Identification of a subpopulation of highly adherent endothelial cells for seeding synthetic vascular grafts.

OBJECTIVE: There is an unmet clinical need for alternatives to autologous vessel grafts. Small-diameter (<6mm) synthetic vascular grafts are not suitable because of unacceptable patency rates. This mainly occurs due to the lack of an endothelial cell (EC) monolayer to prevent platelet activation, thrombosis, and intimal hyperplasia. There are no reliable methods to endothelialize small-diameter grafts, as most seeded ECs are lost due to exposure to fluid shear stress (SS) after implantation. The goal of this work is to determine if EC loss is a random process or if it is possible to predict which cells are more likely to remain adherent. METHODS: In initial studies, we sorted ECs using fluid SS and identified a subpopulation of ECs that are more likely to resist detachment. We use RNA-sequencing (RNA-seq) to examine gene expression of adherent ECs compared to the whole population. Using fluorescence activated cell sorting (FACS), we sorted ECs based on the expression level of a candidate marker and studied their retention in small-diameter vascular grafts in vitro. RESULTS: Transcriptomic analysis revealed that fibronectin leucine rich transmembrane protein 2 (FLRT2), encoding protein FLRT2, is downregulated in the ECs that are more likely to resist detachment. When seeded onto vascular grafts and exposed to SS, ECs expressing low levels of FLRT2 exhibit 59.2±7.4% retention compared to 24.5±6.1% retention for the remainder of the EC population. CONCLUSIONS: For the first time, we show EC detachment is not an entirely random process. This provides validation for the concept that we can seed small-diameter vascular grafts only with highly adherent ECs to maintain a stable endothelium and improve graft patency rates.

3D-bioprinting of patient-derived cardiac tissue models for studying congenital heart disease.

Introduction: Congenital heart disease is the leading cause of death related to birth defects and affects 1 out of every 100 live births. Induced pluripotent stem cell technology has allowed for patient-derived cardiomyocytes to be studied in vitro. An approach to bioengineer these cells into a physiologically accurate cardiac tissue model is needed in order to study the disease and evaluate potential treatment strategies. Methods: To accomplish this, we have developed a protocol to 3D-bioprint cardiac tissue constructs comprised of patient-derived cardiomyocytes within a hydrogel bioink based on laminin-521. Results: Cardiomyocytes remained viable and demonstrated appropriate phenotype and function including spontaneous contraction. Contraction remained consistent during 30 days of culture based on displacement measurements. Furthermore, tissue constructs demonstrated progressive maturation based on sarcomere structure and gene expression analysis. Gene expression analysis also revealed enhanced maturation in 3D constructs compared to 2D cell culture. Discussion: This combination of patient-derived cardiomyocytes and 3D-bioprinting represents a promising platform for studying congenital heart disease and evaluating individualized treatment strategies.

Strategies for Improving Endothelial Cell Adhesion to Blood-Contacting Medical Devices.

The endothelium is a critical mediator of homeostasis on blood-contacting surfaces in the body, serving as a selective barrier to regulate processes such as clotting, immune cell adhesion, and cellular response to fluid shear stress. Implantable cardiovascular devices, including stents, vascular grafts, heart valves, and left ventricular assist devices, are in direct contact with circulating blood and carry a high risk for platelet activation and thrombosis without a stable endothelial cell (EC) monolayer. Development of a healthy endothelium on the blood-contacting surface of these devices would help ameliorate risks associated with thrombus formation and eliminate the need for long-term antiplatelet or anticoagulation therapy. Although ECs have been seeded onto or recruited to these blood-contacting surfaces, most ECs are lost upon exposure to shear stress due to circulating blood. Many investigators have attempted to generate a stable EC monolayer by improving EC adhesion using surface modifications, material coatings, nanofiber topology, and modifications to the cells. Despite some success with enhanced EC retention in vitro and in animal models, no studies to date have proven efficacious for routinely creating a stable endothelium in the clinical setting. This review summarizes past and present techniques directed at improving the adhesion of ECs to blood-contacting devices. Impact statement Clinical success of blood-contacting devices such as vascular grafts, stents, and heart valves has remained limited by postimplantation problems, including thrombosis and loss of patency. Without a stable endothelial cell (EC) monolayer, blood-contacting devices are at risk for platelet activation and thrombosis. Methods to improve EC adhesion on these devices have not translated to long-term in vivo success, as many ECs are lost after exposure to circulating blood. In this study, we summarize methods to improve EC adhesion and retention. Successful endothelialization of blood-contacting devices may improve patient outcomes after device implantation and limit the need for long-term antiplatelet or anticoagulation therapy.