Papers - HATTORI Hidemi
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Effects of Excessive High-fructose Corn Syrup Drink Intake in Middle-aged Mice. Reviewed
Hidaka M, Oshima Y, Hanai Y, Kataoka H, Hattori H
In vivo 38 ( 3 ) 1152 - 1161 2024.5
Authorship：Last author, Corresponding author Language：English Publishing type：Research paper (scientific journal)
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Excessive intake of high-fructose corn syrup drinks induces impaired glucose tolerance Reviewed
Hattori H., Hanai Y., Oshima Y., Kataoka H., Eto N.
Biomedicines 9 ( 5 ) 512 2021.5
Authorship：Lead author, Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Biomedicines
The number of patients with diabetes was approximately 463 million worldwide in 2019, with almost 57.6% of this population concentrated in Asia. Asians often develop type 2 diabetes (T2D), even if they are underweight and consume a smaller amount of food. Soft drinks contain large amounts of sweeteners, such as high-fructose corn syrup (HFCS). Excessive intake of HFCS drinks is considered to be one of the causes of T2D. In the present study, we investigated the effect of excessive consumption of HFCS–water on glucose tolerance and obesity under conditions of controlled caloric intake using a mouse model. Three-week-old male ICR mice were divided into two groups and given free access to 10% HFCS–water or deionized water. The caloric intake was ad-justed to be the same in both groups using a standard rodent diet. The excess HFCS–water intake did not lead to obesity, but led to impaired glucose tolerance (IGT) due to insulin-secretion defect. It affected glucose and fructose metabolism; for example, it decreased the expression of glucoki-nases, ketohexokinase, and glucose transporter 2 in the pancreas. These results suggest that excessive consumption of HFCS drinks, such as soft drinks, without a proper diet, induces nonobese IGT due to insulin-secretion defect.
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Hattori H, Ishihara M.
Cell Biology International 44 ( 5 ) 1124 - 1132 2020.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Cell Biology International
The relationships between eosinophils and adipose tissues are involved in metabolic homeostasis. Eotaxin is a chemokine with potent effects on eosinophil migration. To clarify the mechanisms of eotaxin expression in adipose tissues, we examined the effects of fibroblast growth factor-2 (FGF-2) and interleukin-4 (IL-4) stimulation on eotaxin expression in adipose tissue-derived stromal cells (ASCs), a type of adipocyte progenitor, in vitro. ASCs expressed eotaxin-1 and did not express eotaxin-2 or -3. Eotaxin-1 expression was increased in a concentration-dependent manner following FGF-2 treatment. Additionally, ASCs expressed FGF receptor-1 (FGFR-1) and did not express FGFR-2, -3, or -4. Eotaxin-1 expression was inhibited in cells treated with the FGFR tyrosine kinase inhibitor and extracellular signal-regulated kinase (ERK) inhibitor U0126, even in the presence of FGF-2. Moreover, eotaxin-1 expression was synergistically enhanced by combined treatment with FGF-2 and IL-4 and inhibited in the presence of U0126. Eotaxin-1 expression induced by FGF-2 and IL-4 was involved in ERK activation via FGFR-1 in ASCs. Upregulation of eotaxin expression in adipose tissues could increase eosinophil migration, thereby inducing IL-4 secretion and activation of alternative macrophages and improving glucose homeostasis. These findings provide insights into the mechanisms through which eotaxin mediates metabolic homeostasis in adipose tissues and eosinophils.
DOI： 10.1002/cbin.11309
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Immunostimulatory effect of kumquat (Fortunella crassifolia) and its constituents, β-cryptoxanthin and R-limonene. Reviewed
Terao R, Murata A, Sugamoto K, Watanabe T, Nagahama K, Nakahara K, Kondo T, Murakami N, Fukui K, Hattori H, Eto N
Food & function 10 ( 1 ) 38 - 48 2019.1
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Stability of Weakly Acidic Hypochlorous Acid Solution with Microbicidal Activity. Reviewed
Ishihara M, Murakami K, Fukuda K, Nakamura S, Kuwabara M, Hattori H, Fujita M, Kiyosawa T, Yokoe H.
Biocontrol Science 22 ( 4 ) 223 - 227 2017.11
Language：English Publishing type：Research paper (scientific journal)
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Hattori H., Tsujimoto H., Hase K., Ishihara M.
Carbohydrate Polymers 175 592 - 600 2017.11
Language：English Publishing type：Research paper (scientific journal) Publisher：Carbohydrate Polymers
© 2017 Elsevier Ltd To examine the potential of chitosan-based agents for submucosal injection in endoscopic techniques, a chitosan derivative was prepared with lactose moieties linked to the amino groups of its glucosamine units (CH-LA). After dissolving CH-LA in neutral pH solutions, including physiological saline (CH-LA-S), its response to different concentrations of anionic glycosaminoglycans and proteins in the surrounding environment was examined. The CH-LA-S form changed in the presence of sulfated glycosaminoglycans (heparin, chondroitin sulfate, and mucin) and protein (fibrinogen). High concentrations of sulfated substrates in the solution caused the formation of larger structures. In contrast, in the presence of hyaluronan, 30 mg/mL CH-LA-S did not form any large structures. Submucosal injection of 30 mg/mL CH-LA-S into extracted swine stomachs showed a strong lifting effect of the gastric mucosa. These results indicate the potential utility of CH-LA-S as a submucosal injection for endoscopic techniques such as endoscopic submucosal dissection and mucosal resection of tumors.
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Takabayashi Y., Ishihara M., Kuwabara M., Takikawa M., Nakamura S., Hattori H., Kiyosawa T.
Annals of Plastic Surgery 78 ( 5 ) 562 - 568 2017.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Annals of Plastic Surgery
Activated platelet-rich plasma secrets many growth factors (GFs), and low-molecular weight heparin-protamine micro/nanoparticles (LMWH-P M/NPs) significantly interact with, enhance, and stabilize the secreted GFs. Objective: The purpose of this study was to evaluate the effects of LMWH-P M/NPs and GFs (from platelet-rich plasma) on full-thickness skin graft (FTSG). Methods: A total of 96 inbred male rats were anesthetized and 4-cm2 fullthickness skin wound were created on dorsal skin of rats. LMWH-P M/NPs and GFs, LMWH-P M/NPs, GFs and saline (control) were then injected evenly into cutaneous muscles at the wound. The next day, the rats underwent FTSG. On the indicated days after FTSG, blood flow of FTSG site (wound bed and FTSG) was examined by 2-dimensional laser Doppler blood flowmeter. On 10 days, pictures of FTSG site were taken and FTSG survival rate was evaluated. Histologic analyses of skin samples were performed on 4, 7, and 10 days. Results: Treatment of full-thickness skin wound with LMWH-P M/NPs and GFs effectively promoted survival rate of FTSG and blood flow of FTSG site compared with those treated with GFs, LMWH-P M/NPs, and control. LMWH-P M/NPs and GFs also promoted new vessel formation at FTSG site. Conclusions: The prior injection of LMWH-P M/NPs and GFs into wound bed increases FTSG survival rate, and promotes blood flow and angiogenesis at FTSG site.
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Feasibility of improving platelet-rich plasma therapy by using chitosan with high platelet activation ability. Reviewed
Hattori H, Ishihara M
Experimental and therapeutic medicine 13 ( 3 ) 1176 - 1180 2017.3
Language：English Publishing type：Research paper (scientific journal)
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Cytotoxicity of Silver Nanoparticle and Chitin-Nanofiber Sheet Composites Caused by Oxidative Stress. Reviewed
Kinoda J, Ishihara M, Hattori H, Nakamura S, Fukuda K, Yokoe H
Nanomaterials (Basel, Switzerland) 6 ( 10 ) 2016.10
Language：English Publishing type：Research paper (scientific journal)
DOI： 10.3390/nano6100189
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Kishimoto S., Inoue K., Nakamura S., Hattori H., Ishihara M., Sakuma M., Toyoda S., Iwaguro H., Taguchi I., Inoue T., Yoshida K.
Atherosclerosis 249 132 - 139 2016.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Atherosclerosis
Heparin/protamine micro/nanoparticles (LH/P-MPs) were recently developed as low-molecular weight, biodegradable carriers for adipose-derived stromal cells (ADSCs). These particles can be used for a locally delivered stem cell therapy that promotes angiogenesis. LH/P-MPs bind to the cell surface of ADSCs and promote cell-to-cell interaction and aggregation of ADSCs. Cultured ADSC/LH/P-MP aggregates remain viable. Here, we examined the ability of these aggregates to rescue limb loss in a mouse model of hindlimb ischemia. Methods: Unilateral hindlimb ischemia was induced in adult male BALB/c mice by ligation of the iliac artery and hindlimb vein. For allotransplantation of ADSCs from the same inbred strain, we injected ADSC alone or ADSC/LH/P-MP aggregates or control medium (sham-treated) directly into the ischemic muscles. Ischemic limb blood perfusion, vessel density, and vessel area were recorded. The extent of ischemic limb necrosis or limb loss was assessed on postoperative days 2, 7, and 14. Results: Compared with the sham-treatment control, treatment with ADSCs alone showed modest effects on blood perfusion recovery and increased the number of α-SMA-positive vessels. Response to ADSC/LH/P-MP aggregates was significantly greater than ADSCs alone for every endpoint. ADSC/LH/P-MP aggregates more effectively prevented the loss of ischemic hindlimbs than ADSCs alone or the sham-treatment. Conclusion: The LH/P-MPs augmented the effects of ADSCs on angiogenesis and reversal of limb ischemia. Use of ADSC/LH/P-MP aggregates offers a novel and convenient treatment method and potentially represents a promising new therapeutic approach to inducing angiogenesis in ischemic diseases.
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Takabayashi Y., Nambu M., Ishihara M., Kuwabara M., Fukuda K., Nakamura S., Hattori H., Kiyosawa T.
Clinical, Cosmetic and Investigational Dermatology 9 127 - 134 2016.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Clinical, Cosmetic and Investigational Dermatology
Purpose: Although treatments for alopecia are in high demand, not all treatments are safe and reliable. Dalteparin/protamine nanoparticles (D/P NPs) can effectively carry growth factors (GFs) such as fibroblast GF (FGF)-2. The purpose of this study was to identify the effects of FGF-2-containing D/P NPs (FGF-2&D/P NPs) on hair growth. Patients and methods: In this study, the participants were 12 volunteers with thin hair. One milliliter of FGF-2 (100 ng/mL) and D/P NPs (56 μg/mL) was applied and massaged on the skin of the scalp by the participants twice a day. They were evaluated for 6 months. Participants were photographed using a digital camera for general observation and a hair diagnosis system for measuring hair diameter. Results: The mean diameter of the hairs was significantly higher following the application of FGF-2&D/P NPs for 6 months. Objective improvements in thin hair were observed in two cases. Nine participants experienced greater bounce and hair resilience. Conclusion: The transdermal application of FGF-2&D/P NPs to the scalp can be used as a new treatment for alopecia.
DOI： 10.2147/CCID.S108187
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Takikawa M., Ishihara M., Takabayashi Y., Sumi Y., Takikawa M., Yoshida R., Nakamura S., Hattori H., Yanagibayashi S., Yamamoto N., Kiyosawa T.
Journal of Plastic Surgery and Hand Surgery 49 ( 5 ) 268 - 274 2015.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Plastic Surgery and Hand Surgery
Purpose: The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. Method: Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. Results and conclusion: These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.
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Takikawa M., Nakamura S., Ishihara M., Takabayashi Y., Fujita M., Fujita M., Hattori H., Kushibiki T., Ishihara M.
Journal of Surgical Research 196 ( 2 ) 247 - 257 2015.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Surgical Research
We produced fibroblast growth factor (FGF)-2-containing low-molecular-weight heparin (Fragmin)/protamine nanoparticles (FGF-2 + F/P NPs). The purpose of this study was to evaluate the effectiveness of the local administration of FGF-2 + F/P NPs on repairing crush syndrome (CS)-injured lesions after compression release using a nonlethal and reproducible CS injury rat model. Materials and methods The hind limbs of the anesthetized rats were compressed for 6 h using 3.6 kg blocks, as previously described. The effects of administering FGF-2 + F/P NPs (group A), F/P NPs alone (group B), FGF-2 alone (group C), and saline (control; group D) were examined. Motor function, surface blood flow in the hind limbs, and the wet/dry weight ratio in the tibialis anterior muscle were examined for 1-28 d after the compression release. Histologic analyses were also performed. Results At the middle and late stages (3-28 d after the compression release), group A had higher scores in the motor function, improved blood flow, increased number of blood vessels, and faster recovered muscle tissue, compared with the other groups. There was no significant difference in enhanced edema in the tibialis anterior muscle among all groups. Conclusions The local administration of FGF-2 + F/P NPs to a CS-injured lesion was effective in repairing damaged muscle tissue after compression release.
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Hattori H., Ishihara M.
Stem Cell Research and Therapy 6 ( 1 ) 70 2015.4
Language：English Publishing type：Research paper (scientific journal) Publisher：Stem Cell Research and Therapy
Introduction: Paracrine effects can be exploited in cell-based therapies that secrete factors, such as chemokines and cytokines, and can recruit inflammatory cells to transplants. In this study, mouse adipose tissue-derived stromal cells (ASCs) and bone marrow-derived stromal cells (ST2 cells) were used to examine changes in paracrine interactions with inflammation cells. Methods: Green fluorescent protein positive (GFP<sup>+</sup>) bone marrow cells (BMCs) were injected into an irradiated mouse via the femoral vein, and ASCs and ST2 cells were transplanted intradermally. Subsequently, an in vivo imaging system was used to observe behaviors of GFP<sup>+</sup> BMCs. To detect bone marrow-derived inflammatory cells which migrated to the ASC and ST2 cell transplantation area, the sections were immunostained using antibodies against Gr1, CD11c, and F4/80, and secretory proteins were detected in culture medium using enzyme-linked immunosorbent assay. Results: Many bone marrow-derived inflammatory cells migrated to ASC and ST2 cell transplantation sites. Among these, neutrophils were detected during the early period and macrophages were predominantly detected at a later point in time. Many chemokines, cytokines, growth factors, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were secreted in abundance from ASCs, and the secretion increased by co-culturing with inflammatory cells, except for secretions of insulin-like growth factor-1, MMP-9 and MMP-13. Although secretions from ST2 cells were less than those from ASCs, co-culture with inflammatory cells increased these secretions to levels similar to those of ASCs. However, unlike ASCs, the ST2 cells did not secrete angiostatin, MMP-2, or MMP-3. Finally, ASCs secreted not only proinflammatory cytokines, angiogenic factors and MMPs but also anti-inflammatory cytokines, anti-angiogenesis factors, and TIMPs. Conclusions: The effects of cell-based therapies using ASCs and ST2 cells are depended on paracrine effects that are mediated by chemokines, cytokines, growth factors, MMPs, and TIMPs, which comprise responses to interactions between transplanted cells and inflammatory cells. Moreover, paracrine effects of transplanted cells are influenced by inflammatory cells, and are moderated by a balance of secreted inhibitors.
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Hattori H., Ishihara M.
Biomedical Materials (Bristol) 10 ( 1 ) 015014 2015.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Biomedical Materials (Bristol)
Because the molecular weight (Mw) and degree of deacetylation (DDA) of chitosan can vary depending on the purification method, the identification of appropriate chitosan structures is important for developing more effective hemostatic agents. In this study, the influence of varying Mw and DDA of chitosan on blood aggregation was characterized by 10 types of chitosan with different Mw and DDA, including oligomers. The highest aggregation of whole blood, washed erythrocytes and platelets in platelet-rich plasma (PRP) were observed in chitosan with Mw of 8.6 kDa or more and with DDA of 75 to 88%. However, chitosan with too high Mw (247 kDa) inhibited the aggregation of whole blood, washed erythrocytes and PRP at high chitosan concentration. At certain concentrations, chitosan with 75-85% DDA and 50-190 kDa and chitosan with 87.6% DDA and 247 kDa both aggregated proteins in PRP. Chitosan oligomer did not affect blood aggregation. The results suggested that the aggregation by chitosan depended on the interaction of positively charged chitosan with negatively charged erythrocytes, platelets and plasma protein. It seemed that a suitable balance of positive charge in chitosan and negative charge in hemocytes and some kinds of proteins was important. To develop a hemostatic with effective blood aggregation, the chitosan should not be limited to a single Mw and a single DDA but may be a mixed chitosan with wide range of Mw (8.6-247 kDa) and DDA of 75 to 88%.
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Nguyen V., Ishihara M., Kinoda J., Hattori H., Nakamura S., Ono T., Miyahira Y., Matsui T.
Journal of Nanobiotechnology 12 ( 1 ) 49 2014.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Nanobiotechnology
Background: Chitin nanofibers sheets (CNFSs) with nanoscale fiber-like surface structures are nontoxic and biodegradable biomaterials with large surface-to-mass ratio. CNFSs are widely applied as biomedical materials such as a functional wound dressing. This study aimed to develop antimicrobial biomaterials made up of CNFS-immobilized silver nanoparticles (CNFS/Ag NPs). Materials and methods: CNFSs were immersed in suspensions of Ag NPs (5.17 ± 1.9 nm in diameter; mean ± SD) for 30 min at room temperature to produce CNFS/Ag NPs. CNFS/Ag NPs were characterized by transmission electron microscopy (TEM) and then tested for antimicrobial activities against Escherichia (E.) coli, Pseudomonas (P.) aeruginosa, and H1N1 influenza A virus, three pathogens that represent the most widespread infectious bacteria and viruses. Ultrathin sectioning of bacterial cells also was carried out to observe the bactericidal mechanism of Ag NPs. Results: The TEM images indicated that the Ag NPs are dispersed and tightly adsorbed onto CNFSs. Although CNFSs alone have only weak antimicrobial activity, CNFS/Ag NPs showed much stronger antimicrobial properties against E. coli, P. aeruginosa, and influenza A virus, with the amount of immobilized Ag NPs onto CNFSs. Conclusions: Our results suggest that CNFS/Ag NPs interacting with those microbes exhibit stronger antimicrobial activities, and that it is possible to apply CNFS/Ag NPs as anti-virus sheets as well as anti-infectious wound dressings.
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Takase B., Hattori H., Tanaka Y., Nagata M., Ishihara M.
International Angiology 33 ( 1 ) 27 - 34 2014.2
Language：English Publishing type：Research paper (scientific journal) Publisher：International Angiology
Aim. Assessment of flow-mediated dilation (FMD) and nitroglycerin-mediated dilation (NMD) in the brachial artery by a new device (UNEXEF18G) has been reported to be excellent for evaluating endothelial function, and sympathetic overdrive can accelerate the atherosclerotic process. The purpose of this study was to investigate and confirm whether anti-sympathetic beta-blocking action can enhance the pleiotropic effects of statins. Methods. FMD and NMD were measured using the UNEXEF18G before and after 4-week treatment of rosuvastatin (5 mg/day) with or without atenolol (25 mg/day) in 44 hypercholesterolemic patients (70±8 years old, LDL-C >140 mg/dL) with hypertension. Patients were randomly allocated to two treatment arms: rosuvastatin alone (R-group, N.=22) and rosuvastatin with atenolol (RA-group, N.=22). Results. Baseline FMD was not different between the two treatment arms, and both groups showed improvement in FMD (R-group, 3.48±1.9% to 4.65±2.41%, P<0.05; RAgroup, 3.42±1.48% to 5.46±1.79%, P<0.05), while there were no differences in NMD. The effects on lipid profiles were identical in the two groups. In addition, FMD improvement was greater in the RA-group than in the R-group (Δchange 2.15±1.29% vs. 1.16±1.15%, P<0.05). Conclusion. Beta-blockade enhances the pleiotropic effects of statins on endothelial function. The mechanism should be confirmed by further studies.
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Sumi Y., Ishihara M., Kishimoto S., Takikawa M., Hattori H., Takikawa M., Azuma R., Nakamura S., Fujita M., Kiyosawa T.
Annals of Plastic Surgery 72 ( 1 ) 113 - 120 2014
Language：English Publishing type：Research paper (scientific journal) Publisher：Annals of Plastic Surgery
We investigated the effectiveness of the application of inbred adipose-derived stromal cells (IR-ASCs) in high inbred rat plasma (IRP) (6%)-Dulbecco modified Eagle medium (DMEM) gel with fragmin/protamine microparticles (F/P MPs) (IR-ASCs + IRP-DMEM gel + F/P MPs) on wound healing in streptozotocin-induced diabetic rats. F/P MPs have previously been used as a cell carrier for IR-ASCs in inbred Fisher 344 rats and for preservation and controlled release of various cytokines in IRP-DMEM gel. We applied IR-ASCs + IRP-DMEM gel + F/P MPs to full-thickness skin excisions on the backs of the diabetic rats. The statistical significance of wound closure was evaluated on postwounding days 3, 7, 10, and 14, and the skin area surrounding the wound was removed for histological examination on days 7 and 14. The wound closure rate and histological examination of wounds treated with IR-ASCs + IRP-DMEM gel + F/P MPs demonstrated significantly advanced epithelialization, capillary formation, and granulation tissue formation. When DiI-labeled IR-ASCs + IRP-DMEM gel + F/P MPs were applied to full-thickness skin wounds on the backs of the diabetic rats, histological observation at 2 weeks showed appearances of both DiI-labeled granulation tissue and CD31-immunostained microvessels in the transplant areas. A portion of the transplanted IR-ASCs + IRP-DMEM gel + F/P MPs had been taken up into the granulation tissues to promote wound healing. Thus, IR-ASCs + IRP-DMEM gel + F/P MPs were effective for repairing healing-impaired wounds such as those arising in the diabetic rats. Copyright © 2013 Lippincott Williams & Wilkins.
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Hattori H., Amano Y., Habu-Ogawa Y., Ando T., Takase B., Ishihara M.
Cell Transplantation 22 ( 12 ) 2381 - 2392 2013.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Cell Transplantation
The aim of this study was to identify novel angiogenic mechanisms underlying the regenerative process. To that end, interactions between adipose tissue-derived stromal cells (ASCs) and bone marrow cells (BMCs) were initially investigated using real-time fluorescence optical imaging. To monitor cell behavior in mice, we injected green fluorescent protein-positive (GFP+) BMCs into the tail vein and injected PKH26-labeled ASCs behind the ears. Angiogenesis and inflammation were observed at these sites via an optical imaging probe. Injected GFP+ BMCs migrated from the blood vessels into the tissues surrounding the ASC injection sites. Many of the migrating GFP+ BMCs discovered at the ASC injection sites were inflammatory cells, including Gr-1+, CD11b+, and F4/80+ cells. ASCs cocultured with inflammatory cells secreted increased levels of chemokines such as macrophage inflammatory protein (MIP)-1α, MIP-1β, keratinocyte-derived chemokines, and monocyte chemotactic protein 1. Similarly, these ASCs secreted increased levels of angiogenic growth factors such as hepatocyte growth factor and vascular endothelial growth factor. However, when anti-CXC chemokine receptor type 4 antibody was injected at regular intervals, the migration of GFP+ BMCs (especially Gr-1+ and CD11b+ cells) to ASC injection sites was inhibited, as was angiogenesis. The collective influence of the injected ASCs and BMC-derived inflammatory cells promoted acute inflammation and angiogenesis. Together, the results suggest that the outcome of cell-based angiogenic therapy is influenced not only by the injected cells but also by the effect of intrinsic inflammatory cells. © 2013 Cognizant Comm. Corp.
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Takase B., Nagata M., Hattori H., Tanaka Y., Ishihara M.
Medical Principles and Practice 23 ( 1 ) 59 - 65 2013.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Medical Principles and Practice
Objective: This study evaluated the efficacy of combined therapy with probucol and cilostazol on endothelial function in silent lacunar cerebral infarcts (SLCI) and mild hypercholesterolemia. Subjects and Methods: Flow-mediated vasodilatation (FMD) and nitroglycerin-induced vasodilatation (NMD) were measured before and after 4 weeks of combined therapy with probucol (500 mg/day) and cilostazol (200 mg/day) in 34 patients with a mean age of 72 ± 7 years (range 57-80 years) with SLCI, mild hypercholesterolemia (low-density lipoprotein cholesterol >100 mg/dl) and impaired endothelial function (FMD <6%). Patients were randomly allocated to one of the following two treatment groups: (1) aspirin (100 mg/day) with behavioral modifications, such as diet and/or exercise therapy (A group or control group, n = 17), and (2) probucol and cilostazol treatment (PC group, n = 17), also with behavioral modifications. Results: Although the baseline FMD was not different between the two treatment arms (2.7 ± 1.5 vs. 2.6 ± 1.5%, n.s.), the posttreatment FMD was significantly improved in the PC group (from 2.7 ± 1.5 to 3.5 ± 1.7%, p < 0.05) but not in the A group (from 2.6 ± 1.5 to 2.9 ± 1.4%, n.s.). No differences were observed between baseline and posttreatment NMD in either group. The effects of treatments on lipid profiles were more profound in the PC group. Conclusion: Combined treatment with probucol and cilostazol resulted in subacute improvement in FMD/endothelial function in patients with SLCI with mild hypercholesterolemia. This combination therapy has the potential to reduce the risk of cardiovascular events via improvements in endothelial function and lipid profiles. © 2013 S. Karger AG, Basel.
DOI： 10.1159/000355825
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Kishimoto S., Kishimoto S., Kishimoto S., Ishihara M., Mori Y., Mori Y., Takikawa M., Hattori H., Nakamura S., Sato T.
Journal of Tissue Engineering and Regenerative Medicine 7 ( 12 ) 955 - 964 2013.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Tissue Engineering and Regenerative Medicine
Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet-rich plasma (PRP). In this study, we investigated the capability of F/P NP-coated plates to act as a substratum for the proliferation of human adipose-derived stromal cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP-coated plates and grew optimally, with a doubling time of 30 and 32 h in low-concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor-2 (FGF-2) on the F/P NP-coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF-2 on F/P NP-coated plates pretreated with PRP and FGF-2 in a concentration-dependent manner. Thus, F/P NP-coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low-concentration PRP medium supplemented with FGF-2. No xenogeneic serum is required. © 2012 John Wiley & Sons, Ltd.
DOI： 10.1002/term.1488
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Nguyen V., Ishihara M., Mori Y., Nakamura S., Kishimoto S., Fujita M., Hattori H., Kanatani Y., Ono T., Miyahira Y., Matsui T.
Bio-Medical Materials and Engineering 23 ( 6 ) 473 - 483 2013.11
Language：English Publishing type：Research paper (scientific journal) Publisher：Bio-Medical Materials and Engineering
We previously reported a simple method for the preparation of size-controlled spherical silver nanoparticles (Ag NPs) generated by autoclaving a mixture of silver-containing glass powder and glucose. The particle size is regulated by the glucose concentration, with concentrations of 0.25, 1.0 and 4.0 wt% glucose providing small (3.48±1.83 nm in diameter), medium (6.53±1.78 nm) and large (12.9±2.5 nm) particles, respectively. In this study, Ag NP/chitosan composites were synthesized by mixing each of these three Ag NP suspensions with a 75% deacetylated (DAc) chitosan suspension (pH 5.0) at room temperature. The Ag NPs were homogeneously dispersed and stably embedded in the chitosan matrices. The Ag NP/chitosan composites were obtained as yellow or brown flocs. It was estimated that approximately 60, 120 and 360 μg of the small, medium and large Ag NPs, respectively, were maximally embedded in 1 mg of chitosan. The bactericidal and anti-fungal activities of the Ag NP/chitosan composites increased as the amount of Ag NPs in the chitosan matrix increased. Furthermore, smaller Ag NPs (per weight) in the chitosan composites provided higher bactericidal and anti-fungal activities. © 2013 - IOS Press and the authors.
DOI： 10.3233/BME-130772
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Nguyen V., Ishihara M., Nakamura S., Hattori H., Ono T., Miyahira Y., Matsui T.
Journal of Nanomaterials 2013 2013.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Nanomaterials
Silver nanoparticles (Ag NPs) were 5.17±1.9 nm in diameter, and four <5% deacetylated chitins (A, B, C, and D) differing in size of powder and surface structure properties were used in the study. Chitin/Ag NP composites were synthesized by mixing Ag NP suspensions with each chitin powder at room temperature for 30 min. The Ag NPs were homogenously dispersed and stably adsorbed onto the chitins A and B powders. The resulting chitin/Ag NP composites were brown; darker composites were obtained when larger amounts of Ag NPs were reacted with chitin. Approximately, 26 and 22 g of Ag NPs maximally adsorbed to 1 mg of chitins A and B, respectively, whereas only 2.5 and 1.5 g of Ag NPs maximally adsorbed to chitins C and D, respectively. As the bactericidal and antifungal activities of the chitin/Ag NP composites increased with increasing amounts of Ag NPs adsorbed to the chitin, the antimicrobial activity of chitins A and B/Ag NP composites was much higher than that of chitins C and D/Ag NP composites. These results suggest that the particle size and surface structure of the chitin powder critically influence both the adsorption and antimicrobial activity of Ag NPs. © 2013 Vinh Quang Nguyen et al.
DOI： 10.1155/2013/467534
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Takase B., Hattori H., Tanaka Y., Uehata A., Nagata M., Ishihara M., Fujita M.
Journal of Cardiovascular Ultrasound 21 ( 3 ) 130 - 136 2013.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Cardiovascular Ultrasound
Background: Repeated application of whole-body periodic acceleration (WBPA) upregulates endothelial nitric oxide synthase and improves brachial artery endothelial function (BAEF) as assessed by measurement of flow-mediated vasodilatation (FMD). However, the acute effect of a single application of WBPA on BAEF has not been fully characterized. In addition, although a novel semi-automatic vessel chasing system (UNEXEF18G) has now been developed in Japan, the direct comparison of UNEXEF18G with a conventional method for FMD measures has not been conducted even if UNEXEF18G has already been utilized in a relatively large scale study. Methods: We have developed a novel semi-automatic vessel chasing system (UNEXEF18G) that can measure FMD on-line, identify time to peak vasodilatation (TPV), and determine the area under the vasodilatation curve (AUC). Thus, 45 min of WBPA was applied in 20 healthy volunteers (age, 34 ± 13 years), and BAEF was measured by UNEXEF18G before and after WBPA. Also, UNEXEF18G measured FMD was compared with those of a conventional FMD measurement method at rest in order to validate a novel UNEXEF18G measured FMD. Results: Single WBPA resulted in a significant increase in FMD (from 6.4 ± 3.4 to 10.7 ± 4.3%, p < 0.01), a significant decrease in TPV and a significant increase in AUC. In the validation study for UNEXEF18G, Bland and Altman analysis showed that UNEXEF18G measured FMD was almost identical to those of the conventional method at rest. Conclusion: These data suggest the usefulness of a new UNEXEF18G and that single application of WBPA results in acute improvement in BAEF in humans. © 2013 Korean Society of Echocardiography.
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Tsuda Y., Hattori H., Tanaka Y., Ishihara M., Kishimoto S., Kishimoto S., Amako M., Arino H., Nemoto K.
Journal of Tissue Engineering and Regenerative Medicine 7 ( 9 ) 720 - 728 2013.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Tissue Engineering and Regenerative Medicine
In the field of orthopaedic surgery, an orthopaedic surgeon sometimes requires to suppress excessive bone formation, such as ectopic bone formation, ossifying myositis and radio-ulnar synostosis, etc. Ultraviolet (UV) light irradiation of a photocrosslinkable chitosan (Az-CH-LA) generates an insoluble hydrogel within 30s. The purpose of this study was to evaluate the ability of the photocrosslinked chitosan hydrogel (PCH) to inhibit bone formation in an experimental model of bone defect. Rat calvarium and fibula were surgically injured and PCH was implanted into the resultant bone defects. The PCH implants significantly prevented bone formation in the bone defects during the 4 and 8week observation periods. In the PCH-treated defects, fibrous tissues infiltrated by inflammatory cells were formed by day 7, completely filling the bone defects. In addition to these findings, expression of osteocalcin and runt-related gene 2 (RUNX2) mRNA, both markers of bone formation, was lower in the PCH-treated defects than in the controls. In contrast, collagen type 1α2 and α-smooth muscle actin (α-SMA) mRNA levels were significantly higher in the PCH-treated defects after 1week. PCH stimulated the formation of fibrous tissue in bone defects while inhibiting bone formation. Thus, PCH might be a promising new therapeutic biomaterial for the prevention of bone formation in orthopaedic surgery. © 2012 John Wiley & Sons, Ltd.
DOI： 10.1002/term.1462
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出血性ショック心臓における致死性不整脈の機序に関する実験的検討―活動電位不均一性とConnexin43変化について Invited Reviewed
高瀬凡平, 東村悠子, 木村一生, 田中良弘, 服部秀美, 石原雅之.
Therapeutic Research 34 ( 8 ) 1060 - 6 2013.8
Language：Japanese Publishing type：Research paper (conference, symposium, etc.)
出血性ショックにより平均全身血圧が40mmHg以下に低下遷延すると，不可逆性心筋障害が発生し，いわゆる“出血性ショック心臓”といわれる致死性の病態を呈するとされている。しかし，致死性不整脈の発生機序に関する
検討は少ない。そこでわれわれは，実験的に検討した。
方法：SD rat （n＝32）に30％出血性ショック状態を作製し，非蘇生群，洗浄赤血球蘇生群，生理食塩水蘇生群，5％アルブミン蘇生群の4群間で心筋を摘出Tyrode液で灌流後Na channel感受性色素を用いたoptical mapping system
（OMP）で興奮伝播・活動電位持続時間不均一性（action potential duration dispersion ：APD dispersion），致死性催不整脈性を検討した。また，心筋組織のconnexin43 （Cx43）発現を免疫
組織染色にて検討した。
結果：蘇生群では，3群とも全例蘇生に成功した。しかし，生理食塩水群，5％アルブミン群では OMP で著明な左心室伝導遅延と burst pacingによる心室細動が全例で誘発されたのに対し，洗浄赤血球蘇生群では，伝導遅延・心室細動誘発ともに認められなかった。生理食塩水群，5％アルブミン群では著明にAPD dispersion値が増大したが，洗浄赤血球群では正常に保たれていた。connexin43発現は生理食塩水群，5％アルブミン群では異常が認められたものの，洗浄赤血球群では正常に保たれていた。
結語：出血性ショック心臓では，左心室伝導遅延とAPD dispersion増大およびconnexin43発現異常を惹起し，電気的不安定性から致死性不整脈が誘発されることが示唆された。洗浄赤血球治療はこれら指標の保持と予防効果を有した。 -
Sumi Y., Ishihara M., Kishimoto S., Kishimoto S., Takikawa M., Doumoto T., Azuma R., Nakamura S., Hattori H., Fujita M., Kiyosawa T.
Journal of Biomedical Materials Research - Part B Applied Biomaterials 101 B ( 5 ) 784 - 791 2013.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part B Applied Biomaterials
Fragmin/protamine microparticles (F/P MPs) have been used as a cell carrier for adipose-derived stromal cells (IR-ASCs) in inbred male Fisher 344 rats, and for preservation and controlled-release of fibroblast growth factor (FGF)-2 and various cytokines in inbred rat plasma (IRP)-DMEM (Dulbecco's modified Eagle's medium) gel. In this study, we investigated the capability of an IRP-DMEM gel containing F/P MPs and/or FGF-2, as a three-dimensional (3D)-culture, to expand IR-ASCs. We found that IR-ASCs grow faster under 3D-culture conditions in low IRP (3%)-DMEM gel containing F/P MPs and FGF-2 without any animal serum than those under 2D-culture in low inbred rat serum (3%)-DMEM with F/P MPs and FGF-2. About 0.3 mL of IR-ASCs (about 4,000,000 cells mL-1) grown in IRP (6%)-DMEM gel containing F/P MPs and FGF-2 disappeared 8 days after subcutaneous injection in rats, suggesting that they are rapidly biodegradable. The number of large (diameter ≥200 μm or containing ≥100 erythrocytes), medium (diameter = 20-200 μm or containing 10-100 erythrocytes) and small (diameter ≤20 μm or containing 1-10 erythrocytes) capillaries after injection with IR-ASCs in an IRP-DMEM gel containing both F/P MPs and FGF-2, as well as the thickness of tissue granulation per microphotograph at the injected site, was significantly higher than those after injection with IR-ASCs in an IRP-DMEM gel containing either FGF-2 or F/P MPs. Thus, IRP-DMEM gel containing F/P MPs and FGF-2 are useful and safe IR-ASC carriers that facilitate cell proliferation, vascularization, and tissue granulation locally at injection sites. © 2013 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.b.32882
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血管内皮機能指標の疾患別測定値変動に関する検討 : 心臓リハビリテーション効果判定に関して. Invited Reviewed
高瀬凡平, 永田雅良, 服部秀美, 石原雅之.
心臓リハビリテーション 18 ( 2 ) 231 - 5 2013.6
Language：Japanese Publishing type：Research paper (scientific journal)
[目的] 上腕動脈血管内皮機能検査(Flow-mediated vasodilatation: FMD)は動脈硬化指標として広く用いられている. しかし, FMD測定値に及ぼす因子は多岐にわたるので, 疾患別測定値変動幅を検討する. [方法] 冠動脈危険因子を有する健常者(HV; n=10), 高血圧例(HT; n=34)および冠動脈疾患例(CAD: n=35)に約3年間で少なくとも4回以上のFMD測定を施行し, その変動値につき検討を加えた. この間, 個々のFMD測定は通常のガイドラインに準拠し, FMD測定専用機(UNEXEF)で測定したものの, 期間中の治療内容は主治医の判断により変更可とした. 変動程度は個々の症例の測定値の標準偏差(SD)を求め, 3群間でそのSDの平均±標準偏差を算出して評価した. [結果] 安静時上腕動脈の血管径変動値には3群間で有意差は認められなかった. しかし, FMDはHV群に有意に変動が大きいことが認められた(HV vs HT, CAD; 2.4±1.1 vs 1.4±1.0, 1.0±0.9%, p<0.05). [結論] 上腕動脈血管内皮機能は動脈硬化疾患の進展・予後を規定する検査法として今後ますます重視される項目である. したがって, 上腕動脈血管内皮機能検査法で得られるFMD値の改善基準を, 代表的検査対象である, 健常者例, 高血圧症例, 冠動脈疾患症例で示した. 今後の心臓リハビリテーションなどのinterventionがFMD値に及ぼす影響を検討するうえで有用と考え報告した.
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Development of a Novel Emergency Hemostatic Kit for Severe Hemorrhage Reviewed
Hattori H., Amano Y., Nogami Y., Kawakami M., Yura H., Ishihara M.
Artificial Organs 37 ( 5 ) 475 - 481 2013.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Artificial Organs
Photocrosslinkable chitosan (Az-CH-LA) contains lactose moieties and photoreactive azide groups, and its viscous solution forms an insoluble hydrogel on exposure to UV irradiation. We previously developed an emergency hemostatic kit using the Az-CH-LA solution, calcium alginate, and a UV irradiation apparatus. However, a suitable UV irradiation apparatus is required to effectively convert the Az-CH-LA solution into a hydrogel, and power supply to use the UV irradiation apparatus may not always be available in a disaster area or battlefield. To address this problem, we produced a portable, battery-powered UV irradiation apparatus constituting a novel hemostatic kit for severe hemorrhage. When the hemostatic kit using the UV irradiation apparatus was examined using a rat model of severe hemorrhage, the survival rate increased up to 73%. Hematological values as markers of hemorrhage did not change significantly over the first 3 days. In this study, we describe the characteristics of a portable UV irradiation apparatus and its use in an emergency hemostatic kit prepared using Az-CH-LA and calcium alginate for severe hemorrhage. © 2013, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
DOI： 10.1111/aor.12004
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Nguyen V., Ishihara M., Mori Y., Nakamura S., Kishimoto S., Hattori H., Fujita M., Kanatani Y., Ono T., Miyahira Y., Matsui T.
Journal of Nanomaterials 2013 2013.4
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Nanomaterials
A simple method for the preparation of size-controlled spherical silver nanoparticles (Ag NPs) was reported for their generation by autoclaving a mixture of silver-containing glass powder and glucose. The particle size is regulated by the glucose concentration, with concentrations of 0.25, 1.0, and 4.0 wt% glucose providing small (3.48 ± 1.83 nm in diameter), medium (6.53 ± 1.78 nm), and large (12.9 ± 2.5 nm) particles, respectively. In this study, Ag NP/chitin composites were synthesized by mixing each of these three Ag NP suspensions with a <5% deacetylated (DAc) chitin powder (pH 7.0) at room temperature. The Ag NPs were homogenously dispersed and stably adsorbed onto the chitin. The Ag NP/chitin composites were obtained as yellow or brown powders. Approximately 5, 15, and 20 g of the small, medium, and large Ag NPs, respectively, were estimated to maximally adsorb onto 1 mg of chitin. The bactericidal and antifungal activities of the Ag NP/chitin composites increased as the amount of Ag NPs in the chitin increased. Furthermore, smaller Ag NPs (per weight) in the chitin composites provided higher bactericidal and anti-fungal activities. © 2013 Vinh Quang Nguyen et al.
DOI： 10.1155/2013/693486
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Fujita M., Horio T., Kishimoto S., Kishimoto S., Nakamura S., Takikawa M., Nakayama T., Yamamoto Y., Shimizu M., Hattori H., Tachibana S., Ishihara M.
Journal of Biomedical Materials Research - Part B Applied Biomaterials 101 B ( 1 ) 36 - 42 2013.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part B Applied Biomaterials
The purpose of the study was to evaluate the effects of isogenous platelet-rich plasma (PRP)-containing fragmin/protamine microparticles (F/P MPs) as a delivery system for proteins in PRP on growth of endothelial and smooth muscle cells (SMCs) in vitro and as an alternative treatment for peripheral arterial disease (PAD) and critical limb ischemia. Frozen and thawed PRP contains high concentrations of growth factors that are adsorbed by F/P MPs. Human aorta endothelial cells (AECs) and SMCs were grown in a medium with PRP. Addition of F/P MPs significantly enhanced the proliferative effects of PRP on AECs and SMCs at 37°C for >10 days. Intramuscular administration of phosphate-buffered saline (PBS; 2 mL, control), F/P MPs (12 mg in 2 mL PBS), PRP (2 mL), or PRP (2 mL) containing F/P MPs (12 mg) was then performed in a rabbit model of hindlimb ischemia prepared by resection of the left femoral artery. Blood flow and pressure were measured on days 0, 14, and 28, and angiography to assess arteriogenesis was performed on day 28. PRP-containing F/P MPs strongly induced functional collateral vessels in the rabbit model of hindlimb ischemia, indicating possible use of these microparticles in therapy for PAD. © 2012 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.b.32808
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Pitavastatin subacutely improves endothelial function and reduces inflammatory cytokines and chemokines in patients with hypercholesterolaemia. Reviewed
Takase B, Hattori H, Tanaka Y, Nagata M, Ishihara M
Heart Asia 5 ( 1 ) 204 - 9 2013
Language：English Publishing type：Research paper (scientific journal)
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画像診断と体表面微小電位 心臓MRI法、体表心臓微小電位および非侵襲的心電図予後指標に関する検討 Invited Reviewed
高瀬凡平, 永田雅良, 浜部晃, 上畑 昭美, 服部秀美, 田中良弘, 石原雅之, 栗田明.
心臓 45 ( Suppl. 1 ) 23 - 7 2013
Language：Japanese Publishing type：Research paper (conference, symposium, etc.)
心筋壊死診断に心臓MRI法の遅延造影（delayed enhancement；DE）が有用であり，DEと心筋梗塞（MI）後の予後の関連が報告されているものの，致死性不整脈発生の非侵襲的心電図予測指標との比較検討は少ない．そこで，DEと体表微小心臓電位（LP）やQT dispersion（QTD）と関係および予後予測につき検討した．
陳旧性心筋梗塞症84例（69± 9 歳）に心臓MRI法とLP，QTDを施行した．心臓MRI法は1.5T GE社製Sigma CV/iにてgadlinium投与造影下に左心室短軸 6 断面を撮像した．心筋の壊死形態をmassive（ 1 点）からpatchy（ 3 点）までscore化し（patchy sore；PS），LP陽性およびQTDの結果と比較した．症例を平均35±17カ月追跡調査し心事故との関連を調べた．
LP陽性例22例とLP陰性例62例が認められ，壊死の形態を示すPSはLP陽性例で陰性例に比べ高値を示した（2.3±0.5 vs 1.4±0.6，p＜0.05）．壊死範囲を反映するDE断面数とQTDは相関した．追跡期間中 9 例の心事故が認められDE断面数とQTDが心事故予測に有用であった．
結語：心臓MRI法で求めたDEの形態はLPと相関したものの，予後予測には壊死範囲を示すDE指標やQTDがより有用である可能性が示唆された． -
Nogami Y., Takase B., Kinoshita M., Shono S., Kaneda S., Tanaka Y., Kishimoto S., Hattori H., Ishihara M.
Shock 38 ( 2 ) 159 - 164 2012.8
Language：English Publishing type：Research paper (scientific journal) Publisher：Shock
The use of liposome-encapsulated hemoglobin (LHb), which is a cellular Hb, has been demonstrated to be beneficial in the treatment of hypohemoglobinemic shock. As a molecule of appropriate size (220 nm) that can carry oxygen, LHb may ameliorate cardiac dysfunction during lethal hemodilation. The purpose of this study was to determine the efficacy of LHb transfusion in relieving cardiovascular dysfunction in a rat model of lethal progressive hemodilution. Over the course of 150 min, rats were subjected to blood withdrawal (0.2 mL/min) and simultaneously transfused with LHb, washed rat red blood cells, or 5% albumin. Temporal changes in cardiac function, heart-type fatty acid-binding protein levels, plasma levels of catecholamines, heart rate variability, and hypoxia-inducible factor 1α expression were measured during lethal progressive hemodilution. More than 80% of the rats transfused with either LHb or washed rat red blood cells survived for 8 days. Liposome-encapsulated hemoglobin transfusion suppressed hypoxia-inducible factor 1α expression in the heart, maintained low levels of heart-type fatty acid-binding protein, and attenuated sympathetic nerve activity as reflected by changes in heart rate variability and plasma levels of epinephrine and norepinephrine. The results indicate that LHb attenuates cardiac dysfunction and sympathetic overactivity during lethal hemorrhage. © 2012 by the Shock Society.
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Importance of Platypnea Orthodeoxia in the Differential Diagnosis of Dyspnea Invited
Takase B, Tanaka Y, Hattori H, Ishihara M.
Internal Medicine 51 ( 13 ) 1651 - 2 2012.7
Language：English Publishing type：Research paper (other academic)
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Kumano I., Ishihara M., Nakamura S., Kishimoto S., Fujita M., Hattori H., Horio T., Tanaka Y., Hase K., Maehara T.
Gastrointestinal Endoscopy 75 ( 4 ) 841 - 848 2012.4
Language：English Publishing type：Research paper (scientific journal) Publisher：Gastrointestinal Endoscopy
Background: Hypertonic saline solution (HS) as a submucosal fluid cushion (SFC) for endoscopic submucosal dissection (ESD) has several disadvantages, including a short effect duration and increased risk of bleeding and perforation. Photocrosslinkable chitosan hydrogel in DMEM/F12 medium (PCH) can be converted into an insoluble hydrogel by UV irradiation for 30 seconds. Objective: To evaluate the feasibility, usefulness, and safety of PCH as an SFC for ESD of esophagi, compared with HS and sodium hyaluronate (SH). Design: Survival animal study. Settings: Research laboratory study of 24 pig models in vivo. Interventions: Twenty-four pigs were used in the 2 steps: First, ESD of the esophagus was performed with PCH, SH, or HS (each n = 6) as an SFC, and the effects of these agents on wound healing were examined endoscopically and histologically. Second, in vivo degradation of PCH (n = 3) and HS (n = 3) was examined in independent pig esophagi. Main Outcome Measurements: Outcome measurements included feasibility and safety of PCH-assisted ESD of esophagus, gross and histologic evidence of the treated esophagus, biodegradation of injected PCH, and clinical tolerance by the animals. Result: PCH injection led to a longer-lasting elevation with clearer margins compared with SH and HS, thus enabling precise ESD along the margins of the elevated mucosa without complications such as bleeding and perforation. The aspects of wound repair after PCH-assisted ESD were similar to those of SH- and HS-assisted ESDs. Biodegradation of PCH was confirmed to be almost completed within 8 weeks on the basis of endoscopic and histologic observations. Limitations: In vivo animal model study. Conclusion: PCH permits more reliable ESD of the esophagus without complications than do SH and HS. Furthermore, the applied PCH appeared to be completely biodegraded within 8 weeks. Thus, PCH is a promising agent as an SFC in ESD of the esophagus. © 2012 American Society for Gastrointestinal Endoscopy.
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肺全摘手術術後の右心室負荷による催不整脈性に関する検討. Invited Reviewed
高瀬凡平, 東村悠子, 田中良弘, 服部秀美, 石原雅之, 濱部晃, 永井智雄.
Therapeutic Research 33 ( 11 ) 1616 - 21 2012
Language：Japanese Publishing type：Research paper (conference, symposium, etc.)
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Kishimoto S., Ishihara M., Kanatani Y., Nambu M., Takikawa M., Sumi Y., Nakamura S., Mori Y., Hattori H., Tanaka Y., Sato T.
Journal of Tissue Engineering 2 ( 1 ) 1 - 8 2011.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Tissue Engineering
Low-molecular-weight heparin/protamine microparticles (LH/P MPs) serve as carriers for controlled release of heparin-binding cytokines. LH/P MPs were stably coated onto plastic surfaces by drying. The purpose of this study is to evaluate a culture method for selective expansion of CD34+ cells using LH/P MPs as cytokine-binding matrix. Ficoll-purified mouse bone marrow cells (mouse FP-BMCs) containing CD34+ cells were cultured on LH/P MP-coated plates in the presence of stem cell factor (SCF), thrombopoietin (Tpo), and Flt-3 ligand (Flt-3) in hematopoietic progenitor growth medium (HPGM) supplemented with 4% heat-inactivated fetal bovine serum (FBS). After 8 days of culture, the total cell count increased 4.6-fold, and flow cytometry analyses revealed that 23.8% of the initial cells and 57.4% of the expanded cells were CD34 positive. Therefore, CD34+ cells were estimated to have increased 11.0-fold. In contrast, cultured CD34+ cells on uncoated tissue culture plates increased 5.8-fold in an identical medium. © The Author(s) 2011.
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キトサンの止血機序の解明 Reviewed
山崎敬子, 吉本怜子, 服部秀美, 田中良弘, 石原雅之.
防衛衛生 58 ( 11 ) 221 - 9 2011.11
Language：Japanese Publishing type：Research paper (scientific journal)
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Kumano I., Kishimoto S., Nakamura S., Hattori H., Tanaka Y., Nakata M., Sato T., Fujita M., Maehara T., Ishihara M.
Cellular and Molecular Bioengineering 4 ( 3 ) 476 - 483 2011.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Cellular and Molecular Bioengineering
Mixing a low-molecular-weight heparin (e.g., fragmin) with protamine results in the formation of waterinsoluble fragmin/protamine microparticles (F/P MPs) approximately 0.5-3 lm in diameter. In this study, we investigated the ability of F/P MPs to improve the viability of Lewis lung (3LL) cancer cells, B16 (B16) melanoma cells, and a human hepatoma (Huh7) cell line in a suspension culture, and the ability of F/P MPs to enhance tumor take rate and growth in vivo. F/P MPs rapidly bound to the surface of the cells. The interaction of the cells with F/P MPs induced formations of the tumor cell/F/P MP aggregates and maintained the viability of those cells in suspension for at least 3 days. The addition of F/P MPs with FGF-2 significantly enhanced the growth rate of the cells in a fetal bovine serum (FBS)-free medium. The tumor cell/F/P MP aggregates, which were subcutaneously injected into the backs of mice, significantly stimulated the formation and growth of subcutaneous tumors consisting of 3LL, B16, and Huh7 cells. Furthermore, the tumors produced by injection of 7.5 9 105 Huh7 into nude mice did grow only with F/P MPs. Thus F/P MPs can utilize as cell carrier for tumor cell transplantation. © 2011 Biomedical Engineering Society.
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PRP&F/P MPs improved survival of dorsal paired pedicle skin flaps in rats Reviewed
Takikawa M., Sumi Y., Ishihara M., Kishimoto S., Nakamura S., Yanagibayashi S., Hattori H., Azuma R., Yamamoto N., Kiyosawa T.
Journal of Surgical Research 170 ( 1 ) e189 - 96 2011.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Surgical Research
Background: Skin flap necrosis is a problem encountered postoperatively. The purpose of this study was to evaluate the effects of platelet-rich plasma containing fragmin/protamine microparticles (PRP&F/P MPs) on viability in a rat dorsal paired pedicle skin (DPPS) flap. Materials and Methods: Two symmetrical adjoining rectangular flaps (8 × 2 cm each) were drawn on the rat dorsum. Two days after PRP&F/P MPs-, PRP-, F/P MPs-, and saline (control)-injections (n = 8 each), flaps were elevated as a random pattern flap without the lateral thoracic, posterior intercostal, and deep circumflex iliac vessels. The flaps were immediately sutured back and the flap survival area was measured 7 d after flap elevation. Results: The flap survival rate in PRP&F/P MPs-injected groups (73.1% ± 4.2%) was significantly higher than those in PRP (64.9% ± 4.0%), F/P MPs (59.4 ± 4.5%), and control (61.2% ± 4.2%) groups. Histologic observation of the flaps showed survived thick granulation tissue and neovascularization in PRP&F/P MPs-injected groups. Conclusions: When PRP&F/P MPs are administered 2 d before the flap elevation, the improved flap survivals are observed. The pre-injection of PRP&F/P MPs may thus represent a promising treatment to prevent skin flap necrosis in reconstructive surgery. © 2011 Elsevier Inc. All rights reserved.
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Hemostasis for severe hemorrhage with photocrosslinkable chitosan hydrogel and calcium alginate Reviewed
Hattori H., Amano Y., Nogami Y., Takase B., Ishihara M.
Annals of Biomedical Engineering 38 ( 12 ) 3724 - 3732 2010.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Annals of Biomedical Engineering
In patients with severe hemorrhage, complications such as shock or death may occur if the patient is not treated appropriately and expeditiously. To create a hemostat kit for severe hemorrhage, ultraviolet light irradiation was applied to photocrosslinkable chitosan hydrogel and calcium alginate. As a hemorrhage model, the femoral arteries and veins of anesthetized rats were cut. Hemodynamics and hematological parameters including red blood cell (RBC) count, hemoglobin concentration, hematocrit, white blood cell (WBC) count, and platelet count, and serum parameters including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured as a marker of hemostasis. In rats for which no procedure was used, death occurred within 30 min. By using the hydrogel hemostat, the survival rate rose to 75% or more. RBC count, hematocrit, hemoglobin, and platelet levels were not significantly changed for 3 days. WBC count increased 1 day after hemostasis. AST and ALT increased 1 day after hemostasis, but it decreased 3 days later. The photocrosslinkable chitosan hydrogel and calcium alginate were biodegraded at 3 and 28 days, respectively, by neutrophils and keratinocyte chemoattractant. © 2010 Biomedical Engineering Society.
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Hydrogel blends of chitin/chitosan, fucoidan and alginate as healing-impaired wound dressings Reviewed
Murakami K., Aoki H., Nakamura S., Nakamura S., Takikawa M., Hanzawa M., Kishimoto S., Hattori H., Tanaka Y., Kiyosawa T., Sato Y., Ishihara M.
Biomaterials 31 ( 1 ) 83 - 90 2010.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Biomaterials
In order to create a moist environment for rapid wound healing, a hydrogel sheet composed of a blended powder of alginate, chitin/chitosan and fucoidan (ACF-HS; 60:20:2:4 w/w) has been developed as a functional wound dressing. ACF-HS gradually absorbed DMEM without any maceration, and fluid absorption became constant within 18 h. On application, ACF-HS was expected to effectively interact with and protect the wound in rats, providing a good moist healing environment with exudates. In addition, the wound dressing has properties such as ease of application and removal and good adherence. Full-thickness skin defects were made on the backs of rats and mitomycin C solution (1 mg/ml in saline) was applied onto the wound for 10 min in order to prepare healing-impaired wounds. After thoroughly washing out the mitomycin C, ACF-HS was applied to the healing-impaired wounds. Although normal rat wound repair was not stimulated by the application of ACF-HS, healing-impaired wound repair was significantly stimulated. Histological examination demonstrated significantly advanced granulation tissue and capillary formation in the healing-impaired wounds treated with ACF-HS on day 7, as compared to those treated with calcium alginate fiber (Kaltostat®; Convatec Ltd., Tokyo, Japan) and those left untreated. © 2009.
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Kishimoto S., Oonuma F., Nakamura S., Hattori H., Nakamura S., Mori Y., Tanaka Y., Harada Y., Tagawa M., Ishihara M.
Journal of Biomedical Materials Research - Part B Applied Biomaterials 92 ( 1 ) 32 - 39 2010.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part B Applied Biomaterials
Fragmin (low-molecular-weight heparin)/protamine microparticles (F/P MPs) immobilize to culture plates, thereby retaining the binding of heparin-binding cytokines such as human stem cell factor (SCF). The purpose of this study was to evaluate the ability of F/P MP-coating to immobilize, stabilize, and enhance SCF-activity. Cell assays showed that SCF and preimmobilized SCF on F/P MP-coated plates significantly stimulated the proliferation of human erythroleukemia cell line TF-1 in a concentration-dependent manner. Heparin and fragmin enhanced SCF-induced proliferation of chlorate-treated TF-1 cells, in which the biosynthesis of endogenous sulfated polysaccharides was blocked, on noncoated plates at a range of concentrations (2-8 μg/mL). However, heparin and fragmin had no effect on SCFinduced proliferation of chlorate-treated TF-1 cells on F/P MP-coated plates. The interaction of SCF with fragmin and F/P MPs prolonged the half-life of SCF bioactivity, and immobilized and protected SCF from inactivation, such as from heat and proteolysis. These results suggest that F/P MP-coated plates protect SCF and enhance its activity, and F/P MP-coating provides an excellent biomaterial to immobilize and retain heparin-binding cytokines, including SCF, in bioactive form for optimal expansion of hematopoietic cells. © 2009 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.b.31486
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Controlled release of FGF-2 using fragmin/protamine microparticles and effect on neovascularization Reviewed
Nakamura S., Kanatani Y., Kishimoto S., Nakamura S., Ohno C., Horio T., Masanori F., Hattori H., Tanaka Y., Kiyosawa T., Maehara T., Ishihara M.
Journal of Biomedical Materials Research - Part A 91 ( 3 ) 814 - 823 2009.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part A
Water-insoluble fragmin/protamine microparticles of about 0.5-1 μm in diameter were prepared by simple mixing of low-molecular-weight heparin (fragmin) with protamine. We investigated the capability of these microparticles to immobilize fibroblast growth factor (FGF)-2, to protect FGF-2 against degradation, to enhance FGF-2 activity, and to facilitate controlled release of FGF-2. FGF-2 bound to the fragmin/protamine microparticles with high affinity (Kd = 2.08 × 10-9 M) and the half-life of FGF-2-activity was prolonged substantially through binding of FGF-2 to the microparticles, by protection of FGF-2 from inactivation by heat and proteolysis. After subcutaneous injection into the back of mice, the fragmin/protamine microparticles underwent biodegradation and disappeared in about 2 weeks. A similar injection of FGF-2-containing microparticles resulted in significant neovascularization and fibrous tissue formation near the injection site after 1 week. These results indicate that controlled release of biologically active FGF-2 occurs through both slow diffusion and biodegradation of the microparticles, with subsequent induction of neovascularization. © 2008 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.a.32265
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Enhancement of the Antifungal Activity of Silver Ion Agents by the Addition of Plant Extracts Reviewed
37 ( 11 ) 813 - 9 2009.11
Language：Japanese Publishing type：Research paper (scientific journal)
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Kishimoto S., Hattori H., Nakamura S., Amano Y., Kanatani Y., Tanaka Y., Mori Y., Harada Y., Tagawa M., Ishihara M.
Tissue engineering. Part C, Methods 15 ( 3 ) 523 - 527 2009.9
Language：English Publishing type：Research paper (scientific journal) Publisher：Tissue engineering. Part C, Methods
Fragmin/protamine microparticles (F/P MPs) can be stably coated onto plastic surfaces. A capability of F/P MP-coated plates was investigated to immobilize fibroblast growth factor (FGF)-2 as a substratum to expand human bone marrow-derived mesenchymal stem cells (BMMSCs). FGF-2 molecules in low (2%) human serum (HS) medium were immobilized onto F/P MP-coated plates, and the FGF-2 was gradually released into the medium with a half-releasing time of 4-5 days. BMMSCs adhered well to the F/P MP-coated plates, and grew at a doubling time of about 28 h in low (2%) HS medium with FGF-2 (5 ng/mL), while the cells grew at a doubling time of about 30 and 38 h in high (10%) HS medium and in low (2%) HS medium with FGF-2, respectively, without F/P MP coating. The expanded BMMSCs on the F/P MP-coated plates in low (2%) HS medium with FGF-2 maintained their multilineage potential for differentiation into adipocytes and osteoblasts.
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Usefulness of automatic QT dispersion measurement for detecting exercise-induced myocardial ischemia Reviewed
Takase B., Masaki N., Hattori H., Ishihara M., Kurita A.
Anadolu Kardiyoloji Dergisi 9 ( 3 ) 189 - 195 2009.8
Language：English Publishing type：Research paper (scientific journal) Publisher：Anadolu Kardiyoloji Dergisi
Objective: The electrocardiographic index of QT dispersion (QTd) is related to the occurrence of arrhythmia. In patients with suspected or known coronary artery disease, QTd may be affected by exercise. We investigated whether QTd that is automatically calculated by a newly developed computer system could be used as a marker of exercise-induced myocardial ischemia. Methods: The design of this study was prospective and observational. Eighty-three consecutive patients were enrolled in this study. Their QTd was measured at rest and after 3 min of exercise during exercise-stress Thallium-201 scintigraphy and compared with conventional ST-segment changes. The patients were classified into 4 groups (normal group, redistribution group, fixed defect group, redistribution with fixed defect group) based on the result of single photon emission computed tomography. As statistical analysis, one-way ANOVA with post-hoc Scheffe's method, receiver-operating characteristics (ROC) and multiple logistic regression analysis were performed. Results: At rest, QTd was significantly greater (p<0.05) in the fixed defect group (52±21 ms) and the redistribution with fixed defect group (53±20 ms) than in the normal group (32±14 ms) and the redistribution group (31±16 ms). However, QTd tended to increase after exercise in the redistribution group, while QTd tended to decrease in the normal group, the fixed defect group, and the redistribution with fixed defect group (QTd after exercise, normal group, 28±17 ms, redistribution group, 35±19 ms, fixed defect group, 43±25 ms, redistribution with fixed defect group, 49±27 ms). Exercise significantly increased QTcd (RR interval-corrected QT dispersion) in the redistribution group. The best cut-off values of QTd and QTcd obtained from ROC curves for exercise-induced myocardial ischemia were 41.6 ms and 40.4 ms, respectively (Qtd - AUC 0.68, 95%CI 0.53- 0.83 and QTcd - AUC 0.67, 95%CI 0.55-0.80). Using these values as cut-off ones, QTd, QTcd, and conventional ST-segment change had comparable sensitivities and specificities for detecting exercise-induced myocardial ischemia (sensitivity - 60%, 58% and 49%, respectively; specificity - 78%, 80% and 83%, respectively). In addition, multiple logistic regression analysis showed that QTd (OR=2.01, 95%CI 1.15-4.10, p<0.05), QTcd (OR=2.12, 95% CI 1.02-4.30, p<0.05) and ST-segment change (OR=1.89, 95%CI 1.03-3.40, p<0.05), were the significantly associated with exercise-induced myocardial ischemia. Conclusion: QT dispersion and/or QTcd after exercise could be a useful marker for exercise-induced myocardial ischemia in routine clinical practice. © Copyright 2009 by AVES Yayincilik Ltd.
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Periodic recurrence of wide QRS tachycardia in myocardial infarction and vasospasm: utility of heart rate variability to assess autonomic nervous system activity on vasospasm-induced lethal arrhythmia. Reviewed
Takase B, Kimura K, Hamabe A, Uehata A, Hidemi H, Ishihara M, Kurita A
Anadolu kardiyoloji dergisi 9 ( 4 ) 345 - 7 2009.8
Language：English Publishing type：Research paper (scientific journal)
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Kishimoto S., Nakamura S., Nakamura S., Kanatani Y., Hattori H., Tanaka Y., Harada Y., Tagawa M., Mori Y., Maehara T., Ishihara M.
Artificial Organs 33 ( 6 ) 431 - 438 2009.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Artificial Organs
Fragmin/protamine microparticles (F/P MPs) have been shown to bind to culture plates, thereby retaining heparin-binding cytokines. Most protocols for in vitro cultures of human microvascular endothelial cells (hMVECs), human dermal fibroblast cells (hDFCs), and hematopoietic cell line (TF-1) include high fetal bovine serum (FBS) (10%) medium as a nutritional supplement. Growth rates of those cells on the F/P MP-coated plates were higher in low FBS (1%) medium containing fibroblast growth factor (FGF)-2 (for hMVECs and hDFCs) and interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor (for TF-1 cells) than without coating. The cytokines in low FBS medium were shown to be immobilized on the F/P MP-coated plate and released into the culture medium with a half releasing time of 4-5 days. Furthermore, those cells grew well on each cytokine-preimmobilized F/P MP-coated plate in low FBS medium. Thus, the F/P MP-coated matrix with adequate heparin-binding cytokines may provide biomaterials for controlling cellular growth and differentiation. © 2009, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
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Hattori H., Higuchi K., Nogami Y., Amano Y., Ishihara M., Takase B.
Circulation: Cardiovascular Imaging 2 ( 3 ) 277 - 278 2009.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Circulation: Cardiovascular Imaging
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Human stem cell factor (SCF) is a heparin-binding cytokine Reviewed
Kishimoto S., Nakamura S., Hattori H., Nakamura S., Oonuma F., Kanatani Y., Tanaka Y., Mori Y., Harada Y., Tagawa M., Ishihara M.
Journal of Biochemistry 145 ( 3 ) 275 - 278 2009.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biochemistry
Binding affinities of chemically modified heparins for human stem cell factor (SCF) were examined using fragmin/protamine microparticles (F/P MPs) and an enzyme-linked immunosorbent assay (ELISA). The binding of SCF to F/P MP-coated plates was inhibited with high concentrations of heparin and fragmin, but not others. The binding of SCF was also inhibited with 0.55 M or higher concentrations of NaCl in the medium. These results suggested that a high content of all three sulfate groups in repeating disaccharide units is required for interaction with SCF. Furthermore, pre-immobilized SCF on F/P MP-coated plates significantly stimulated proliferation of a human erythroleukemia cell line. © The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.
DOI： 10.1093/jb/mvn169
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Cytokine-immobilized microparticle-coated plates for culturing hematopoietic progenitor cells Reviewed
Kishimoto S., Nakamura S., Nakamura S., Hattori H., Oonuma F., Kanatani Y., Tanaka Y., Harada Y., Tagawa M., Maehara T., Ishihara M.
Journal of Controlled Release 133 ( 3 ) 185 - 190 2009.2
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Controlled Release
The purpose of this study was to provide a culture method for an effective expansion of human CD 34 positive hematopoietic progenitor cells (CD 34 (+) HCs) utilizing low molecular weight heparin/protamine microparticles (LH/P MPs) which can be stably coated onto plastic surfaces and cytokines. CD 34 (+) HCs optimally proliferated on LH/P MP-coated plates in the presence of stem cell factor (SCF; 5 ng/ml), thrombopoietin (Tpo; 10 ng/ml), and Flt-3 ligand (Flt-3; 10 ng/ml) in hematopoietic progenitor growth medium (HPGM). After 6 days, the total cells expanded 16.5-fold. Those cytokines were shown to be partially immobilized on the LH/P MP-coated plates, and the immobilized cytokines were gradually released into the medium with half releasing time of 3-4 days. Since flow cytometry analyses revealed that 90% of initial cells and 44.5% of expanded cells were CD 34 positive, CD 34 (+) HCs were estimated to have increased 8.0-fold after 6 days, and to have increased to over 31.9-fold after 12 days. In contrast, cultured CD 34 (+) HCs on non-coated tissue culture plates increased only 2.9-fold in the identical medium after 6 days, and only 5.2-fold after 12 days. © 2008 Elsevier B.V. All rights reserved.
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Ishizuka T., Ishihara M., Aiko S., Nogami Y., Nakamura S., Kanatani Y., Kishimoto S., Hattori H., Horio T., Tanaka Y., Maehara T.
Endoscopy 41 ( 1 ) 25 - 28 2009.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Endoscopy
Background and study aims: Saline as an injection solution for endoscopic resection techniques has several disadvantages such as a short-lasting effect leading to a potentially higher risk of bleeding and perforation. The new substance of photocrosslinkable chitosan hydrogel in a DMEM/F12 medium (PCH) can be converted into an insoluble hydrogel by ultraviolet irradiation for 30 s, and was evaluated in two sets of animal experiments. Methods: 18 pigs were used in the two parts of the study. First, mucosal resections were done with either PCH or hypertonic saline; the effects of both agents on wound healing were examined endoscopically and histologically. Second, in vivo degradation of PCH was examined using six pig stomachs. Result: PCH injection led to a longer-lasting elevation with clearer margins, compared with hypertonic saline, thus enabling precise endoscopic submucosal dissection (ESD) along the margins of the elevated mucosa. The endoscopic appearance after ESD was similar in both groups. PCH biodegradation was completed within 8 weeks according to endoscopic and histologic analyses. Conclusion: PCH is a promising agent for submucosal injection prior to various techniques of endoresection. It should be evaluated in clinical trials after biocompatibility testing for PCH is completed. © Georg Thieme Verlag KG Stuttgart.
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FGF-2含有フラグミンプロタミンマイクロキャリア(F/P MPs)による虚血改善効果の検討. Reviewed
片桐彰男, 橋谷華世, 中村伸吾, 服部秀美, 岸本聡子, 前原正明, 石原雅之.
防衛衛生 56 ( 1 ) 17 - 24 2009.1
Language：Japanese Publishing type：Research paper (scientific journal)
塩基性線維芽細胞増殖因子(fibroblast growth factor-2:FGF-2)には、例えば、血管新生作用や創傷治癒作用などの様々な重要な生理作用があることが知られている。FGF-2は、立体構造の不安定さなどから生体内における活性半減期が非常に短いが、ヘパリンやヘパラン硫酸といったヘパリノイドと相互作用することにより、構造を安定化したり受容体との結合を効率よく進めたりする。我々はこれまでに、低分子化ヘパリンであるフラグミンとその中和剤であるプロタミンを混合することで容易に作製できるフラグミンプロタミンマイクロキャリア(F/P MPs;fragmin/protamine micro particles)についての報告を行ってきた。このF/P MPsは、FGF-2を様々な不活化要因から保護して活性を維持延長させることができる。また、FGF-2含有F/P MPsをマウスの背部皮下へ注入すると、当該部位において血管新生が促進される。そこで本研究では、後肢虚血モデル動物を作製し、ここにFGF-2含有F/P MPsを投与することで虚血状態が改善し治療効果が得られるかどうかの基礎検討を行った。その結果、FGF-2含有F/P MPsを投与した後肢虚血モデル動物では壊死が防がれ、未処置群と比べて酸素飽和度も大幅な改善がみられた。したがって、FGF-2含有F/P MPsは虚血性疾患に対する治療に有用なマテリアルになると考えられた。
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Tsuda Y., Ishihara M., Amako M., Arino H., Hattori H., Kanatani Y., Yura H., Nemoto K.
Artificial Organs 33 ( 1 ) 74 - 77 2009.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Artificial Organs
UV light irradiation to a photocrosslinkable chitosan (Az-CH-LA) resulted in an insoluble and flexible hydrogel within 30 s. The purpose of this study was to evaluate the ability of the photocrosslinkable chitosan to inhibit bone formation in the bone defects. A 5-mm-diameter defect was made in the rat calvarium, and then photocrosslinkable chitosan was implanted and irradiated with UV for 30 s. Furthermore, a 2-mm defect was made in the fibula of a rat hind leg, and then photocrosslinkable chitosan was implanted and irradiated with UV. Bone formations in the rat skull and fibula defects with photocrosslinkable chitosan hydrogel were significantly prevented for 8 weeks. Thus, the chitosan hydrogel has an inhibitory effect on bone formation. © 2009, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
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Hattori H., Nogami Y., Tanaka T., Amano Y., Fukuda K., Kishimoto S., Kanatani Y., Nakamura S., Takase B., Ishihara M.
Journal of Biomedical Materials Research - Part B Applied Biomaterials 87 ( 1 ) 229 - 236 2008.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part B Applied Biomaterials
Adipose tissue contains a population of cells that have extensive self-renewal capacity and the ability to differentiate along multiple lineages. In addition, adipose tissue-derived stromal cells (ATSCs) are able to differentiate into various cell types that may be useful for autologous cell transplantation for defects of bone, cartilage, adipose, and tendon, etc. Most protocols for in vitro cultures of ATSCs include fetal bovine serum (FBS) as a nutritional supplement. However, in some cell cultures, it involves multiple doses of FBS, which raises a concern over possible infections as well as immunological reactions that are caused by medium-derived FBS proteins, sialic acid, etc. In this study, we were able to expand mouse ATSCs using low mouse serum media containing collagen type I, heparin-carrying polystyrene, and fibroblast growth factor (FGF)-2. These expanded mouse ATSCs maintained their multilineage potential for differentiation into adipocytes, osteoblasts, and chondrocytes. Therefore, this method, which uses autologous cells and low serum media, may be able to be utilized for clinical cell therapies. © 2008 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.b.31101
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虚血性心疾患の予後予測における上腕動脈内皮機能の役割に関する検討. Invited
高瀬凡平, 上畑昭美, 木村一生, 服部秀美, 石原雅之, 栗田明.
血圧 15 ( 9 ) 762 - 5 2008.9
Language：Japanese Publishing type：Research paper (scientific journal)
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Nakamura S., Nambu M., Ishizuka T., Hattori H., Kanatani Y., Takase B., Kishimoto S., Amano Y., Aoki H., Kiyosawa T., Ishihara M., Maehara T.
Journal of Biomedical Materials Research - Part A 85 ( 3 ) 619 - 627 2008.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part A
We produced a chitosan/fucoidan micro complex-hydrogel as a carrier for controlled release of heparin binding growth factors such as fibroblast growth factor (FGF)-2. Material consisting of a soluble chitosan (CH-LA) mixed with fucoidan yielded a water-insoluble and injectable hydrogel with filamentous particles. In this study, we examined the ability of the chitosan/fucoidan complex-hydrogel to immobilize FGF-2 and to protect its activity, as well as the controlled release of FGF-2 molecules. The chitosan/fucoidan complex-hydrogel has high affinity for FGF-2 (Xd = 5.4 × 10-9 M). The interaction of FGF-2 with chitosan/fucoidan complex-hydrogel substantially prolonged the biological half-life time of FGF-2. It also protected FGF-2 from inactivation, for example by heat and proteolysis, and enhance FGF-2 activity. When FGF-2-containing complex-hydrogel was subcutaneously injected into the back of mice, significant neovascularization and fibrous tissue formation were induced near the site of injection at 1 week, and the complex-hydrogel was biodegraded and disappeared by 4 weeks. These findings indicate that controlled release of biologically active FGF-2 molecules is caused by both slow diffusion and biodegradation of the complex-hydrogel, and that subsequent induction of vascularization occurs. FGF-2-containing chitosan/fucoidan micro complex-hydrogel is thus useful and convenient for treatment of ischemic disease. © 2007 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.a.31563
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フラグミン/プロタミンマイクロキャリアによるFGF-2の活性保護効果と血管新生効果. Reviewed
大野千寿子, 中村伸吾, 南部正樹, 野上弥志郎, 岸本聡子, 天野嘉子, 服部秀美, 高瀬凡平, 前原正明, 石原雅之.
防衛衛生 54 ( 10 ) 259 - 67 2007.10
Language：Japanese Publishing type：Research paper (scientific journal)
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Nambu M., Ishihara M., Nakamura S., Mizuno H., Yanagibayashi S., Kanatani Y., Hattori H., Takase B., Ishizuka T., Kishimoto S., Amano Y., Yamamoto N., Azuma R., Kiyosawa T.
Wound Repair and Regeneration 15 ( 4 ) 505 - 510 2007.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Wound Repair and Regeneration
The aim of this study was to evaluate the potential accelerating effects of an adipose tissue-derived stromal cells (ATSC)-containing atelocollagen matrix with silicone membrane (ACMS) for repairing mitomycin C-treated healing-impaired wounds. Mitomycin C was applied to full-thickness skin incisions in this study to create a healing-impaired wound model in rat. After thoroughly washing out the mitomycin C from the wound, ACMS alone or ATSC-containing ACMS was applied to the wounds. Histological sections of the wounds were then prepared at indicated time periods after the treatments. These results indicated significantly advanced granulation tissue and capillary formations in the healing-impaired wounds treated with ATSC-containing ACMS compared with those treated with ACMS alone. Thus, this study suggested that transplantation of inbred ATSC-containing ACMS is effective for repairing healing-impaired wounds. © 2007 by the Wound Healing Society.
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異なった抗不整脈薬剤の心拍変動指標に及ぼす影響. Invited Reviewed
高瀬凡平, 野上弥志郎, 服部秀美, 石原雅之, 浜部晃, 原幹, 荒川宏, 楠原正俊, 大鈴文孝, 栗田明.
Therapeutic Research 28 ( 2 ) 208 - 10 2007.2
Language：Japanese Publishing type：Research paper (conference, symposium, etc.)
特発性心室性期外収縮患者14例(男9例・女5例)を無作為にdisopyramide投与7例(平均59歳:A群)とaprindine投与7例(平均61歳:B群)の2群に分け、24時間ホルター心電図により心拍変動(HRV)指標に及ぼす影響を比較した。HRV時系列解析指標としてmean間隔、SDANN、SD index、rMSSD、pNN50を、HRV周波数解析指標はtotal power、low frequency spectra(LF)、high frequency spectra(HF)、LF/HFを、また非線形HRV指標としてfractal component(β)(フラクタル成分)を求めた。両群とも4週間の投薬で心室性期外収縮頻度は有意に低下した。A群は時系列・周波数解析指標には有意な変化がなかったが、フラクタル成分は有意に減少した。B群では時系列解析指標のうちSD index、周波数解析指標ではtotal power、LFが有意に増加し、他の各指標も増加傾向を示した。フラクタル成分は有意な変化がなかった。HRV指標に対しaprindine投与は改善、disopyramide投与は悪化傾向を示すことが示唆された。
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Short QT症候群型心電図の頻度と予後に関する検討. Invited Reviewed
高瀬凡平, 服部秀美, 石原雅之, 浜部晃, 原幹, 荒川宏, 大鈴文孝, 永井知雄, 上畑昭実, 栗田明.
Therapeutic Research 28 ( 1 ) 91 - 4 2007.1
Language：Japanese Publishing type：Research paper (conference, symposium, etc.)
2002～2005年に防衛医大病院を受診し心電図がファイリングシステムに記録された55000例および1999～2000年に自衛隊仙台病院で行われた自衛隊循環器検診において記録された心電図4054例を対象に、Short QT症候群型心電図の頻度と同心電図を呈した症例の予後について検討した。心拍数が≦60bpmでQT<320msを「definite Short QT症候群型心電図」、心拍数にかかわらずQT<320msを「probable Short QT症候群型心電図」、心拍数にかかわらずQT>320msかつQT<350msを「possible Short QT症候群型心電図」と定義した。防衛医大病院においてdefinite Short QT症候群型心電図の頻度は0%、probable Short QT症候群型は0.01%、possible Short QT症候群型は0.24%であり、予後は2.5±1.3年の追跡調査で心臓性突然死は1例もなかった。自衛隊仙台病院においてdefinite Short QT症候群型は0%、probable Short QT症候群型は0.24%、possible Short QT症候群型は9.1%であり、4.2±0.8年の追跡調査で心臓性突然死はなかった。
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Effect of lateral body position on heart rate variability in patients with sleep apnea syndrome. Reviewed
Urabe T, Takase B, Tomiyama Y, Maeda Y, Ishikawa Y, Hattori H, Uehata A, Ishihara M.
Journal of Arrhythmia 23 ( 2 ) 140 - 5 2007
Language：English Publishing type：Research paper (scientific journal)
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Masuoka K., Asazuma T., Hattori H., Yoshihara Y., Sato M., Matsumura K., Matsui T., Takase B., Nemoto K., Ishihara M.
Journal of Biomedical Materials Research - Part B Applied Biomaterials 79 ( 1 ) 25 - 34 2006.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part B Applied Biomaterials
Adipose tissue derived stromal cells (ATSCs), which were isolated from adipose tissue of rabbit, have shown to possess multipotential, that is, they differentiate into osteoblasts and adipocytes in plate-culturing and into chondrocytes in an established aggregate culture using defined differentiation-inductive medium. The aim of this study was to evaluate the utility of ATSCs in tissue engineering procedures for repair of articular cartilage-defects using the atelocollagen honeycomb-shaped scaffold with a membrane sealing (ACHMS-scaffold). We intended to repair full-thickness articular cartilage defects in rabbit knees using autologously cultured ATSCs embedded in the ACHMS-scaffold. ATSCs were incubated within the ACHMS-scaffold to allow a high density and three-dimensional culture with control medium. An articular cartilage defect was created on the patellar groove of the femur, and the defect was filled with the ATSCs-containing ACHMS-scaffold, ACHMS-scaffold alone, or empty (control). Twelve weeks after the operation, the histological analyses showed that only the defects treated with the ATSCs-containing ACHMS-scaffold were filled with reparative hyaline cartilage, highly expressed Type II collagen. These results indicate that transplantation of autologous ATSCs-containing ACHMS-scaffold is effective in repairing articular cartilage defects. © 2006 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.b.30507
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高血圧症例におけるアテノロールの運動負荷時の心拍変動指標および血圧反応に及ぼす影響 Reviewed
高瀬凡平, 阿部良行, 永田雅良, 服部秀美, 大鈴文孝, 栗田明, 石原雅之.
血圧 13 ( 10 ) 1137 - 42 2006.10
Language：Japanese Publishing type：Research paper (scientific journal)
血圧コントロールが不良な本態性高血圧症患者11例を対象にアテノール(25mg/日)投与を4週間行い、運動負荷中の血圧反応と心拍変動指数(HRV)を測定した。その結果、アテノールの4週間投与で、運動負荷時の血圧上昇は有意に抑制され、また、安静時および運動負荷時のHRVを有意に増加させることが確認された。
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Nakamura S., Ishihara M., Obara K., Masuoka K., Ishizuka T., Kanatani Y., Takase B., Matsui T., Hattori H., Sato T., Kariya Y., Maehara T.
Journal of Biomedical Materials Research - Part A 78 ( 2 ) 364 - 371 2006.8
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part A
We prepared a 6-O-desulfated (DS-) heparin (Hep) hydrogel as an excellent carrier for the controlled release of Hep-binding growth factors, such as fibroblast growth factor (FGF)-2. This material, which is partially derived from photoreactive groups, such as cinnamate, is easily crosslinked upon ultraviolet light (UV)-irradiation, resulting in a water-insoluble, viscous, and injectable hydrogel. In the present study, we examined the capacity of 6-O-DS-Hep hydrogel to immobilize FGF-2, as well as the controlled release of FGF-2 molecules from this hydrogel in vitro and in vivo. Only 10% of FGF-2 was gradually released from the FGF-2-containing 6-O-DS-Hep hydrogel (photocrosslinked 6-O-DS-Hep (4%; w/w) hydrogel containing 50 μg/mL FGF-2) into PBS (phosphate-buffered saline) within first 7 days. The 6-O-DS-Hep hydrogel in vitro maintained the original form through 1 weeks incubation in PBS, but it was gradually fragmented and could not maintain the original form by 2-3 week-washing. When the FGF-2-containing 6-O-DS-Hep hydrogel was subcutaneously injected into the back of rats, significant neovascularization and fibrous tissue formation were induced near the injected site from day 3 after the injection. And, the hydrogel had been biodegraded and completely disappeared from the injected sites in vivo within about 15-20 days after the injection. These findings indicate a controlled release of biologically active FGF-2 molecules together with fragmentation and biodegradation of 6-O-DS-Hep hydrogel and the subsequent induction of neovascularization in vivo. © 2006 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.a.30688
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Noncontact vital sign monitoring system for isolation unit (casualty care system) Reviewed
Matsui T., Gotoh S., Arai I., Hattori H., Fujita M., Obara K., Masuoka K., Nakamura S., Takase B., Ishihara M., Kikuchi M.
Military Medicine 171 ( 7 ) 639 - 643 2006.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Military Medicine
For measuring the vital signs of casualties inside an isolation unit, we developed a noncontact vital sign monitoring system using a microwave radar. The system was tested on eight healthy volunteers ranging in age from 30 to 48 years. The heart and respiratory rates derived by the microwave radar correlated with the heart and respiratory rates determined by electrocardiogram and respiratory sensor (r = 0.98, p < 0.0001 for heart rate; r = 0.84, p < 0.01 for respiratory rate). The exhaled CO and CO2, as a measure of trauma injury, were measured using an exhaled gas analyzer. The CO and CO 2 concentrations were found to average 3.8 ± 4.3 ppm and 2.9 ± 0.4%, respectively, The expired air temperature and body temperature, as indicators of hemorrhagic hypothermia, averaged 31.8 ± 1.7°C and 36.2 ± 0.4°C, respectively. The results show that our system is promising for future prehospital application in determining casualty conditions for fluid infusions by the Casualty Care System intravenous lines. Copyright © by Association of Military Surgeons of U.S., 2006.
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Hattori H., Ishihara M., Fukuda T., Suda T., Katagiri T.
Biochemical and Biophysical Research Communications 343 ( 4 ) 1118 - 1123 2006.5
Language：English Publishing type：Research paper (scientific journal) Publisher：Biochemical and Biophysical Research Communications
In the clinical field, cell-based therapies are used to treat bone defects. Adipose tissues contain many osteoblast progenitors, among other cell types. We separated mouse adipose tissue-derived stromal cells (ATSCs) according to their cell adhesive properties. Cells in a fraction adherent to the culture dishes 0.5 h after inoculation (AF-0.5) had a potent ability to differentiate into both osteoblasts and adipocytes in vitro. Their differentiation pathways depended on the culture conditions. In these cells, the expression of marker genes for osteoblast differentiation was induced in osteogenic medium. Moreover, the AF-0.5 cells, which were induced to differentiate into osteoblasts in vitro, formed abundant bone tissues in vivo. These results suggest that the AF-0.5 cells have been enriched with bi-potential progenitor cells destined for either osteoblasts or adipocytes. This simple and efficient method for preparing osteoblast progenitor cells from ATSCs may be utilized for bone defect treatment clinically. © 2006 Elsevier Inc. All rights reserved.
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2006239379 虚血性心疾患予後予測における上腕動脈内皮機能および頸動脈硬化指標の役割 運動負荷心電図との比較検討 Invited Reviewed
高瀬凡平, 松島吉宏, 上畑昭美, 浜部晃, 河野浩章, 服部秀美, 荒川宏, 大鈴文孝, 矢野捷介, 石原雅之, 栗田明.
心臓 38 ( Suppl.2 ) 3 - 6 2006.5
Language：Japanese Publishing type：Research paper (scientific journal)
冠動脈疾患の疑いで冠動脈造影を施行した患者103例(男79例・女24例,平均62歳)を対象に,症候限界性treadmill運動負荷試験,上腕動脈flow-mediated vasodilation(FMD),頸動脈超音波法による頸動脈内膜の肥厚(IMT)を測定し,これらの関係を検討した.患者の臨床的背景は,高血圧症64例,糖尿病38例,高脂血症71例,喫煙62例,冠動脈疾患有家族歴22例であった.冠動脈造影の結果,70%以上の有意狭窄を73例に認めた.動脈硬化指数(CSI)とIMT,FMDとの間には有意な相関を認めたが,nitroglycerin依存性内皮非依存性血管拡張能との間には認めなかった.平均49.8ヵ月間の追跡期間で,心事故は15例に認め,致死性心筋梗塞1例,非致死性心筋梗塞2例,不安定狭心症10例,急性左心不全2例であった.心事故発症と各測定値との関連をみると,虚血性運動負荷心電図変化およびFMDは心事故予測に有用であったが,IMTとは関連を認めなかった
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Yura H., Ishihara M., Kanatani Y., Takase B., Hattori H., Suzuki S., Kawakami M., Matsui T.
Journal of Biochemistry 139 ( 4 ) 637 - 643 2006.4
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biochemistry
Flow cytometric analysis of synthetic galactosyl polymers, asialofetuin and LDL derivatives labeled with FITC (Fluorescein Isothiocyanate) was carried out to determine the phenotypes of endocytic receptors, such as asialoglycoprotein (ASPG) and the LDL receptor, on various types of cells. When FITC-labeled galactosyl polystyrene (GalCPS), being a synthetic ligand of ASPG, was applied to rat hepatocytes and human cancer cells (Hep G2 and Chang Liver), surface fluorescence intensities varied according to receptor expression on the cells. The fluorescence intensity originates from the calcium-dependent binding of the FITC-labeled GalCPS. Although unaltered by pre-treatment with glucosyl polystyrene (GluCPS), fetuin and LDL, the fluorescence intensity was suppressed by pre-treatment with (non-labeled) GalCPS and asialofetuin. Flow cytometry allowed us to demonstrate that the calcium-dependent binding of FITC-labeled LDL (prepared from rabbits) upon the addition of 17α-ethinyl estradiol enhances LDL receptor expression, and the expression is suppressed upon the addition of a monoclonal antibody to the LDL receptor. The binding efficiency based on the combination of FITC-labeled ligands suggests a possible application for the classification of cell types and conditions corresponding to endocytic receptor expression without the need for immuno-active antibodies or radiolabeled substances. Furthermore, the synthetic glycoconjugate (GalCPS) is shown to be a sensitive and useful marker for classification based on cell phenotype using flow cytometry. © 2006 The Japanese Biochemical Society.
DOI： 10.1093/jb/mvj077
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Matsui T., Hattori H., Takase B., Ishihara M.
Journal of Medical Engineering and Technology 30 ( 2 ) 97 - 101 2006.3
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Medical Engineering and Technology
In order to conduct non-contact estimation of arterial blood pH after massive haemorrhage, we calculated the arterial pH based on linear-regression analysis of exhaled gas concentrations (CO and CO2) and vital signs (heart rate, respiratory rate, and surface temperature) measured using non-contact methods in hypovolemic animals. © 2006 Taylor & Francis.
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Bone formation using human adipose tissue-derived stromal cells and a biodegradable scaffold Reviewed
Hattori H., Masuoka K., Sato M., Ishihara M., Asazuma T., Takase B., Kikuchi M., Nemoto K., Ishihara M.
Journal of Biomedical Materials Research - Part B Applied Biomaterials 76 ( 1 ) 230 - 239 2006.1
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part B Applied Biomaterials
Human adipose tissue, obtained by liposuction, was processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). The ATSCs, as well as bone marrow-derived mesenchymal stem cells (BMSCs), have the capacity for renewal and the potential to differentiate into multiple lineages of mesenchymal tissues. These cells are capable of forming bone when implanted ectopically in an appropriate scaffold. The aim of this study was to evaluate a β-tricalcium phosphate (β-TCP) as a scaffold and to compare the potential of osteogenic differentiation of ATSCs with BMSCs. Both cell types were loaded into β-TCP disk and cultured in an osteogenic induction medium. Optimal osteogenic differentiation in ATSCs in vitro, as determined by secretion of osteocalcin, scanning electron microscope, and histology, were obtained in the culturing with the β-TCP disk. Furthermore, bone formation in vivo was examined by using the ATSC- or BMSC-loaded scaffolds in nude mice. The present results show that ATSCs have a similar ability to differentiate into osteoblasts and to synthesize bone in β-TCP disk as have BMSCs. © 2005 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.b.30357
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Obara K., Ishihara M., Ozeki Y., Ishizuka T., Hayashi T., Nakamura S., Saito Y., Yura H., Matsui T., Hattori H., Takase B., Ishihara M., Kikuchi M., Kikuchi M., Maehara T.
Journal of Controlled Release 110 ( 1 ) 79 - 89 2005.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Controlled Release
A photocrosslinkable chitosan (Az-CH-LA) aqueous solution containing paclitaxel resulted in an insoluble hydrogel within 30 s of ultrtaviolet light (UV)-irradiation. About 35-40% of the paclitaxel was released from the paclitaxel-incorporated chitosan hydrogel into phosphate buffered saline (PBS) within 1 day, after which gradual release occurred during 3 days under in vitro non-degradation conditions of the hydrogel. The paclitaxel remaining in the chitosan hydrogel retained its biological activity in vitro for at least 21 days, and was released from the chitosan hydrogel in vivo upon degradation of the hydrogel. The paclitaxel-incorporated Az-CH-LA hydrogel inhibited the growth of subcutaneously induced tumors with Lewis lung cancer (3LL) cells more effectively than those treated with only Az-CH-LA, only paclitaxel, and a non-treated group (control) for at least 11 days. Furthermore, paclitaxel-incorporated chitosan hydrogel markedly reduced the number of CD34-positive vessels in subcutaneous 3LL tumors, indicating a strong inhibition of angiogenesis. These results suggested that application of paclitaxel-incorporated Az-CH-LA hydrogel has an inhibitory activity on angiogenesis and tumor growth. © 2005 Elsevier B.V. All rights reserved.
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Matsui T., Arai I., Gotoh S., Hattori H., Takase B., Kikuchi M., Ishihara M.
Biomedicine and Pharmacotherapy 59 ( SUPPL. 1 ) S188 - S191 2005.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Biomedicine and Pharmacotherapy
Background. - The impaired balance of the low-frequency/high-frequency ratio obtained from spectral components of RR intervals can be a diagnostic test for sepsis. In addition, it is known that a reduction of heart rate variability (HRV) is useful in identifying septic patients at risk of the development of multiple organ dysfunction syndrome (MODS). We have reported a non-contact method using a microwave radar to monitor the heart and respiratory rates of a healthy person placed inside an isolator or of experimental animals exposed to toxic materials. Apparatus design and testing. - With the purpose of preventing secondary exposure of medical personnel to toxic materials under biochemical hazard conditions, we designed a novel apparatus for non-contact measurement of HRV using a 1215 MHz microwave radar, a high-pass filter, and a personal computer. The microwave radar monitors only the small reflected waves from the subject's chest wall, which are modulated by the cardiac and respiratory motion. The high-pass filter enhances the cardiac signal and attenuates the respiratory signal. In a human trial, RR intervals derived from the non-contact apparatus significantly correlated with those derived from ECG (r=0.98, P<0.0001). The non-contact apparatus showed a similar power spectrum of RR intervals to that of ECG. Conclusions. - Our non-contact HRV measurement apparatus appears promising for future pre-hospital monitoring of septic patients or for predicting MODS patients, inside isolators or in the field for mass casualties under biochemical hazard circumstances. © 2005 Elsevier SAS. All rights reserved.
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Nogami Y., Takase B., Matsui T., Hattori H., Hamabe A., Fujita M., Ohsuzu F., Ishihara M., Maekara T.
Biomedicine and Pharmacotherapy 59 ( SUPPL. 1 ) S169 - S173 2005.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Biomedicine and Pharmacotherapy
The cardiac arrhythmic suppression trial (CAST) reported that antiarrhythmic treatments in post-myocardial infarction (MI) patients resulted in poor outcome and decreased in heart rate variability indices (HRV). The goal of the present study was to determine whether aprindine and procainamide, antiarrhythmic agents that increase HRV, result in beneficial effects in post-MI rabbits. Four weeks before experiment, MI was induced in four rabbits by ligating the major branch of left coronary artery. A total of eight rabbits (four post-MI and four normal rabbits) were randomly assigned to treatment with either intravenous aprindine (1 mg/kg) or intravenous procainamide (15 mg/kg). Frequency domain HRV (low frequency spectra, LF, 0.04-0.15 Hz; high frequency spectra, HF, 0.15-0.40 Hz) were assessed by MemCalc software. Aprindine significantly increased HF and LF in both MI and normal rabbits, whereas procainamide tended to decrease HF and LF in MI and normal rabbits (in total rabbits; aprindine, LF, from 6.3 ± 7.9 to 16.5 ± 15.0 ms 2/Hz, P < 0.05; HF, from 8.0 ± 11.7 to 17.5 ± 15.0 ms2/Hz, P < 0.05; procainamide, LF, from 4.9 ± 7.4 to 4.8 ± 8.5 ms2/Hz, NS; HF, from 11.1 ± 23.0 to 5.1 ± 10.6 ms2/Hz, NS). Under pharmacological denervation with propranolol (0.1 mg/kg) and atropine (0.04 mg/kg), aprindine increased LF and HF (LF, from 0.2 ± 0.2 to 0.8 ± 0.7 ms2/Hz, P < 0.05; HF, from 0.1 ± 0.0 to 0.2 ± 0.0 ms2/Hz, P < 0.05). These data suggest that aprindine can increase HRV in post-MI rabbits. Further experiments in human subjects would be of benefit. © 2005 Elsevier SAS. All rights reserved.
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Masuoka K., Asazuma T., Ishihara M., Sato M., Hattori H., Ishihara M., Yoshihara Y., Matsui T., Takase B., Kikuchi M., Nemoto K.
Journal of Biomedical Materials Research - Part B Applied Biomaterials 75 ( 1 ) 177 - 184 2005.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Biomedical Materials Research - Part B Applied Biomaterials
The aim of this study was to investigate with tissue engineering procedures the possibility of using atelocollagen honeycomb-shaped scaffolds sealed with a membrane (ACHMS scaffold) for the culturing of chondrocytes to repair articular cartilage defects. Chondrocytes from the articular cartilage of Japanese white rabbits were cultured in ACHMS scaffolds to allow a high-density, three-dimensional culturing for up to 21 days. Although the DNA content in the scaffold increased at a lower rate than monolayer culturing, scanning electron microscopy data showed that the scaffold was filled with grown chondrocytes and their produced extracellular matrix after 21 days. In addition, glycosaminoglycan (GAG) accumulation in the scaffold culture was at a higher level than the monolayer culture. Cultured cartilage in vitro for 14 days showed enough elasticity and stiffness to be handled in vivo. An articular cartilage defect was initiated in the patellar groove of the femur of rabbits and was subsequently filled with the chondrocyte-cultured ACHMS scaffold, ACHMS scaffold alone, or nonfilled (control). Three months after the operations, histological analysis showed that only defects inserted with chondrocytes being cultured in ACHMS scaffolds were filled with reparative hyaline cartilage, and thereby highly expressing type II collagen. These results indicate that implantation of allogenic chondrocytes cultured in ACHMS scaffolds may be effective in repairing articular cartilage defects. © 2005 Wiley Periodicals, Inc.
DOI： 10.1002/jbm.b.30284
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Takase B., Abe Y., Nagata M., Matsui T., Hattori H., Ohsuzu F., Ishihara M., Kurita A.
Biomedicine and Pharmacotherapy 59 ( SUPPL. 1 ) S158 - S162 2005.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Biomedicine and Pharmacotherapy
Betaxolol hydrochloride is a β1-selective antagonist that produces vasodilation in patients with hypertension and ischemic heart disease. The goal of the present study was to characterize the effect of betaxolol on heart rate variability indices (HRV), a well-established prognostic marker. Symptom limited-treadmill exercise testing was performed in 17 hypertensive patients (60.9 ± 14.8 years-old) before and immediately a 3 weeks course of betaxolol hydrochloride (5 mg daily). Frequency domain HRV (high frequency spectra, HF; 0.15-0.40 Hz: low frequency spectra, LF; 0.04-0.15 Hz) was measured during exercise treadmill testing using MemCalc software. Betaxolol hydrochloride significantly decreased the maximal systolic blood pressure and heart rate (184 ± 29 vs. 156 ± 26 mmHg, P < 0.01; 132 ± 21 vs. 113 ± 15 bpm, P < 0.01) and significantly increased HF and LF during exercise treadmill testing (HF, 32 ± 36 vs. 56 ± 55 men/Hz, P < 0.01; LF, 64 ± 58 vs. 95 ± 86 men/Hz, P < 0.01). Thus, treatment with betaxolol hydrochloride resulted in a decrease in blood pressure during exercise treadmill testing and in an increase in HRV. This suggests that this agent could have beneficial effects on long-term prognosis in patients with hypertension. © 2005 Elsevier SAS. All rights reserved.
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Obara K., Ishihara M., Fujita M., Kanatani Y., Hattori H., Matsui T., Takase B., Ozeki Y., Nakamura S., Ishizuka T., Tominaga S., Hiroi S., Kawai T., Maehara T.
Wound Repair and Regeneration 13 ( 4 ) 390 - 397 2005.7
Language：English Publishing type：Research paper (scientific journal) Publisher：Wound Repair and Regeneration
Application of ultraviolet light irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution including fibroblast growth factor-2 (FGF-2) results within 30 seconds in an insoluble, flexible hydrogel. The FGF-2 molecules retained in the chitosan hydrogel remain biologically active and are released from the chitosan hydrogel upon in vivo biodegradation of the hydrogel. To evaluate the accelerating effect on wound healing of this hydrogel, full-thickness skin incisions were made in the backs of healing-impaired diabetic (db/db) mice and their normal (db/+) littermates. The mice were later killed, and histological sections of the wound were prepared. The degree of wound healing was evaluated using several histological parameters such as the rate of contraction, epithelialization, and tissue filling. Application of the chitosan hydrogel significantly advanced the rate of contraction on Days 0 to 2 in db/db and db/+ mice, Although the addition of FGF-2 into the chitosan hydrogel in db/+ mice had little effect, application of the chitosan hydrogel-containing FGF-2 further accelerated the adjusted tissue filling rate (Days 2 to 4 and Days 4 to 8) in db/db mice. Furthermore, the chitosan hydrogel-containing FGF-2 markedly increased the number of CD-34-positive vessels in the wound areas of db/db mice on Day 4, Thus, the application of chitosan hydrogel-containing FGF-2 onto a healing-impaired wound induces significant wound contraction and accelerates wound closure and healing. Copyright © 2005 by the Wound Healing Society.
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Fujita M., Ishihara M., Morimoto Y., Simizu M., Saito Y., Yura H., Matsui T., Takase B., Hattori H., Kanatani Y., Kikuchi M., Maehara T.
Journal of Surgical Research 126 ( 1 ) 27 - 33 2005.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Journal of Surgical Research
Background. Therapeutic angiogenesis in ischemic myocardium has been shown to be an effective strategy to improve regional blood flow and myocardial function. However, no effective delivery system for growth factor administration is yet known to induce important therapeutic angiogenic responses in ischemic myocardium. Materials and methods. FGF-2-incorporated chitosan (FGF-2/chitosan) hydrogels were immobilized on the surface of ischemic myocardium of rabbit models of chronic myocardial infarction by UV-irradiation. After 4 weeks, cardiac functional analyses by noradrenalin challenge and histopathological analyses were performed to evaluate the efficacy of a controlled release of FGF-2 from FGF-2/chitosan hydrogel immobilized on the surface of ischemic myocardium. Results. Significant improvement by application of FGF-2/chitosan hydrogels was found in systolic pressure at the left ventricle, +dp/dt maximum, and -dp/dt maximum during noradrenalin challenge at a dose of 1 μg/kg/min. Histological observations showed that a significantly larger amount of viable myocardium and CD 31 immunostained blood vessels were found in the FGF-2/chitosan hydrogel-applied group than only the chitosan-applied and control groups. Conclusions. These preliminary results indicate that the controlled release of biologically active FGF-2 molecules from FGF-2/chitosan hydrogel induces angiogenesis and possibly collateral circulation in ischemic myocardium, thereby protecting the myocardium. © 2005 Elsevier Inc. All rights reserved.
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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.