Direct, Broad-Spectrum Antimicrobial Activity of Ag+-Doped Hydroxyapatite against Fastidious Anaerobic Periodontal and Aerobic Dental Bacteria

Abstract

Periodontitis and caries, while seemingly innocuous medical conditions, actually pose significant challenges because of their potential etiology with far more serious conditions. Efficacious treatment is hindered by bacterial antibiotic resistance. Standard AgNPs are ineffective against periodontal anaerobic bacteria, because they require oxidative dissolution to release Ag⁺ ions, which are the actual antimicrobial agents, but oxidation is not possible under anaerobic conditions. Prior studies on Ag-based periodontal antimicrobial materials either did not confirm a silver oxidation state or did not use strictly anaerobic growth media or both, causing spurious antimicrobial efficacy estimates. Here, we prove that silver ion-doped hydroxyapatite nanoparticles (AgHAp NPs) synthesized at various pHs contain an Ag⁺ oxidation state and directly release Ag⁺ even in a strictly anerobic medium. Thus, these AgHAp NPs exhibit direct antimicrobial activity against the fastidious anaerobic Gram-negative periodontal bacterium Fusobacterium nucleatum (F. nucleatum) and against caries-causing aerobic, Gram-positive bacterium Streptococcus mutans (S. mutans). The synthesis pH (6-11) correlates inversely with the Ag⁺ content (4.5-0.45 wt %) of AgHAp NPs and, hence, with antimicrobial efficacy, thus providing tunable efficacy for the target application. AgHAp NPs had greater antimicrobial efficacy than Ag⁰-containing AgNPs and were less cytotoxic to the mouse fibroblast L929 cell line. Thus, AgHAp NPs (especially AgHAp7) are superior to AgNPs as effective, broad-spectrum, biocompatible antimicrobials against both anaerobic periodontal and aerobic dental bacteria. AgHAp NP synthesis is also inexpensive and scalable, which are significant factors for treating large global populations of indigent people affected by periodontitis and dental caries.

Keywords: anaerobic bacteria; broad-spectrum antimicrobial; hydroxyapatite; oxidation state; silver ion.

Figure 1

Characterization of Ag0, Hap, and AgHAp NPs. (A) TEM images of (a) AgNPs, (b) HAp, (c) AgHAp9.5, (d) AgHAp7, and (e) AgHAp6; (B) XRD patterns of the NPs; (C) Total silver content obtained from ICP-OES analysis for the AgHAp NPs synthesized at different pHs. Error bar represents the standard deviation from the mean.

Figure 2

XPS analysis. (A) Survey spectrum for (a) AgNPs, (b) AgHAp9.5, and (c) AgHAp7. (B) High-resolution scan of Ag3d XPS spectra: (a) AgNPs, (b) AgHAp9.5, and (c) AgHAp7. Open circles: raw spectra; black curve: fitted peak; filled area: fitted component peak.

Figure 3

Inhibition zone antimicrobial test results for AgNPs, HAp, AgHAp9.5, AgHAp7, and AgHAp6 against F. nucleatum and S. mutans. (A) Agar plate images for F. nucleatum (top panel) and S. mutans (middle panel). (B,C) Inhibition zone diameters against F. nucleatum (B) and S. mutans (C). Student t-test was selected for statistical analysis (* < 0.05, **** < 0.0001, and ns = no significant difference).

Figure 4

Conductivity test under aerobic (cyan) and anaerobic (magenta) conditions with L-Cysteine (solid line) added and without L-Cysteine (dashed line).

Figure 5

Cell viability by the LIVE/DEAD assay of murine fibroblast L-929 cells in the presence of various concentrations of AgHAp NPs compared to AgNPs and pure HAp NPs on (A) Day 1 and (B) Day 3. All images were taken at 50× magnification with the same scale bar of 100 µm. Images shown have not been adjusted for differences in contrast.

Figure 6

Fibroblast L-929 cell viability by MTT assay on Day 1 (A) and Day 3 (B) of exposure to various NPs at different concentrations. (C) IC50 values of AgNPs, HAp, AgHAp9.5, and AgHAp7. Curves indicate fits (see Section 2) to the experimental data (solid circles): AgNPs (orange), HAp (Black), AgHAp9.5 (Blue), and AgHAp7 (Green). IC50 values were obtained from curve fits.