Biomedical subjects

Hiroo Iwata

Publications and source records attributed to Hiroo Iwata.

At least 37 records · Page 2Linked to original sources

PubMed2005

Cell microarray for screening feeder cells for differentiation of embryonic stem cells.

Microarrays are currently recognized as one of major tools in the assessment of gene expression via cDNA or RNA analysis and are now accepted as a powerful experimental tool for high-throughput screening of a large number of samples, such as cDNA and siRNAs. In this study, we examined the potential of the microarray methodology for high-throughput screening of candidate cells as feeder cells which effectively differentiate embryonic stem (ES) cells to the specific lineage. Cell arrays were prepared by applying three kinds of cells, PA6, human umbilical vein endothelial, and COS-1 cells, to circular spots, 2 mm in diameter, on a glass plate, followed by the application of mouse ES cells to the cell microarray. After 8 d in culture, TuJ1 (neuron-specific class III beta-tubulin) immunocytochemical staining clearly demonstrated that only PA6 cell spots had the capability to induce ES cells to neuronal differentiation. Although this is a model experiment, these findings clearly indicate that the cell microarray will become a powerful tool for high-throughput screening large numbers of candidate feeder cells for specific differentiation.

Animals↗

PubMed2005

Immobilization of basic fibroblast growth factor on a platinum microcoil to enhance tissue organization in intracranial aneurysms.

OBJECT: To enhance tissue organization in an aneurysm lumen, the authors prepared a platinum microcoil carrying basic fibroblast growth factor (bFGF) and analyzed its effectiveness in the treatment of aneurysms. METHODS: Ultrathin multiorganic layers were assembled on a platinum coil through successive deposition of cationic polyethylenimine and anionic heparin, and then bFGF was immobilized through an affinity interaction with heparin. The bFGF was effectively immobilized on the surface of the platinum coil without deterioration of the coil's mechanical properties. Coil embolization of aneurysms constructed using a canine common carotid artery was performed via the endovascular approach. The aneurysms together with parent arteries were harvested 2 weeks after coil embolization. Platinum coils unmodified, coated with heparin, or immobilized with heparin and bFGF were examined. The percentage of occlusion at the aneurysm orifice in animals treated with bFGF-immobilized coils (92.99+/-7.94%) was significantly greater than that in animals treated with heparin-coated coils (57.26+/-10.76%) or unmodified coils (52.86+/-8.54%). The histological score of the aneurysms treated with bFGF-immobilized coils was also significantly greater than the scores in the control group. CONCLUSIONS: These results indicated that bFGF-immobilized microcoils may be beneficial in the obliteration of aneurysms.

Animals↗

PubMed2005

Tenascin-C-coated platinum coils for acceleration of organization of cavities and reduction of lumen size in a rat aneurysm model.

OBJECT: Detachable platinum coils are widely used in the endovascular treatment of intracranial aneurysms. The use of coil placement produces a higher incidence of aneurysm recurrence compared with surgical clipping. To reduce the incidence of recurrence by promoting clot organization, the authors designed a platinum coil coated with tenascin-C (TNC), an extracellular matrix glycoprotein, and then histologically examined tissue responses. METHODS: Platinum coils were prepared by successive coatings with cationic polyethyleneimine and anionic heparin and then TNC or basic fibroblast growth factor (bFGF) was immobilized by affinity binding to the heparin. Six unmodified, six heparin-coated, six bFGF-coated, or eight TNC-coated platinum coils were inserted into ligated common carotid arteries (CCAs) of adult male rats, and CCA segments were harvested after 14 or 28 days. The percentages of organized areas occupying the luminal cavity in unmodified, heparin-coated, bFGF-coated, and TNC-coated groups were 4.8 +/- 4.6, 1.6 +/- 1.1, 17.9 +/- 10.7, and 93.4 +/- 6.9%, respectively. In addition, the mean lumen size in the TNC-coated group (0.35 +/- 0.23 mm2) was reduced to less than half that of the unmodified group (0.72 +/- 0.21 mm2). Immunohistochemical analysis revealed that alpha-smooth muscle actin-positive cells were a major cellular component of the organized tissue within the TNC-coated coils but not in the bFGF group. Collagen fibrils in the organized areas were also much thicker and denser with TNC-coated coils than with bFGF-coated coils. CONCLUSIONS: Placement of TNC-coated coils can remarkably accelerate organization of luminal cavities and reduce their volume, providing improved efficacy of these coils for endovascular embolization.

Animals↗

PubMed2005

Functional evaluation of bioartificial liver using RT-PCR.

In order to evaluate functions of a bioartificial liver (BAL), containing porcine hepatocytes in a hollow fiber cartridge, some chemical loading tests have been employed. However the kinds of functions that can be evaluated by chemical loading tests are limited. We desire versatile methods to estimate various BAL functions. The purpose of this report is to propose a method using the reverse transcription polymerase chain reaction (RT-PCR) for functional evaluation of a BAL. In vitro perfusion culture of a BAL cartridge was carried out using 20% of human whole blood. At pre-determined periods of perfusion culture, hepatocytes were taken from the cartridge and mRNA was extracted from the hepatocytes. The mRNA expression levels of albumin and cytochrome P450 (CYP3A29) were determined by RT-PCR method. In order to quasi-quantitatively determine the time courses of mRNA expression levels during the perfusion culture, PCR of target DNA was carried out by co-amplification with its competitor DNA that was constructed by means of the partial deletion of target DNA. The results showed that the amounts of both albumin and cytochrome P450 mRNA rapidly decreased during the initial few days' perfusion culture and remained at a constant level for the following week. This fact corresponded with the analysis of lidocaine metabolic functions via pharmacokinetics in our previous study. Additionally, the RT-PCR is so sensitive that we can measure much more minute quantity of various proteins. Consequently, this method is useful for comprehensive evaluation of BAL functions.

Albumins↗

PubMed2005

Development of a polyvinyl alcohol hydrogel for temperature measurement by MRI.

This paper describes the development of a Polyvinyl alcohol hydrogel (PVA-H) model for calibration and measurement of temperature using image contrast on MRI using DMSO/H_2O as a solvent to alter the freezing and melting points of PVA-H. Tissues exposed to temperature changes above 41 degrees C or below 0 degrees C exhibit increasingly extensive and irreversible damage, depending on the exposure duration. MR images can provide a map of temperature if there is sufficient tissue signal. To evaluate treatment principles using temperature changes (hyperthermia, cryotherapy), a model simulating tissue may be useful to provide a reproducible test environment. PVA-H is water soluble and can be seen on MRI. It can be used to construct complicated shapes such as vascular structure, soft tissues, and so on. Therefore, PVA-H can be useful to measure temperatures and assume the distribution of temperature under treatment. In this paper, we applied PVA-H as a temperature detector and calibrated temperature from image contrast. The results exhibit good capability as a temperature detector not only of high temperature (around 41 degrees C), but also of low temperature (as low as -23 degrees C).

Calibration↗

PubMed2004

Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.

Functional characterization of human genes is one of the most challenging tasks in current genomics. Owing to a large number of newly discovered genes, high-throughput methodologies are greatly needed to express in parallel each gene in living cells. To develop a method that allows efficient transfection of plasmids into adherent cells in spatial- and temporal-specific manners, we studied electric pulse-triggered gene transfer using a plasmid-loaded electrode. A plasmid was loaded on a gold electrode surface having an adsorbed layer of poly(ethyleneimine), and cells were then plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, resulting in efficient gene expression, even in primary cultured cells. The location of transfected cells could be restricted within a small area on a micropatterned electrode, showing the versatility of the method for spatially controlled transfection. Plasmid transfection could also be performed in a temporally controlled manner without a marked loss of the efficiency when an electric pulse was applied within 3 days after cell plating. The method described here will provide an efficient means to transfer multiple genes, in parallel, into cultured mammalian cells for high-throughput reverse genetics research.

Animals↗

PubMed2004

Micropatterned, self-assembled monolayers for fabrication of transfected cell microarrays.

The aim of this study was to develop the cell microarray that allows efficient transfer of multiple genes into mammalian cells cultured on the microarray in a high-throughput fashion. A microarray was fabricated using a gold-coated glass plate having a micropatterned, self-assembled monolayer of alkanethiols carrying ionic and nonionic terminal groups. Plasmid DNA and a cationic lipid were loaded by alternate electrostatic adsorption to the microspots to obtain a plasmid DNA microarray. The loading and the release of lipid-DNA complex were studied by, respectively, the fluorescence staining of DNA and the imaging of the microarray with a surface plasmon resonance (SPR) apparatus. The transfection efficiency was evaluated by directly plating and culturing human embryonic kidney cells onto the microarray. The results demonstrated that cells which adhered to the DNA-loaded spots were transfected to express the encoded model proteins for several days. The chemistry of the monolayers and the number of alternate adsorption cycles had large effects on the efficiency of transfection. This may be explained from the availability of the lipid-DNA complex to the cells directly contacted. We conclude that the micropatterned, self-assembled monolayers greatly facilitate regionally defined loading of DNAs and expression of the encoded protein in mammalian cells.

Adsorption↗

PubMed2004

Surface modification of poly(ethylene-co-vinyl alcohol): hydroxyapatite immobilization and control of periodontal ligament cells differentiation.

To reveal and control the differentiation and proliferation of the periodontal ligament (PDL) cells and to develop a highly organized hybrid implant possessing periodontium, hydroxyapatite (HAP) was immobilized on the poly(ethylene-co-vinyl alcohol) (EVA) by alternate soaking method following with carboxyl groups' introduction through ozone exposure. Human PDL cells were cultured on the ozone-exposed EVA, collagen-immobilized EVA, HAP-immobilized EVA, HAP plate, tricalcium phosphate plate, and conventional tissue culture dish. Cell proliferation was highest on the collagen-immobilized EVA and lowest on the HAP-immobilized EVA. Alkaline phosphatase activity and osteocalcin secretion were highest on the HAP-immobilized EVA. These results suggest that PDL cells were differentiated toward bone-like cells on the HAP-immobilized EVA.

Adsorption↗

PubMed2004

Bioartificial pancreas research in Japan.

The bioartificial pancreas (BAP), a medical device enclosing insulin-secreting cells in a semipermeable membrane, is expected to physiologically control glucose levels, and thus to be able to inhibit development of serious chronic complications in diabetic patients. In this brief review, we introduce research activities on the development of the BAP in Japan, including membrane preparation for the BAP, evaluation of in vitro and in vivo BAP functions, and challenges for production of insulin-secreting cells from stem cells.

Animals↗

PubMed2004

Pharmacokinetic considerations in development of a bioartificial liver.

We consider the pharmacokinetics of bioartificial livers (BALs) prepared using a hollow fibre module with respect to two key functions, detoxification and plasma protein supply, and present the results in a simple form. We then discuss the advantages and disadvantages of BAL therapy in comparison with the non-biological therapies of haemodialysis and plasma exchange. Nitrogenous and other potentially toxic compounds, such as ammonia, mercaptans, short-chain fatty acids and gamma-aminobutyric acid, are produced in the bowels and accumulate in the systemic blood because of impaired elimination by the ailing liver. Adrenal and gonadal steroids, including corticosteroids, estrogens, progestins and androgens, are biosynthesised, and high concentrations of these hormones become harmful. All these endogenously produced toxins require effective metabolism. In haemodialysis, toxins that permeate through the hollow fibre membrane are rapidly removed by the dialysate flow, and their concentrations decrease to almost zero. In a BAL bioreactor, the toxins are slowly metabolised by hepatocytes in the hollow fibres, and decreased to concentrations that are inversely proportional to the number of hepatocytes in the BAL, even after a long-term assist. It is difficult to rationalise the clinical usage of BAL systems containing small amounts of hepatocytes (70-100g) to remove the toxins. Concentrations of plasma proteins in a patient after long-term BAL treatment are proportional to the number of hepatocytes in the device. BAL reactors prepared using porcine hepatocytes supply porcine proteins, not human proteins, to the recipient. Plasma exchange increases protein concentrations much more effectively than BAL as long as a sufficient amount of plasma is available. The blood inflow rate to the liver is about 1500 mL/min in a normal adult. On the other hand, blood draw rates to a BAL system are restricted to the range of 100-300 mL/min. Toxins that are rapidly cleared by the liver (for example, the ammonia clearance of the normal human liver is several hundred mL/min) cannot be effectively eliminated from the systemic blood by BAL systems currently under clinical evaluation. Hepatocytes are the only elements in a BAL reactor that can metabolise toxins and synthesise proteins, and thus BAL performance increases with the increasing number of hepatocytes in the bioreactor. The human liver weighs about 1500 g and contains about 80% hepatocytes, i.e. about 1200 g of hepatocytes. The blood flow through the liver is about 1500 mL/min in a normal adult. To effectively replace liver functions would require a BAL reactor containing the functional equivalent of several hundreds of grams of human hepatocytes with an extracorporeal perfusion rate of more than 1000 mL/min. At this point, only orthotopic liver transplantation can meet these criteria.

Algorithms↗

PubMed2004

Beta-tricalcium phosphate combined with recombinant human bone morphogenetic protein-2: a substitute for autograft, used for packing interbody fusion cages in the canine lumbar spine.

Beta-tricalcium phosphate (beta-TCP) combined with recombinant human bone morphogenetic protein-2 (BMP-2) was examined as a substitute for autograft for packing into interbody fusion cages in the canine lumbar spine model. Discectomy and interbody cage fusion were performed at three disc spaces in eight dogs. Examination of microradiographs and histological sections of the lumbar spine at 16 weeks postsurgery revealed three fusions in the autograft cages (Group A), three in the beta-TCP cages (Group B), and five in the beta-TCP-BMP-2 cages (Group C). The mean percentage of trabecular bone area in the cages was 51.9% in Group A, 48.8% in Group B, and 65.6% in Group C. Mean percentage of trabecular bone formation and mechanical stiffness were highest in the cages filled with beta-TCP and BMP-2. Combination of BMP to beta-TCP may act as an osteoconductive and osteoinductive bone graft substitute in clinical spine surgery.

Animals↗

PubMed2004

Vascular endothelial growth factor immobilized on platinum microcoils for the treatment of intracranial aneurysms: experimental rat model study.

Platinum microcoils coated with immobilized recombinant human vascular endothelial growth factor (rhVEGF) were prepared and the effectiveness for the embolization of aneurysms was investigated using a rat model. Platinum coils were prepared by successive deposition of cationic polyethyleneimine and anionic heparin, and VEGF was immobilized through affinity interaction with heparin. Unmodified, heparin-coated, or rhVEGF-immobilized platinum coil segments were inserted into the ligated external carotid arteries at the bifurcation of the common carotid artery (CCA) of adult female rats. The bifurcation segments of the CCA were harvested 2 weeks after the coil placement. rhVEGF-immobilized coils showed significantly greater endothelial formation at the aneurysm orifice and cell infiltration in the aneurysm body compared with the unmodified and heparin-coated coils. The percentage of sac occlusion was significantly greater in the rhVEGF-immobilized group (77.53 +/- 27.58%) than in the heparin-coated group (44.81 +/- 38.30%) and unmodified group (34.99 +/- 28.15%). Scanning electron microscopy showed a tendency for more fibrotic and cellular collections on the coil surface and more tissue mass filling in the coil lumen in the rhVEGF-immobilized group. Platinum microcoils coated with immobilized rhVEGF may be effective for the obliteration of aneurysms.

Animals↗

PubMed2004

Development of gold stents for the treatment of intracranial aneurysms: an experimental study in a canine model.

BACKGROUND AND PURPOSE: Gold has often been used in medicine because of its radiopacity and flexibility. To perform stent-supported coil embolization of intracranial aneurysms, we prepared a gold stent and examined its flexibility, radiopacity, and thrombogenic properties in comparison with a stainless steel device implanted in vitro and in vivo. METHODS: Gold stents were prepared by plating gold on stainless steel stents as a template. Their mechanical properties and trackability in vitro were determined and compared with those of stainless steel stents of the same design. Twenty gold stents and two stainless steel stents were implanted in canine external carotid, vertebral, and renal arteries, as a muscle branch of the maxillary arteries, to examine their performance in vivo. RESULTS: The gold stent exhibited much less radial force and greater flexibility than the stainless steel stent. It also demonstrated superior trackability and radiopacity in the experimental endovascular procedures in canines. Histologic examination showed good patency of the stented artery with slight endothelial hypertrophy. CONCLUSION: Although there is still room for more radial strength, less influence on intimal hypertrophy, a more suitable flexibility, and a smoother surface, the superior trackability and radiopacity of gold stents seem to support use of this device for the endovascular treatment of intracranial aneurysms.

Animals↗

PubMed2004

Poly-vinyl alcohol hydrogel vascular models for in vitro aneurysm simulations: the key to low friction surfaces.

PURPOSE: To develop a vessel model with poly-vinyl alcohol hydrogel (PVA-H) with low surface friction for in vitro simulations of vascular pathologies. MATERIALS AND METHODS: Simulations of variable vascular pathologies were based on human vessel casts reproduced in wax. Constructions of vascular models were then obtained by lost wax techniques. As materials, liquid silicon or PVA in organic solvents were used. After solidification, the wax was drained by heating. Comparison of PVA-H with silicone models included evaluation of surface characteristics (friction coefficient), of visibility (transparence and compatibility with medical imaging techniques), and of stiffness (pulsatility of vascular lumen). Both model types were integrated in a circulation circuit allowing for flow simulations and testing with endovascular medical devices. RESULTS AND CONCLUSION: Both model types were transparent and compatible with current medical imaging methods. PVA-H models exhibited much lower friction characteristics. PVA-H stiffness was closer to soft tissue when compared to silicone, allowing for pulsations during flow simulations.

Friction↗

PubMed2003

Investigation on light-addressable potentiometric sensor as a possible cell-semiconductor hybrid.

This article reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible biological cell-semiconductor hybrid that will enable us to make an interface between the physical and biological system. To increase the surface potential sensitivity, we used a LAPS structure with single insulator (SiO2) coated with poly-L-ornithine and laminin (PLOL) on Si. Efficient culturing of PC-12 and nerve cells of Lymnaea stagnalis on PLOL-coated Si3N4 and SiO2 was achieved. The thickness of the PLOL layer was found to be about 4 nm by the atomic force microscope (AFM) measurement. Using the advantage of this thin layer of PLOL, we compared the performance of a novel structure to the previously reported "PLOL-coated Si3N4/SiO2/Si" structure. Due to high insulating capacitance, the photocurrent response of the novel LAPS was found to be very steep. As a result, higher sensitivity was achieved. This steepness did not degrade during 10 days when the sensor surface was kept in contact with the cell culture medium and environment. The thickness of PLOL layer, its ability to improve the biological cell adhesion, enhanced sensitivity, and experiment with simulated neural action potential (AP) applied to the novel LAPS show a good promise for LAPS to be a biological cell-semiconductor hybrid.

Action Potentials↗

PubMed2003

Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance.

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.

Autoantibodies↗

PubMed2003

Indefinite islet protection from autoimmune destruction in nonobese diabetic mice by agarose microencapsulation without immunosuppression.

BACKGROUND: The recurrence of autoimmunity and allograft rejection act as major barriers to the widespread use of islet transplantation as a cure for type 1 diabetes. The aim of this study was to evaluate the feasibility of immunoisolation by use of an agarose microcapsule to prevent autoimmune recurrence after islet transplantation. METHODS: Highly purified islets were isolated from 6- to 8-week-old prediabetic male nonobese diabetic (NOD) mice and microencapsulated in 5% agarose hydrogel as a semipermeable membrane. Islet function was evaluated by a syngeneic islet transplantation model, in which islets were transplanted into spontaneously diabetic NOD mice. RESULTS: The nonencapsulated islet grafts were destroyed and diabetes recurred within 2 weeks after transplantation in all 12 mice. In contrast, 13 of the 16 mice that underwent transplantation with microencapsulated islets maintained normoglycemia for more than 100 days after islet transplantation. Histologic examination of the nonencapsulated islet grafts showed massive mononuclear cellular infiltration with beta-cell destruction. In contrast, the microencapsulated islets showed well-granulated beta cells with no mononuclear cellular infiltration around the microcapsules or in the accompanying blood capillaries between the microcapsules. CONCLUSIONS: Agarose microcapsules were able to completely protect NOD islet isografts from autoimmune destruction in the syngeneic islet transplantation model.

Animals↗

PubMed2003

Targeting a complex transcriptome: the construction of the mouse full-length cDNA encyclopedia.

We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5'-end clusters identify regions that are potential promoters for 8637 known genes and 5'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Animals↗