Nitinol-based nanotubular coatings for the modulation of human vascular cell function

Abstract

In this study, we describe the synthesis of an upright nanotubular coating with discrete, exposed nanotubes on top of superelastic Nitinol via anodization and characterization of the surface elemental composition and nickel release rates. We demonstrate, for the first time, that this coating could improve re-endothelialization by increasing the cell spreading and migration of primary human aortic endothelial cells on Nitinol. We also show the potential for reducing neointimal hyperplasia by decreasing the proliferation and expression of collagen I and MMP-2 in primary human aortic smooth muscle cells (HASMC). Furthermore, we did not observe the nanotubular surface to induce inflammation through ICAM-1 expression in HASMC as compared to the flat control. This coating could be used to improve Nitinol stents by reducing restenosis rates and, given the extensive use of Nitinol in other implantable devices, act as a generalized coating strategy for other medical devices.

Keywords: Human Aortic Endothelial Cells; Human Aortic Smooth Muscle Cells; Nanotubes; Nitinol; Restenosis; Stents.

Figure 1

SEM images of the flat control Nitinol (A) and the upright nanotubular coating that was synthesized on top of superelastic Nitinol via an anodization process at 85 V (B and C) and 70 V (D).

Figure 2

Energy dispersive X-ray spectroscopy (EDS) data of the nanotubular coated Nitinol (A) and the flat Nitinol control (B). Surface elemental analysis data is presented in the tables and EDS spectra. The nanotubular coating was found to consist of both NiO and TiO₂, whereas the control substrates had undetectable levels of a surface oxide layer.

Figure 3

(A) Cumulative nickel released from nanotubular coated Nitinol (Nanotubes 1, 2, and 3) and flat control Nitinol (Control 1, 2, and 3) as compared to the intravenous nickel contamination limit (Safety Limit). (B) The same data enlarged, excluding the intravenous nickel contamination limit.

Figure 4

Nanotubular coated Nitinol increased the migration of HAEC. The schematic (A) describes the migration assay used, whereas the data (B) shows a significant increase in migration of HAEC from the collagen gel onto the nanotubular coated Nitinol as compared to flat control Nitinol. * = p < 0.001, N = 5.

Figure 5

Fluorescence microscopy images of HAEC on nanotubular coated Nitinol (A) and flat control Nitinol (B) after 7 days of culture. FITC-Phalloidin staining of F-actin is shown in red, whereas DAPI staining of cell nuclei is shown in blue. Scale bars are 50 μm. Cell spreading is represented by the average cell surface area (C) normalized to that of the control, while HAEC growth (D) is represented in number of cells per cm² over a period of 7 days. * = p < 0.01, N = 5.

Figure 6

Fluorescence microscopy images of HASMC on nanotubular coated Nitinol (A) and flat control Nitinol (B) after 7 days of culture. FITC-Phalloidin staining of F-actin is shown in red, whereas DAPI staining of cell nuclei is shown in blue. Scale bars are 50 μm. HASMC growth (C) is represented in number of cells per cm² over a period of 7 days. * = p < 0.01, N = 5.

Figure 7

Immunofluorescent staining of Col1A (green) and DAPI staining of nuclei (blue) in HASMC grown on nanotubes-coated Nitinol (A) and the control (B). Relative mRNA expression levels of Collagen 1 (Col1), Collagen 3 (Col3) and Matrix Metalloproteinase 2 (MMP2) genes in HASMC (C) is presented as fold change using delta–delta CT values with GAPDH as the reference gene. # = p < 0.05, N = 5.

Figure 8

Flow cytometry histogram plots of HASMC stained for ICAM-1 and cultured on nanotubes-coated (Nanotubes) or flat control Nitinol (Control) surfaces. ICAM-1 induction with 2 ng/mL (A) and 10 ng/mL (B) of TNF-alpha is shown as a comparison to the endogenous level of the HASMC cultured on both surfaces (No TNF).