Abstract
Native blood vessels contain both an antiaggregatory intimal layer, which prevents platelet activation in the intact vessel, and a proaggregatory medial layer, which stimulates platelet aggregation upon vascular damage. Yet, current techniques for assessing the functional properties of tissue-engineered blood vessels may not be able to assess the relative effectiveness of both these pro- and antiaggregatory properties of the vessel construct. In this study, we present a novel technique for quantitatively assessing the pro- and antiaggregatory properties of different three-dimensional blood vessel constructs made using a layered fabrication method. This technique utilizes real-time measurements of cytosolic Ca2+ signaling to assess platelet activation in fluorescently labeled human platelet suspensions using fluorescence spectrofluorimetry, while also permitting examination of thrombus formation upon the surface of the construct using fluorescent imaging of DiOC6-labeled platelets. Experiments using this method demonstrated that type I collagen hydrogels, commonly used as scaffolds for vascular tissue engineering, were unable to support significant platelet activation, while type I and III neo-collagen secreted from human coronary artery smooth muscle cells cultured within these hydrogels as the medial layer were able to support thrombus formation. The incorporation of an intimal layer consisting of human umbilical vein endothelial cells on top of the medial layer inhibited platelet activation and aggregation. These data demonstrate that the methodology presented here is able to quantitatively compare the capacity of different constructs to trigger or prevent platelet activation. As such, this technique may provide a useful tool for standardizing the assessment of the functional properties of tissue-engineered blood vessel constructs developed using different culturing techniques.
Highlights
The integrity of the high-pressure closed circulatory system of mammals is constantly maintained by a hemostatic system aiming to prevent excessive blood loss upon vascular injury.[1]
These data demonstrate that the methodology presented here is able to quantitatively compare the capacity of different constructs to trigger or prevent platelet activation
Primary human coronary artery smooth muscle cells (HCASMCs), human umbilical vein endothelial cells (HUVECs) pooled, medium 200, medium 231, low-serum growth supplement (LSGS), smooth muscle growth supplement (SMGS), cell tracker (CMAC), and 5-(and-6)carboxyfluorescein diacetate, succinimidyl ester, mixed isomers (CFSE) were all obtained from GIBCO, Life Technologies
Summary
The integrity of the high-pressure closed circulatory system of mammals is constantly maintained by a hemostatic system aiming to prevent excessive blood loss upon vascular injury.[1]. Through studying the processes, which regulate platelet activation in (patho)physiological conditions, it should be possible to better prevent and treat patients suffering from these cardiovascular disorders.[2] Currently, the favored research system in which to study in vivo thrombus formation is through the use of intravital microscopy to study clotting elicited by damage to the mouse mesenteric artery.[3,4,5] there are many known differences in both the platelet transciptome and proteome, as well as the hemodynamics in mice and humans.[6] In addition, intravital microscopy requires the use of general anesthetics, which have been shown to interfere with various aspects of blood clotting in previous in vitro studies.[7] there appears a strong need to create a physiologically relevant ex vivo human blood vessel model to study the normal processes of thrombus formation. We hypothesized that a three-dimensional (3D) tissue-engineered human blood vessel construct may provide a useful model system to complement current animal studies of thrombus formation
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