Preparation of a Nanoscaled Poly(vinyl alcohol)/Hydroxyapatite/DNA Complex Using High Hydrostatic Pressure Technology for In Vitro and In Vivo Gene Delivery

Our previous research showed that poly(vinyl alcohol) (PVA) nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps) prepared by high hydrostatic pressurization (980 MPa), which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity

ハイブリッド型人工臓器に用いる高分子材料の基礎的研究

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである京都大学0048新制・課程博士博士(工学)甲第5709号工博第1355号新制||工||947(附属図書館)UT51-94-J141京都大学大学院工学研究科高分子化学専攻(主査)教授 筏 義人, 教授 升田 利史郎, 教授 今西 幸男学位規則第4条第1項該当Doctor of EngineeringKyoto UniversityDFA

Complete Cell Killing by Applying High Hydrostatic Pressure for Acellular Vascular Graft Preparation

Pressure treatment has been developed in tissue engineering application. Although the tissue scaffold prepared by a ultrahydrostatic pressure treatment has been reported, an excessive pressure has a potential to disrupt a structure of extracellular matrix through protein denaturation. It is important to understand the suitable low-pressure condition and mechanisms for cell killing. In this study, cellular morphology, mitochondria activity, and membrane permeability of mammalian cells with various pressure treatments were investigated with in vitro models. When the cells were treated with a pressure of 100 MPa for 10 min, cell morphology and adherence were the same as an untreated cells. Dehydrogenase activity in mitochondria was almost the same as untreated cells. On the other hand, when the cells were treated with the pressure of more than 200 MPa, the cells did not adhere, and the dehydrogenase activity was completely suppressed. However, green fluorescence was observed in the live/dead staining images, and the cells were completely stained as red after above 500 MPa. That is, membrane permeability was disturbed with the pressure treatment of above 500 MPa. These results indicated that the pressure of 200 MPa for 10 min was enough to induce cell killing through inactivation of mitochondria activity

Effect of luminal surface structure of decellularized aorta on thrombus formation and cell behavior.

Due to an increasing number of cardiovascular diseases, artificial heart valves and blood vessels have been developed. Although cardiovascular applications using decellularized tissue have been studied, the mechanisms of their functionality remain unknown. To determine the important factors for preparing decellularized cardiovascular prostheses that show good in vivo performance, the effects of the luminal surface structure of the decellularized aorta on thrombus formation and cell behavior were investigated. Various luminal surface structures of a decellularized aorta were prepared by heating, drying, and peeling. The luminal surface structure and collagen denaturation were evaluated by immunohistological staining, collagen hybridizing peptide (CHP) staining, and scanning electron microscopy (SEM) analysis. To evaluate the effects of luminal surface structure of decellularized aorta on thrombus formation and cell behavior, blood clotting tests and recellularization of endothelial cells and smooth muscle cells were performed. The results of the blood clotting test showed that the closer the luminal surface structure is to the native aorta, the higher the anti-coagulant property. The results of the cell seeding test suggest that vascular cells recognize the luminal surface structure and regulate adhesion, proliferation, and functional expression accordingly. These results provide important factors for preparing decellularized cardiovascular prostheses and will lead to future developments in decellularized cardiovascular applications

Elastic Modulus of ECM Hydrogels Derived from Decellularized Tissue Affects Capillary Network Formation in Endothelial Cells

Recent applications of decellularized tissue have included the use of hydrogels for injectable materials and three-dimensional (3D) bioprinting bioink for tissue regeneration. Microvascular formation is required for the delivery of oxygen and nutrients to support cell growth and regeneration in tissues and organs. The aim of the present study was to evaluate the formation of capillary networks in decellularized extracellular matrix (d-ECM) hydrogels. The d-ECM hydrogels were obtained from the small intestine submucosa (SIS) and the urinary bladder matrix (UBM) after decellularizing with sodium deoxycholate (SDC) and high hydrostatic pressure (HHP). The SDC d-ECM hydrogel gradually gelated, while the HHP d-ECM hydrogel immediately gelated. All d-ECM hydrogels had low matrix stiffness compared to that of the collagen hydrogel, according to a compression test. D-ECM hydrogels with various elastic moduli were obtained, irrespective of the decellularization method or tissue source. Microvascular-derived endothelial cells were seeded on d-ECM hydrogels. Few cells attached to the SDC d-ECM hydrogel with no network formation, while on the HHP d-ECM hydrogel, a capillary network structure formed between elongated cells. Long, branched networks formed on d-ECM hydrogels with lower matrix stiffness. This suggests that the capillary network structure that forms on d-ECM hydrogels is closely related to the matrix stiffness of the hydrogel