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Toshie Tsuchiya

Publications and source records attributed to Toshie Tsuchiya.

At least 19 recordsLinked to original sources

Markedly different effects of hyaluronic acid and chondroitin sulfate-A on the differentiation of human articular chondrocytes in micromass and 3-D honeycomb rotation cultures.

A source of morphologically and functionally available human cartilagenous tissue for implantation is required in the field of tissue engineering. To achieve this goal, we evaluated the effects of hyaluronic acid (HA-810 and 1680 kDa), and chondroitin sulfate (CS-A 16 and C-34 kDa) on human articular chondrocytes (HC) in micromass and rotation culture conditions. Cell proliferation was increased by CS-A 16 kDa under micromass and rotation cultures, while cell differentiation was increased under rotation but not micromass conditions. Proliferation and differentiation due to CS-C 34 kDa were very similar to the control under both culture conditions. With HA, cell proliferation was increased depending on the molecular weight under micromass and rotation conditions. In contrast, chondrocyte differentiation was enhanced under rotation conditions, but decreased under micromass conditions depending on the molecular weight of HA. In both culture conditions, aggrecan gene was continuously expressed. However, the collagen type II gene was more weakly expressed in rotation than the micromass culture conditions. Thus, the chemical structures of polysaccharides, and the culture condition, rotation or micromass, caused differences in chondrogenesis.

Aggrecans↗

Effects of sulfated hyaluronan on keratinocyte differentiation and Wnt and Notch gene expression.

Sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan (Hya), has high activity on and biocompatibility with cells. When normal human epidermal keratinocytes (NHEKs) were incubated in dishes coated with SHya, cell proliferation was suppressed in a dose-dependent manner. The expression levels of keratin 1 and loricrin mRNAs, as detected by real-time RT-PCR, were increased significantly. The expressions of Wnt mRNAs, which play important roles in cell proliferation and differentiation, were modulated. Wnt4 and Wnt6 mRNA expressions were increased compared to controls, while expression of Wnt5a was similar to the control and that of Wnt7a mRNA was decreased. In addition, the expression of Notch mRNAs, which play a critical role in keratinocyte differentiation, were affected. Notch3 mRNA was increased significantly, while Notch1 mRNA was decreased compared to controls, and expression of Notch2 was similar to that of control. These results suggested that a SHya-coated scaffold might be useful for regulating cell activity in tissue engineering.

Cell Differentiation↗

Effects of a biodegradable polymer synthesized with inorganic tin on the chondrogenesis of human articular chondrocytes.

Recent study has shown that biodegradable polymers are attractive candidates for chondrocyte fixation and further transplantation in cartilage tissue engineering. Poly (glycolic acid) (PGA), a polymer of glycolic acid, is widely used in orthopedic applications as a biodegradable polymer. Organotin, lead, antimony, and zinc are catalysts commonly used in synthesizing PGA. Here, we investigated the biocompatibility of PGA, synthesized with and without inorganic tin as a catalyst in chondrogenesis of human articular chondrocytes in a micromass culture system. Significant enhancement of chondrocyte proliferation and expression of the collagen type II protein gene were observed in cultures treated with PGA synthesized with a tin catalyst. However, aggrecan gene expression was very similar to the control culture. Amount of collagen type II protein was also increased in the same group of cultured chondrocytes. In contrast, PGA without a catalyst caused overall inhibition of chondrogenesis. Despite several positive findings, extensive investigations are essential for the feasibility of this PGA(Sn) in future clinical practice.

Absorbable Implants↗

A mouse strain difference in tumorigenesis induced by biodegradable polymers.

The use of poly-L-lactic acid (PLLA) surgical implants for repair of bone fractures has gained popularity in the past decade. The aim of this study was to evaluate the in vivo effect of PLLA plates on subcutaneous tissue in two mouse strains, BALB/cJ and SJL/J, which have higher and lower tumorigenicity, respectively. Gap-junctional intercellular communication and protein expression of connexin 43 were significantly suppressed, whereas secretion of transforming growth factor-beta1 and expression of extracellular matrix, insulin-like growth factor binding protein 3, and cysteine-rich intestinal protein 2 were significantly increased in PLLA-implanted BALB/cJ mice when compared with BALB/cJ controls. Finally, tumors were formed after implantation of cultured cells from the more-tumorigenic BALB/cJ, but not SJL/J, mice into nude mice.

Animals↗

Changes in expression of genes related to cell proliferation in human mesenchymal stem cells during in vitro culture in comparison with cancer cells.

We investigated the expression levels of several genes related to cell proliferation in human mesenchymal stem cells (hMSCs) during in vitro culture for use in clinical applications. In this study, we focused on the relationship between hMSC proliferation and transforming growth factor beta (TGFbeta) signaling during in vitro culture. The proliferation rate of hMSCs gradually decreased and marked changes in hMSC morphology were not observed in 3 months of in vitro culture. The mRNA expressions of TGFbeta1, TGFbeta2, and TGFbeta receptor type I (TGFbetaRI) in hMSCs increased with the length of cell culture. There had been no change in the TGFbeta3, TGFbetaRII, and TGFbetaRIII mRNA expressions by the 12th passage from the primary culture (at about 3 months). The mRNA expression of Smad3 increased, but those of c-myc and nucleostemin decreased with the length of hMSC in vitro culture. In addition, the expression profiles of the genes that regulate cellular proliferation in hMSCs were significantly different from those of cancer cells. In conclusion, hMSCs derived from bone marrow seldom underwent spontaneous transformation during 1-2 months of in vitro culture for use in clinical applications. In hMSCs as well as in epithelial cells, growth might be controlled by the TGFbeta family signaling.

Activin Receptors, Type I↗

[Nano -level imaging for analyzing protein structure and function].

The present manuscript outlines the nano-level imaging project, which is under promotion by the three national research institutes and supported by a research grant from the Ministry of Health, Labor and Welfare (nano-001). This research project targets collecting fundamental information regarding comprehensive understanding of cardiovascular, neurological and the other disorders, developing new diagnostic and therapeutic methods by visualizing protein structure and function in atomic(sub-nano level) or molecular (nano-level) resolution. The results of the current projects will be extended into drug design, clinical diagnostic technology and medical materials in near future.

Nanotechnology↗

In vitro induction of polyploidy and chromatid exchanges by culture medium extracts of natural rubbers compounded with 2-mercaptobenzothiazole as a positive control candidate for genotoxicity tests.

We tested extracts of custom-made natural rubber samples for cytotoxicity using V79 cells and for chromosome aberration (CA) induction using CHL cells in compliance with the Japanese guidelines for basic biological tests of medical materials and devices. The samples were formulated with a high level of 2-mercaptobenzothiazole (MBT) (A); a low level of MBT (B); or zinc dibutyldithiocarbamate (ZDBC) (C). In the CA test, MBT induced mainly polyploidy, including endoreduplication, and ZDBC induced structural CAs. In the cytotoxicity test, culture medium extracts of A, B, and C suppressed colony formation to 50% of the control value at 53.1%, 94.3%, and >100%, respectively. Culture medium extracts of sample A induced polyploidy and structural CAs in the absence of an exogenous metabolic activation system (S9 mix), but at lower concentrations in its presence, indicating the existence of other leachable promutagens. The extracts of sample B induced structural CAs at the highest concentration and only with S9 mix. Sample C was negative. The facts suggest that sample A may be a candidate for a positive control for genotoxicity tests. The high frequency of polyploidy induced by sample A was not predicted by MBT, suggesting the usefulness of the test for safety evaluation of medical devices. Numerical CAs induced by MBT and sample A are discussed.

Animals↗

Reducing the migration of di-2-ethylhexyl phthalate from polyvinyl chloride medical devices.

We attempted to determine the processing conditions for decreasing the migration of phthalate esters, particularly di-2-ethylhexyl phthalate (DEHP), from polyvinyl chloride (PVC) products using a drug solvent after dilution based on the package insert. PVC sheets and PVC tubing were subjected to optical irradiation (ultraviolet (UV), visible light irradiation) and heat treatment to determine whether they are deteriorated by these treatments. UV irradiation to one side of the PVC sheet decreased the levels of DEHP migration from the sheets by almost 50%, although the amount of DEHP content in PVC sheet was observed no significant change. On the other hand, the levels of DEHP migrating from the inner surface of PVC tubing UV-irradiated from the outer surface were not decreased compared with the control. Therefore, the surface structure was examined by conducting Fourier transform infrared spectroscopy (FT-IR), electron spectroscopy for chemical analysis (ESCA) and static angle of contact measurement. In FT-IR analysis, we found that the UV-irradiated PVC sheets were exhibited broadened absorption bands with time. In ESCA analysis, the chlorine content was decreased and the oxygen content was increased with time in UV-irradiated PVC sheets. Moreover, the other treated PVC sheets shows no significant change compared with the non-UV-irradiated PVC sheet. Therefore, the surface structure of the UV-irradiated PVC sheet was changed. As a result, the migration of DEHP from PVC products can be decreased with simple treatment, such as UV-irradiation. This could be a useful method to develop novel PVC products.

Cyclosporine↗

The effect of hyaluronic acid on insulin secretion in HIT-T15 cells through the enhancement of gap-junctional intercellular communications.

The transplantation of bioartificial pancreas has the potential to restore endogenous insulin secretion in type I diabetes. The bioartificial pancreas is constructed in vitro from cells and a support matrix. Hyaluronic acid (HA) is an extremely ubiquitous polysaccharide of extracellular matrix in the body and plays various biological roles. It has been suggested that high molecular weight (HMW) HA increases in the function of gap-junctional intercellular communications (GJIC) and the expression of connexin-43 (Cx43). To determine whether the function of pancreatic beta-cells is affected by gap junctions after HMW HA-treatment, we exposed HIT-T15, a clonal pancreatic beta-cell line, in various concentrations of HA for 24h, and then detected the insulin secretion and content, using an insulin assay kit by ELISA technique. The cellular functions of GJIC were assayed by dye-transfer method using the dye solution of Lucifer yellow. HA-treatment resulted in the enhancement of GJIC function, the increase of insulin release and insulin content. The results obtained in this study suggest that HA-coating increases the insulin secretion of HIT-T15 cells by the enhancement of Cx43-mediated GJIC. The results give useful information on design biocompatibility of HA when is used as a biomaterial for bioartificial pancreas.

Animals↗

Purification and aqueous phase atomic force microscopic observation of recombinant P2X2 receptor.

Recombinant P2X2 receptor was observed by atomic force microscope in the aqueous phase. The P2X2 receptor was expressed in an insect cell line, and recombinant proteins were prepared under native conditions. The membrane fractions were extracted, and histidine-tagged receptor protein was purified from the fractions by column chromatography. When the purified protein fraction was diluted with water and served for atomic force microscopy, dispersed particles of about 3 nm in height were observed. In the presence of 1 mM ATP, the assembly-like images of the particles were obtained. More densely assembled images of the particles were achieved when the protein was dissolved in a Tris buffer containing 1 mM ATP. Under this condition, imaging of the surface of the particles exhibited a circular structure with a diameter of about 10 nm having a pore-like structure. These results suggest that atomic force microscopy provides structural information about P2X2 receptor in aqueous phase.

Animals↗

Hydroxy apatite microspheres enhance gap junctional intercellular communication of human osteoblasts composed of connexin 43 and 45.

The aseptic loosening of artificial joints with associated periprosthetic bone resorption may be partly due to the suppression of osteoblast function to form new bone by wear debris from the joint. To assess the effect of wear debris on osteoblasts, effects of model wear debris on gap junctional intercellular communication (GJIC) of normal human osteoblasts were estimated. The GJIC activity of the osteoblasts after a 1-day incubation with the microspheres was similar to that of normal osteoblasts. However, hydroxy apatite particles, which have been reported to enhance the differentiation of osteoblasts in contact with them, enhanced the GJIC function of the osteoblasts. From RT-PCR studies, not only connexin 43 but also connexin 45 is suggested to play a role in the GJIC of the osteoblasts in an early stage of coculture with the microspheres, although it is still unclear how these connexins work and are regulated in the GJIC and differentiation. However, this study suggests that there is a relationship between the early levels of GJIC and the differentiation of the cells. Therefore, estimating the effect of biomaterials, even in the microsphere form, on the GJIC of model cells, with which the biomaterials may be in contact in vivo, can provide important information about their biocompatibility.

Base Sequence↗

Development of a simple method for predicting the levels of di(2-ethylhexyl) phthalate migrated from PVC medical devices into pharmaceutical solutions.

This study deals with the development of a simple method for predicting the elution levels of di-2-ethylhexyl phthalate (DEHP) from medical devices made of polyvinyl chloride (PVC) by using the physicochemical properties of pharmaceutical injections as a marker. GC-MS analysis showed that the release of DEHP from medical grade PVC product was concentration-dependently increased by extraction with two kinds of lipophilic injections (Sandimmun and Prograf) and three kinds of surfactants (HCO-60, Tween 80, and SDS). The solubility of lipophilic pigments such as Sudan III, methyl yellow, and 1,4-diamino-anthraquinone against these solutions were also increased in a concentration-dependent manner, in which methyl yellow showed the highest response regarding the increase of optical density (O.D.). Further, electrical conductivity and static contact angle to the PVC sheet of the solutions were also increased or decreased in the same manner. As a result of the comparative study, significant correlation was found between DEHP release levels and these three physicochemical properties, particularly methyl yellow solubility, of the solutions tested. To evaluate the relationship in detail, DEHP release levels from PVC tubing and methyl yellow solubility of 53 injections used in gynecologic and obstetric fields were determined. None of the hydrophilic medicines showed any significant release of DEHP, and all showed low solubility of methyl yellow. On the other hand, the lipophilic medicines releasing a large amount of DEHP showed high solubility of methyl yellow (greater than O.D. 0.8). These results indicate that a significant proportional relationship exists between DEHP release potency and methyl yellow solubility of pharmaceutical solutions, and the risk of DEHP exposure to the patients administered pharmaceuticals through transfusion set could be easily predicted by the solubility test without complicated elution tests of DEHP using GC-MS or LC-MS.

Diethylhexyl Phthalate↗

Monitoring of polycyclic aromatic hydrocarbons and water-extractable phenols in creosotes and creosote-treated woods made and procurable in Japan.

The recycling of disused railway sleepers treated with wood preservatives such as creosote as exterior wood for use in gardens has recently become popular in Japan. Creosote contains high quantities of polycyclic aromatic hydrocarbons (PAHs), and can lead to skin irritation and disease. In this work we have determined the amount of PAHs and water-extractable phenols in creosote and creosote-treated wood products such as railway sleepers and stakes for agricultural use that are either made or are procurable in Japan. PAHs were extracted with dichloromethane and analyzed by gas chromatography-mass spectrometry. Among carcinogenic PAHs, benz(a)anthracene was detected in the highest concentration, varying between 228 and 6328 microg/g in creosotes. Benzo(b)fluoranthene, benzo(k)fluoranthene and benzo(a)pyrene (BaP) were found in the range of 67-3541 microg/g. Almost all creosotes contained more than 50 microg/g of BaP, which is the upper limit level that is permitted in the European Union (EU). Creosote-impregnated wood products, such as brand-new or secondhand railway sleepers and foundations, contained large amounts of BaP (58-749 microg/g) and benz(a)anthracene (250-1282 microg/g). Concentrations of between 692 and 2489 microg/g of phenols were determined in the water extracts from creosotes, but the level was considerably less than the EU control value (3% by mass), and there was no correlation between the amount of water-extractable phenols and the amount of PAHs detected in each sample. The situation that consumers are free to use the creosotes containing a high concentration of carcinogens such as BaP may cause unacceptable damage to the health of persons handling these creosote products.

Benzo(a)pyrene↗

Biodegradable polymers in chondrogenesis of human articular chondrocytes.

The aim of this study was to evaluate the potential role of polyglycolic acid (PGA), poly(glycolic acid-epsilon-caprolactone) (PGCL), poly(L-lactic acid-glycolic acid) (PLGA), poly(L-lactic acid-epsilon-caprolactone, 75:25 (w/w)) [P(LA-CL)25], poly-epsilon-caprolactone (tetrabutoxy titanium) [PCL(Ti)], and fullerene C-60 dimalonic acid (DMA) in cartilage transplants. After 4 weeks of culture of human articular cartilage, the levels of cell proliferation and differentiation and the expression of cartilage-specific matrix genes were estimated. The relationship between cell differentiation and gap junction protein connexin 43 (Cx43) was also evaluated. All materials except PCL(Ti) retained cell proliferation activities similar to the controls. Cell differentiation levels from the highest to the lowest were in the following order: PGA >> PLGA > PGCL > Control = DMSO > P(LA-CL)25 = PCL(Ti) >> fullerene C-60 DMA. Expression of the collagen type II gene was selectively upregulated for PGA, PGCL, and PLGA and slightly increased for P(LA-CL)25 polymers but was downregulated for fullerene C-60 DMA. Aggrecan gene expression was strongest with PGA and was consistently expressed with other matrices, especially with PGCL and PLGA. However, the expression patterns of the connexin 43 gene were different from the former two genes. Multiple regression analysis revealed a high correlation between cartilage proteoglycans production and expression levels of these three genes.

Biodegradation, Environmental↗

The response of normal human osteoblasts to anionic polysaccharide polyelectrolyte complexes.

Polyelectrolyte complexes (PEC) were prepared from chitosan as the polycation and several synthesized functional anion polysaccharides, and their effects on cell attachment, morphology, proliferation and differentiation were estimated using normal human osteoblasts (NHOst). After a 1-week incubation, PEC made from polysaccharides having carboxyl groups as polyanions showed low viability of NHOst on it although the NHOst on it showed an enhancement in their differentiation level. On the other hand, NHOst on PEC made from sulfated or phosphated polysaccharides showed similar attachment and morphology to those on the collagen-coated dish. When the number of NHOst was estimated after 1 week, the number on the PEC was ranged from 70% to 130% of those on the collagen-coated dish, indicating few effects of these PEC on cell proliferation. In addition, NHOst on PEC films made from sulfated polysaccharides differentiated to a level very similar to that observed on the collagen-coated dish, indicating that these PEC films maintain the normal potential of NHOst to both proliferate and differentiate. Measurement of gap junctional intercellular communication of NHOst on PEC revealed that PEC did not inhibit communication, suggesting that PEC films have few effects on cell homeostasis. Thus, PEC made from the sulfated polysaccharide may be a useful material as a new scaffold for bone regeneration.

Alkaline Phosphatase↗

[Cytotoxicity of chemicals used in household products: 1997- 2004].

The cytotoxicities of chemicals used in household products were evaluated using a neutral red (NR) uptake assay. The chemicals tested during 1997-2004 were rubber additives (accelerators, antioxidants and retarders), solvents, plasticizers and biocides, such as antimicrobials, fungicides, preservatives used in paints, paper, wood and plastic products. The cytotoxicity potential of each chemical was classified by determining the concentrations inducing 50% reduction of NR uptake into Chinese hamster fibroblast V79 cells compared to control (IC50). In vivo eye irritancy of each chemical was estimated by the IC50 value. Most biocides tested showed strong cytotoxicity and had a high probability of inducing strong eye irritation.

Animals↗

Novel mechanism of tumorigenesis: increased transforming growth factor-beta 1 suppresses the expression of connexin 43 in BALB/cJ mice after implantation of poly-L-lactic acid.

Poly-L-lactic acid (PLLA) is a widely used promising material for surgical implants such as tissue-engineered scaffolds. In this study, we aimed to determine the in vivo effect of PLLA plates on the cellular function of subcutaneous tissue in the two mouse strains, BALB/cJ and SJL/J, higher and lower tumorigenic strains, respectively. Gap-junctional intercellular communication (GJIC) and the expression of connexin 43 (Cx43) protein were significantly suppressed, whereas the secretion of transforming growth factor-beta 1 (TGF-beta 1) level was significantly increased in PLLA-implanted BALB/cJ mice compared with BALB/cJ controls. However, no significant difference in TGF-beta 1 secretion was observed between the SJL/J-implanted and SJL/J control mice. We found for the first time that a significant difference was observed between the two strains; thus, the PLLA increased the secretion of TGF-beta 1 and suppressed the mRNA expression of Cx43 at the earlier stage after implantation into the higher-tumorigenic strain, BALB/cJ mice. This novel mechanism might have a vital role in the inhibition of GJIC and promote the tumorigenesis in BALB/cJ mice.

Animals↗

Proteomic analysis of putative latex allergens.

BACKGROUND: Extensive analysis of allergenic proteins is generally time-consuming and labor-intensive. Accordingly, a rapid and easy procedure for allergen identification is required. As sequence information on proteins and genes is accumulated in databases, it is becoming easier to identify a candidate protein using proteomic strategies, i.e. two-dimensional gel electrophoresis, site-specific fragmentation, mass spectrometry and then database search. In this study, we evaluated the usefulness of a proteomic strategy for identifying putative allergens through its application to latex proteins. METHODS: Latex proteins were separated with two-dimensional gel electrophoresis, and putative allergens were visualized by IgE immunoblotting using pooled serum from latex-sensitive patients. The IgE-interactive proteins were cut out from the negatively stained two-dimensional gel and subjected to in-gel digestion by trypsin. Then the resulting peptides were analyzed with mass spectrometry. Based on the mass spectrometric data we obtained, the allergen candidates were assigned by a database search. RESULTS: Five previously reported allergens and five new allergen candidates were identified with the proteomic approach without isolating the individual proteins. Less than 1 mg of crude latex protein was sufficient for the entire protocol. Because plural proteins can be processed in parallel, analysis of about 50 IgE-interactive proteins was accomplished within 1 week. CONCLUSIONS: Analysis of putative allergens with proteomic strategies (allergenomics) is a promising avenue for rapid and exhaustive research. The high resolving power of two-dimensional gel electrophoresis is superior to conventional gel electrophoresis. Moreover, the notable sensitivity and speed of mass spectrometry have pronounced advantages over the N-terminal sequencing that has generally been used for protein identification.

Adult↗