Proliferation of osteoblast precursor cells on the surface of TiO2 nanowires anodically grown on a β-type biomedical titanium alloy

Abstract

Studies have shown that anodically grown TiO₂ nanotubes (TNTs) exhibit excellent biocompatibility. However, TiO₂ nanowires (TNWs) have received less attention. The objective of this study was to investigate the proliferation of osteoblast precursor cells on the surfaces of TNWs grown by electrochemical anodization of a Ti-35Nb-7Zr-5Ta (TNZT) alloy. TNT and flat TNZT surfaces were used as control samples. MC3T3-E1 cells were cultured on the surfaces of the samples for up to 5 days, and cell viability and proliferation were investigated using fluorescence microscopy, colorimetric assay, and scanning electron microscopy. The results showed lower cell proliferation rates on the TNW surface compared to control samples without significant differences in cell survival among experimental conditions. Contact angles measurements showed a good level of hydrophilicity for the TNWs, however, their relatively thin diameter and their high density may have affected cell proliferation. Although more research is necessary to understand all the parameters affecting biocompatibility, these TiO₂ nanostructures may represent promising tools for the treatment of bone defects and regeneration of bone tissue, among other applications.

Figure 1

XRD analysis of the TNZT alloy used as the substrate for the anodic growth of TNTs and TNWs. All the reflections observed are from the body-centered cubic (β) phase of titanium.

Figure 2

FEG-SEM micrographs of a TNZT sample anodized in the organic electrolyte at 20 V for 12 h, which resulted in the formation of TNWs on top. (a) Top-view image, (b) angled-view image, and (c) top-view image at a higher magnification.

Figure 3

FEG-SEM micrographs of a TNZT sample anodized in the aqueous electrolyte at 20 V for 1 h. (a,b) Top-view and (c) side-view images.

Figure 4

XPS analyses for the TNTs (anodization in the aqueous electrolyte) (blue line) and TNWs (anodization in the organic electrolyte) (red line). (a) Full survey spectrum and high-resolution spectra of (b) Ti 2p, (c) Nb 3d, (d) Zr 3d, (e) Ta 4f, and (f) O 1s.

Figure 5

Fluorescence microscopy images of calcein-stained MC3T3-E1 cells on days 1 to 5 of culture on the surfaces of the chemically polished material, TNTs, and TNWs.

Figure 6

Proliferation and viability of MC3T3-E1 cells after different times of culture on the surfaces of the TNW and control samples (flat TNZT and TNT), as assessed by fluorescence microscopy (a) and by the MTT assay (b). Values represent the mean ± SEM of three independent experiments. *P ≤ 0.05, **P ≤ 0.001 vs. flat TNZT samples (two-way analysis of variance, followed by Dunnett’s post-hoc test).

Figure 7

Scanning electron micrographs of MC3T3-E1 cells after 48 h of culture on the surfaces of the (a) flat TNZT, (b) TNT, and (c) TNW samples.

Figure 8

Scanning electron micrographs of MC3T3-E1 cells after 48 h of culture. (a) Flat TNZT, (b) TNTs, and (c) TNWs.

Figure 9

Wettability (contact angle) measurements of the flat TNZT, TNT, and TNW surfaces. The influence of the drying method after anodization for the TNW samples is shown.