Valve replacement is an important therapeutic approach for management of valvular heart disease, accounting for over 200,000 surgeries annually worldwide. Deficiencies of current heart valve prostheses led the NHLBI cardiac surgery working group to highlight the need for improved heart valve leaflet biomaterials. Recently, our group has engineered a novel method to eliminate antigens from bovine pericardium (BP) extracellular matrix (ECM) scaffolds, while retaining biomaterial structure-function properties and regenerative capacity. However, despite reducing BP-ECM scaffold antigen burden by >92%, graft-specific humoral IgG response persisted at ~30% of the level associated with native BP in a rabbit model. In this study, we sought to determine the identities of residual antigens present in antigen removed BP-ECM scaffolds. New Zealand White Rabbits were subpannicularly implanted with either native, glutaraldehyde fixed, or antigen-removed BP-ECM scaffolds (n = 6 per group). Rabbit serum, collected pre- and at 56 d post-implantation, was used to generate IgG affinity chromatography columns. Native BP protein extracts were loaded onto columns, and captured antigens were identified by LC-MS/MS. A total of 259 antigens were identified in native BP rabbits. The glutaraldehyde fixed and antigen-removed rabbits responded to ~4% and ~12% of these same antigens, respectively. A small subset of antigens were uniquely identified in each of the treatment groups, not identified in native BP. These results inform ongoing efforts to eliminate antigens from xenogeneic tissues in development of ECM scaffolds, and may provide insight into the relative immunogenicity of individual xenoantigens in the context of tissue engineering.

Chronic vascular diseases affect over 25 million patients in the U.S, alone. While non-invasive therapies are available, approximately 4.5 million individuals are estimated to require a vascular bypass annually, worldwide. Autologous vascular grafts remain the standard of care; yet the absence of a suitable donor vessel results in approximately one third of patients being ineligible for autologous grafting. “Off-the-shelf” alternatives have been proposed, but have had limited pre-clinical success, primarily due to graft failure via thrombosis. Thrombotic failure risk has been shown to be dramatically reduced by the formation of a luminal quiescent endothelial cell monolayer. Recently, our group engineered an unfixed, antigen-removed (AR) extracellular matrix (ECM) scaffold from bovine saphenous vein (SV) which is minimally immunogenic and avoids in vivo thrombosis. However, the mechanism by which AR-ECM SV scaffolds prevent thrombosis remains unknown. In this study, we utilized cell culture, immunofluorescence, and RNA-seq to assess the hypothesis that hAECs seeded on the basement membrane (BM) surface of AR-ECM SV scaffolds adopt a quiescent phenotype. Cells seeded on the BM surface proliferated and underwent significantly greater XY migration than those seeded on the non-BM surface. Additionally, unlike non-BM seeded cells, BM seeding resulted in quiescent hAEC phenotype (i.e., apical polarization of podocalyxin and adherence junction protein (i.e., cadherin and β-catenin) colocalization). Finally, the transcriptional phenotype of hAECs seeded on the BM surface was similar to cells treated with simvastatin (i.e. “quiescent”) and significantly different from those treated with TNFα (i.e. “activated”) (n=6/group), as assessed by gene set enrichment and t-SNE analysis. We conclude that seeding hAECs on the BM surface of AR-SV ECM scaffolds induces a favorable “quiescent” phenotype, while an unfavorable “activated” phenotype is avoided. Ultimately, these results show that the BM surface of AR-SV-ECM scaffolds possesses inherent characteristics which promote quiescent hAEC behavior and resultant vascular homeostasis and will help inform future pre-clinical studies of this novel small diameter vascular grafting biomaterial.

No 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.

No abstract