Background: Ex vivo vascular bioreactors that enable interventions in arteries from slaughterhouse surplus hearts present valuable alternatives to animal models to test cardiovascular stents. However, the knowledge for stent implantation during ex vivo culture in slaughterhouse coronary arteries is limited. The objective of the study is two-fold: first, to determine culture settings, the time point and optimal conditions for in-culture stent implantation using surplus right coronary arteries (RCAs) from swine with known in vivo RCA diameters; and second, to implement the gained insights to culture and stent RCAs obtained from slaughterhouse hearts (unknown in vivo diameter). Methods: Swine RCAs were mounted, cultured and stented in an ex vivo vascular bioreactor (VABIO) under conditions of flow and pressure. The bioreactor culture and stenting protocols were optimized using a step wise approach. In Step 1, the RCAs dissected from in-house swine hearts, with known diameters, were cultured until endothelialized as the ideal time point for stenting, and the stent implantation procedure was optimized. In Step 2, the successful ex vivo stent implantation procedure was repeated in slaughterhouse RCAs. Structural changes of the RCAs were assessed by ultrasound imaging during culture. The morphology of the RCAs at the end of culture was assessed by histology. Results: The RCAs adapted to the ex vivo environment, stabilizing their diameter in the range of the in vivo diameter after day 3, which was selected as the earliest time point for stenting. Because stent implantations caused mural dissections in the RCAs, visible with ultrasound imaging and confirmed by histology, we developed an external support for the RCA. This was found to be critical for better physiological intravascular pressures and to minimize dissections upon stent implantation. Finally, the stent implantation procedure was successfully replicated in slaughterhouse arteries. Conclusions: Our study demonstrates the feasibility of in-culture ex vivo stent implantation in the VABIO, providing important requirements and useful insights for in vivo mimicking stent implantation, for future investigations in slaughterhouse arteries.

Coronary atherosclerosis is caused by plaque build-up, with lipids playing a pivotal role in its progression. However, lipid composition and distribution within coronary atherosclerosis remain unknown. This study aims to characterize lipids and investigate differences in lipid composition across disease stages to aid in the understanding of disease progression. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize lipid distributions in coronary artery sections (n ¼ 17) from hypercholesterolemic swine. We performed histology on consecutive sections to classify the artery segments and to investigate colocalization between lipids and histological regions of interest in advanced plaque, including necrotic core and inflammatory cells. Segments were classified as healthy (n ¼ 6), mild (n ¼ 6), and advanced disease (n ¼ 5) artery segments. Multivariate data analysis was employed to find differences in lipid composition between the segment types, and the lipids' spatial distribution was investigated using non-negative matrix factorization (NMF). Through this process, MALDI-MSI detected 473 lipid-related features. NMF clustering described three components in positive ionization mode: triacylglycerides (TAG), phosphatidylcholines (PC), and cholesterol species. In negative ionization mode, two components were identified: one driven by phosphatidylinositol(PI)(38:4), and one driven by ceramidephosphoethanolamine(36:1). Multivariate data analysis showed the association between advanced disease and specific lipid signatures like PC(O-40:5) and cholesterylester(CE)(18:2). Ether-linked phospholipids and LysoPC species were found to colocalize with necrotic core, and mostly CE, ceramide, and PI species colocalized with inflammatory cells. This study, therefore, uncovers distinct lipid signatures correlated with plaque development and their colocalization with necrotic core and inflammatory cells, enhancing our understanding of coronary atherosclerosis progression.

Safety and efficacy of coronary drug-eluting stents (DES) are often preclinically tested using healthy or minimally diseased swine. These generally show significant fibrotic neointima at follow-up, while in patients, incomplete healing is often observed. The aim of this study was to investigate neointima responses to DES in swine with significant coronary atherosclerosis. Adult familial hypercholesterolemic swine (n = 6) received a high fat diet to develop atherosclerosis. Serial OCT was performed before, directly after, and 28 days after DES implantation (n = 14 stents). Lumen, stent and plaque area, uncovered struts, neointima thickness and neointima type were analyzed for each frame and averaged per stent. Histology was performed to show differences in coronary atherosclerosis. A range of plaque size and severity was found, from healthy segments to lipid-rich plaques. Accordingly, neointima responses ranged from uncovered struts, to minimal neointima, to fibrotic neointima. Lower plaque burden resulted in a fibrotic neointima at follow-up, reminiscent of minimally diseased swine coronary models. In contrast, higher plaque burden resulted in minimal neointima and more uncovered struts at follow-up, similarly to patients’ responses. The presence of lipid-rich plaques resulted in more uncovered struts, which underscores the importance of advanced disease when performing safety and efficacy testing of DES.

Atherosclerotic arteries are commonly treated using drug-eluting stents (DES). However, it remains unclear whether and how the properties of atherosclerotic plaque affect drug transport in the arterial wall. A limitation of the currently used atherosclerotic animal models to study arterial drug distribution is the unpredictability of plaque size, composition, and location. In the present study, the aim is to create an artificial atherosclerotic plaque—of reproducible and controllable complexity and implantable at specific locations—to enable systematic studies on transport phenomena of drugs in stented atherosclerosis-mimicking arteries. For this purpose, mixtures of relevant lipids at concentrations mimicking atherosclerotic plaque are incorporated in gelatin/alginate hydrogels. Lipid-free (control) and lipid-rich hydrogels (artificial plaque) are created, mounted on DES and successfully implanted in porcine coronary arteries ex-vivo. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) is used to measure local drug distribution in the arterial wall behind the prepared hydrogels, showing that the lipid-rich hydrogel significantly hampers drug transport as compared to the lipid-free hydrogel. This observation confirms the importance of studying drug transport phenomena in the presence of lipids and of having an experimental model in which lipids and other plaque constituents can be precisely controlled and systematically studied.