Endothelial responses of magnesium and other alloying elements in magnesium-based stent materials

Abstract

Biodegradable tailored magnesium (Mg) alloys are some of the most promising scaffolds for cardiovascular stents. During the course of degradation after implantation, all the alloying elements in the scaffold will be released to the surrounding vascular tissues. However, fundamental questions regarding the toxicity of alloying elements towards vascular cells, the maximum amount of each element that could be used in alloy design, or how each of the alloying elements affects vascular cellular activity and gene expression, are still not fully answered. This work systematically addressed these questions by revealing how application of different alloying elements commonly used in Mg stent materials influences several indices of human endothelial cell health, i.e., viability, proliferations, cytoskeletal reorganizations, migration, and the gene expression profile. The overall cell viability and proliferation showed a decreasing trend with increasing concentrations of the ions, and the half maximal effective concentrations (EC50) for each element were determined. When applied at a low concentration of around 10 mM, Mg had no adverse effects but improved cell proliferation and migration instead. Mg ions also altered endothelial gene expression significantly in a dose dependent manner. Most of the changed genes are related to angiogenesis and the cell adhesion signaling pathways. Findings from this work provide useful information on maximum safe doses of these ions for endothelial cells, endothelial responses towards these metal ions, and some guidance for future Mg stent design.

Fig. 1

MTT viability of HCAECs after treating with ECM supplemented with different metal chloride solutions for 24 h. The dashed lines indicated the half maximal effective concentration (EC₅₀). Stars indicate that the cell viability was significantly decreased compared to control (n = 3, P < 0.05).

Fig. 2

LDH release from HCAECs after treating with ECM supplemented with the different ion solutions. Stars indicate that the LDH release was significantly increased compared to control (n = 3, P < 0.05).

Fig. 3

HCAECs proliferation rate measured by BrdU assay. Stars indicate that cell proliferation rates are significantly changed compared to control (n = 3, P < 0.05).

Fig. 4

Optical images of HCAECs migration at 0, 6 and 24 h by scratch wound assay. A straight line in a cell monolayer was created by scratching the surface with a p200 pipette tip. Cells were treated by ECM supplemented with gradient concentrations of MgCl₂. The gap width (GW) of the line was calculated by Image Pro software. Recovery rate (RR) and recovery speed (RS) are shown on the top left corner of the image (n = 18, P < 0.05).

Fig. 5

HCAECs recovery ratio after treating with individual REE (500 μM) for 24 h. All the groups were significantly different from each other except for NdCl₃ and YCl₃ at 6 h. C represents the control group treated with normal culture media (n = 18, P < 0.05).

Fig. 6

Fluorescent images of HCAECs after treating with different concentrations of MgCl₂ for 24 h. Cell nucleus (blue) was stained by Slow-fade Gold anti-fade Reagent with DAPI. Microtubule (red) was stained by mouse anti-β tubulin followed by Alexa Fluor 546 rabbit anti-mouse IgG. Microfilament (green) was stained by Actin Green 488 Ready Probes Reagent.

Fig. 7

Normalized green fluorescence intensity (GFI) of HCAECs microfilament. Stars indicate that the GFIs were significantly different from the control (n = 12, P < 0.05).

Fig. 8

HCAECs gene expression profile obtained by the RT-PCR profiling kit (including 84 functional genes) after treating with ECM supplemented with 10 mM MgCl₂ (A) and 50 mM MgCl₂ (B). Gene functions were classified into 7 different groups (Vaso C&D represents vasoconstriction & vasodilation). X-axis represents different gene functions and Y-axis represents the number of genes significantly changed. The bars above the X-axis are the up-regulated genes and below are the down-regulated genes (n = 3, P < 0.05).