Basement Membrane of Tissue Engineered Extracellular Matrix Scaffolds Modulates Rapid Human Endothelial Cell Recellularization and Promote Quiescent Behavior After Monolayer Formation

Abstract

Off-the-shelf small diameter vascular grafts are an attractive alternative to eliminate the shortcomings of autologous tissues for vascular grafting. Bovine saphenous vein (SV) extracellular matrix (ECM) scaffolds are potentially ideal small diameter vascular grafts, due to their inherent architecture and signaling molecules capable of driving repopulating cell behavior and regeneration. However, harnessing this potential is predicated on the ability of the scaffold generation technique to maintain the delicate structure, composition, and associated functions of native vascular ECM. Previous de-cellularization methods have been uniformly demonstrated to disrupt the delicate basement membrane components of native vascular ECM. The antigen removal (AR) tissue processing method utilizes the protein chemistry principle of differential solubility to achieve a step-wise removal of antigens with similar physiochemical properties. Briefly, the cellular components of SV are permeabilized and the actomyosin crossbridges are relaxed, followed by lipophilic antigen removal, sarcomeric disassembly, hydrophilic antigen removal, nuclease digestion, and washout. Here, we demonstrate that bovine SV ECM scaffolds generated using the novel AR approach results in the retention of native basement membrane protein structure, composition (e.g., Collagen IV and laminin), and associated cell modulatory function. Presence of basement membrane proteins in AR vascular ECM scaffolds increases the rate of endothelial cell monolayer formation by enhancing cell migration and proliferation. Following monolayer formation, basement membrane proteins promote appropriate formation of adherence junction and apicobasal polarization, increasing the secretion of nitric oxide, and driving repopulating endothelial cells toward a quiescent phenotype. We conclude that the presence of an intact native vascular basement membrane in the AR SV ECM scaffolds modulates human endothelial cell quiescent monolayer formation which is essential for vessel homeostasis.

Influence of Substrate Structure and Associated Properties on Endothelial Cell Behavior in the Context of Behaviors Associated with Laminar Flow Conditions

“…26,40,44 As endothelial activation is a pro-inflammatory and procoagulant state of the ECs, which causes thrombosis and intimal hyperplasia. 28,45 So, a smooth substrate surface may take more time for a complete endothelial monolayer formation. Still, it may reduce endothelial activation, leading to less chance of thrombosis and intimal hyperplasia in the long run.…”

“…26,40,44 As endothelial activation is a proinflammatory and procoagulant state of the endothelial cells, which causes thrombosis and intimal hyperplasia. 28,45 Moreover, the roughness of the random substrate could potentially increase endothelial cell activation because cells need to proliferate and produce more intercellular connections to cover the surface. However, the random cell-cultured substrate was still significantly rougher than the aligned cellcultured substrate, indicating that cell activation, proliferation, and intercellular junction formation cannot adequately lead to monolayer formation as those observed on aligned substrates.…”

“…Laminin, engaging with cellular integrins, is critical for endothelial cell adhesion, motility, and functionality. 16,28 Finally, VEGF is the primary growth factor that regulates endothelial cell production of ECM components (collagen and laminin) and many homeostatic regulators. 40,41 Moreover, all the proteins discussed above are expressed more in endothelial cells in laminar flow conditions than in static conditions.…”

“…11,15 Moreover, not assessing these factors has significant consequences, including thrombosis and intimal hyperplasia in grafts, which result in poor patency. 11,15,28 For example, autologous grafts mimic many physical and biological attributes of native blood vessels; they often exhibit positive results and good long-term patency postimplantation. 5,13 This success is primarily attributed to the endothelial cells entering a quiescent state after the endothelialization of the graft.…”

“…This oversight leaves a significant gap in understanding whether endothelial cells grown on such substrates adequately mimic the native endothelium by expressing critical regulators involved in cell–cell junction formation, cytoskeletal reorganization, autophagy, and endothelial cell quiescence. , Moreover, not assessing these factors has significant consequences, including thrombosis and intimal hyperplasia in grafts, which result in poor patency. ,, For example, autologous grafts mimic many physical and biological attributes of native blood vessels; they often exhibit positive results and good long-term patency postimplantation. , This success is primarily attributed to the endothelial cells entering a quiescent state after the endothelialization of the graft . Conversely, allogeneic grafts and tissue-engineered autografts, while exhibiting physical features similar to native vessels, fare poorly, with many failing within a year postimplantation. , …”