(I-352) A Better Benchtop model for atherosclerosis using Tissue Engineered Vascular

Introduction:: Atherosclerosis costs $431.8 billion yearly and accounting for nearly 1 in every 5 deaths (Khera et al., 2020; Ohsfeldt et al., 2010). The progression of atherosclerosis is seen over decades, though a benchtop model is still unavailable (Bäck et al., 2019; Kattoor et al., 2017; Libby et al., 2016). Pharmacotherapy has not changed significantly for decades. We hypothesize that a lack of human-relevant disease models is responsible. Our lab proposes a benchtop model and for atherosclerosis and coronary artery disease utilizing our laboratory’s tissue engineering methods. We propose to create vessels from patient tissues for benchtop testing of disease progression in response to pharmaceuticals.

Materials and Methods:: For initial experiments, we used our lab’s tissue engineering technique to form vessels from HASMC (human aortic smooth muscle cells) and PtFb (patient fibroblast) cells (Pinnock et al., 2017). Cells are cultured in relevant media until 90% confluence and centrifugation. The pellet is mixed into a hydrogel of fibrin and thrombin into each well of a 6 well plate pre-coated with PDMS with a central 4mm post. Cells will proliferate in the hydrogel and gradually dissolve the hydrogel, leading it to contract to this central post over a 2-4 day period, creating a tissue ring segment with 4mm lumen that will be assembled into a complete vessel. The vessel was then seeded with endothelial cells to represent the tunica intima. Oxidized Low-density-lipoprotein (OxLDL) was infused at 80, 100, and 120 ng/mL concentrations over a 1-week period before and after formation to determine the effect and visual density of OxLDL uptake into PtFb vessels. HASMC segments were formed without OxLDL and once the vessel was assembled the inner lumen was seeded with endothelial cells to create an inner layer to replicate the tunica intima. Next, OxLDL was introduced to determine the effect and visual density of OxLDL infiltration into each layer. Several SMC experimental vessels were also infused with macrophages at 100ng/dL to determine their effects on OxLDL uptake into the endothelial cell layer over a 1-week period. These SMC vessels were preserved in Optimal cutting temperature (OCT) compound in preparation for frozen sectioning and histologic analysis.


Results, Conclusions, and Discussions:: PtFb segment formation with OxLDL added demonstrated a slower rate of ring segment formation and a greater rate of failure as seen on daily gross examination. Time to contraction and ring segment formation was 3-4d instead of 1-2d for controls without OxLDL. PtFb vessel segments with OxLDL exposure were more likely to exhibit an inconsistent lumen diameter and vessel thickness. Failure for ring segments to form a 4mm lumen as measured via caliper was deemed to be a failure. SMC vessels with endothelial seeding exhibited no gross malformations when exposed to OxLDL with and without macrophages.

PtFb cells exposed to OxLDL contract to form ring segments at a slower rate and with a higher rate of failure. OxLDL disrupts vessel segment formation, implying cytotoxicity that could be linked to the early stages of atherosclerosis. Our next steps will investigate cell viability and density via histologic analysis to quantify OxLDL’s localization and effects on cell viability. These methods will be extended to the SMC vessels to determine the effect of macrophages in OxLDL uptake into the endothelial layer. Once this groundwork is established, we will develop testing conditions to more closely resemble atherosclerotic vessels seen in patients to further develop a benchtop model.


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