Humanization of porcine pulmonary valvulated conduits after decellularization and repopulation with human endothelial cells

Introduction. In heart valve surgical replacement, the most suitable hemodynamic properties and mechanical performances depend on the preservation of cusp anatomical shape and stromal structure. In addition, post-operatory valve calcification can be avoided only after removing all native cells from bioprostheses. Previously, complete cell extraction was achieved from porcine valve leaflets with concurrent preservation of their extracellular matrix (ECM) (1). These acellular scaffolds allowed "in vitro" repopulation with homologous valve interstitial cells, which also re-differentiated into all four cell phenotypes existing in heart valves (2). Methods. Porcine pulmonary valvulated segments (PVCs) were decellularized using combined non -denaturating neutral detergents Triton X-100 and Cholate, followed by Benzonase® digestion. Acellular PVCs were (i) orthotopically implanted in recipient pigs for 1-2 months, or (ii) "in vitro" seeded with endothelial cells derived from human umbilical cord (HUVEC), and incubated for 1-2 weeks. Histological and TEM-SEM ultrastructural analysis was performed, also after histochemical reactions for glycosaminoglycan (GAG) localization and laminin immuno-localization. Results. The treated PVCs exhibited complete cell remotion, good ECM preservation and surface reactivity for laminin. (i) After 2-month implantation, "in vivo" cell colonization spontaneously occurred by two distinct cell populations: endothelial-like cells, adhering to PVC luminal areas, and mesenchimal-like cells, migrating through PVC interstitium. (ii) After cell seeding and 1-week incubation, monolayers of human endothelial cells completely covered PVC luminal surfaces. Cell adhesion to the retained basal "lamina" and cell junction formation were also observed. In addition, valve interstitium was enriched by newly secreted GAGs at the subendothelial aspects. After cell seeding and 2-week incubation, micropinocytotic activity by endothelium and increased GAG-reactivity were observed. Conclusions. The decellularized PVCs are propensive for both (i) "in vivo" homologous cell repopulation, and (ii) "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 anti-thrombogenic bioprostheses. 1. Spina M., Ortolani F., El Messlemani A., Gandaglia A., Bujan J., Garcia-Honduvilla N., Vesely I., Gerosa G., Casarotto D., Petrelli L., Marchini M.: Isolation of intact aortic valve scaffolds for heart valve bioprostheses: extracellular matrix structure, prevention from calcification and cell repopulation features. J. Biomed. Mater. Res., 67, 1338-1350, 2003. 2. Bertipaglia B., Ortolani F., Petrelli L., Gerosa G., Spina M.,, Pauletto P., Casarotto D., Marchini M., Sartore S.: Cell characterization of porcine aortic valve and decellularized leaflets repopulated with aortic valve interstitial cells. Ann. Thorac. Surg., 75, 1274-1282, 2003.