ABSTRACT: American Heart Association estimates a 2.5% prevalence of valvular heart disease in the US, requiring over 100,000 valve replacements annually. Current replacement heart valves are far from ideal, leading the NHLBI cardiac surgery working group to recommend increased support for heart valve biomaterial research. Although glutaraldehyde fixed xenogeneic tissue valves (e.g., bovine pericardium (BP)) improve short-term survival, chronic graft-specific immune responses persist, resulting in long-term biomaterial damage, calcification and ultimately failure (~2-10 yr depending on age at implantation). Indeed, NHLBI xenotransplantation working group noted that biomaterial antigenicity represents the primary translational barrier to expanding the use of xenogeneic tissues in clinical practice. Unfixed BP in which human-relevant antigens are eliminated has potential to serve as an immunologically-acceptable extracellular matrix (ECM) scaffold for heart valve bioprostheses. However, identifying human-relevant BP antigens and facilitating their removal from candidate ECM scaffolds represent critical translational barriers for development of such biomaterials. We hypothesize that elimination of human- relevant antigens can be achieved by employing targeted antigen solubilization steps during BP ECM scaffold production. This proposal seeks to define primary BP antigens responsible for initiating graft-specific immune responses in human patients (Aim 1, Phase 1), quantify removal (Aim 1, Phase 2) and target elimination of such human-relevant antigens from BP ECM scaffolds (Aim 1, Phase 3). Unfixed ECM scaffolds that avoid destructive graft-specific adaptive immune responses have potential to modulate constructive pro-regenerative recipient innate immune responses. Our group has previously demonstrated that retention of native tissue ECM niche in BP scaffolds is critical to promoting pro-regenerative in vivo recipient responses. However, extent to which exposure of natural matricryptic sites can further enhance pro-regenerative innate immune polarization towards intact BP ECM scaffolds remains unknown. We hypothesize that ECM niche and matricryptic signal exposure are critical factors in modulating human macrophage polarization and ultimate in vivo scaffold fate. This proposal aims to determine mechanisms (i.e., ECM niche component and macrophage receptor) by which differing sources of matricryptic signal exposure modulate human macrophage polarization (Aim 2, Phase 1) and combine optimal levels of each matricryptic exposure source toward maximizing pro-regenerative polarization (Aim 2, Phase 2). Aim 1 and 2 factors identified as having potential to positively modulate in vivo scaffold fate in humans will be validated using an in vivo ovine heart valve replacement model (Aim 3). Completion of this proposal will provide mechanistic insights into human-relevant antigens responsible for initiating graft-specific immune response towards current clinicall...