Differentiation patterns of human mesenchymal cells after seeding on homograft-derived acellular pulmonary valve scaffolds

Calcification risk and immune response are reduced after cardiac valve decellulariztion and subsequent repopulation with recipient cells can provide durable, autogenic-like implants. Previously, we showed that porcine aortic valve leaflets subjected to decellularization (Spina et al., 2003) allowed proper in vitro colonization by valve-derived porcine fibroblasts/myofibroblasts with additional re-expression of endothelial cells and smooth muscle cells (Bertipaglia et al., 2003). Here, we used human mesenchymal stem cells (hBM-MSC) to repopulate human valve homografts that had undergone decellularization with Triton-X-100/sodium-cholate and aspecific benzonase treatments and incubation in fetal bovine serum (FBS) and bovine fibronectin (50 ug/ml in PBS). hBM-MSC were isolate from bone marrow by Ficoll density-gradient centrifugation, selected for plastic adhesion, expanded in MEMalpha plus FBS, and characterized by FACS-analyius and cytocentrifugation. Seeding with 2 x 106 cell/cm2 was performed on ventricularis aspect of leaflets under incubation in modified DMEM Hepes plus FBS for 30 days. Immunohistochemical analysis revealed the reconstitution of endothelial cells (von Willebrand factor), myofibroblasts (smooth muscle actin, platelet myosin, vimentin and SM22), and matrix synthesis (collagen I, III and elastin), as well as for native valves. A minor percentage of the seeded cells maintained stem cell markers (OCT4, SSEA4). The differentiated cells showed no osteogenic phenotype. Proper cell differentiation was also revealed by TEM analysis. These data showed that our decellularization procedure allows suitable in vitro cell repopulation by human mesenchymal stem cells and suggests the feasibility of a novel, autogenic-like valve bioprosthesis.