Antibacterial nanocomposite with calcium phosphate and quaternary ammonium

Abstract

Secondary caries is a frequent reason for restoration failure, resulting from acidogenic bacteria and their biofilms. The objectives of this study were to: (1) develop a novel nanocomposite containing nanoparticles of amorphous calcium phosphate (NACP) and quaternary ammonium dimethacrylate (QADM); and (2) investigate its mechanical and antibacterial durability. A spray-drying technique yielded NACP with particle size of 116 nm. The nanocomposite contained NACP and reinforcement glass fillers, with QADM in the resin. Two commercial composites were tested as controls. Composites were inoculated with Streptococcus mutans. After 180-day water-aging, NACP+QADM nanocomposite had flexural strength and elastic modulus matching those of commercial controls (p > 0.1). NACP+QADM nanocomposite reduced the biofilm colony-forming units (CFU) by 3-fold, compared with commercial composites (p < 0.05). Metabolic activity and lactic acid production of biofilms on NACP+QADM were much less than those on commercial composites (p < 0.05). The antibacterial properties of NACP+QADM were maintained after water-aging for 30, 90, and 180 d (p > 0.05). In conclusion, the novel NACP-QADM nanocomposite greatly decreased biofilm metabolic activity, CFU, and lactic acid, while matching the load-bearing capability of commercial composites without antibacterial properties. The NACP-QADM nanocomposite with strong and durable antibacterial properties, together with its previously reported Ca-PO(4) release capability, may render it useful for caries-inhibiting restorations.

Figure 1.

Mechanical properties of composites in water immersion. (A) Flexural strength. (B) Elastic modulus. Each value is the mean of 6 measurements, with the error bar showing one standard deviation (mean ± SD; n = 6). In each plot, values with dissimilar letters are significantly different (p < 0.05). There was a moderate decrease during the first 30 d, with little decrease from 30 to 180 d. At 180 d, the NACP-QADM nanocomposite had values similar to those of the commercial composites (p > 0.1).

Figure 2.

Live/dead staining of 3-day biofilms on composites. Live bacteria were stained green, and dead bacteria were stained red. Live and dead bacteria in close proximity showed yellow/orange colors. The images shown in (A-I) are representative of each group. CompositeR was covered by a dense biofilm with green staining. CompositeF had some compromised bacteria. NACP+QADM had much more dead bacteria staining than the controls. The area fraction of live bacteria staining is plotted in (J) (mean ± SD; n = 6). There was little difference in biofilm viability vs. aging time, indicating that the antibacterial activity of NACP-QADM nanocomposite was not lost in water immersion.

Figure 3.

SEM micrographs of typical biofilms. (A-C) Lower magnification. (D-F) Higher magnification. Each type of composite, aged for 1 to 180 d, had a similar biofilm appearance. The images shown here are for composites aged for 180 d, to demonstrate the long-term antibacterial activity of the NACP-QADM nanocomposite. CompositeR and CompositeF had dense biofilms. NACP-QADM had much less biofilm coverage. In (C) and (F), “R” indicates the resin composite surface not covered by biofilms. Arrows indicate the chain structure of S. mutans biofilms. The chains are much shorter on NACP-QADM in (F), along with individual cells that did not form a chain. Each bacterial cell had the shape of a short rod with a length of about 1 µm (arrow in F).

Figure 4.

Biofilm viability, growth, and acid production. (A) MTT metabolic activity, (B) CFU counts, and (C) lactic acid production of 3-day biofilms on the composites water-aged for 1 to 180 d. Each value is mean ± SD (n = 6). In each plot, values with dissimilar letters are significantly different (p < 0.05). For the MTT assay, a higher absorbance indicates a higher formazan concentration, which in turn indicates a higher metabolic activity in the biofilm. The NACP-QADM nanocomposite had biofilm metabolic activity and lactic acid production that were about 1/2 of those on the commercial composites, and CFU counts about 1/3 of those on commercial composites (p < 0.05). Aging for 1 to 180 d did not reduce the antibacterial potency of the NACP-QADM nanocomposite (p > 0.1).