Antibacterial and Cytotoxicity Evaluation of New Hydroxyapatite-Based Granules Containing Silver or Gallium Ions with Potential Use as Bone Substitutes

Abstract

The aim of the current work was to study the physicochemical properties and biological activity of different types of porous granules containing silver or gallium ions. Firstly, hydroxyapatites powders doped with Ga³⁺ or Ag⁺ were synthesized by the standard wet method. Then, the obtained powders were used to fabricate ceramic microgranules (AgM and GaM) and alginate/hydroxyapatite composite granules (AgT and GaT). The ceramic microgranules were also used to prepare a third type of granules (AgMT and GaMT) containing silver or gallium, respectively. All the granules turned out to be porous, except that the AgT and GaT granules were characterized by higher porosity and a better developed specific surface, whereas the microgranules had very fine, numerous micropores. The granules revealed a slow release of the substituted ions. All the granules except AgT were classified as non-cytotoxic according to the neutral red uptake (NRU) test and the MTT assay. The obtained powders and granules were subjected to various antibacterial test towards the following four different bacterial strains: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli. The Ag-containing materials revealed high antibacterial activity.

Keywords: antibacterial activity; biomaterials; calcium phosphates; gallium; silver.

Figure 1

Powder X-ray diffractograms (a) and FT-IR spectra (b) of the synthesized powder samples.

Figure 2

Representative SEM images of the samples: ceramic microgranules (AgM and GaM) (a–c); composite granules (AgT and GaT) (d–f); composite granules AgMT and GaMT (g–i).

Figure 3

The results of the release study of silver and gallium ions from: AgT and GaT granules (a); AgMT and GaMT granules (b).

Figure 4

The NRU (blue bars) and MTT (orange bars) tests results obtained for granules in the whole range of tested extracts concentrations. Black stars indicate the decrease in the cells’ viability under 70%, which classifies the sample as cytotoxic.

Figure 5

Antibacterial activity of hydroxyapatite powders against 4 bacterial strains: Staphylococcus aureus (a), Staphylococcus epidermidis (b), Escherichia coli (c) and Pseudomonas aeruginosa (d). (ʹ) and (ʺ) symbols accompanying powders designation on X axes indicate their concentration in mixtures (0.1 mg/mL and 1 mg/mL, respectively). (*) symbol indicates statistically significant differences between the samples and control, (#) symbol indicates statistically significant results between HAʹ and the samples, ($) symbol indicates statistically significant results between HAʺ and the samples, (^) symbol indicates statistically significant results between Ag-HAʹ and the samples, (@) symbol indicates statistically significant results between Ag-HAʺ and the samples, (~) symbol indicates statistically significant results between Ga-HAʹ and the samples; according to one-way ANOVA with post-hoc Dunnett’s test or post-hoc Tukey’s test (p < 0.05).

Figure 6

Antibacterial activity according to the AATCC 100-2004 test method (a) and bacterial adhesion (b) of hydroxyapatite-based granules against 4 bacterial strains. (*) symbol indicates statistically significant differences between the samples and control, (#) symbol indicates statistically significant results between the samples and HAT granules, ($) symbol indicates statistically significant results between AgT and all doped samples, (^) symbol indicates statistically significant results between GaT and all doped samples; according to one-way ANOVA with post-hoc Dunnett’s test or post-hoc Tukey’s test (p < 0.05).