Cardiac valves are dynamic structures, exhibiting a highly specialized architecture consisting of cells and extracellular matrix with a relevant proteoglycan and glycosaminoglycan content, collagen and elastic fibers. Biological valve substitutes are obtained from xenogenic cardiac and pericardial tissues. To overcome the limits of such non viable substitutes, tissue engineering approaches emerged to create cell repopulated decellularized scaffolds. This study was performed to determine the glycosaminoglycans content, distribution, and disaccharides composition in porcine aortic and pulmonary valves and in pericardium before and after a detergent-based decellularization procedure. The fine structural characteristics of galactosaminoglycans chondroitin sulfate and dermatan sulfate were examined by FACE. Furthermore, the mechanical properties of decellularized pericardium and its propensity to be repopulated by in vitro seeded fibroblasts were investigated. Results show that galactosaminoglycans and hyaluronan are differently distributed between pericardium and valves and within heart valves themselves before and after decellularization. The distribution of glycosaminoglycans is also dependent from the vascular district and topographic localization. The decellularization protocol adopted resulted in a relevant but not selective depletion of galactosaminoglycans. As a whole, data suggest that both decellularized porcine heart valves and bovine pericardium represent promising materials bearing the potential for future development of tissue engineered heart valve scaffolds. 1. Introduction Heart valve disease has a deep impact worldwide related with the large number of valvular replacement operations performed every year. Typical valve substitutes are mechanical prostheses and bioprostheses obtained from cardiac-valvulated conduits (aortic and pulmonary root) or pericardial tissue of porcine and bovine origin. Bioprosthetic valves, although associated with a lower risk of thromboembolism with respect to the mechanical ones, possess limited longevity due to dystrophic calcification consequent to glutaraldehyde (GA) treatment used for preventing rejection  and suffer for many of the same degenerative processes that afflict native valves . In the last years, tissue engineering (TE) approaches raised in response to limitations associated with tissue and organ transplantation and with the scarcity of available donors. Three are the components essential for a TE substitute: cells, scaffolds (designed to maintain the cells in a three-dimensional environment),
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