and NAg had much more dead bacteria. There was little difference in biofilm appearance vs ageing time, indicating that the antibacterial activity was not lost during water-ageing. NACP, nanoparticles of amorphous calcium phosphate; NAg, nanoparticles of silver; QADM, quaternary ammonium dimethacrylate.

Figure 3

Quantification of biofilm viability. (a) MTT metabolic activity; (b) lactic acid production of 2-day biofilms on the composites after being water-aged for 1 day to 12 months. Each value represents the mean±standard deviation; n=6. In each plot, values with dissimilar letters are significantly different from each other (P<0.05). Water-ageing for 12 months did not reduce the antibacterial properties of the composite containing QADM and NAg (P>0.1). MTT, 3-[4,5–dimethylthiazol–2–yl]-2,5-diphenyltetrazolium bromide; NACP, nanoparticles of amorphous calcium phosphate; NAg, nanoparticles of silver; QADM, quaternary ammonium dimethacrylate.

Figure 4

CFUs of 2-day biofilms on composites after water-ageing for 12 months. (a) Total microorganisms; (b) total streptococci; (c) mutans streptococci. Combining QADM and NAg together in NACP composite achieved a stronger antibacterial activity than using QADM or NAg alone. Water-ageing for 12 months did not reduce the antibacterial potency of composite containing QADM and NAg (P>0.1). Each value represents the mean±standard deviation; n=6. Values with dissimilar letters are significantly different from each other (P<0.05). CFU, colony-forming unit; NACP, nanoparticles of amorphous calcium phosphate; NAg, nanoparticles of silver; QADM, quaternary ammonium dimethacrylate.