Cardiovascular disease is a leading cause of death in the US, Europe and Japan and is comprised of a wide range of pathologies. One of the most common procedures is heart valve replacement and is required when the valve fails due to regurgitation or is unable to open fully during the cardiac cycle. Causes include congenital defects, calcification and prolapse, but regardless of origin there are limited options to repair valves and surgical treatments are focused primarily on replacement. It is estimated that each year more than 150,000 patients receive heart valve replacements at a mean cost of ~$200,000 per procedure, corresponding to >$30B cost to the healthcare system. The heart valve market has continued to grow over the past decade due to advances in surgical and minimally-invasive technologies associated with heart valve placement. However, current valves represent some compromise in fit, biological performance, durability and surgical procedure, with unique advantages and disadvantages associated with current mechanical and bioprosthetic heart valves. In this proposal our objective is to develop a new bioprosthetic heart valve using advanced manufacturing approaches that has the durability of mechanical valves, the non-thrombogenicity of biologic valves, the soft deformability for minimally-invasive transcatheter delivery, and the ability to custom fit the anatomy of any patient. To do this FluidForm, Inc in collaboration with Carnegie Mellon University will develop a new freeform reversible embedding of suspended hydrogels (FRESH) 3D printed heart valve using collagen type I that recreates the laminar and anisotropic extracellular matrix (ECM) architecture in native valves. Our preliminary data shows that FRESH 3D printing can be used to manufacture functional tri-leaflet heart valves entirely from collagen and can support physiologic flow rates and pressure for short periods of time. Here we will improve valve performance by recreating the collagen fiber arrangement and mechanical properties in native valve leaflets via two research aims. First, we will demonstrate that FRESH 3D printing of collagen type I can recreate the collagen fiber architecture in the different layers of the native aortic valve leaflets with <10% difference in mean orientation angle. Second, we will prove that FRESH 3D printed collagen valve leaflets can be engineered to have radial and circumferential elastic modulus, non-linear stress-strain response, creep, and fatigue life within 75% of native aortic valve leaflets. Phase I proof-of-concept success will provide a strong foundation for a Phase II SBIR project that will validate the complete FRESH printed, bioprosthetic aortic valve in an in vitro flow system that simulates human pressure and flow rate and in a pre-clinical ovine model to assess hemocompatibility and biological response.