Yoshihide Hashimoto

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.

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.

Therapeutic Effect of Nanogel-Based Delivery of Soluble FGFR2 with S252W Mutation on Craniosynostosis

Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIcS252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIcS252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIcS252W, and FGFR2IIIbS252W. Administration of sFGFR2IIIcS252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIcS252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIcS252W could be effective for treating this disorder.

Cycloamylose-nanogel drug delivery system-mediated intratumor silencing of the vascular endothelial growth factor regulates neovascularization in tumor microenvironment

RNAi enables potent and specific gene silencing, potentially offering useful means for treatment of cancers. However, safe and efficient drug delivery systems (DDS) that are appropriate for intra‐tumor delivery of siRNA or shRNA have rarely been established, hindering clinical application of RNAi technology to cancer therapy. We have devised hydrogel polymer nanoparticles, or nanogel, and shown its validity as a novel DDS for various molecules. Here we examined the potential of self‐assembled nanogel of cholesterol‐bearing cycloamylose with spermine group (CH‐CA‐Spe) to deliver vascular endothelial growth factor (VEGF)‐specific short interfering RNA (siVEGF) into tumor cells. The siVEGF/nanogel complex was engulfed by renal cell carcinoma (RCC) cells through the endocytotic pathway, resulting in efficient knockdown of VEGF. Intra‐tumor injections of the complex significantly suppressed neovascularization and growth of RCC in mice. The treatment also inhibited induction of myeloid‐derived suppressor cells, while it decreased interleukin‐17A production. Therefore, the CH‐CA‐Spe nanogel may be a feasible DDS for intra‐tumor delivery of therapeutic siRNA. The results also suggest that local suppression of VEGF may have a positive impact on systemic immune responses against malignancies.

Fabrication of a heterostructural fibrillated collagen matrix for the regeneration of soft tissue function

Most collagen matrices used in tissue engineering and regenerative medicine are gels or sponges. Our goal is to prepare an artificial extracellular matrix (ECM) that possesses similar structure to the native ECM of soft tissue in order to replicate its physical and biological properties. Here, we report the preparation of collagen matrices with fibrillated structures based on the fibrillogenesis/gelling method. We focused on regenerating the function of native ECM of soft tissues by creating a multifunctional collagen. A collagen matrix is a sponge/gel composite structure which allows the cell to infiltrate only on one side of the matrix without shrinkage. This results in healing and regeneration of the tissue surrounding the matrix, similar to the native ECM in which cell infiltration occurs from one side during implantation. Furthermore, high mechanical strength can prevent deformation or shrinkage, and decelerate the degradation during the early phase of implantation, which eventually cause device failure.

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.

Ultrastructural analysis of the decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty in a rabbit model

The decellularized cornea has received considerable attention for use as an artificial cornea. The decellularized cornea is free from cellular components and other immunogens, but maintains the integrity of the extracellular matrix. However, the ultrastructure of the decellularized cornea has yet to be demonstrated in detail. We investigated the influence of high hydrostatic pressure (HHP) on the decellularization of the corneal ultrastructure and its involvement in transparency, and assessed the in vivo behaviour of the decellularized cornea using two animal transplantation models, in relation to remodelling of collagen fibrils. Decellularized corneas were prepared by the HHP method. The decellularized corneas were executed by haematoxylin and eosin and Masson’s trichrome staining to demonstrate the complete removal of corneal cells. Transmission electron microscopy revealed that the ultrastructure of the decellularized cornea prepared by the HHP method was better maintained than that of the decellularized cornea prepared by the detergent method. The decellularized cornea after interlamellar keratoplasty and microkeratome-assisted anterior lamellar keratoplasty using a rabbit model was stable and remained transparent without ultrastructural alterations. We conclude that the superior properties of the decellularized cornea prepared by the HHP method were attributed to the preservation of the corneal ultrastructure.

Effects of a polysaccharide nanogel-crosslinked membrane on wound healing.

INTRODUCTION Wound-dressing materials that promote wound healing while protecting wounds from infections are advantageous for clinical applications. Hence, we developed a cholesterol-bearing pullulan (CHP) nanogel that stimulated wound healing; however, it was mechanically weak and difficult to handle. Thus, the purpose of this study was to examine precisely the effects of a mechanically reinforced nanogel-crosslinked (NanoClik) membrane on wound healing. MATERIALS AND METHODS NanoClik was prepared by mixing a thiol-terminated polyethylene glycol solution and an acryloyl group-modified CHP nanogel solution. A thin silicone sheet membrane, which was combined with NanoClik, was prepared. The NanoClick membranes and both positive and negative control membranes (collagen combined with silicone membrane and silicone membrane alone, respectively) were tested in vivo using a dorsal skin defect rat model. The rate and extent of wound healing was compared between groups after 7 and 14 days of implantation. RESULTS In the NanoClik membrane group, the wound area was significantly reduced and neoepithelialization was promoted, compared with that observed in the other groups. In addition, extension and accumulation of collagen fibers were evident in the NanoClik membrane group. CONCLUSION The NanoClik membrane is a strong candidate for use as an effective and safe wound-dressing material.

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.

Capture and Release of Target Cells Using a Surface That Immobilizes an Antibody via Desthiobiotin-Avidin Interaction

We designed and synthesized a polyethylene film that immobilizes antibodies via desthiobiotin– avidin interaction by grafting poly(acrylic acid) (PAAc) and biotin to its surface. The desthiobiotinmodified antibodies (anti-mouse CD45 and CD25 antibodies) were immobilized on the film through desthiobiotin–avidin interaction. Mouse bone marrow (CD45-positive) and HL60 (CD45negative) cells were seeded on the films with and without immobilizing the anti-mCD45 antibody, and the selective adhesion of cells was observed. Also, mouse spleen cells (CD25-positive) were selectively captured on the anti-mCD25-antibody-immobilized film. These captured cells were completely released by the addition of biotin-conjugated water-soluble polymers, indicating that the dissociation of desthiobiotin–avidin complexes occurred owing to the different association constants of desthiobiotin and biotin to avidin. From these results, it was suggested that target cells could be selectively captured and collected using a film having a surface that immobilizes an antibody via desthiobiotin–avidin interaction.