Toshiya Fujisato

The use of high-hydrostatic pressure treatment to decellularize blood vessels.

A decellularization method using high-hydrostatic pressure (HHP) technology (>600MPa) is described. The HHP disrupts the cells inside the tissue. The cell debris can be eliminated with a simple washing process, producing clean, decellularized tissue. In this study, porcine aortic blood vessel was decellularized by HHP. The mechanical properties and in vivo performance of the decellularized tissue were evaluated. Mechanical properties of the decellularized tissue were not altered by the HHP treatment. Reduced inflammation of the decellularized tissue was confirmed by xenogenic transplant experimentation. An allogenic transplantation study showed that decellularized blood vessel endured the arterial blood pressure, and there was no clot formation on the luminal surface. In addition, cellular infiltration into the vessel wall was observed 4 weeks after implantation, suggesting that HHP treatments could be applied widely as a high-quality decellularization method.

Novel adhesion prevention membrane based on a bioresorbable copoly(ester-ether) comprised of poly-L-lactide and Pluronic: in vitro and in vivo evaluations.

Block copolymers consisting of poly(L-lactide) (PLLA) and poly(oxyethylene-co-oxypropylene), with various compositions, were synthesized and characterized in vitro and in vivo for their application as postoperative adhesion prevention membranes. It was found that the flexibility and degradability of the cast films of the block copolymers grew with increasing Pluronic F68 [PN; poly(oxyethylene-co-oxypropylene] composition. The receding contact angle of the copolymer films against water became lower than that of the PLLA film, because the surface was predominantly covered with more hydrophilic PN segments in a wet state. This surface property significantly affects the cell attachment property of the copolymer films, and the fibroblasts cultured on the films exhibit a spheroid-like morphology. The copolymer films subcutaneously implanted in the back of rats induced milder tissue responses compared with PLLA homopolymers, because of the increased surface hydrophilicity in the former. In vivo evaluation using a uterus horn model in rats revealed that the performance of these copolymer films as an adhesion-prevention membrane is comparable to that of a conventionally utilized membrane of oxidized regenerated cellulose. These results indicate that the copolymer films are biocompatible materials with controllable mechanical properties and biodegradability as adhesion-prevention membranes.

The effect of decellularized bone/bone marrow produced by high-hydrostatic pressurization on the osteogenic differentiation of mesenchymal stem cells.

Decellularized bone/bone marrow was prepared to provide a microenvironment mimicking that of the bone marrow for three-dimensional culture in vitro. Bone/bone marrows were hydrostatically pressed at 980 MPa at 30 °C for 10 min to dismantle the cells. Then, they were washed with EGM-2 and further treated in an 80% EtOH to remove the cell debris and lipid, respectively. After being rinsed and shaken with PBS again, treated bone/bone marrows were stained with hematoxylin and eosin (H-E) to assess the efficacy of decellularization. Cells were determined to have been completely removed through H-E staining of their sections and DNA quantification. Rat mesenchymal stem cells (rMSCs) were seeded on the decellularized bone/bone marrows and cultured for 21 days. The adhesion of rMSCs on or into decellularized bone/bone marrows was confirmed and proliferated over time in culture. The osteogenic differentiation effect of decellularized bone/bone marrows on rMSCs in the presence or absence of dexamethasone was investigated. Decellularized bone/bone marrows without dexamethasone significantly increased alkaline phosphatase (ALP) activity, indicating promoted osteogenic differentiation of rMSCs. In an animal study, when decellularized bone/bone marrows were implanted into the rat subcutaneous, no immune reaction occurred and clusters of the hematopoietic cells could be observed, suggesting the decellularized bone/bone marrows can provide a microenvironment in vivo.

Tissue-engineered acellular small diameter long-bypass grafts with neointima-inducing activity

Researchers have attempted to develop efficient antithrombogenic surfaces, and yet small-caliber artificial vascular grafts are still unavailable. Here, we demonstrate the excellent patency of tissue-engineered small-caliber long-bypass grafts measuring 20-30 cm in length and having a 2-mm inner diameter. The inner surface of an acellular ostrich carotid artery was modified with a novel heterobifunctional peptide composed of a collagen-binding region and the integrin α4β1 ligand, REDV. Six grafts were transplanted in the femoral-femoral artery crossover bypass method. Animals were observed for 20 days and received no anticoagulant medication. No thrombogenesis was observed on the luminal surface and five cases were patent. In contrast, all unmodified grafts became occluded, and severe thrombosis was observed. The vascular grafts reported here are the first successful demonstrations of short-term patency at clinically applicable sizes.

High-hydrostatic pressure technique is an effective method for the preparation of PVA–heparin hybrid gel

To develop an antithrombotic material for preparation of small-diameter vascular graft, we describe a novel method to prepare a poly(vinyl alcohol) (PVA)-heparin hydrogels prepared by high-hydrostatic pressure (HHP, 980 MPa), which is designed for sustained release of heparin. Antithrombogenic test revealed that HHP method would not affect the antithrombin III (ATIII) activity of the released heparin. The distribution of heparin in the polymer matrix was homogeneous than freeze-thawing gel, due to the fast gelling affect of PVA which takes approximately 10 min for gel formation. The formation of intra- and intermolecular hydrogen bonds between PVA chains has trapped the heparin inside, suppressing the phase separation between PVA and heparin. Furthermore, evenly distribution of heparin induced the formation of heparin and PVA molecular complex, which brought the sustained release of heparin from the PVA despite the high swelling ratio. Our results show that it is possible to prepare a PVA-heparin hybrid gel which can be applied as an effective material for an antithrombotic system without using any chemical agent.

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.

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.

Decellularized dermis–polymer complex provides a platform for soft-to-hard tissue interfaces

To develop a soft-to-hard tissue interface, we made a decellularized dermis/poly(methyl methacrylate) (PMMA) complex by soaking the decellularized dermis in methyl methacrylate (MMA) and an initiator, and then polymerizing the MMA. The decellularized tissue was chosen because of its good biocompatibility and the easiness of suturing it, and MMA because of its hard tissue compatibility and wide use in the biomedical field. The MMA filled the cavities in the dermis and polymerized within 10 min. No leaking or polymer aggregation was observed, implying that a homogenous tissue-polymer complex had formed. The cell infiltration and the integration between the tissue and the dermis occurred in vivo, whereas the cells could not infiltrate the tissue-polymer complex. This implies that the interface tissue should possess both complex and noncomplex parts, where the cells infiltrate the noncomplex part and stop when they encounter the complex part, integrating the soft and hard tissue or hard polymer.

Study on the physical properties of tissue-engineered blood vessels made by chemical cross-linking and polymer-tissue cross-linking.

In this study, we attempted to chemically cross-link decellularized blood vessel tissue and to perform cross-linking with a polymer in order to control its stability and functionalization. For this purpose, we cross-linked tissue by intrahelical, interhelical, and intermolecular cross-linking between the polymer and the collagen helix, which is a component of the native tissue. The intrahelically cross-linked tissue showed weaker stability against heat and degradation caused by collagenase compared to the interhelically cross-linked tissue. The tissue intermolecularly cross-linked with polymer showed the highest stability against heat and degradation caused by collagenase. The mechanical strength test showed that the Young’s moduli were different for the intra/interhelically and intermolecularly cross-linked tissues, with the latter being stiffer. This is thought to be because the cross-linked polymer functions in the same way as elastin, whereas simple collagen cross-linking provides a supportive matrix that holds the collagen and elastin together.

In Vivo Characterization of a Decellularized Dermis-Polymer Complex for Use in Percutaneous Devices

To develop a method for making percutaneous devices that have high biocompatibility and do not induce downgrowth of epidermal cells, we prepared a partial decellularized dermis (DD)/poly(methyl methacrylate) (PMMA) complex (PDPC) with a PMMA rod firmly stabilized inside. The porcine decellularized tissue was chosen because of its high biocompatibility and mechanical properties, and MMA was used because it would adhere firmly to a polymer such as a catheter. The MMA filled the cavities in the dermis and polymerized, anchoring to the collagenous fibrils inside the porcine DD. The PDPC was cemented to the PMMA rod tightly and it was integrated with the surrounding tissue within 12 weeks of implantation. Furthermore, no downgrowth of the epidermis, which may cause clinical problems, was observed. We consider that the tissue-polymer complex may be a suitable candidate for use in percutaneous devices.