"To Be Microbiocidal and Not to Be Cytotoxic at the Same Time…"-Silver Nanoparticles and Their Main Role on the Surface of Titanium Alloy Implants

Abstract

The chemical vapor deposition (CVD) method has been used to produce dispersed silver nanoparticles (AgNPs) on the surface of titanium alloy (Ti6Al4V) and nanotubular modified titanium alloys (Ti6Al4V/TNT5), leading to the formation of Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs systems with different contents of metallic silver particles. Their surface morphology and silver particles arrangement were characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS), and atomic force microscopy (AFM). The wettability and surface free energy of these materials were investigated on the basis of contact angle measurements. The degree of silver ion release from the surface of the studied systems immersed in phosphate buffered saline solution (PBS) was estimated using inductively coupled plasma ionization mass spectrometry (ICP-MS). The biocompatibility of the analyzed materials was estimated based on the fibroblasts and osteoblasts adhesion and proliferation, while their microbiocidal properties were determined against Gram-positive and Gram-negative bacteria, and yeasts. The results of our works proved the high antimicrobial activity and biocompatibility of all the studied systems. Among them, Ti6Al4V/TNT5/0.6AgNPs contained the lowest amount of AgNPs, but still revealed optimal biointegration properties and high biocidal properties. This is the biomaterial that possesses the desired biological properties, in which the potential toxicity is minimized by minimizing the number of silver nanoparticles.

Keywords: antimicrobial activity; biointegration; silver ions release; silver nanoparticles; titanium alloy; titanium dioxide nanotubes.

Figure 1

The scanning electron microscopy (SEM) images of Ti6Al4V/0.9AgNPs; d = 18 ± 8 nm (a), Ti6Al4V/1.1AgNPs; d = 45 ± 15 nm (b), Ti6Al4V/1.3AgNPs; d = 68 ± 32 nm (c), Ti6Al4V/2.3AgNPs; d = 53 ± 18 nm (d).

Figure 2

Scanning electron microscopy (SEM) images of Ti6Al4V/TNT5/0.6AgNPs; d = 38 ± 14 nm (a), Ti6Al4V/TNT5/1.0AgNPs; d = 43 ± 10 nm (b), Ti6Al4V/TNT5/1.6AgNPs; d = 57 ± 24 nm (c), Ti6Al4V/TNT5/2.3AgNPs; d = 115 ± 49 nm (d).

Figure 3

Atomic forces microscopy (AFM) images and R_a parameters determined for the Ti6Al4V, Ti6Al4V/AgNPs, Ti6Al4V/TNT5, and Ti6Al4V/TNT5/AgNPs samples.

Figure 4

The energy dispersive X-ray spectroscopy (EDS) spectra and maps images of Ti6Al4V/0.9AgNPs, Ti6Al4V/2.3AgNPs, Ti6Al4V/TNT5/0.6AgNPs, and Ti6Al4V/TNT5/2.3AgNPs (AgNPs are marked as the green dots on the presented map images).

Figure 5

The contact angles values for Ti6Al4V, Ti6Al4V/AgNPs, Ti6Al4V/TNT5, and Ti6Al4V/TNT5/AgNPs.

Figure 6

The surface free energy values for Ti6Al4V, Ti6Al4V/AgNPs, Ti6Al4V/TNT5, and Ti6Al4V/TNT5/AgNPs.

Figure 7

The release amount of Ag⁺ ions from Ti6Al4V/AgNPs and Ti6Al4V/TNT5/AgNPs samples (containing 2.3 and 1.0–1.1 wt% of AgNPs) immersed in a phosphate buffered saline (PBS) and measured by inductively coupled plasma ionization mass spectrometry (ICP-MS).

Figure 8

The L929 murine fibroblasts (A) and human osteoblasts MG-63 (B) adhesion (measured after 24 h) and proliferation (evaluated after 72 h and 120 h) on the surface of Ti6Al4V/AgNPs, detected by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The absorbance values are expressed as means ± SEM of five independent experiments. Hash marks indicate significant differences at the appropriate incubation time between the cells incubated on the reference Ti6Al4V alloy foils (Ti6Al4V) compared to the specimen coatings doped by the different concentrations of Ag (# p < 0.05, ## p < 0.01). Tables below Figure 8A,B presented relative L929 cells or MG-63 cell viability (%) compared to the Ti6Al4V reference sample measured after 120 h of incubation.

Figure 9

The effect of TNT5/AgNPs coatings on the L929 fibroblasts (A) and MG-63 osteoblasts (B) adhesion (measured after 24 h) and proliferation (evaluated after 72 h and 120 h), detected by the MTT assay. The absorbance values are expressed as means ± SEM of five independent experiments. Asterisks indicate significant differences at the appropriate incubation time when the level of cell proliferation on the surface of specimens coating doped by the different concentrations of Ag was higher compared to the reference Ti6Al4V alloy foils (Ti6Al4V) (*** p < 0.001). Hash marks denote significant differences at the appropriate incubation time when the level of cell proliferation on the samples enriched with AgNPs was lower in comparison with the reference Ti6Al4V alloy foils (### p < 0.001). Tables below Figure 9A,B presented relative L929 or MG-63 cells viability (%) compared to the Ti6Al4V reference sample measured after 120 h of incubation.

Figure 10

The scanning electron microscopy (SEM) images presenting adhesion (after 24 h) and proliferation (after 72 h and 120 h) of the murine L929 fibroblasts growing on the surface of reference Ti6Al4V alloy foils (a–c), (Ti6Al4V/0.9AgNPs) (d–f) or Ti6Al4V/TNT5/0.6AgNPs (g–i). Arrows in the micrographs indicate numerous filopodia spreading between the fibroblasts (j–k) or filopodia, which attached the cells to the surface of nanocoatings (l).

Figure 11

The scanning electron microscopy (SEM) micrographs showing the human osteoblast-like MG-63 cells adhesion (after 24 h) and proliferation (after 72 h and 120 h) growing on the surface of references Ti6Al4V alloy foils (a–c), (Ti6Al4V/0.9AgNPs) (d–f) or Ti6Al4V/TNT5/0.6AgNPs (g–i). The micrograph (j) presents the multilayer growth of cells on the surface of Ti6Al4V/TNT5/0.6AgNPs sample. Arrows indicate numerous filopodia, which attached the osteoblasts to the nanocoatings surface (l) and filopodia spreading between the cells (k).