There is a great need for living valve replacements for patients of all ages. Such constructs could be built by tissue engineering, with perspective of the unique structure and biology of the aortic root. The aortic valve root is composed of several different tissues, and careful structural and functional consideration has to be given to each segment and component. Previous work has shown that immersion techniques are inadequate for wholeroot decellularization, with the aortic wall segment being particularly resistant to decellularization. The aim of this study was to develop a differential pressure gradient perfusion system capable of being rigorous enough to decellularize the aortic root wall while gentle enough to preserve the integrity of the cusps. Fresh porcine aortic roots have been subjected to various regimens of perfusion decellularization using detergents and enzymes and results compared to immersion decellularized roots. Success criteria for evaluation of each root segment (cusp, muscle, sinus, wall) for decellularization completeness, tissue integrity, and valve functionality were defined using complementary methods of cell analysis (histology with nuclear and matrix stains and DNA analysis), biomechanics (biaxial and bending tests), and physiologic heart valve bioreactor testing (with advanced image analysis of open-close cycles and geometric orifice area measurement). Fully acellular porcine roots treated with the optimized method exhibited preserved macroscopic structures and microscopic matrix components, which translated into conserved anisotropic mechanical properties, including bending and excellent valve functionality when tested in aortic flow and pressure conditions. This study highlighted the importance of (1) adapting decellularization methods to specific target tissues, (2) combining several methods of cell analysis compared to relying solely on histology, (3) developing relevant valve-specific mechanical tests, and (4) in vitro testing of valve functionality.
Glutaraldehyde-crosslinked bovine pericardium is widely used in bioprosthetic heart valve fabrication. In an attempt to set a scientific basis for more reproducible tissue selection, we produced and analyzed topographical maps of glutaraldehyde-treated bovine pericardium. Whole pericardia were divided into specific anatomical areas and their thickness was measured and mapped on templates. In each area, the suture holding power was determined in both parallel and perpendicular (to the base-apex line) directions; analyses of the tearing patterns in each fragment were used to evaluate predominant fiber orientation, and observations were confirmed by polarized light microscopy. Complete maps were superimposed graphically to aid in the selection of certain areas that would have known fiber orientation, high suture holding power, and suitable thickness. Our results describe regional heterogeneity of bovine pericardial structure and mechanical properties, specifically demonstrating variations in thickness, suture holding power, and collagen fiber orientation. Two areas of choice (representing about 35% of the total) were described as suitable for use in bioprosthetic heart valve fabrication.
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