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Tissue engineered heart valves (TEHVs) that can grow and remodel have the potential to serve as permanent replacements of the current non-viable prosthetic valves particularly for pediatric patients. A major challenge in designing functional TEHVs is to mimic both structural and anisotropic mechanical characteristics of the native valve leaflets. To establish a more biomimetic model of TEHV, we fabricated tri-layered scaffolds by combining electrospinning and microfabrication techniques. These constructs were fabricated by assembling microfabricated poly(glycerol sebacate) (PGS) and fibrous PGS/poly(caprolactone) (PCL) electrospun sheets to develop elastic scaffolds with tunable anisotropic mechanical properties similar to the mechanical characteristics of the native heart valves. The engineered scaffolds supported the growth of valvular interstitial cells (VICs) and mesenchymal stem cells (MSCs) within the 3D structure and promoted the deposition of heart valve extracellular matrix (ECM). MSCs were also organized and aligned along the anisotropic axes of the engineered tri-layered scaffolds. In addition, the fabricated constructs opened and closed properly in an ex vivo model of porcine heart valve leaflet tissue replacement. The engineered tri-layered scaffolds have the potential for successful translation towards TEHV replacements.

Tissue engineered heart valves (TEHV) can be useful in the repair of congenital or acquired valvular diseases due to their potential for growth and remodeling. The development of biomimetic scaffolds is a major challenge in heart valve tissue engineering. One of the most important structural characteristics of mature heart valve leaflets is their intrinsic anisotropy, which is derived from the microstructure of aligned collagen fibers in the extracellular matrix (ECM). In the present study, a directional electrospinning technique is used to fabricate fibrous poly(glycerol sebacate):poly(caprolactone) (PGS:PCL) scaffolds containing aligned fibers, which resemble native heart valve leaflet ECM networks. In addition, the anisotropic mechanical characteristics of fabricated scaffolds are tuned by changing the ratio of PGS:PCL to mimic the native heart valve's mechanical properties. Primary human valvular interstitial cells (VICs) attach and align along the anisotropic axes of all PGS:PCL scaffolds with various mechanical properties. The cells are also biochemically active in producing heart-valve-associated collagen, vimentin, and smooth muscle actin as determined by gene expression. The fibrous PGS:PCL scaffolds seeded with human VICs mimick the structure and mechanical properties of native valve leaflet tissues and would potentially be suitable for the replacement of heart valves in diverse patient populations.

Purpose: Shear-mediated thrombosis is a clinically relevant phenomenon that underlies excessive arterial thrombosis and device-induced thrombosis. Red blood cells are known to mechanically contribute to physiological hemostasis through margination of platelets and vWF, facilitating the unfurling of vWF multimers, and increasing the fraction of thrombus-contacting platelets. Shear also plays a role in this phenomenon, increasing both the degree of margination and the near-wall forces experienced by vWF and platelets leading to unfurling and activation. Despite this, the contribution of red blood cells in shear-induced platelet aggregation has not been fully investigated—specifically the effect of elevated hematocrit has not yet been demonstrated. Methods: Here, a microfluidic model of a sudden expansion is presented as a platform for investigating platelet adhesion at hematocrits ranging from 0 to 60% and shear rates ranging from 1000 to 10,000 s−1. The sudden expansion geometry models nonphysiological flow separation characteristic to mechanical circulatory support devices, and the validatory framework of the FDA benchmark nozzle. PDMS microchannels were fabricated and coated with human collagen. Platelets were fluorescently tagged, and blood was reconstituted at variable hematocrit prior to perfusion experiments. Integrin function of selected blood samples was inhibited by a blocking antibody, and platelet adhesion and aggregation over the course of perfusion was monitored. Results: Increasing shear rates at physiological and elevated hematocrit levels facilitate robust platelet adhesion and formation of large aggregates. Shear-induced platelet aggregation is demonstrated to be dependent on both αIIbβIII function and the presence of red blood cells. Inhibition of αIIbβIII results in an 86.4% reduction in overall platelet adhesion and an 85.7% reduction in thrombus size at 20-60% hematocrit. Hematocrit levels of 20% are inadequate for effective platelet margination and subsequent vWF tethering, resulting in notable decreases in platelet adhesion at 5000 and 10,000 s-1 compared to 40% and 60%. Inhibition of αIIbβIII triggered dramatic reductions in overall thrombus coverage and large aggregate formation. Stability of platelets tethered by vWF are demonstrated to be αIIbβIII-dependent, as adhesion of single platelets treated with A2A9, an anti-αIIbβIII blocking antibody, is transient and did not lead to sustained thrombus formation. Conclusions: This study highlights driving factors in vWF-mediated platelet adhesion that are relevant to clinical suppression of shear-induced thrombosis and in vitro assays of platelet adhesion. Primarily, increasing hematocrit promotes platelet margination, permitting shear-induced platelet aggregation through αIIbβIII-mediated adhesion at supraphysiological shear rates. [ABSTRACT FROM AUTHOR]