Papers - HATTORI Hidemi
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The interaction of chitosan with fibroblast growth factor-2 and its protection from inactivation Reviewed
Masuoka K., Ishihara M., Asazuma T., Hattori H., Matsui T., Takase B., Kanatani Y., Fujita M., Saito Y., Yura H., Fujikawa K., Nemoto K.
Biomaterials 26 ( 16 ) 3277 - 3284 2005.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Biomaterials
Application of ultraviolet light (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) results within 30 s in an insoluble, flexible hydrogel. The retained FGF-2 molecules in the chitosan hydrogel remain biologically active, and are released from the chitosan hydrogel upon the in vivo biodegradation of the hydrogel. In view of these findings, we here tested the interaction of chitosan with FGF-2, thereby modifying and stabilizing the FGF-2 activity from inactivations. The photocrosslinkable chitosan hydrogel has a low affinity for FGF-2 (Kd=6.12×10 -7 m). Soluble chitosan (CH-LA; Az-CH-LA without photocrosslinkable azide group) substantially prolonged the biological half-life time of FGF-2. Furthermore, CH-LA could protect the FGF-2 activity from inactivation, such as heat, proteolysis, and acid. The effect of chitosan on the FGF-2 activity is of a protective nature, since it had no effect of modifying the FGF-2 activity directly on growth of human umbilical vein endothelial cells (data not shown). Thus, one of the ways by which the chitosan potentiated the FGF-2 activity could be through protecting it from inactivations by the interaction between FGF-2 and chitosan molecules. © 2004 Elsevier Ltd. All rights reserved.
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Hattori H., Sato M., Masuoka K., Ishihara M., Kikuchi T., Matsui T., Takase B., Ishizuka T., Kikuchi M., Fujikawa K., Ishihara M.
Cells Tissues Organs 178 ( 1 ) 2 - 12 2004.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Cells Tissues Organs
Adult bone marrow contains mesenchymal stem cells (bone marrow-derived mesenchymal stem cells; BMSCs) which contribute to the generation of mesenchymal tissue such as bone, cartilage, muscle and adipose. However, using bone marrow as a source of stem cells has the limitation of a low cell number. An alternate source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Human adipose tissue obtained by liposuction was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). In this study, we compared the osteogenic differentiation of ATSCs with that of BMSCs. Both cell types were cultured in atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS scaffold) for three-dimensional culturing in a specific osteogenic induction medium. Optimal osteogenic differentiation in both cell types, as determined by alkaline phosphatase cytochemistry, secretion of osteocalcin, mineral (calcium phosphate) deposition and scanning electron microscopy, was obtained with the same three-dimensional culture. Furthermore, osteoblastic lining in vivo was examined using ATSC-seeded or BMSC-seeded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts to that of BMSCs. Copyright © 2004 S. Karger AG, Basel.
DOI： 10.1159/000081088
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Inhibition of vascular prosthetic graft infection using a photocrosslinkable chitosan hydrogel Reviewed
Fujita M., Kinoshita M., Ishihara M., Kanatani Y., Morimoto Y., Simizu M., Ishizuka T., Saito Y., Yura H., Matsui T., Takase B., Hattori H., Kikuchi M., Maehara T.
Journal of Surgical Research 121 ( 1 ) 135 - 140 2004.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Surgical Research
Despite improvements in surgical techniques and antimicrobial therapies, prosthetic aortic graft infections remain a clinical problem. It is well known that chitosan has strong antibacterial activities to a wide variety of bacteria including Staphylococcus aureus, epidermidis and Escherichia coli (E. coli). The antibacterial activity by adhering a photocrosslinkable chitosan hydrogel to Dacron grafts was investigated in vitro and in vivo using a rabbit model. The photocrosslinkable chitosan hydrogel (50 μl) coated grafts (3 x 2 mm fragments) were evaluated on a resistance against E. coli in vitro. The graft infections in vivo were also initiated through implantation of a Dacron graft fragment into the infrarenal aorta of a rabbit, followed by a topical inoculation with 10 6 colony-forming units of E. coli. The graft infection was allowed to develop over the following 1 week. The photocrosslinkable chitosan hydrogel-coated grafts exhibited a resistance against E. coli in vitro. Furthermore, application of 0.1 ml photocrosslinkable chitosan hydrogel on the Dacron implant in vivo substantially inhibited graft infection with E. coli. These preliminary results suggested the potential use of a photocrosslinkable chitosan hydrogel in directing graft infection prophylaxis. © 2004 Elsevier Inc. All rights reserved.
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ヒト吸引脂肪組織由来の多能性幹細胞を分化させた骨芽細胞の検討 Reviewed
服部秀美, 佐藤正人, 増岡一典, 石原美弥, 菊地寿幸, 松井岳巳, 菊地眞, 冨士川恭輔, 石原雅之.
防衛医科大学校雑誌 28 ( 4 ) 128 - 36 2003.12
Language：Japanese Publishing type：Research paper (scientific journal)
脂肪組織中に間葉系幹細胞に類似した多能性幹細胞が発見された.これはヒトの皮下に存在する脂肪組織を吸引することにより採取され,PLA細胞と名付けられた.ヒトの吸引脂肪組織由来の多能性幹細胞すなわちPLA細胞と既に確立されているヒト骨髄間葉系幹細胞(MSCs)を用いて骨芽細胞に分化する能力の比較・検討を行った.PLA細胞はMSCsと同等の骨芽細胞への分化能力を示した.よって,骨再生医療の細胞供給源として有用であることが示唆された.
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Obara K., Ishihara M., Ishizuka T., Fujita M., Ozeki Y., Maehara T., Saito Y., Yura H., Matsui T., Hattori H., Kikuchi M., Kurita A.
Biomaterials 24 ( 20 ) 3437 - 3444 2003.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Biomaterials
Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) resulted within 30s in an insoluble, flexible hydrogel. About 20% of the FGF-2molecules were released from the FGF-2-incorporated chitosan hydrogel into phosphate buffered saline (PBS) within 1 day, after which no further significant release occurred under in vitro non-degradation conditions of the hydrogel. The FGF-2molecules retained in the chitosan hydrogel remained biologically active, and were released from the chitosan hydrogel upon the in vivo biodegradation of the hydrogel. In order to evaluate its accelerating effect on wound healing, full thickness skin incisions were made on the back of healing-impaired diabetic (db/db) mice and their normal (db/+) littermates. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure in both db/db and db/+ mice. However, the addition of FGF-2 in the chitosan hydrogel further accelerated wound closure in db/db mice, although not in db/+ mice. Histological examination also has demonstrated an advanced granulation tissue formation, capillary formation and epithelialization in wounds treated with FGF-2-incorporated chitosan hydrogels in db/db mice. © 2003 Elsevier Science Ltd. All rights reserved.
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Sato M., Kikuchi M., Ishihara M., Ishihara M., Asazuma T., Kikuchi T., Masuoka K., Hattori H., Fujikawa K.
Medical and Biological Engineering and Computing 41 ( 3 ) 365 - 371 2003.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Medical and Biological Engineering and Computing
The objective of the study was to investigate the regeneration of intervertebral discs after laser discectomy using tissue engineering procedures. Annulus fibrosus (AF) cells from the intervertebral discs of Japanese white rabbits were cultured in an atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS scaffold), to produce a high-density, three-dimensional culture for up to 3 weeks. Although the DNA content in the scaffold increased at a lower rate than that in the monolayer culture, expression of type II collagen and glycosaminoglycan accumulation in the scaffold were at higher levels than in the monolayer. The AF cells that had been cultured in the scaffold for 7 days were allografted into the lacunae of intervertebral discs of recipients (40 rabbits, 14-16 weeks old; average weight, 3.2kg), whose nucleus pulposus (NP) had been vaporised with an ICG dye-enhanced laser. The allografted cultured AF cells survived and produced hyaline-like cartilage. Furthermore, the narrowing of the intervertebral disc space of the cell-containing scaffold insertion groups was significantly inhibited after 12 post-operative weeks.
DOI： 10.1007/BF02348444
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Ishihara M., Obara K., Ishizuka T., Fujita M., Sato M., Masuoka K., Saito Y., Yura H., Matsui T., Hattori H., Kikuchi M., Kurita A.
Journal of Biomedical Materials Research - Part A 64 ( 3 ) 551 - 559 2003.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part A
Application of ultraviolet (UV) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted within 10 s in an insoluble, flexible hydrogel. A low molecular weight acidic molecule like trypan blue and various high molecular weight molecules such as bovine serum albumin (BSA), heparin and protamine were all retained within the hydrogel, while a low molecular weight basic molecule like toluidine blue was rapidly released from the hydrogel. In the present work, we examined the retaining capability of the chitosan hydrogel for growth factors and controlled release of growth factors from the chitosan hydrogel in vitro and in vivo. Fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor165 (VEGF165), heparin-binding epidermal growth factor (HB-EGF) in phosphate buffered saline (PBS) were mixed with Az-CH-LA aqueous solution to form growth factor-incorporated chitosan hydrogels. About 10-25% of the growth factor was released from a growth factor-incorporated chitosan hydrogel into PBS within the first day, after which no further substantial release took place. The growth factors interacted with Az-CH-LA molecules poly-ion complexation, and probably were unable to be released after the first day under the in vitro nondegradation conditions of the hydrogel. Although the FGF-1, FGF-2, and VEGF165-incorporated chitosan hydrogels on a culture plate significantly stimulated HUVEC growth, the stimulating activity of the growth factor-incorporated chitosan hydrogel was completely cancelled out by washing the hydrogel with PBS solution for 3 days or more. The stimulating activity on the HUVEC growth were however highly recovered by treating the washed growth factor-incorporated chitosan hydrogel during 7 days with chitinase and chitosanase to partly degrade the hydrogel, strongly suggesting that the growth factors within the hydrogel retained their biologically active forms. The chitosan hydrogel (100 μl) when implanted into the back of a mouse was biodegraded in about 10-14 days. When FGF-1- and FGF-2-incorporated chitosan hydrogels were subcutaneously implanted into the back of a mouse, significant neovascularization was induced near the implanted site of the FGF-1- and FGF-2-incorporated chitosan hydrogels. Furthermore, addition of heparin with either FGF-1 or FGF-2 into the hydrogel resulted in a significantly enhanced and prolonged vascularization effect. These results indicate that the controlled release of biologically active FGF-1 and FGF-2 with heparin is caused by biodegradation of the chitosan hydrogel, and subsequent induction of vascularization. © 2003 Wiley Periodicals, Inc.
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Adsorption of inflammatory cytokines using a heparin-coated extracorporeal circuit Reviewed
Fujita M., Ishihara M., Ono K., Hattori H., Kurita A., Shimizu M., Mitsumaru A., Segawa D., Hinokiyama K., Kusama Y., Kikuchi M., Maehara T.
Artificial Organs 26 ( 12 ) 1020 - 1025 2002.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Artificial Organs
Cardiopulmonary bypass (CPB) surgeries cause an increase in plasma inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) along with whole-body inflammatory responses. The inflammatory responses during a CPB treatment are reduced when using a heparin-coated extracorporeal circuit. Because many cytokines, growth factors, and complements are known to interact with heparin, the reduction of inflammatory responses by a heparin-coated circuit is likely to depend on this heparin-binding nature of the inflammatory cytokines. In this study, the inflammatory cytokines, TNF-α and IL-6, in fetal bovine serum (FBS) bound to a heparin-agarose beads (heparin beads)-column and the adsorptions were competitively inhibited on addition of heparin in a concentration-dependent manner. TNF-α in FBS required a higher concentration of heparin (50% concentration inhibition [IC50] > 20μg/ml) to inhibit adsorption to the heparin beads-column compared with IL-6, probably because of a stronger interaction between TNF-α and heparin-beads. TNF-α and IL-6 concentrations in human heparinized blood significantly increased after a CPB treatment. Although the adsorbed amount of IL-6 onto the heparin-coated circuit was low (less than 6% of free circulating IL-6), a significant amount of TNF-α adsorbed onto the circuit (23.9-755% of free circulating TNF-α). Therefore, the adsorption of inflammatory cytokines, especially TNF-α, onto the inner heparin-coated surface of an extracorporeal circuit may partly account for a reduction in inflammatory responses. © 2002 International Society for Artificial Organs.
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Photocrosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process Reviewed
Ishihara M., Nakanishi K., Ono K., Sato M., Kikuchi M., Saito Y., Yura H., Matsui T., Hattori H., Uenoyama M., Kurita A.
Biomaterials 23 ( 3 ) 833 - 840 2002
Language：English Publishing type：Research paper (scientific journal) Publisher：Biomaterials
Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 s. The chitosan hydrogel could completely stop bleeding from a cut mouse tail within 30 s of UV-irradiation and could firmly adhere two pieces of sliced skins of mouse to each other. In order to evaluate its accelerating effect on wound healing, full thickness-skin incisions were made on the back of mice and subsequently an Az-CH-LA aqueous solution was added into the wound and irradiated with UV light for 90 s. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing. Histological examinations also have demonstrated an advanced granulation tissue formation and epithelialization in the chitosan hydrogel treated wounds. The chitosan hydrogel due to its accelerating healing ability is considered to become an excellent dressing for wound occlusion and tissue adhesive in urgent hemostasis situations. © 2001 Elsevier Science Ltd. All rights reserved.
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Acceleration of wound contraction and healing with a photocrosslinkable chitosan hydrogel Reviewed
Ishihara M., Ono K., Sato M., Nakanishi K., Saito Y., Yura H., Matsui T., Hattori H., Fujita M., Kikuchi M., Kurita A.
Wound Repair and Regeneration 9 ( 6 ) 513 - 521 2001.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Wound Repair and Regeneration
Application of ultraviolet light irradiation to a photocrosslinkable chitosan aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 seconds. In order to evaluate its accelerating effect on wound healing, full-thickness skin incisions were made on the backs of mice and subsequently a photocrosslinkable chitosan aqueous solution was added into the wound and irradiated with UV light for 90 seconds. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing compared with the untreated controls. Histological examination also showed an advanced contraction rate on the first 2 days and tissue fill rate on days 2 to 4 in the chitosan hydrogel-treated wounds. Furthermore, in cell culture studies, chitosan hydrogel culture medium supplemented with 5% fetal-bovine serum was found to be chemoattractant for human dermal fibroblasts in an invasion chamber assay using filters coated with Matrigel and in a cell migration assay. Due to its ability to accelerate wound contraction and healing, chitosan hydrogel may become accepted as an occlusive dressing for wound management.
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Photocrosslinkable chitosan: an effective adhesive with surgical applications. Invited Reviewed
Ishihara M, Ono K, Saito Y, Yura H, Hattori H, Matsui T, Kurita A.
International Congress Series 1223 251 - 7 2001.11
Language：English Publishing type：Research paper (international conference proceedings)
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Ishihara M., Sato M., Hattori H., Saito Y., Yura H., Ono K., Masuoka K., Kikuchi M., Fujikawa K., Kurita A.
Journal of Biomedical Materials Research 56 ( 4 ) 536 - 544 2001.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research
Heparin-carrying polystyrene (HCPS), consisting of low molecular weight heparin chains linked to a synthetic polystyrene core, is able to attach to polymeric surfaces. In this study, HCPS has efficiently bound to collagencoated micro-plates and collagen membranes thereby retaining the binding of heparin-binding growth factors, such as vascular endothelial growth factor (VEGF)165 or fibroblast growth factor (FGF)-2. Both human skin fibroblast cells and human umbilical vein endothelial cells have shown a good adherence to both collagen- and HCPS-bound collagen substrata. The growth rate of the fibroblast cells on the HCPS-bound collagen substratum in the presence of low concentrations of FGF-2 is higher than on a collagen surface. The fibroblast cells grow at a significantly higher rate on the HCPS-bound collagen substratum retained with FGF-2. Similarly, the growth rate of the endothelial cells on the HCPS-bound collagen substrata in the presence of low concentrations of either FGF-2 or VEGF165 is higher than on collagen. The endothelial cells also grow at a significantly higher rate on the HCPS-bound collagen substratum retained with either FGF-2 or VEGF165. These results indicate that HCPS-bound collagen substrata with various bioactive heparin-binding molecules may provide novel biomaterials controlling cellular activities such as growth and differentiation. © 2001 John Wiley & Sons, Inc.
DOI： 10.1002/1097-4636(20010915)56:4<536::AID-JBM1125>3.0.CO;2-#
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Ishihara M., Ono K., Ishikawa K., Hattori H., Saito Y., Yura H., Akaike T., Ozeki Y., Tanaka S., Mochizuki H., Kurita A.
Journal of Biochemistry 127 ( 5 ) 797 - 803 2000.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biochemistry
Heparin-carrying polystyrene (HCPS) consists of low-molecular-weight heparin chains enriched in trisulfated disaccharide structures linked to a polystyrene core. In this study, the interactions between HCPSs of various molecular weights and heparin-binding growth factors, VEGF165, FGF-2, and HGF, were compared to the interactions of the same factors with native heparin, periodate-oxidized heparin (IO4-heparin) and periodate-oxidized alkaline-degraded heparin (IO4-LMW-heparin). The binding of each growth factor to heparin-agarose beads (heparin-beads) was more strongly inhibited by HCPSs in a molecular weight-dependent manner than by native heparin or the modified heparins, indicating a stronger interaction between HCPS and these growth factors. HCPSs also inhibit heparin-binding growth factor-induced endothelial cell growth in a molecular weight-dependent manner much more strongly than the native or modified heparins. However, HCPSs did not inhibit the mitogenic activity of VEGF121, which has a non-heparin-binding nature. Thus, HCPSs exhibit enhanced abilities to interact with each of the heparin- binding growth factors studied and to inhibit heparin-binding growth factor- induced endothelial cell proliferation in a molecular weight-dependent manner. These effects might be ascribed to the heparin-clustering effect of HCPSs.
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Ishihara M., Saito Y., Yura H., Ono K., Ishikawa K., Hattori H., Akaike T., Kurita A.
Journal of Biomedical Materials Research 50 ( 2 ) 144 - 152 2000.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research
Various sugar-carrying polystyrenes (PSs), which consist of synthetic styrene and sugar moieties, are glycoconjugates that are able to attach to polymeric surfaces. Heparin-carrying PS (HCPS) is especially able to retain the binding of heparin-binding growth factors (GFs) such as vascular endothelial GF 165 (VEGF165) or fibroblast GF 2 (FGF-2). Human skin fibroblast cells, human coronary smooth muscle cells, and human coronary endothelial cells have good adherence to the HCPS-coated plate. The growth rate of fibroblast cells on HCPS-coated plates is higher than or comparable to fibronectin-coated, gelatin-coated, or tissue culture treated plates, and the HCPS coating inhibits the growth of smooth muscle cells. On the other hand, the growth rate of endothelial cells on HCPS-coated plates in the presence of either VEGF165 or FGF-2 is comparable to that on fibronectin- coated, gelatin-coated, and tissue culture treated plates. Endothelial cells grow at a higher rate on HCPS-coated plates retained with either VEGF165 or FGF-2 than on the other coated plates. These results indicate that growth of various cells can be controlled by the HCPS coating, thereby retaining the bioactivity of molecules such as heparin-binding GFs. Thus, HCPS-coated surfaces control selective growth of various cells. (C) 2000 John Wiley and Sons, Inc.
DOI： 10.1002/(SICI)1097-4636(200005)50:2<144::AID-JBM8>3.0.CO;2-S
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Structural features in heparin that interact with VEGF165 and modulate its biological activity Reviewed
Ono K., Hattori H., Takeshita S., Kurita A., Ishihara M., Ishihara M.
Glycobiology 9 ( 7 ) 705 - 711 1999.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Glycobiology
The 165 amino acid form of vascular endothelial growth factor (VEGF165) is a heparin-binding growth factor with mitogenic activity for vascular endothelial cells. We examined activities of various heparin derivatives toward their interactions with VEGF165 using an enzyme-linked immunosorbent assay and elucidated the structural features in heparin for the interactions. Native heparin interacted with VEGF165, whereas N-desulfated, N-acetylated (N-DS, N-Ac-) heparin, and 6-O-desulfated (6-O-DS-) heparin did not. The 2-O-desulfated (2-O-DS-) heparin retained the ability for the interaction with VEGF165. In contrast, the 2-O-DS-heparin exhibited no ability for the interaction with FGF-2 and HGF. Thus, structural requirements in heparin for the specific interaction with VEGF165 are distinct from those with FGF-2 and HGF which require a high content of 2-O-sulfate groups. In a cell proliferation assay, native heparin and 2-O-DS-heparin exhibited inhibitory abilities for VEGF165-induced proliferation of human umbilical vein endothelial cells (HUVECs) with their high concentrations (more than 64 μg/ml), while only native heparin could enhance the proliferation of the chlorate-treated cells. These results suggested that a high content of 2-O-sulfate groups is not required for the specific interaction with VEGF165 alone, although it is essential for the mitogenic activity of the growth factor.
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ヘパリノイドとヘパリン結合性タンパク質との相互作用を評価するためのELISA様検定法 Reviewed
小野克明, 石原雅之, 服部秀美, 栗田明.
防衛医科大学校雑誌 23 ( 3 ) 175 - 81 1998.9
Language：Japanese Publishing type：Research paper (scientific journal)
ヘパリン結合性タンパク質とヘパリノイドの相互作用を評価するELISA様検定法を開発した.ヘパリンはFGF-1やFGF-2のヘパリン固定化ビーズへの結合に対する強い阻害活性をもつが,コンドロイチン硫酸-A,コンドロイチン硫酸-C,デルマタン硫酸,ヒアルロン酸はこの阻害活性を全く示さなかった.過ヨウ素酸酸化還元ヘパリンは,化学的修飾をしていないヘパリンと同等の阻害活性を示すが,N-脱硫酸化N-アセチル化ヘパリンにはこの阻害活性がない.2-O-脱硫酸化ヘパリンのFGF-1及びFGF-2の両者に対する阻害活性と6-O-脱硫酸化ヘパリンのFGF-1に対する阻害活性は,大きく減少したが,6-O-脱硫酸化ヘパリンのFGF-2に対する阻害活性はごく僅かの減少にとどまった.