“In vivo” homologous cell repopulation and “in vitro” heterologous re-endothelization of pulmonary valvulated conduits after ECM-preserving decellularization

Introduction: Fundamental prerequisites for prosthetic valvulated conduits (VVSs) consist in preservation of anatomical shape and stromal texture as well as absence of native cells, major cause of post-operatory calcification. Previously, we achieved complete extraction of cytomembranes and nuclear chromatin form porcine valve leaflets with concurrent good preservation of their extracellular matrix (ECM). These acellular valve scaffolds allowed "in vitro" repopulation with homologous valve interstitial cells, which also re-differenziated into all four cell phenotypes characterizing the heart valves. Methods: Porcine pulmonary VVSs were decellularized using combined non-denaturating neutral detergents Triton X-100 and Cholate (TRI-COL), followed by Benzonase® digestion. Acellular VVSs were (i) orthotopically implanted in recipient pigs for 1-2 months, and (ii) "in vitro" seeded with human umbilical cord endothelium cells, and incubated for 1-2 weeks. Histological and TEM-SEM ultrastructural analyses were performed, including reactions for glycosaminoglycan ultrastructural localization and laminin immuno-localization. Results: Complete remotion of cell components and ECM preservation were observed in treated VVSs. In addition, VVSs luminal surfaces showed positivity for laminin. After 2-month implantation, an "in vivo" cell colonization occurred spontaneously by two distinct cell populations: endothelial-like cells, adhering to a number of VVSs luminal areas, and mesenchimal-like cells, migrating through VVS interstitium. After 1-week seeding, human endothelial cells completely covered VVS luminal surfaces, closely adhering to the pre-existing endothelium basal "lamina", and forming cell junctions. Conclusions: Unaltered ECM texture of these acellular VVSs warrant appropriate mechanical properties in the short term. Moreover, VVSs propensity for both "in vivo" homologous cell re-population and "in vitro" heterologous re-endothelization suggests the feasibility of manifacturing grafts which should acquire autologous-like features after "in vitro" pre-treatment or not.