Antibacterial amorphous calcium phosphate nanocomposites with a quaternary ammonium dimethacrylate and silver nanoparticles

Abstract

Objectives: Calcium and phosphate ion-releasing resin composites are promising for remineralization. However, there has been no report on incorporating antibacterial agents to these composites. The objective of this study was to develop antibacterial and mechanically strong nanocomposites incorporating a quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP).

Methods: The QADM, bis(2-methacryloyloxyethyl) dimethylammonium bromide (ionic dimethacrylate-1), was synthesized from 2-(N,N-dimethylamino)ethyl methacrylate and 2-bromoethyl methacrylate. NAg was synthesized by dissolving Ag 2-ethylhexanoate salt in 2-(tert-butylamino)ethyl methacrylate. Mechanical properties were measured in three-point flexure with bars of 2 mm×2 mm×25 mm (n=6). Composite disks (diameter=9 mm, thickness=2 mm) were inoculated with Streptococcus mutans. The metabolic activity and lactic acid production of biofilms were measured (n=6). Two commercial composites were used as controls.

Results: Flexural strength and elastic modulus of NACP+QADM, NACP+NAg, and NACP+QADM+NAg matched those of commercial composites with no antibacterial property (p>0.1). The NACP+QADM+NAg composite decreased the titer counts of adherent S. mutans biofilms by an order of magnitude, compared to the commercial composites (p<0.05). The metabolic activity and lactic acid production of biofilms on NACP+QADM+NAg composite were much less than those on commercial composites (p<0.05). Combining QADM and NAg rendered the nanocomposite more strongly antibacterial than either agent alone (p<0.05).

Significance: QADM and NAg were incorporated into calcium phosphate composite for the first time. NACP+QADM+NAg was strongly antibacterial and greatly reduced the titer counts, metabolic activity, and acid production of S. mutans biofilms, while possessing mechanical properties similar to commercial composites. These nanocomposites are promising to have the double benefits of remineralization and antibacterial capabilities to inhibit dental caries.

[1]

Representative TEM micrographs of the size and dispersion of silver nanoparticles in the resin matrix: (A) lower and (B) high magnification. Silver 2-ethylhexanoate salt was dissolved in 2-(tert-butylamino) ethyl methacrylate and incorporated into a BisGMA-TEGDMA resin at a silver salt mass fraction of 0.08% in the resin. The silver nanoparticles were formed in the resin by simultaneous reduction of the silver salt and photopolymerization of the dimethacrylates. The particle size was measured (mean ± sd; n = 100) to be (2.7 ± 0.6) nm. Arrows indicate the silver nanoparticles, which were well dispersed in the resin with minimal appearance of nanoparticle aggregates.

[2]

Mechanical properties. (A) Flexural strength, and (B) elastic modulus for CompositeNoF, CompositeF, NACP composite, NACP+QADM composite, NACP+NAg composite, and NACP+QADM+NAg composite. Each value is the mean of six measurements with the error bar indicating one standard deviation (mean ± sd; n = 6).

[3]

Live/dead fluorescence images of S. mutans biofilms on composites at 1 d: (A) CompositeNoF, (B) CompositeF, (C) NACP composite, (D) NACP+QADM composite, (E) NACP+NAg composite, (F) NACP+QADM+NAg composite. Live bacteria were stained green, and the compromised bacteria were stained red. When the live and dead bacteria were close to each other or on the top of each other, the green staining was mixed with the red, resulting in yellowish or orange colors. Arrows in D, E and F indicate areas of the compromised bacteria.

[4]

Live/dead fluorescence images of S. mutans biofilms on composites at 3 d: (A) CompositeNoF, (B) CompositeF, (C) NACP composite, (D) NACP+QADM composite, (E) NACP+NAg composite, (F) NACP+QADM+NAg composite. Arrows in D, E and F indicate areas of the compromised bacteria. A–C had mature biofilms in which the staining was mostly green, hence the bacteria were primarily alive. NACP+QADM+NAg composite had the most red/orange staining, indicating the greatest amount of compromised bacteria.

[5]

The metabolic activity of S. mutans biofilms adherent on CompositeNoF, CompositeF, NACP composite, NACP+QADM composite, NACP+NAg composite, and NACP+QADM+NAg composite. Metabolic activity was measured via the MTT assay at (A) 1 d, and (B) 3 d. In each plot, values (mean ± sd; n = 6) with dissimilar letters are significantly different (p < 0.05).

[6]

CFU counts of S. mutans biofilms adherent on the composites at (A) 1 d, and (B) 3 d, with the y-axis units being 10⁶ bacteria per composite disk. In each plot, the values (mean ± sd; n = 6) indicated with dissimilar letters are significantly different from each other (p < 0.05). The NACP+QADM+NAg composite had the least CFU which was an order of magnitude less than that of CompositeNoF.

[7]

Lactic acid production by S. mutans biofilms adherent on disks of CompositeNoF, CompositeF, NACP composite, NACP+QADM composite, NACP+NAg composite, and NACP+QADM+NAg composite. Each value is mean ± sd; n = 6. Dissimilar letters indicate values that are significantly different from each other (p < 0.05).