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ABSTRACT: Many types of decellularized tissues have been studied and some have been commercially used in clinics. In this study, small-diameter vascular grafts were made using HHP to decellularize porcine radial arteries. One decellularization method, high hydrostatic pressure (HHP), has been used to prepare the decellularized porcine tissues. Low-temperature treatment was effective in preserving collagen and collagen structures in decellularized porcine carotid arteries. The collagen and elastin structures and mechanical properties of HHP-decellularized radial arteries were similar to those of untreated radial arteries. Xenogeneic transplantation (into rats) was performed using HHP-decellularized radial arteries and an untreated porcine radial artery. Two weeks after transplantation into rat carotid arteries, the HHP-decellularized radial arteries were patent and without thrombosis. In addition, the luminal surface of each decellularized artery was covered by recipient endothelial cells and the arterial medium was fully infiltrated with recipient cells. Copyright © 2012 John Wiley & Sons, Ltd.
Journal of Tissue Engineering and Regenerative Medicine 12/2012; · 3.28 Impact Factor
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ABSTRACT: We have previously proposed a new method of adhesion using the integrated low-level energy sources heat, vibration, and pressure. This adhesion method can be used to attach biological tissue to a metal object. Effects of surface roughness and energy of the metal subject on adhesion performance were studied by using commercially pure titanium (cpTi) and stainless steel (SUS304). Surface roughness and energy were changed by sandblast treatment and heat treatment, respectively. A porcine aorta was adhered to sandblast-treated SUS304 by use of an adhesion temperature of 80 °C, a vibration amplitude of 15 μm, a pressure of 2.5 MPa, an adhesion time of 120 s, and a surface roughness of an Ra 0.25 μm. The shear tensile strength of the adhesion was 0.45 MPa. The adhesion performance was improved by roughening the surface of the metal specimen. Surface energy has an insignificant effect on adhesive strength. The adhesion performance varied depending on metal material for the same surface roughness, Ra, and energy. Results from analysis of the surface roughness profile suggested that the size of surface asperity has an effect on adhesion performance.
Journal of Artificial Organs 08/2012; · 1.59 Impact Factor
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ABSTRACT: Fifteen Isodon diterpenoids (1-15) were evaluated for their cytotoxic activity against HeLa and HL-60 human cancer cell lines, and against murine vincristine (VCR)-resistant P388 cells. Kamebanin (14) showed efficient cytotoxic activity against HeLa and HL-60 cells. In addition, although dihydroenmein (2) and trichorabdal B (7) were inactive against several tested cell types, they were found to have cytotoxic-enhancing activity of VCR against VCR-resistant P388 cells.
Natural product communications 08/2012; 7(8):977-8. · 1.24 Impact Factor
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ABSTRACT: Decellularized bone/bone marrow was prepared to provide a microenvironment mimicking that of the bone marrow for three-dimensional culture in vitro. Bone/bone marrows were hydrostatically pressed at 980 MPa at 30 °C for 10 min to dismantle the cells. Then, they were washed with EGM-2 and further treated in an 80% EtOH to remove the cell debris and lipid, respectively. After being rinsed and shaken with PBS again, treated bone/bone marrows were stained with hematoxylin and eosin (H-E) to assess the efficacy of decellularization. Cells were determined to have been completely removed through H-E staining of their sections and DNA quantification. Rat mesenchymal stem cells (rMSCs) were seeded on the decellularized bone/bone marrows and cultured for 21 days. The adhesion of rMSCs on or into decellularized bone/bone marrows was confirmed and proliferated over time in culture. The osteogenic differentiation effect of decellularized bone/bone marrows on rMSCs in the presence or absence of dexamethasone was investigated. Decellularized bone/bone marrows without dexamethasone significantly increased alkaline phosphatase (ALP) activity, indicating promoted osteogenic differentiation of rMSCs. In an animal study, when decellularized bone/bone marrows were implanted into the rat subcutaneous, no immune reaction occurred and clusters of the hematopoietic cells could be observed, suggesting the decellularized bone/bone marrows can provide a microenvironment in vivo.
Biomaterials 07/2011; 32(29):7060-7. · 7.40 Impact Factor
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ABSTRACT: Decellularized tissues have attracted a great deal of attention as regenerating transplantation materials. A decellularizing method based on high hydrostatic pressure (HHP) has been developed, and the preparation of many types of decellularized tissues has been investigated, including aorta, cornea, and dermis. The preparation of a small-diameter vascular graft was studied using a carotid artery from the viewpoint of collagen denaturation and leakage. After HHP, the carotid artery was washed at two washing temperatures (37 and 4°C). Histological evaluation, collagen content measurement and circular dichroism (CD) measurement indicated that the washing temperatures clearly affected the collagen structure of the decellularized carotid artery. The amount of collagen decreased in the carotid artery decellularized by HHP washed at 37°C (HHP/37°C). On the other hand, the amount and structure of collagen were preserved in the carotid artery washed at 4°C after HHP (HHP/4°C). In rat carotid artery syngeneic transplantation, the HHP/37°C decellularized carotid artery occluded after 2 weeks, but the HHP/4°C decellularized one did not. These results indicate that collagen denaturation and leakage of the decellularized carotid artery affect the in vivo performance of the carotid artery.
Journal of Artificial Organs 05/2011; 14(3):223-31. · 1.59 Impact Factor
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ABSTRACT: Four new Erythrinan alkaloids (1-4) were isolated from the seeds of Erythrina velutina. The structures of these new compounds 1-4 were elucidated by spectroscopic methods including 2D-NMR. Three of four were found to be novel sulfated Erythrinan alkaloids.
Chemical & pharmaceutical bulletin 01/2011; 59(5):564-7. · 1.70 Impact Factor
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ABSTRACT: Extracellular physical features of underlying the adhesive substrate affect cell adhesion to it substrate. In this study, the effects of vibration, a type of artificial physical stimulation, on the adhesion of mouse L929 cells, mouse embryonic fibroblasts (MEFs), HeLa cells and human umbilical vein endothelial cells (HUVECs) are reported. A nano-vibration system was designed to produce nanometer-scale vibration. When L929 cells, HeLa cells, and HUVECs were subjected to vibration at 100 Hz and 1 kHz, they were not affected. On the other hand, in MEFs, the adherent cells were increased and changed their shape remarkably in response to 1 kHz vibration. To investigate the MEFs' sensitivity to vibration in detail, the cells shape was classified into four types; round, stellate, filopodia-formed and lamellipodia-formed. In serum addition culture, 1 kHz vibration increased the number of filopodia-formed cells but decreased lamellipodia cells. Furthermore, the preliminary cDNA microarray experiments showed that expression of genes which regulate cytoskeleton were changed by vibration stimulation. These results suggest that vibration could affect cell adhesion and the determination cell shape.
Bio-medical materials and engineering 01/2011; 21(3):149-58. · 1.23 Impact Factor
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ABSTRACT: Our previous research showed that poly(vinyl alcohol) (PVA) nanoparticles incorporating DNA with hydrogen bonds obtained by high hydrostatic pressurization are able to deliver DNA without any significant cytotoxicity. To enhance transfection efficiency of PVA/DNA nanoparticles, we describe a novel method to prepare PVA/DNA nanoparticles encapsulating nanoscaled hydroxyapatites (HAps) prepared by high hydrostatic pressurization (980 MPa), which is designed to facilitate endosomal escape induced by dissolving HAps in an endosome. Scanning electron microscopic observation and dynamic light scattering measurement revealed that HAps were significantly encapsulated in PVA/HAp/DNA nanoparticles. The cytotoxicity, cellular uptake, and transgene expression of PVA/HAp/DNA nanoparticles were investigated using COS-7 cells. It was found that, in contrast to PVA/DNA nanoparticles, their internalization and transgene expression increased without cytotoxicity occurring. Furthermore, a similar level of transgene expression between plasmid DNA and PVA/HAp/DNA nanoparticles was achieved using in vivo hydrodynamic injection. Our results show a novel method of preparing PVA/DNA nanoparticles encapsulating HAp nano-crystals by using high hydrostatic pressure technology and the potential use of HAps as an enhancer of the transfection efficiency of PVA/DNA nanoparticles without significant cytotoxicity.
Journal of drug delivery. 01/2011; 2011:962743.
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ABSTRACT: To develop an antithrombotic material for preparation of small-diameter vascular graft, we describe a novel method to prepare a poly(vinyl alcohol) (PVA)-heparin hydrogels prepared by high-hydrostatic pressure (HHP, 980 MPa), which is designed for sustained release of heparin. Antithrombogenic test revealed that HHP method would not affect the antithrombin III (ATIII) activity of the released heparin. The distribution of heparin in the polymer matrix was homogeneous than freeze-thawing gel, due to the fast gelling affect of PVA which takes approximately 10 min for gel formation. The formation of intra- and intermolecular hydrogen bonds between PVA chains has trapped the heparin inside, suppressing the phase separation between PVA and heparin. Furthermore, evenly distribution of heparin induced the formation of heparin and PVA molecular complex, which brought the sustained release of heparin from the PVA despite the high swelling ratio. Our results show that it is possible to prepare a PVA-heparin hybrid gel which can be applied as an effective material for an antithrombotic system without using any chemical agent.
European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 12/2010; 41(5):617-22. · 2.61 Impact Factor
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Kenji Yamamoto,
Tsuyoshi Kimura,
Kwangwoo Nam,
Seiichi Funamoto,
Yukiko Ito,
Kumiko Shiba,
Ayako Katoh,
Shigeru Shimizu,
Kimio Kurita,
Tetsuya Higami,
Toru Masuzawa, Akio Kishida
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ABSTRACT: Interface phenomena such as encapsulation and formation of dead space around implanted biomaterials lower biomaterial performance. To advance tissue adhesive technology, understanding the interactions between tissue (collagen) and polymer is indispensable. Adhesion between tissue and polymer was studied using an ultrasonically activated scalpel (UAS).
The Harmonic Scalpel was used as an ultrasonically activated scalpel for polymer and tissue adhesion. A piece of porcine aorta and a polymeric film were layered and placed between the blades of the Harmonic Scalpel. Then the samples were gripped with 20 kgf of force for 1-10 s to adhere the porcine aorta and polymeric films. The adhesion was characterized by macro- and microscopic observation, thermographic analysis, and measurement of bonding strength, static contact angle (SCA), and surface properties.
Cellulose, vinylon, polyethylene terephthalate, nylon, and Pellethane could be bonded to the aorta. Bonding was not observed for the polyethylene, polypropylene, polyvinyl chloride, or polytetrafluoroethylene. This suggests that the existence of functional groups such as hydroxyl, carbonyl, carboxyl, and amide groups in the polymer structure are key factors in adhesion. Harmonic Scalpel modification of the polyethylene surface during corona discharge treatment further indicated that the functional groups of the polymers are one of the essential factors for tissue adhesion. The temperatures of adhesion were 90-150 °C for the polymers, and the melting temperatures (Tm) were 193-306 °C. This suggests that adhesion was formed by the interaction between the melted polymer surface and the tissue collagen.
Both polar functional groups and adequate thermal characteristics are necessary for polymers to bond with tissues. These findings should be useful for the development of novel polymers that can be bonded to living tissues with UAS treatment, which can be applied for endoscopic surgery.
Surgical Endoscopy 10/2010; 25(4):1270-5. · 4.01 Impact Factor
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ABSTRACT: Different types of physiological-mechanical stress, such as shear stress in vascular endothelial cells or hydrostatic pressure in chondrocytes are well known as regulators of cell function. In this study, the effects of vibration, a type of non-physiological mechanical stimulation, on differentiation of rat pheochromocytoma (PC12) cells are reported. A nano-vibration system was designed to produce nanometer-scale vibration. The frequency and amplitude of the nano-vibrations were monitored by a capacitance displacement sensor connected to an oscilloscope. When PC12 cells exposed to nerve growth factor were subjected to vibration at 10 kHz, differentiation and elongation of their neurites were promoted earlier in the culture. Vibration promoted differentiation of PC12 cells. This approach could therefore also be promising for determining of the effects of the physical environment on cell differentiation.
Biotechnology and Bioengineering 10/2010; 108(3):592-9. · 3.95 Impact Factor
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ABSTRACT: In this study, we aimed to replicate the function of native tissues that can be used in tissue engineering and regenerative medicine. The key to such replication is the preparation of an artificial collagen matrix that possesses a structure resembling that of the extracellular matrix. We, therefore, prepared a collagen matrix by fibrillogenesis in a NaCl/Na(2)HPO(4) aqueous solution using a dialysis cassette and investigated its biological behavior in vitro and in vivo. The in vitro cell adhesion and proliferation did not show any significant differences. The degradation rate in the living body could be controlled according to the preparation condition, where the collagen matrix with high water content (F-collagen matrix, >98%) showed fast degradation and collagen matrix with lower water content (T-collagen matrix, >80%) showed no degradation for 8 weeks. The degradation did not affect the inflammatory response at all and relatively faster wound healing response was observed. Comparing this result with that of collagen gel and decellularized cornea, it can be concluded that the structural factor is very important and no cell abnormal behavior would be observed for quaternary structured collagen matrix.
Journal of Biomaterials Science Polymer Edition 10/2010; 22(15):1963-82. · 1.69 Impact Factor
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ABSTRACT: Two new Erythrinan alkaloids, cristanines A and B (1, 2), were isolated from the bark of Erythrina crista-galli L. together with nine known Erythrinan alkaloids (3-5, 7-12) and an indole alkaloid (13). The structures of the compounds, cristanines A (1) and B (2), were elucidated by spectroscopic methods. Three of the twelve compounds isolated showed significant inhibitory activity on lipopolysaccharide induced nitric oxide (NO) production.
Chemical & pharmaceutical bulletin 08/2010; 58(8):1119-22. · 1.70 Impact Factor
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ABSTRACT: We previously reported a novel technology for the engineering of a capillary network using an optical lithographic technique. To apply this technology to the therapy of ischemic diseases, we tested human omental microvascular endothelial cells (HOMECs) as an autologous cell source and decellularized human amniotic membranes (DC-AMs) as a pathogen-free and low immunogenic transplantation scaffold.
Human umbilical vein endothelial cells were aligned on a patterned glass substrate and formed a capillary structure when transferred onto an amniotic membrane (AM). In contrast, HOMECs were scattered and did not form a capillary structure on AMs. Treatment of HOMECs with sphingosine 1-phosphate (S1P) inhibited HOMEC migration and enabled HOMEC formation of a capillary structure on AMs. Using quantitative RT-PCR and Western blot analyses, we demonstrated that the main S1P receptor in HOMECs is S1P(2), which is lacking in human umbilical vein endothelial cells, and that inhibition of cell migration by S1P is mediated through an S1P(2)-Rho-Rho-associated kinase signaling pathway. Implantation of capillaries engineered on DC-AMs into a hindlimb ischemic nude mouse model significantly increased blood perfusion compared with controls.
A capillary network consisting of HOMECs on DC-AMs can be engineered ex vivo using printing technology and S1P treatment. This method for regeneration of a capillary network may have therapeutic potential for ischemic diseases.
Arteriosclerosis Thrombosis and Vascular Biology 07/2010; 30(7):1300-6. · 6.37 Impact Factor
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ABSTRACT: We have developed a method that allows biological tissues to be adhered together with minimal invasion by delivering integrated low-level energies from heat, pressure, and vibration. Tensile tests on adhered slices of porcine aorta were performed to determine the relationships between adhesive strength and conditions such as adhesion temperature, time, pressure, and vibration. The maximal adhesive shear tensile strength using the proposed method was 0.2MPa, which is half the strength of the porcine aorta and stronger than surgical tissue adhesive. Adhesion strength increased in proportion to temperature, time, and pressure, and also in the presence of vibration, indicating that vibrational energy contributes to the adhesive mechanism and strength. Adhesive stability, the effect of heat on adhesive strength, and the ability of tissue to adhere to artificial materials were also clarified.
Medical Engineering & Physics 05/2010; 32(4):304-11. · 1.62 Impact Factor
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Kei Oya,
Yuta Tanaka,
Yoshihisa Moriyama,
Yuki Yoshioka,
Tsuyoshi Kimura,
Yusuke Tsutsumi,
Hisashi Doi,
Naoyuki Nomura,
Kazuhiko Noda, Akio Kishida,
Takao Hanawa
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ABSTRACT: The roughness and cleanness of a titanium surface must be controlled in order to investigate the expression mechanism of hard tissue compatibility on titanium. In this study, osteogenic MC3T3-E1 cells were cultured and differentiation-induced on bulk and sputter-deposited titanium specimens, and the osteogenesis were investigated. For the preparation of bulk specimens, titanium discs were mirror-polished. On the other hand, titanium was sputter-deposited on smooth and clean cover glasses as sputter-deposited specimens. As a result, no significant difference was observed in the cell morphology and attached number. On the other hand, the time showing maximum activity in the alkaline phosphatase and gene expressions, which are related to bone differentiation on the bulk titanium, were superior to those on the sputter-deposited titanium. From the surface observation of the specimens with a scanning electron microscope and a scanning probe microscope, the surface on the sputter-deposited titanium was more uniform and cleaner than that on the bulk titanium. According to X-ray photoelectron spectroscopy, the thickness of surface oxide film on the sputter-deposited titanium was smaller than that on the bulk titanium. In addition, the proportions of TiO and Ti(2)O(3) in the surface oxide film on the sputter-deposited titanium were larger than those on the bulk titanium. These differences might influence the differentiation of osteoblastic cells.
Journal of Biomedical Materials Research Part A 03/2010; 94(2):611-8. · 2.63 Impact Factor
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ABSTRACT: A decellularization method using high-hydrostatic pressure (HHP) technology (>600MPa) is described. The HHP disrupts the cells inside the tissue. The cell debris can be eliminated with a simple washing process, producing clean, decellularized tissue. In this study, porcine aortic blood vessel was decellularized by HHP. The mechanical properties and in vivo performance of the decellularized tissue were evaluated. Mechanical properties of the decellularized tissue were not altered by the HHP treatment. Reduced inflammation of the decellularized tissue was confirmed by xenogenic transplant experimentation. An allogenic transplantation study showed that decellularized blood vessel endured the arterial blood pressure, and there was no clot formation on the luminal surface. In addition, cellular infiltration into the vessel wall was observed 4 weeks after implantation, suggesting that HHP treatments could be applied widely as a high-quality decellularization method.
Biomaterials 02/2010; 31(13):3590-5. · 7.40 Impact Factor
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Yoshihide Hashimoto,
Seiichi Funamoto,
Shuji Sasaki,
Takako Honda,
Shinya Hattori,
Kwangwoo Nam,
Tsuyoshi Kimura,
Manabu Mochizuki,
Toshiya Fujisato,
Hisatoshi Kobayashi, Akio Kishida
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ABSTRACT: To prepare acellular corneal scaffold, we used high-hydrostatic pressurization (HHP) to decellularize porcine cornea. The HHP method disrupts cells by hydrostatic pressurization, and then the disrupted cells' components are removed by washing with a cell culture medium. Porcine corneas were hydrostatically pressed at 980 MPa at 10 or 30 degrees C for 10 min to make them opaque. There was no change in the thickness of the corneas immediately after the pressurization, but they swelled during the washing process. The cornea swelling caused by HHP was suppressed when medium containing 3.5% w/v dextran was used. For H-E staining of the cornea decellularized with the HHP method, the complete removal of corneal cells was confirmed. Furthermore, when the corneas were immersed in glycerol for 1 hour, their optical properties were restored to those of native corneas. In an animal study, when acellular porcine corneas were implanted into rabbit cornea, no immune reaction occurred and the turbid corneas became clear. The decellularized corneas obtained through HHP could be useful as a corneal scaffold for tissue regeneration.
Biomaterials 02/2010; 31(14):3941-8. · 7.40 Impact Factor
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ABSTRACT: The drawback with collagen/2-methacryloyloxyethyl phosphorylcholine (MPC) polymer hybrid gels (collagen/phospholipid polymer hybrid gels) prepared in alkaline morpholinoethane sulfonic acid (MES) aqueous solution is that the cross-linking rate between the polymer and the collagen is low. To solve this problem, ethanol has been adopted as the reaction solvent, to prevent 1-ethyl-3-(3-dimethylaminopropyl)-1-carbodiimide hydrochloride (EDC) hydrolysis. Alterations in the ethanol mole concentration changed the cross-linking rate between the MPC polymer and the collagen gel. Prevention of EDC hydrolysis is clearly observed; protonation of carboxyl groups implies that the ratio of ethanol to water should be controlled. The polymer shows signs of penetration into the collagen gel layer, thus forming a totally homogeneous phase gel. This affects the mechanical strength of the collagen gel, making the gel much stiffer and brittle with an increase in the swelling ratio, as compared with that prepared in MES buffer. However, it is possible to obtain a collagen/phospholipid polymer hybrid gel with a high polymer portion and the cross-linking rate can be successfully controlled.
Acta biomaterialia 07/2009; 6(2):403-8. · 3.98 Impact Factor
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ABSTRACT: The aim of the present study was to evaluate the biological properties of a collagen-phospholipid polymer hybrid gel (MiC30 gel) designed for use as a tissue membrane. The following four types of collagen gels were synthesized and tested in vitro and in vivo: physically cross-linked collagen gel (Uc gel), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC)/N-hydroxysuccinimide-cross-linked collagen gel (EN gel), MiC30 gel and glutaraldehyde-cross-linked collagen gel (G gel). The cell adhesivity and proliferation rate were observed to be lowest for the MiC30 gel and highest for the Uc gel, indicating that the phospholipid-polymer-covered surface of the collagen gel interacted weakly with cells. The collagen gel was implanted into rats subcutaneously and was observed 1, 2 and 8 weeks after implantation. The Uc gel and G gel were degraded and induced an inflammatory response. Granulation was not observed for 8 weeks after implantation and the formation of foreign body giant cells was observed around both the Uc and G gels. On the other hand, cell infiltration and degradation were not observed in the case of the EN and MiC30 gels. The formation of foreign body giant cells was suppressed and the healing process was accelerated. The MiC30 gel is suitable for use as a biomaterial that is stable in vivo because it suppresses the foreign body response and accelerates the healing process.
Acta biomaterialia 07/2009; 6(2):409-17. · 3.98 Impact Factor
