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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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Crosslinking of collagenous biomaterials currently employs the use of glutaraldehyde. The putative enhancement of glutaraldehyde crosslinking by lysine was investigated in three model systems: bovine pericardium, collagen membranes, and bovine serum albumin. Repetitive sequential treatment of bovine pericardium with glutaraldehyde and lysine and finally with formaldehyde produced a matrix which, by the two criteria used (shrinkage temperature and urea/SDS soluble collagen), was shown to be more highly crosslinked than pericardium fixed in glutaraldehyde alone. Essentially the same results were obtained when membranes prepared from pepsin-soluble pericardial collagen were subjected to sequential glutaraldehyde and lysine treatments, reaching shrinkage temperatures of more than 90 degrees C. Heart valves prepared from lysine-enhanced glutaraldehyde crosslinked bovine pericardium were tested in vitro in an accelerated fatigue tester and have been shown to behave satisfactorily after 300 million cycles. These additional crosslinks proved to be stable in saline at 37 degrees C. Studies on bovine serum albumin attempted to get an insight into the mechanisms of lysine enhancement of glutaraldehyde crosslinking by treating sequentially albumin with glutaraldehyde and lysine and analysis of the products by gel filtration and SDS-PAGE. These studies suggest that free amino groups exposed by proteins are initially reacted with glutaraldehyde and then bridged by the diamino compound (lysine) producing more extensive intermolecular crosslinking than glutaraldehyde alone.

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The presence and activity of proteolytic enzymes has been investigated in vitro on soluble and insoluble preparations obtained from both unimplanted and implanted glutaraldehyde-treated bovine parietal pericardium. Using detection by colorimetric techniques, soluble preparations were shown to hydrolyze enzyme substrates that are characteristic for trypsin-like proteases, cathepsin-like proteases, and collagenase. As detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gradient gels and gel filtration on Sepharose CL-6B, insoluble (pellet) preparations degraded denatured type I collagen in a time-dependent pattern, producing low-molecular-weight fragments. These activities were partially inhibited by phenylmethylsulfonyl fluoride, N-ethyl maleimide, soybean trypsin inhibitor, para-chloromercuribenzoic acid, or ethylenediaminetetraacetic acid, suggesting the presence of a heterogeneous enzymatic mixture. Insoluble preparations incubated with pure pericardial dermatan sulfate proteoglycan detached the glycosaminoglycan chains from their core protein carrier, producing a digestion pattern similar to Cathepsin C. These findings demonstrate the presence of active proteases in glutaraldehyde-fixed bovine pericardium per se and in explanted pericardial bioprosthetic cardiac valves, an additional factor that might contribute to intrinsic extracellular matrix degeneration in pericardial bioprosthetic devices.