ZnO Nanosheet-Coated TiZrPdSiNb Alloy as a Piezoelectric Hybrid Material for Self-Stimulating Orthopedic Implants

Abstract

A Ti-based alloy (Ti₄₅Zr₁₅Pd₃₀Si₅Nb₅) with already proven excellent mechanical and biocompatibility features has been coated with piezoelectric zinc oxide (ZnO) to induce the electrical self-stimulation of cells. ZnO was grown onto the pristine alloy in two different morphologies: a flat dense film and an array of nanosheets. The effect of the combined material on osteoblasts (electrically stimulable cells) was analyzed in terms of proliferation, cell adhesion, expression of differentiation markers and induction of calcium transients. Although both ZnO structures were biocompatible and did not induce inflammatory response, only the array of ZnO nanosheets was able to induce calcium transients, which improved the proliferation of Saos-2 cells and enhanced the expression of some early differentiation expression genes. The usual motion of the cells imposes strain to the ZnO nanosheets, which, in turn, create local electric fields owing to their piezoelectric character. These electric fields cause the opening of calcium voltage gates and boost cell proliferation and early differentiation. Thus, the modification of the Ti₄₅Zr₁₅Pd₃₀Si₅Nb₅ surface with an array of ZnO nanosheets endows the alloy with smart characteristics, making it capable of electric self-stimulation.

Keywords: TiZrPdSiNb alloy; differentiation; nanogenerators; osteoblast; piezoelectric; proliferation; self-stimulating.

Figure 1

Schematic illustration of the coating process to obtain ZnO thin films (sputtering) and ZnO nanosheets (hydrothermal synthesis) on TiZrPdSiNb alloy.

Figure 2

SEM images of (a) pristine alloy, (b) ZnO thin film, and (c) ZnO nanosheets deposited on top. EDX patterns of (d) pristine alloy and (e) ZnO thin film samples. (f) Grazing incidence X-ray diffraction (GIXRD) patterns of pristine alloy and ZnO nanosheets.

Figure 3

Proliferation of Saos-2 cells grown on pristine alloy, ZnO thin film, and ZnO nanosheets at 3 and 7 days in culture. Results were normalized by day 1. Different superscripts on top of the columns denote significant differences (p < 0.05) among the materials at the same time-point.

Figure 4

Logarithmic representation of cytokine release by macrophages cultured in the presence of lipopolysaccharide (LPS) (positive control) and the three different samples. Secretion was analyzed by the cytometric bead array (CBA) test at 24 h of culture. Error bars indicate the standard error of the mean of three replicas.

Figure 5

Saos-2 cells adhered to the surface of the pristine alloy (a), ZnO thin film (b), and ZnO nanosheets (c) after 3 days in culture. Stress fibers (actin; red), vinculin (green) and nuclei (DNA; blue) can be observed. Focal contacts (yellow arrows) appear as yellow spots due to the overlay of actin and vinculin signals.

Figure 6

SEM images of Saos-2 cells cultured for 7 days on pristine alloy (a,b), ZnO thin film (c,d), or ZnO nanosheets (e,f). Several nucleoli can be seen inside nuclei (yellow arrows). Long projections are anchored directly on the nanosheets (green arrow).

Figure 7

Percentage of activated osteoblastic (Saos-2) cells which underwent transient changes in intracellular calcium concentration when grown on top of the different samples. Different superscripts on top of the columns denote significant differences (p < 0.05) among the materials.

Figure 8

Quantification of mRNA levels. Relative expression of osteoblast differentiation markers COL1, ALP, BGLAP, IBSP, SPARC and SPP1 (under the protein encoded) in Saos-2 cells on day 7, 14 and 21 after seeding on the three samples. The target gene levels are expressed as a relative value. Different superscripts on top of the columns denote significant differences (p < 0.05) among the materials at the same time-point.