Reendothelialization of decellularized swine fasciocutaneous flap: a proof-of-concept study
Victor Pozzo1,2,3,4, Aylin Acun1,3, Elise Lupon1,2,3,4, Ruben Oganesyan1,3, Golda Romano1,2,3,4, Marion Goutard1,2,3,4, Alec R. Andrews1,2, Pierre Tawa1,2,3,4, Mark A. Randolph1,2, Alexandre G. Lellouch1,2,3,4, Curtis L. Cetrulo Jr1,2, Basak E. Uygun1,3.
1Harvard Medical School, Boston, MA, United States; 2Department of Plastic Surgery, Massachusetts General Hospital, Boston, MA, United States; 3Department of Surgery, Shriners Hospitals for Children, Boston, MA, United States; 4Department of Plastic Surgery, Hôpital Européen Georges Pompidou , Paris, France
Background: The main limiting factor in vascularized composite allograft (VCA) transplantation remains the necessity of lifelong immunosuppression. A promise exists with the de novo creation of a patient specific graft via tissue engineering using perfusion decellularization-recellularization (DE/RE) technology. Bioartificial organs (liver, lungs, kidneys) have been generated with that technology, but VCA remains challenging due to the multiplicity of cell types. Reendothelialization is the first step of RE process. In this study we report our experience to reendothelialize a swine fasciocutaneous flap.
Methods: We harvested saphenous fasciocutaneous flaps from Yorkshire pigs and decellularized them by perfusion (Fig-1). We started with Sodium Dodecyl Sulfate (0.2%, for 120 hours) followed by washing with water (24 hours), Triton X-100 (1%, 24 hours), and PBS (48 hours). We asserted the integrity of the vasculature by comparative X-RAY before and after decellularization. Prior to reendothelialization we functionalized the flaps with REDV fused elastin-like peptide via covalent bonding to increase endothelial cell attachment. Then, we injected the endothelial cells (HUVECs) manually through the artery and perfused the flap either for 24 hours or 48 hours. We evaluated the reendothelialization during perfusion by cell counting in the perfusate outflow and at the end by histology with H&E staining.
Results: Macroscopic aspect of the flaps showed a partial reendothelialization with a coloration of the main vessels. Cell count during the perfusion demonstrates a low-rate (<5%) cells in perfusate/cells injected proving the entrapment of the cells in the scaffold. Finally, histological analysis confirmed success in partial reendothelialization of our flaps with endothelial cells attached and aligned in the main artery and microcirculation (Fig-2).
Conclusion: In this study, we have shown successful repopulation of the vasculature with endothelial cells in engineered scaffolds. In ongoing work, we will test the functionality of endothelialized grafts in blood perfusion experiments. We will then validate our results using induced pluripotent stem cell-derived endothelial cells and complete the recellularization with the addition of other skin cells into the scaffolds.
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