The Morphology, Structure, Mechanical Properties and Biocompatibility of Nanotubular Titania Coatings before and after Autoclaving Process

Abstract

The autoclaving process is one of the sterilization procedures of implantable devices. Therefore, it is important to assess the impact of hot steam at high pressure on the morphology, structure, and properties of implants modified by nanocomposite coatings. In our works, we focused on studies on amorphous titania nanotubes produced by titanium alloy (Ti6Al4V) electrochemical oxidation in the potential range 5⁻60 V. Half of the samples were drying in argon stream at room temperature, and the second ones were drying additionally with the use of immersion in acetone and drying at 396 K. Samples were subjected to autoclaving and after sterilization they were structurally and morphologically characterized using Raman spectroscopy, diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) and scanning electron microscopy (SEM). They were characterized in terms of wettability, mechanical properties, and biocompatibility. Obtained results proved that the autoclaving of amorphous titania nanotube coatings produced at lower potentials (5⁻15 V) does not affect their morphology and structure regardless of the drying method before autoclaving. Nanotubular coatings produced using higher potentials (20⁻60 V) require removal of adsorbed water particles from their surface. Otherwise, autoclaving leads to the destruction of the architecture of nanotubular coatings, which is associated with the changing of their mechanical and biointegration properties.

Keywords: autoclaving; biocompatibility; mechanical properties; nanotubes; titanium alloy; titanium dioxide; wettability.

Scheme 1

The scheme of experimental procedure applied in the preparation of the samples.

Figure 1

Scanning Electron Microscopy (SEM) images of Ti6Al4V/TNT5-15/Ar, Ti6Al4V/TNH5-TNH15, and Ti6Al4V/TNT5-15 samples surface (selected destruction sites of TiO₂ nanotubes were marked with circles).

Figure 2

SEM images of Ti6Al4V/TNT20-60/Ar, Ti6Al4V/TNH20-TNH60, and Ti6Al4V/TNT20-60 samples surface.

Figure 3

Raman spectra of Ti6Al4V/TNT20-60 (a) and Ti6Al4V/TNH20-TNT60 (b) samples (A—TiO₂ anatase form, R—TiO₂ rutile form).

Figure 4

Infrared (IR) spectra (DRIFT) of (a) Ti6Al4V/TNT20/Ar and (c) Ti6Al4V/TNT50/Ar samples (the samples after drying in the Ar stream) and Ti6Al4V/TNT20/Ac (b) and Ti6Al4V/TNT50/Ac (d) the samples immersed in acetone and dried at 396 K by 1 h.

Figure 5

IR DRIFT spectra of (a) Ti6Al4V/TNT20-60/Ar and (b) Ti6Al4V/TNT20-60/Ac systems.

Figure 6

Atomic force microscopy (AFM) topography of Ti6Al4V/TNH20-60 and Ti6Al4V/TNT20-60 systems.

Figure 7

L929 murine fibroblasts (A) and human osteoblasts MG-63 (B) proliferation (after 24-, 72- and 120 h) on the surface of Ti6Al4V/TNH20-TNH60 and Ti6Al4V/TNT20-TNT60 samples, detected by MTT assay. The absorbance values are expressed as means ± SEM of five independent experiments. Asterisk and hash mark indicate significant differences between the cells incubated with the alloy references samples (Ti6Al4V) compared to the TNH and TNT specimens after 72 h (* p < 0.05, ** p < 0.01, *** p < 0.001) or 120 h (^# p < 0.05, ^## p < 0.01, ^### p < 0.001) of incubation time, respectively. Tables below the graphs A and B present the statistical differences in the proliferation of the cells, between TNH and TNT coatings produced by the electrochemical anodization of the Ti6Al4V foil at the same selected potential (^$ p < 0.05, ^$$ p < 0.01, ^$$$ p < 0.001).

Figure 8

Scanning electron microscopy (SEM) images presenting proliferation of the murine L929 fibroblasts growing on the surface of the titanium alloy references sample (Ti6Al4V; (a–c) in comparison with Ti6Al4V/TNH40 sample; (d–f) and Ti6Al4V/TNT40 sample; (g–i). Arrows in the image (j) show the cells growing in layers on top of each other. Arrows in micrographs (k) and (l) indicate filopodia spread between cells or penetrating deep into the TNT40 nanolayers, respectively

Figure 9

Scanning electron microscopy (SEM) micrographs showing the human osteoblast-like MG-63 cell proliferation on the surface of the references sample (Ti6Al4V; (a–c) compared to Ti6Al4V/TNH40 sample; (d–f) and the Ti6Al4V/TNT40 sample; (g–i). Micrograph (j) presents the multilayer growth of cells. Arrows indicate numerous filopodia spreading between cells (k) or filopodia, which attached osteoblasts to the nanocoatings surface (l).