Jun Negishi

Effect of treatment temperature on collagen structures of the decellularized carotid artery using high hydrostatic pressure

Decellularized tissues have attracted a great deal of attention as regenerating transplantation materials. A decellularizing method based on high hydrostatic pressure (HHP) has been developed, and the preparation of many types of decellularized tissues has been investigated, including aorta, cornea, and dermis. The preparation of a small-diameter vascular graft was studied using a carotid artery from the viewpoint of collagen denaturation and leakage. After HHP, the carotid artery was washed at two washing temperatures (37 and 4°C). Histological evaluation, collagen content measurement and circular dichroism (CD) measurement indicated that the washing temperatures clearly affected the collagen structure of the decellularized carotid artery. The amount of collagen decreased in the carotid artery decellularized by HHP washed at 37°C (HHP/37°C). On the other hand, the amount and structure of collagen were preserved in the carotid artery washed at 4°C after HHP (HHP/4°C). In rat carotid artery syngeneic transplantation, the HHP/37°C decellularized carotid artery occluded after 2xa0weeks, but the HHP/4°C decellularized one did not. These results indicate that collagen denaturation and leakage of the decellularized carotid artery affect the in vivo performance of the carotid artery.

Therapeutic Angiogenesis by Implantation of a Capillary Structure Constituted of Human Adipose Tissue Microvascular Endothelial Cells

Objective—We previously reported a novel technology for the engineering of a capillary network using an optical lithographic technique. To apply this technology to the therapy of ischemic diseases, we tested human omental microvascular endothelial cells (HOMECs) as an autologous cell source and decellularized human amniotic membranes (DC-AMs) as a pathogen-free and low immunogenic transplantation scaffold. Methods and Results—Human umbilical vein endothelial cells were aligned on a patterned glass substrate and formed a capillary structure when transferred onto an amniotic membrane (AM). In contrast, HOMECs were scattered and did not form a capillary structure on AMs. Treatment of HOMECs with sphingosine 1-phosphate (S1P) inhibited HOMEC migration and enabled HOMEC formation of a capillary structure on AMs. Using quantitative RT-PCR and Western blot analyses, we demonstrated that the main S1P receptor in HOMECs is S1P2, which is lacking in human umbilical vein endothelial cells, and that inhibition of cell migration by S1P is mediated through an S1P2–Rho–Rho-associated kinase signaling pathway. Implantation of capillaries engineered on DC-AMs into a hindlimb ischemic nude mouse model significantly increased blood perfusion compared with controls. Conclusion—A capillary network consisting of HOMECs on DC-AMs can be engineered ex vivo using printing technology and S1P treatment. This method for regeneration of a capillary network may have therapeutic potential for ischemic diseases.

Corneal Regeneration by Deep Anterior Lamellar Keratoplasty (DALK) Using Decellularized Corneal Matrix

The purpose of this study is to demonstrate the feasibility of DALK using a decellularized corneal matrix obtained by HHP methodology. Porcine corneas were hydrostatically pressurized at 980 MPa at 10°C for 10 minutes to destroy the cells, followed by washing with EGM-2 medium to remove the cell debris. The HHP-treated corneas were stained with H-E to assess the efficacy of decellularization. The decellularized corneal matrix of 300 μm thickness and 6.0 mm diameter was transplanted onto a 6.0 mm diameter keratectomy wound. The time course of regeneration on the decellularized corneal matrix was evaluated by haze grading score, fluorescein staining, and immunohistochemistry. H-E staining revealed that no cell nuclei were observed in the decellularized corneal matrix. The decellularized corneal matrices were opaque immediately after transplantation, but became completely transparent after 4 months. Fluorescein staining revealed that initial migration of epithelial cells over the grafts was slow, taking 3 months to completely cover the implant. Histological sections revealed that the implanted decellularized corneal matrix was completely integrated with the receptive rabbit cornea, and keratocytes infiltrated into the decellularized corneal matrix 6 months after transplantation. No inflammatory cells such as macrophages, or neovascularization, were observed during the implantation period. The decellularized corneal matrix improved corneal transparency, and remodelled the graft after being transplanted, demonstrating that the matrix obtained by HHP was a useful graft for corneal tissue regeneration.

Porcine radial artery decellularization by high hydrostatic pressure

Many types of decellularized tissues have been studied and some have been commercially used in clinics. In this study, small‐diameter vascular grafts were made using HHP to decellularize porcine radial arteries. One decellularization method, high hydrostatic pressure (HHP), has been used to prepare the decellularized porcine tissues. Low‐temperature treatment was effective in preserving collagen and collagen structures in decellularized porcine carotid arteries. The collagen and elastin structures and mechanical properties of HHP‐decellularized radial arteries were similar to those of untreated radial arteries. Xenogeneic transplantation (into rats) was performed using HHP‐decellularized radial arteries and an untreated porcine radial artery. Two weeks after transplantation into rat carotid arteries, the HHP‐decellularized radial arteries were patent and without thrombosis. In addition, the luminal surface of each decellularized artery was covered by recipient endothelial cells and the arterial medium was fully infiltrated with recipient cells. Copyright

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.

Effect of decellularized tissue powders on a rat model of acute myocardial infarction.

Many research groups are currently investigating new treatment modalities for myocardial infarction. Numerous aspects need to be considered for the clinical application of these therapies, such as low cell integration and engraftment rates of cell injection techniques. Decellularized tissues are considered good materials for promoting regeneration of traumatic tissues. The properties of the decellularized tissues are sustained after processing to powder form. In this study, we examined the use of decellularized tissue powder in a rat model of acute myocardial infarction. The decellularized tissue powders, especially liver powder, promoted cell integration and neovascularization both in vitro and in vivo. Decellularized liver powder induced neovascularization in the infarct area, resulting in the suppression of myocardial necrosis. The results of this study suggest that decellularized liver powder has good potential for application as a blood supply material for the treatment of myocardial infarction.

Evaluation of Small-Diameter Vascular Grafts Reconstructed from Decellularized Aorta Sheets.

Following small-diameter vascular grafting, blood vessels fail to retain excellent antithrombotic function and therefore require application of antithrombogenic drugs. We have previously reported early attachment of endothelial cells to the luminal surface of high hydrostatic pressure (HHP)-decellularized arteries after transplantation. In addition, the graft retained antithrombotic function by endothelialization and remained open for several weeks. To fabricate tube grafts of optimal size and shape for small-diameter vascular grafting, we evaluated decellularized porcine aorta sheets as a suitable antithrombogenic material. Porcine aortic sheets were decellularized using detergent-based or HHP methods. The HHP-, but not detergent-based-, decellularized aortic sheets were verified to be acellular, and the mechanical properties of the native aortic sheet remained intact. To fabricate vascular grafts, the decellularized aortic sheets were rolled into tubes and secured using fibrin glue bonding. After implantation into a rat carotid artery model, the tubular grafts withstood normal blood pressure, mechanical beating, and pulsatile flow. After 3 weeks, the tubular grafts remained patent and recipient cell infiltration and cell attachment were observed on the luminal surface. These results indicate that HHP-decellularized aortic sheets may be useful as an antithrombogenic material for tubular vascular grafts.

Fabrication of a heparin–PVA complex hydrogel for application as a vascular access

A high hydrostatic pressure method, which can apply over 600 MPa pressure was employed for preparing a hydrogel of poly(vinyl alcohol) (PVA) loaded with heparin. The aim of this study was to fabricate a heparin-PVA hydrogel conduit and evaluate its potential for vascular access. Heparin-PVA complex hydrogel showed suppressed heparin release and prevented clot formation, depending on the molecular weight of the PVA. Strength of the hydrogel conduit was increased by embedding a Dacron mesh between two PVA layers. The tubular heparin-PVA complex hydrogel displayed a burst pressure of 750 mmHg. The tubular heparin-PVA complex hydrogel did not show any occlusion or burst for 2 weeks after implantation, implying that this heparin-PVA complex hydrogel shows high potential for use as a vascular access. This is the first report on the preparation of a multilayered PVA hydrogel with heparin embedded on one side only. The proposed approach could be expanded to the fabrication of various biomaterials for specific purposes.

Tissue Engineering with Natural Tissue Matrices

Natural tissue, especially autologous tissue is one of ideal materials for tissue regeneration. Decellularized tissue could be assumed as a second choice because the structure and the mechanical properties are well maintained. Decellularized human tissues, for instance, heart valve, blood vessel, and corium, have already been developed and applied clinically. Nowadays, decellularized porcine tissues are also investigated. These decellularized tissues were prepared by detergent treatment. The detergent washing is easy but sometime it has problems. We have developed the novel decellularization method, which applied the high-hydrostatic pressure (HHP). As the tissue set in the pressurizing chamber is treated uniformly, the effect of the high-hydrostatic pressurization does not depend on the size of tissue. We have reported the HHP decellularization of heart valve, blood vessel, bone, and cornea. Furthermore, HHP treatments are reported to have the ability of the extinction of bacillus and the inactivation of virus. So, the HHP treatment is also expected as the sterilization method. We are investigating efficient processes of decellularization and recellularization of biological tissues to have bioscaffolds keeping intact structure and biomechanical properties. Our recent studies on tissue engineering using HHP decellularized tissue will be reported here.

A fibrin-coated pericardial extracellular matrix prevented heart adhesion in a rat model: FIBRIN-COATED PERICARDIAL EXTRACELLULAR MATRIX

As most surgical treatments pose a risk of tissue adhesion, methods to prevent adhesion are needed across various surgical fields. In this study, we investigated the use of a decellularized pericardium with fibrin glue to prevent rat heart adhesion. Porcine pericardia were decellularized by a high-hydrostatic pressure method. Cells adhered to the resulting pericardial extracellular matrix (ECM) during an in vitro cell-seeding test, but fibrin-coated pericardial ECM showed reduced cell adhesion. In a rat surgical model of heart adhesion, the fibrin-coated pericardial ECM did not adhere to the heart and mesothelial cell adhesion was observed on the ECM surface. Notably, the anti-adhesion effect of fibrin-coated pericardial ECM was observed 4 weeks after surgery. These results support the utility of fibrin-coated pericardial ECM as an adhesion prevention material for cardiovascular surgery.