Comprehensive Evaluation of the Biological Properties of Surface-Modified Titanium Alloy Implants

Abstract

An increasing interest in the fabrication of implants made of titanium and its alloys results from their capacity to be integrated into the bone system. This integration is facilitated by different modifications of the implant surface. Here, we assessed the bioactivity of amorphous titania nanoporous and nanotubular coatings (TNTs), produced by electrochemical oxidation of Ti6Al4V orthopedic implants' surface. The chemical composition and microstructure of TNT layers was analyzed by X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD). To increase their antimicrobial activity, TNT coatings were enriched with silver nanoparticles (AgNPs) with the chemical vapor deposition (CVD) method and tested against various bacterial and fungal strains for their ability to form a biofilm. The biointegrity and anti-inflammatory properties of these layers were assessed with the use of fibroblast, osteoblast, and macrophage cell lines. To assess and exclude potential genotoxicity issues of the fabricated systems, a mutation reversal test was performed (Ames Assay MPF, OECD TG 471), showing that none of the TNT coatings released mutagenic substances in long-term incubation experiments. The thorough analysis performed in this study indicates that the TNT5 and TNT5/AgNPs coatings (TNT5-the layer obtained upon applying a 5 V potential) present the most suitable physicochemical and biological properties for their potential use in the fabrication of implants for orthopedics. For this reason, their mechanical properties were measured to obtain full system characteristics.

Keywords: Ti6Al4V implants; XPS; anodization process; anti-inflammatory properties; antimicrobial activity; genotoxicity assessment; mechanical properties.

Figure 1

(a) Photography of the orthopedic implant produced using selective laser sintering of Ti6Al4V powder, SEM images of (b) the implant surface obtained, (c) implant surface after grinding and polishing, (d) surface modification of the implant by anodic oxidation using a 5 V potential, (e) the morphology of the TNT5 coating, (f) the morphology of the TNT15 coating, (g) the morphology of the TNT5/AgNPs coating, and (h) the morphology of the TNT5/AgNPs coating.

Figure 2

Proliferation of human osteoblast-like MG 63 cells (A), L929 murine fibroblast cells (B), and murine macrophage cell line RAW 264.7 (C) on the surface of TiO₂ nanotube coatings analyzed by the MTT assay (a colorimetric assay for assessing cell metabolic activity). MG-63 osteoblasts and L929 fibroblasts were cultured on the specimens for 24, 72, and 120 h, whereas RAW 264.7 macrophages were cultivated for 24 and 48 h in the presence or absence of LPS (Lipopolysaccharide). The absorbance values are expressed as means ±SEM of five independent experiments. Asterisks indicate significant differences comparing to the reference Ti6Al4V alloy foils (Ti6Al4V) (*** p < 0.001, * p < 0.05). Hash marks denote significant differences when the level of cell proliferation was lower in comparison with the reference Ti6Al4V alloy foils (### p < 0.001, ## p < 0.01, # p <0.05).

Figure 3

Scanning electron microscopy (SEM) images showing human osteoblast-like MG-63 cells that grow on the surface of the tested titania coatings and the reference Ti6Al4V alloy foils enriched or not with silver nanograins. Micrographs (m,n) present the cells grown on the surface of Ti6Al4V orthopedic implants, which were produced using selective laser sintering 3D technology. Arrows in image (l) indicate filopodia spread between cells and those in image (q,r) present filopodia penetrating deep into the samples and attaching the cells to the surface. The type of sample, cell incubation time, and scale of the images are shown in the figures as indicated.

Figure 4

Alkaline phosphatase activity (ALP) of MG-63 osteoblasts growing on TiO₂ nanotube coatings produced by electrochemical anodic oxidation at potentials of 5 (TNT5) or 15 V (TNT15) and enriched with silver nanoparticles in comparison with the reference Ti6Al4V alloy foils and enriched or not with silver nanograins. The cells were cultured on the surface of the tested specimens for 24, 72, and 120 h. ALP activity [units] was calculated per µg of protein and it is expressed as the means ± SEM of five independent experiments. Asterisks indicate significant differences at the appropriate incubation time when the ALP activity of the cells growing on the tested specimens was higher compared to the reference Ti6Al4V alloy foils (Ti6Al4V) (*** p < 0.001, * p < 0.05). Hash marks denote significant differences at the appropriate incubation time when the ALP activity of osteoblasts cultivated on the tested samples was lower in comparison with the reference Ti6Al4V alloy foils (### p < 0.001, ## p < 0.01, # p < 0.05).

Figure 5

Secretion of pro-inflammatory (A–C) and anti-inflammatory (D) cytokines or total nitric oxide (E) by RAW 264.7 macrophages in the standard and LPS-stimulated conditions. The cells were cultured on the tested specimens for 24 and 48 h. Cytokine and nitric oxide (NO) production was normalized to a number of 105 cells. Data are expressed as mean ± SE (n = 3). Asterisks indicate significant differences at the appropriate incubation time when the amounts of cytokines and NO produced by the cells growing on the tested specimens were higher in comparison with the reference Ti6Al4V alloy foils (Ti6Al4V) (*** p < 0.001, ** p < 0.01, * p < 0.05). Hash marks denote significant differences at the appropriate incubation time when the levels of cytokines and NO secreted by the cells cultivated on the tested samples were lower in comparison with the reference Ti6Al4V alloy foils (### p < 0.001, ## p < 0.01, # p < 0.05).

Figure 6

Assessment of implants’ genotoxicity by the Ames assay performed in five genetically modified bacteria strains: (a) TA98, (b) TA100, (c) TA1535, (d) TA1537, and (e) E.coli; to improve the readability of Ames assay results of TA98 (a), TA1535 (c), and TA1537 (d), their enlarged versions are added.

Figure 7

Bacterial biofilm on TNT- and TNT/AgNPs-modified Ti6Al4V surfaces assessed using Alamar Blue staining. The results are presented as the mean percentage ± standard deviation (SD) of the bacterial biofilm formed on the tested layers compared to a control biofilm formed on the reference biomaterial (Ti6Al4V) considered as 100%. Statistical analysis was estimated with nonparametric Kruskal–Wallis one-way ANOVA test (* significant differences, p < 0.05).

Figure 8

Fungal biofilm on TNT- and TNT/AgNPs-modified Ti6Al4V surfaces assessed using FDA (fluorescein diacetate) staining. The results are presented as the mean percentage ± standard deviation (SD) of the fungal biofilm formed on the tested layers compared to a control biofilm formed on the reference biomaterial (Ti6Al4V) considered as 100%. Statistical analysis was estimated with nonparametric Kruskal–Wallis one-way ANOVA test (* significant differences, p < 0.05).

Figure 9

Antimicrobial effect of the supernatants obtained after 24 h (a), 2 weeks (b), and 4 weeks of (c) TNT- and TNT/AgNPs-modified Ti6Al4V surfaces’ incubation in PBS, tested using the culture method and colony forming unit (CFU) counting. The results are presented as the mean microbial suspension density [CFU/mL] ± standard deviation (SD) after 24 h of culture in the presence of the tested supernatants.

Figure 10

The topography of TNT5 and TNT5/Ag implants with Sa (Average Roughness) parameter values, which was determined using atomic force microscope (AFM).

Figure 11

The nanomechanical properties (nanohardness and Young’s modulus) of TNT5/Ag implant in the studied area II.