BACKGROUND: Pre-clinical evidence suggests that structural valve deterioration (SVD) of bioprosthetic xenogenic tissue heart valves (XTHV) is a result of chronic immune rejection. Recellularization of XTHVs with human autologous cells represents one potential strategy to prevent this xenoreactive immune response and mitigate SVD. The purpose of this study was to directly address the role of recellularization of decellularized bovine pericardial XTHVs with autologous human mesenchymal stem cells (hMSCs) on the activation of the xenoreactive immune response.
METHODS AND RESULTS: Bone marrow, human pericardium and heparinized whole blood was collected from adult patients undergoing elective cardiac surgery. Decellularized bovine pericardium underwent recellularization with hMSCs, which were isolated and cultured from the bone marrow, via co-incubation to allow for cell seeding. To examine the cell mediated immune response, we performed immunohistochemical staining for CD68+ macrophages, CD3+ T-cells, CD20+ B-cells and performed a proliferation assay to quantify T-cell proliferation. To investigate the humoral immune response, we performed an enzyme linked immunosorbent assay for pro-inflammatory cytokines TNF-a and INF-y. Human peripheral blood mononuclear cells (PBMCs) were isolated, stained with proliferation dye and cultured for 3-days with autologous recellularized tissue. Serum and PBMCs were collected for subsequent biochemical and flow cytometric analysis. We show that decellularized bovine pericardium, exposed to human serum, had significantly decreased T-cell activation, represented by TNF-a and INF-y expression, as well as a significant decrease in T-cell proliferation, when compared to wild-type bovine pericardium (p < 0.01). Moreover, when decellularized bovine pericardium was recellularized with autologous hMSCs and exposed to human serum, there was an additional decrease in TNF-a, INF-y expression along with a significant increase in CD4+ T-cell proliferation and decrease in CD8+ T-cell proliferation, when compared to both wild-type and decellularized bovine pericardium (p < 0.01). Importantly, recellularized XTHVs exposed to human serum had an equivalent expression of TNF-a, INF-y and T-cell proliferation when compared to native human pericardium exposed to autologous human serum.
CONCLUSION: Taken together, our data suggest that autologous human MSC recellularization of decellularized bovine pericardium abrogates the xenoreactive immune response and may promote naïve T-cells to preferentially take on a Treg phenotype as seen by the increase in CD4+ T-cell proliferation with subsequent reduction in pro-inflammatory cytokine production. As such, autologous hMSC recellularization of an acellular xenogenic scaffolds may be an effective approach to decrease the progression of bioprosthetic SVD.