Decellularization and in vivo and in vitro repopulation with endothelial cells of porcine heart valve leaflets

Bioprostheses are widely used to replace cardiac valves. Besides favorable hemodynamic properties, however, these prostheses suffer from the major disadvantage of limited duration linked to sclerosis and dystrophic calcification events. These are linked, at least in part, to defective extracellular matrix (ECM) preservation and incomplete removal of native cells in currently glutaraldehyde-treated scaffolds. In addition, the absence of the endothelial coat may represent a relevant pathogenetic factor of the graft sclerotic process. As previously reported in Spina et al. (2003), we achieved complete cell extraction from porcine valve leaflets with concurrent preservation of their extracellular matrix. Moreover, as reported by Bertipaglia et al. (2003), these acellular scaffolds allowed in vitro repopulation with homologous valve interstitial cells, which also redifferentiated into all four cell phenotypes existing in heart valves. Porcine pulmonary valvulated segments (PVCs) were decellularized using combined nondenaturating neutral detergents Triton X-100 and cholate, followed by Benzonase® digestion. Acellular PVCs were othotopically implanted in recipient pigs for 1-2 months, or in vivo seeded with endothelial cells derived from human umbilical cord (HUVEC), and incubated for 2 weeks. Histological and TEM-SEM ultrastructural analysis was performed, also after histochemical reactions for glycosaminoglycan (GAG) localization, laminin immunolocalization, immune reactions for endothelial cell inflammatory or thrombotic phenotype. The treated PVCs exhibited complete cell remotion, good ECM preservation, and surface reactivity for laminin. After 2-month implantation, in vivo cell colonization spontaneously occurred by two distinct cell populations: endothelial-like cells, adhering to PVC luminal areas, and mesenchymal-like cells, migrating through PVC interstitium. After cell seeding and 2-week incubation, monolayers of antiinflammatory and anti-thrombogenic human endothelial cells completely covered PVC luminal surfaces. Cell adhesion to the retained basal lamina and cell junction formation also were observed. In addition, valve interstitium was enriched by newly secreted GAGs. After cell seeding and 2-week incubation, micropinocytotic activity by endothelium and increased GAG-reactivity were observed. The decellularized PVCs are propensive for both in vivo homologous cell repopulation and in vitro heterologous endothelization with HUVEC. In addition, PVC stroma acquired more and more hybrid character because human-endothelium-generated GAGs were added to the native ECM macromolecules retained within the treated porcine PVCs. Thus, these engineered PVCs appear as promising autologous-like, glutaraldehyde-free, and antithrombogenic bioprostheses.