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Allograft tissue is used to reconstruct cardiac birth defects but induces an immune response resulting in allo-sensitization. Decellularization reduces the immune response, however, acellular vascular tissue is thrombogenic. In-vitro endothelialization may attenuate thrombogenicity. Here we offer our work, which determines a novel method of endothelial cell attachment using Arginine-Glycine-Aspartic Acid (RGD) peptides. We show that an RGD-FITC peptide can be bound to a decellularized ovine cardiac scaffold. RGD modification increases HUVEC cell adhesion to the surface at 3 days of static incubation in-vitro compared to decellularized tissue alone. Repetition using a decellularized human scaffold shows similar results. Cleavage of the potentially immunogenic FITC label retains our RGD peptide. In summary, we determine that decellularized allografts show enhanced HUVEC cell adhesion when modified with an RGD peptide under static conditions. This may increase cell retention in-vivo leading to a decellularized cardiac allograft repopulated with functional autologous cells from the recipient. / Experimental Surgery

Engineering a complex tissue that exceeds 100-200 [mu]m requires a vascular connection. Methods to enhance vascularization include the delivery of angiogenic factors, and the use of scaffolds that encourage vascular ingrowth. However, these techniques rely on the host to vascularize the construct upon implantation, which is often too slow to provide nutrients to the entire construct. Hence, recent research has focused on creating de novo vascular networks prior to implantation. Such technologies would enable faster anastomosis with the host vascular system, as well as fully perfused constructs that can increase cell viability. Many techniques have been investigated to create de novo vascular networks with varying levels of success. Our approach was to utilize native vascular extracellular matrix (ECM) obtained from decellularizing highly vascularized tissue as a substrate for re-endothelialization and thus to create a three-dimensional vascular bed for ultimate use with various implant and tissue engineering applications. We have demonstrated a method of chemical decellularization that effectively removes cellular material while leaving behind an organized patent vascular network down to the capillary scale. Standard histological methods, DNA quantification, as well as vascular corrosion casting demonstrated this efficacy. Subsequent subcutaneous implantation then explantation of the scaffold at 7 and 28 days was used to assess the immunogenicity of the graft by analyzing the presence of immune cells. This scaffold was then re-endothelialized with human dermal microvascular endothelial cells (HDMECs) and conditioned with peristaltic flow for 60 hours to help improve vascular patency. Cellular distribution was determined qualitatively by first incubating the HDMECs with gold nanotracers, then imaging their presence upon implantation through ultrasound-guided photoacoustic (US/PA) imaging. Following the culture process, the scaffolds were analyzed for vascular patency through vascular corrosion casting, and cellular phenotype through histological methods---demonstrating a decrease in vascular damage. The re-endothelialized scaffolds were then assessed for functional vascular performance by perfusing whole blood through them. Results demonstrated better blood clearance in re-endothelialized scaffolds compared to scaffolds without cells. These results point to the ability of the optimized acellular (OA) scaffold to be used in future experiments focused on vascular and tissue engineering. / text

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21297号 / 医博第4386号 / 新制||医||1030(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 妹尾 浩, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thrombosis and restenosis are two major complications associated with current commercial vascular stents. In situ regeneration of a healthy endothelium has been recognized as a promising strategy to address these issues. Numerous strategies have been explored for this goal. However, in most of the cases, they only focused on enhancing endothelial cell growth, ignoring antithrombotic requirements and the competition between smooth muscle cells (SMCs) and endothelial cells (ECs) for their growth. This resulted in non-satisfying clinical results. In this study, we created a multifunctional surface that meets the need of antithrombosis and re-endothelialization. A nanotubular titanium oxide (TiO₂) system has been developed, which elutes the direct thrombin inhibitor, bivalirudin (BVLD); moreover, polydopamine (PDAM) is used to tailor the surface functionality of TiO₂ nanotubes (NTs) for controlling the elution of BVLD. PDAM-functionalized TiO₂ NTs controls the BVLD for more than two months. BVLD eluted from NTs was bioactive and showed a substantial inhibitory effect on thrombin bioactivity, platelet adhesion and activation. In addition, the BVLD-eluting nanotubular TiO₂ system has high selectivity to enhance human umbilical vein endothelial cell (HUVEC) growth, while it inhibits human umbilical artery smooth muscle cell (HUASMC) proliferation. Our design strategy for the BVLD-eluting nanotubular TiO₂ system creates a favorable microenvironment for durable thromboresistance and the promotion of reendothelialization, and thus it is suitable for the long-term treatment of cardiovascular diseases.