mimic the tunica media, and human aortic endothelial cells (HAEC) as a substitute for the tunica intima. Cells were cultured for 48 hours, and then insulted with a ...
European Cells and Materials Vol. 30. Suppl. 3, 2015 (page 71)
ISSN 1473-2262
In vitro cytotoxicity evaluation of vascular cells after exposure to zinc ER Shearier1, PK Bowen2, J Drelich2, J Goldman1, F Zhao1 1 Department of Biomedical Engineering 2 Department of Materials Science and Engineering, Michigan Technological University, Houghton, Michigan, USA INTRODUCTION: Endovascular stent research in the last decade has focused on the development of bioabsorbable materials of both polymeric and metallic composition. When considering metallic base materials, zinc (Zn) and its alloys have been proposed as a promising material in comparison to more traditional bioabsorbable materials such as iron or magnesium [1]. The low corrosion rate does not appear to damage adjacent tissue in vivo, which can serve as a benchmark for an in vitro evaluation of Zn. Human vascular cell types are used here for focused in vitro evaluation. These include endothelial cells, smooth muscle cells, and fibroblasts. Aqueous insult with Zn2+ using a method similar to that described by Feyerabend et al. [2] and direct metallic contact experiments were performed on these key cell types. In vitro experiments help to determine how these cells will react to Zn in a controlled environment, allowing the determination of detailed cellular mechanisms. METHODS: The specific cell types used in these cytotoxicity tests included human dermal fibroblasts (hDF) to represent adventitial tissue, human aortic smooth muscle cells (AoSMC) to mimic the tunica media, and human aortic endothelial cells (HAEC) as a substitute for the tunica intima. Cells were cultured for 48 hours, and then insulted with a ZnCl2 solution buffered with MES with concentrations ranging from 1 to 500 µM Zn2+ for four hours, and allowed to recover for 24 hours. An XTT assay was performed to determine the relative number of viable cells. Live/dead stains were performed on samples ranging from 1 to 200 µM Zn2+ in full media. Cells were also seeded directly on Zn foils, as well as a modified surface with an attached sheet of collagen. Scanning electron microscopy (SEM) and fluorescent microscopy were then performed. RESULTS: Cell viability following Zn2+ insult, recovery, and analysis using the XTT assay was performed. The median lethal dose (LD50) values were 50 μM for hDF, 70 μM for AoSMC, and 265 μM for HAEC. HAEC exhibited the greatest tolerance for Zn2+. A similar result was seen in the live/dead imaging, as shown in Figure 1. A distinct
Fig. 1: Live/dead imaging of the three vascular cell types. Cells were exposed to media containing 1 to 200 µM Zn2+ in full media. Living cells show only green, whereas dead cells also fluoresce red. change in morphology occurs in all cells, with HAEC exhibiting the highest viability in the presence of Zn2+. When cells were seeded directly on Zn discs for six hours and visualized with SEM and fluorescent imaging there were no living cells identifiable. To mimic the protein layer/proto-thrombus seen in vivo, Zn discs were then coated with a 200 µm layer of collagen gelatin. After 2 hours, living cells were still visible, albeit with a rounded morphology. DISCUSSION & CONCLUSIONS: Zinc has already been demonstrated to be a possible biodegradable endovascular stent material. In this work, the response of key vascular cell types— HAEC, AoSMC, and hDF—to Zn in vitro was evaluated. HAEC appear to have the highest tolerance to both ionic Zn2+ and metallic Zn. This is important in vivo, as they play vital role in the formation of a neoendothelium on an implanted stent. The evaluation of these vascular cell types helps to further our understanding of cellular reactions in vivo. REFERENCES: 1 P.K. Bowen et al. (2013) Adv Mater 25:2577-82. 2 F. Feyerabend et al. (2010) Acta Biomater 6:1834-42. ACKNOWLEDGEMENTS: PKB was supported by an AHA fellowship. Project support from NIH NHLBI award no. 1R15HL129199-01.
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