A unique hybrid-structured surface produced by rapid electrochemical anodization enhances bio-corrosion resistance and bone cell responses of β-type Ti-24Nb-4Zr-8Sn alloy

Abstract

Ti-24Nb-4Zr-8Sn (Ti2448), a new β-type Ti alloy, consists of nontoxic elements and exhibits a low uniaxial tensile elastic modulus of approximately 45 GPa for biomedical implant applications. Nevertheless, the bio-corrosion resistance and biocompatibility of Ti2448 alloys must be improved for long-term clinical use. In this study, a rapid electrochemical anodization treatment was used on Ti2448 alloys to enhance the bio-corrosion resistance and bone cell responses by altering the surface characteristics. The proposed anodization process produces a unique hybrid oxide layer (thickness 50-120 nm) comprising a mesoporous outer section and a dense inner section. Experiment results show that the dense inner section enhances the bio-corrosion resistance. Moreover, the mesoporous surface topography, which is on a similar scale as various biological species, improves the wettability, protein adsorption, focal adhesion complex formation and bone cell differentiation. Outside-in signals can be triggered through the interaction of integrins with the mesoporous topography to form the focal adhesion complex and to further induce osteogenic differentiation pathway. These results demonstrate that the proposed electrochemical anodization process for Ti2448 alloys with a low uniaxial tensile elastic modulus has the potential for biomedical implant applications.

Figure 1

Surface FE-SEM morphologies of the test Ti and Ti2448 specimens. (a) Ti-M: Ti specimen mechanically polished with SiC paper up to #1200; (b) Ti2448-M: Ti2448 specimen mechanically polished with SiC paper up to #1200; (c) Ti2448-A1: Ti2448-M treated through electrochemical anodization with current A1; (d) Ti2448-A2: Ti2448-M treated with current A2. Surface AFM topography and roughness of the test Ti and Ti2448 specimens: (e) Ti-M; (f) Ti2448-M; (g) Ti2448-A1; (h) Ti2448-A2. Cross-sectional TEM images of the anodized Ti2448 specimens: (i) Ti2448-A1; (j) Ti2448-A2; (k) Higher magnification of (j) with the diffraction patterns of the selected area.

Figure 2

Contact angle and surface free energy of the test Ti and Ti2448 specimens.

Figure 3

Polarization curves of the test Ti and Ti2448 specimens in different electrolytes: (a) SBP; (b) AS.

Figure 4

Protein adsorption analysis, in terms of the N1s spectra, obtained using an XPS on the test Ti and Ti2448 specimens: (a) albumin; (b) fibronectin.

Figure 5

Cell distribution and adhesion morphologies, observed using fluorescence microscopy and FE-SEM, of hMSCs-GFP cultured on the test Ti and Ti2448 specimens for 1 h: (a) Ti-M; (b) Ti2448-M; (c) Ti2448-A1; (d) Ti2448-A2. Immunofluorescence images of focal adhesion complex formation and cytoskeletal arrangement, observed using fluorescence microscopy, of the hMSCs cultured on the test Ti and Ti2448 specimens for 6 h: (e,i) Ti-M; (f,j) Ti2448-M; (g,k) Ti2448-A1; (h,l) Ti2448-A2 (green: vinculin; red: F-actin; blue: nucleus). (m) Cell migration, observed using fluorescence microscopy, of the hMSCs-GFP on the test Ti and Ti2448 specimens. (n) Cell proliferation and (o) cell mineralization analyzed using MTT assay and Alizarin Red S staining, respectively, of the hMSCs on the test Ti and Ti2448 specimens. Data are shown as the mean ± SD. (^#p < 0.05 indicates a statistically significant difference compared to Ti-M).

Figure 6

Osteogenic protein marker expression, assessed using Western blotting analysis, of the hMSCs on the test Ti and Ti2448 specimens: (a) Representative electrophoresis images of osteogenic-related protein expression after incubation of 7, 14 and 21 days; (b) the quantitative levels of osteogenic-related protein expression in (a). The data are shown as the mean ± SD. (*p < 0.05 indicates a statistically significant difference compared to Ti2448-M; ^#p < 0.05 and ^##p < 0.01 indicate a statistically significant difference compared to Ti-M).

Figure 7

Illustration of how the hybrid surface oxide layer, produced by electrochemical anodization treatment, enhanced the bio-corrosion resistance and cell responses of the Ti2448 alloy: (a) a hybrid oxide layer with outer mesoporous topography and inner dense section could be produced using a simple and rapid electrochemical anodization treatment; (b) the thicker and dense inner section of the hybrid oxide layer reduced the release of metal ions. This decreased the potential risk of metal ion accumulation and cytotoxicity from the Ti248 alloy. Moreover, the outer mesoporous topography of the hybrid oxide layer improved the surface wettability and protein adsorption ability of the Ti2448 alloy. This enhanced the formation of the focal adhesion complex and cytoskeletal arrangement of bone cell, which, in turn, induced cell migration and osteogenic differentiation. The scale bar in the upper-left micrograph of (b) is 20 nm.