Tissue Engineering Of Decellularized Rat Aortic Valvular Conduit Coated By Heparin-SDF-1α Multilayer

PartⅠ Fabricating of decellularized rat aortic valvular conduitsObjectiveNowadays, heart valve replacement remains the most effective treatment of heart valve disease. However heart valve prostheses used clinically still have their limitations in biocompatibility or durability. Tissue-engineered heart valves(TEHVs) might be a promising substitution for valve prostheses used currently for their potential to self-repair, remodel, and grow. In this section, to seek a better decellarization protocol for tissue engineering heart valves, we tested different decellularization procedures for comparison the potential of cell removal and ECM protection.Materials and methodsRat aortic valvular conduits were harvested and randomly divided into four groups: 0.25% Triton-X100+0.25% Sodium Deoxycholate(SD) group(TS group, n=9), 1% Sodium Dodecyl Sulfate group(SDS group, n=9), 0.5% Triton-X100 +0.5% SD + 0.5% SDS(STS group, n=9)and native conduits(control group, n=9). To confirm the best decellarization, H&E staining was performed to evaluate the decellularization effect and the fiber structure maintenance, and DNA content assay was performed to evaluate the DNA residual. At last, scanning electron microscope(SEM) was performed to further evaluate decellularization effect and arrangement of fiber structure.ResultsTS group and SDS group showed incompletely cell removal and collagen fiber structural disruption. However, STS group demonstrated completely cell removal and extracellular structure maintenance.ConclusionsRat aortic valvular conduits decellularized by combined application of Triton-X100, SD and SDS showed completely cell removal and intact fiber structure, which would be the scaffolds for following studies.Part Ⅱ Deposition of PEM on rat decellularized valvular conduits and biocompatibility assayObjectiveDecellularized heart valvular scaffolds have been demonstrated to be a promising solution to overcome the limitations of current heart valvular prostheses. However, residual immunogenicity, thrombogenicity and slow recolonizing of the graft in vivo might be the major reasons leading early graft failure. To overcome these drawbacks and improve recolonizing, we coated decellularized rat aortic valvular grafts with Heparin-SDF-1α polyelectrolyte multilayer films(PEM) and biocompatibility assay was performed.Materials and methodsThe donor U-shaped aortic valvular conduits were dip-coated with heparin and SDF-1α alternately to form PEM. Coating of valvular conduits with heparin-SDF-1α PEM was proved by immunofluorescence. Platelet adhesion and LDH assay were used to evaluate the antiplatelet property. Bone marrow stem cells(BMSCs) were cultured, and the adhesion, growth and migration of BMSCs to the modified valvular scaffolds were assessed.ResultsImmunofluorescence results showed that heparin-SDF-1α PEM were coated on rat decellularized valvular conduits. In vitro studies demonstrated Heparin-SDF-1α PEM improved haemocompatibility with respect to a substantial reduction of platelet adhesion on the modified decellularized graft. BMSCs also achieved better adhesion, proliferation and migration on the modified graft.ConclusionsHeparin-SDF-1α PEM could be used to coat the decellularized aortic valvular graft and demonstrated excellent haemocompatibility and improve stem cells attachment, proliferation and migration to the graft.Part Ⅲ Recellularization of decellularized rat aortic valvular conduits coated with heparin-SDF-1α PEM in vivoObjectiveOur previous study had confirmed the biocompatibility of heparin-SDF-1α PEM modified decellularized rat aortic valvular conduits while the recellularization property of PEM-DVC remained unknown. In this section, we implanted the modified decellularized valvular conduits into rat infrarenal aorta and evaluation of function and recellularization in vivo was performed.Materials and methodsThe grafts were implanted to rat infrarenal aorta, without or with heparin- SDF-1α multilayer. 12 rats were divided into 2 groups: PEM-DVC group(n=6) were implanted heparin-SDF-1α PEM modified decellularized rat aortic valvular conduits while Un DVC group(n=6) were implanted unmodified decellularized rat aortic valvular conduits. Functional assessment was performed by Doppler echography at day 1 and 4-week time points and micro CT at 2- and 4-week time points after implantation. Four weeks after implantation, the valvular conduits were explanted and histology and immunohistochemistry were performed to evaluate the fiber structure and recellularization.ResultsFor in vivo studies, using ultrasound and micro-CT, we found that both PEM-DVC group and Un DVC group were confirmed to remain patent with no signs of thrombus, stenosis and aneurysm. Explanted implants showed the structural integrity of the collagen and elastic ?bers of both groups. However, it was demonstrated that the modified decellularized grafts had better self-endothelialization and recruitment of endothelial progenitor cells(EPCs) after one-month implantation.ConclusionsTissue engineered aortic valvular conduit modified with heparin-SDF-1α PEM could improve the intimal re-endothelialization, and it is of optimal haemocompatibility and function. It provides a new theoretical foundation for the construction of tissue engineered heart valve.