Biomedical subjects

Teruo Okano

Publications and source records attributed to Teruo Okano.

At least 109 records · Page 6Linked to original sources

PubMed2002

Aqueous chromatography utilizing hydrophobicity-modified anionic temperature-responsive hydrogel for stationary phases.

A new pH-/temperature-responsive poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) (poly(IPAAm-co-AAc-co-tBAAm)) hydrogel grafted on silica beads was evaluated as column matrix for a cation-exchange thermoresponsive chromatography. The stationary phase showed simultaneous changes in temperature-responsive surface charge density and hydrophobicity by incorporation of anionic AAc and hydrophobic tBAAm into IPAAm sequences. Thermoresponsive polymer property alterations were confirmed by temperature-responsive phase transition and shift in apparent pKa values. Catecholamine derivatives were retained on poly(IPAAm-co-AAc-co-tBAAm)-modified column at pH 7.0. Analyte retention was primarily due to the electrostatic interaction. It was noted that the temperature-induced phase transition of poly(IPAAm-co-AAc-co-tBAAm) hydrogel layer on the stationary phases was evidenced by the apparent inflection point in van't Hoff plots around 36 degrees C. This suggests that solute interactions should be changed below and above the stationary phase transition temperature, reducing electrostatic interaction above the transition temperature.

Anions↗

PubMed2002

Time-programmed pulsatile release of dextran from calcium-alginate gel beads coated with carboxy-n-propylacrylamide copolymers.

Time-programmed release of macromolecular drugs was achieved by utilization of calcium-alginate gel beads modified with coated copolymer layers. Modified calcium-alginate gel beads coated with poly(carboxy-n-propylacrylamide-co-dimethylacrylamide) [poly(CNPAAm-co-DMAAm)] (22.7 mol% of CNPAAm) of varying coating thickness from 25 to 125 microm were developed as drug carriers. Model macromolecular drugs used were fluorescein isothiocyanate (FITC)-labeled dextrans with different molecular weights ranging from 9400 to 145000. FITC-dextran release was strongly dependent on both copolymer coating thicknesses and the dextran molecular weights. Release of FITC-dextran (MW 9400) followed Fickian diffusion according to t(1/2) dependence, indicating that the drug diffusion is the main driving force for release of dextran MW 9400. Release of higher molecular weight FITC-dextrans (71,00 and 145,00) exhibited a burst-effect preceded by a preset lag time. These release profiles were governed by the dissociation of calcium ions from polyguluronate sequences in alginate molecules along with the diffusion of sodium ions into the gel bead core. This created osmotic pressure inside the gel, inducing breakage of the coated copolymer layer and accelerated drug release. Burst release of macromolecular drugs thus occurred after a certain lag period. The lag time was regulated by the copolymer coat thickness. A pulsatile release of FITC-dextran was demonstrated by combining a series of modified alginate gel beads in a single batch.

Acrylic Resins↗

PubMed2002

Fabrication of pulsatile cardiac tissue grafts using a novel 3-dimensional cell sheet manipulation technique and temperature-responsive cell culture surfaces.

Recent progress in cell transplantation therapy to repair impaired hearts has encouraged further attempts to bioengineer 3-dimensional (3-D) heart tissue from cultured cardiomyocytes. Cardiac tissue engineering is currently pursued utilizing conventional technology to fabricate 3-D biodegradable scaffolds as a temporary extracellular matrix. By contrast, new methods are now described to fabricate pulsatile cardiac grafts using new technology that layers cell sheets 3-dimensionally. We apply novel cell culture surfaces grafted with temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), from which confluent cells detach as a cell sheet simply by reducing temperature without any enzymatic treatments. Neonatal rat cardiomyocyte sheets detached from PIPAAm-grafted surfaces were overlaid to construct cardiac grafts. Layered cell sheets began to pulse simultaneously and morphological communication via connexin43 was established between the sheets. When 4 sheets were layered, engineered constructs were macroscopically observed to pulse spontaneously. In vivo, layered cardiomyocyte sheets were transplanted into subcutaneous tissues of nude rats. Three weeks after transplantation, surface electrograms originating from transplanted grafts were detected and spontaneous beating was macroscopically observed. Histological studies showed characteristic structures of heart tissue and multiple neovascularization within contractile tissues. Constructs transplanted into 3-week-old rats exhibited more cardiomyocyte hypertrophy and less connective tissue than those placed into 8-week-old rats. Long-term survival of pulsatile cardiac grafts was confirmed up to 12 weeks. These results demonstrate that electrically communicative pulsatile 3-D cardiac constructs were achieved both in vitro and in vivo by layering cardiomyocyte sheets. Cardiac tissue engineering based on this technology may prove useful for heart model fabrication and cardiovascular tissue repair. The full text of this article is available at http://www.circresaha.org.

Actin Cytoskeleton↗

PubMed2002

Process design for efficient and controlled drug incorporation into polymeric micelle carrier systems.

For the efficient and well-controlled incorporation of the anti cancer drug adriamycin (ADR) into the inner core of a thermo-responsive polymeric micelle carrier system, we have analyzed and optimized the incorporation procedure in this paper. A dialysis method was used for preparing the micelle solution and ADR incorporation simultaneously. Quantities of ADR and triethylamine (TEA) were varied and the effects of their quantities were analyzed. Solvent composition at the starting time of dialysis was also varied. The initial dialysis condition, solvent with 40% water, brought about the largest amount and yield of ADR incorporation. With the initial 40% water content, it was considered that the block polymers formed a micelle-like association with a swollen hydrophobic core. This swollen core may be suitable for a large amount of ADR incorporation, since this core, swollen by an organic solvent-water mixture, is expected to show a liquid-state character to allow ADR molecules entry into the cores. By starting the dialysis procedure at this 40% water content, this swollen core suitable for the ADR incorporation is considered to be maintained for a much longer period than a case starting with a polymer-ADR solution in a solvent with a water content of less than 40%, and, therefore, ADR is expected to be incorporated efficiently. Preparation temperature of 20-25 degrees C was found to provide the most effective ADR incorporation in this thermo-responsive polymeric micelle system. These results indicate that the efficient incorporation of ADR can be achieved in consideration of the dynamic micelle formation and drug incorporation processes.

Doxorubicin↗

PubMed2002

Pulsatile drug release control using hydrogels.

Current research in the field of drug delivery devices, by which pulsed and/or pulsatile release is achieved, has been intensified. In this article several types of drug delivery systems using hydrogels are discussed that showed pulsed and/or pulsatile drug delivery characteristics. As is frequently found in the living body, many vital functions are regulated by pulsed or transient release of bioactive substances at a specific site and time. Thus it is important to develop new drug delivery devices to achieve pulsed delivery of a certain amount of drugs in order to mimic the function of the living systems, while minimizing undesired side effects. Special attention has been given to the thermally responsive poly(N-isopropylacrylamide) and its derivative hydrogels. Thermal stimuli-regulated pulsed drug release is established through the design of drug delivery devices, hydrogels, and micelles. Development of modified alginate gel beads with pulsed drug delivery characteristic is also described in this article.

Animals↗

PubMed2002

Adhesion behavior of monocytes, macrophages, and foreign body giant cells on poly (N-isopropylacrylamide) temperature-responsive surfaces.

Monocyte and macrophage adhesion and foreign body giant cell (FBGC) formation has been observed on surfaces with a wide range of properties. In this study we have utilized novel, temperature-responsive surfaces (TRS) with dynamic surface properties to investigate inflammatory cell adhesion behavior. With temperature changes, grafted chains of poly-N-isopropylacrylamide pass through their lower critical solution temperature (LCST) and can either extend (hydrate), creating a hydrophilic surface at 20 degrees C, or contract (dehydrate), creating a hydrophobic surface at 37 degrees C. Isolated human monocytes and monocyte-derived macrophages were able to adhere, spread, and form FBGC on the hydrophobic surface. Decreasing the temperature below the lower critical solution temperature induced a change in the surface wettability, creating a hydrophilic surface, that induced a differential detachment of adherent cells that decreased with time, ranging from 98% after 2 h of culture to 30% at day 10. These detached cells remained viable, and were recultured onto TCPS for 3, 7, and 10 days. These novel surfaces allow investigation of the adhesive behavior of adherent inflammatory cells in a temporal manner, and the effects of surface conformation and wettability changes on cell adhesion and detachment.

Cell Adhesion↗

PubMed2002

Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured cardiomyocyte sheets.

Recent progress in stem cell biology is likely to provide implantable sources of human cardiomyocytes in the near future. This possibility has encouraged cardiac tissue engineering. To construct heart-like tissue, we have exploited the capabilities of novel cell culture surfaces grafted with a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), to produce intact viable monolayer cell sheets simply by reducing culture temperature. Cultured chick embryonic cardiomyocyte sheets detached from PIPAAm-grafted surfaces were layered into tissue-like laminate stacks using hydrophilic support and transfer membranes. The layered cell sheets rapidly adhered to each other, establishing cell-to-cell connections characteristic of heart tissue, including desmosomes and intercalated disks. Bilayer cell sheets pulsed spontaneously and synchronously, altering their characteristic pulsing frequency with applied electric stimulation transmitted across the sheets. These results demonstrate that electrically communicative three-dimensional cardiac constructs can be achieved by stacking monolayer cardiomyocyte sheets. Cardiac tissue engineering based on this technology will facilitate new in vitro heart models and may prove useful for in vivo cardiovascular tissue repair.

Acrylic Resins↗

PubMed2002

Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture.

Tissue engineering constructs that effectively duplicate natural tissue function must also maintain tissue architectural and organization features, particularly the integration of multiple cell types preserving distinct, integrated phenotypes. Cell-cell communication and biochemical cross-talk have been shown to be essential for the maintenance of differentiated cell functions in tissues and organs. Current limitations of cell-culture hinder progress in understanding the features and dynamics of heterotypic cell communication pathways critical to developing more sophisticated or effective tissue-engineered devices. We describe a method to conveniently electron-beam pattern cell culture surfaces with thermo-responsive polymer chemistry that exploits changes in cell-polymer adhesive interactions over a temperature window amenable for high-throughput cell culture. Cells seeded on these patterned surfaces at 20 degrees C adhere only to surface areas lacking thermo-responsive grafting chemistry; grafted domains at 20 degrees C are hydrophilic and non-cell adhesive. The culture temperature is then increased to 37 degrees C collapsing the hydrated grafted chemistry. A second cell type is added to the culture and adheres only to these exposed relatively hydrophobic grafted patterns. Both cell types can then be effectively co-cultured at 37 degrees C under multiple conditions. Long-term cell pattern fidelity and differentiated cell functions characteristic of each co-planar cell type are observed. This method is simple and has few limitations, compared with other existing co-culture methods.

Animals↗

PubMed2002

Two-dimensional cell sheet manipulation of heterotypically co-cultured lung cells utilizing temperature-responsive culture dishes results in long-term maintenance of differentiated epithelial cell functions.

Here we report two-dimensional cell sheet manipulation (2D CSM) of heterotypically co-cultured lung cell sheets and the maintenance of differentiated phenotypes of lung epithelial cells over prolonged periods of up to 70 days. This was facilitated by poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture dishes. PIPAAm-grafted dishes are responsive to temperature changes and offer a unique surface on which cells adhere and multiply like on ordinary tissue culture dishes under the permissive temperature of 37 degrees C, but on lowering of temperature resulting in changes in hydration of the polymer the cells spontaneously detach from the surface without use of enzymes like trypsin which is the common procedure. It has been well documented that type II pneumocytes of the lung lose many of their special features rapidly in culture. The culture system detailed here comprises random co-culture of epithelial and mesenchymal cells of lung. The heterotypic cell culture system promotes cell-cell interactions maintaining a harmonized physiology. When this heterotypic monolayer on PIPAAm-grafted dishes was subjected to lower temperature of 20 degrees C and 2D CSM we were able to transfer the monolayer as a single contiguous sheet with cell-cell connections intact to other surfaces. This non-invasive transfer of cell sheet resulted in shrinkage of the monolayer, enabling the type II cells to regain their cuboidal morphology and specialized characters like Maclura pomifera lectin binding and surfactant protein A (SP-A) expression. The active dome formation also observed subsequent to transfer reaffirms the uniqueness of the culture conditions and 2D CSM in future for developing tissue like architecture in vitro.

Acrylic Resins↗

PubMed2002

Incorporation of the anticancer agent KRN5500 into polymeric micelles diminishes the pulmonary toxicity.

KRN5500 is a highly active new semi-synthetic water-insoluble anticancer agent. The only mechanism of anticancer activity of KRN5500 described so far is an inhibitory effect on protein synthesis. At the time of writing, a phase I clinical trial is under way at the National Cancer Center Hospital, Tokyo, and at the National Cancer Institute in the USA. Although preclinical data did not indicate lung toxicity, some cases of severe pulmonary disorder were reported in the phase I clinical trials. This study has been conducted to examine whether incorporation of KRN5500 into polymeric micelles (KRN/m) could reduce the toxic effects caused by the current formulation of KRN5500. The in vitro and in vivo antitumor activities of KRN5500 and KRN/m were compared. Pulmonary toxicity of KRN5500 and KRN/m was studied using a bleomycin (BLM)-induced lung injury rat model. In BLM-rats, extensive pulmonary hemorrhage with diapedesis was observed with KRN5500 i.v. bolus injection at the dose of 3 mg/kg, which is equivalent to 21.0 mg/m2 (level 5) of the Japanese phase I trial. However, toxicity was not observed when rats were administered KRN / m at the equivalent dose to KRN5500 in potency. Electron microscopy of the lung treated with KRN5500 showed disruption of the alveolar type II membrane with release of lamellar debris. Furthermore, in vivo, KRN/m showed similar antitumor activity to KRN5500. These results indicate that KRN/m may be useful for reducing the pulmonary toxicity associated with the current formulation of KRN5500, while fully maintaining its antitumor activity.

Animals↗

PubMed2002

Temperature-responsive chromatography using poly(N-isopropylacrylamide) hydrogel-modified silica.

Poly(N-isopropylacrylamide) (PNIPAAm) has the sharpest phase transition of the class of thermo-sensitive N-alkyl acrylamide polymers. We developed a new method of HPLC using packing materials modified with cross-linked poly(N-isopropylacrylamide) (PNIPAAm) hydrogel. A temperature-responsive surface was prepared by polymerization of NIPAAm in the presence of a cross-linker on the silica support. The surface properties and functions of the stationary phases change in response to the external temperature. Therefore it easily changes the interaction of a solute with the surface with a constant aqueous mobile phase. A temperature-responsive elution behavior was observed on the separation of steroids and PTH-amino acids. The method is expected to be applicable to separation in the pharmaceutical and biomedical fields.

Acrylamides↗

PubMedDate not supplied

Copolymerization of 2-carboxyisopropylacrylamide with N-isopropylacrylamide accelerates cell detachment from grafted surfaces by reducing temperature.

Acrylic acid (AAc) has been utilized to introduce reactive carboxyl groups to a temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm). However, AAc introduction shifts the copolymer phase transition temperatures higher and dampens the steep homopolymer phase transition with increasing AAc content. We previously synthesized 2-carboxyisopropylacrylamide (CIPAAm) having both a similar side chain structure to IPAAm and a functional carboxylate group in order to overcome these shortcomings. In the present study, these copolymers, grafted onto cell culture plastic, were assessed for cell adhesion control using their phase transition. AAc introduction to PIPAAm-grafted surfaces resulted in excessive surface hydration and hindered cell spreading in culture at 37 degrees C. In contrast, CIPAAm-containing copolymer-grafted surfaces exhibited relatively weak hydrophobicity similar to both homopolymer PIPAAm-grafted surfaces as well as commercial ungrafted tissue culture polystyrene dish surfaces. Cells adhered and spread well on these surfaces at 37 degrees C in culture. As observed previously on PIPAAm-grafted surfaces, cells were spontaneously detached from the copolymer-grafted surfaces by reducing culture temperature. Cell detachment was accelerated on the CIPAAm copolymer-grafted surfaces compared to pure IPAAm surfaces, suggesting that hydrophilic carboxyl group microenvironment in the monomer and polymer is important to accelerate grafted surface hydration below the lower critical solution temperature, detaching cells.

Acrylamides↗

PubMedDate not supplied

Preparation of comb-type N-isopropylacrylamide hydrogel beads and their application for size-selective separation media.

A series of the comb-type poly(N-isopropylacrylamide) (NIPAM) gel beads were prepared by inverse suspension polymerization techniques. The comb-type NIPAM gel beads exhibited large volume change at 30 degrees C, and their deswelling rate, defined as the time required for half-shrinking, was 10 times faster than that of the normal-type NIPAM gel beads. The gel beads were utilized to concentrate dilute aqueous solutions of albumin, gamma-globulin, and vitamin B(12). The separation efficiencies of albumin and gamma -globulin with the comb-type NIPAM gel were 80% and 85%, respectively. Whereas those with normal-type NIPAM gel were 55% and 60%, respectively. The incorporation of grafted chains into gel makes the effective mesh size smaller. Therefore it induces the additional obstruction effects between the solutes and network and excludes the high molecular weight solutes. After they have extracted water, their rapid deswelling property makes the gel regenerate effectively by warming to release the absorbed water.

Acrylamides↗

PubMedDate not supplied

Temperature-responsive cell culture surfaces enable "on-off" affinity control between cell integrins and RGDS ligands.

In this study, specific interactions between immobilized RGDS (Arg-Gly-Asp-Ser) cell adhesion peptides and cell integrin receptors located on cell membranes are controlled in vitro using stimuli-responsive polymer surface chemistry. Temperature-responsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) (P(IPAAm-co-CIPAAm)) copolymer grafted onto tissue culture grade polystyrene (TCPS) dishes permits RGDS immobilization. These surfaces facilitate the spreading of human umbilical vein endothelial cells (HUVECs) without serum depending on RGDS surface content at 37 degrees C (above the lower critical solution temperature, LCST, of the copolymer). Moreover, cells spread on RGDS-immobilized surfaces at 37 degrees C detach spontaneously by lowering culture temperature below the LCST as hydrated grafted copolymer chains dissociate immobilized RGDS from cell integrins. These cell lifting behaviors upon hydration are similar to results using soluble RGDS in culture as a competitive substitution for immobilized ligands. Binding of cell integrins to immobilized RGDS on cell culture substrates can be reversed spontaneously using mild environmental stimulation, such as temperature, without enzymatic or chemical treatment. These findings are important for control of specific interactions between proteins and cells, and subsequent "on-off" regulation of their function. Furthermore, the method allows serum-free cell culture and trypsin-free cell harvest, essentially removing mammalian-sourced components from the culture process.

Cell Adhesion↗

PubMedDate not supplied

Polymer terminal group effects on properties of thermoresponsive polymeric micelles with controlled outer-shell chain lengths.

Well-defined amphiphilic diblock copolymers comprising thermoresponsive polymer segments of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PID) and hydrophobic polymer segments, poly(benzyl methacrylate) (PBzMA), were synthesized by controlled living radical polymerization. Terminal derivatization of PID segments to either hydroxyl or phenyl groups was achieved through reactions of coupling agents with thiol groups exposed by cleavage of terminal dithiobenzoate groups. Diblock copolymers formed core-shell type polymeric micelles with thermoresponsive outer shells. Hydrodynamic micellar diameters ranged from 12 to 31 nm, controlled by varying PID chain lengths. Differences in PID terminal groups did not affect the critical micelle concentration or micellar diameters. However, these groups demonstrated a significant influence on the micellar thermoresponses. Hydroxylated PID/PBzMA micelles exhibited a phase transition of approximately 40 degrees C, independent of PID molecular weights. Even though molecular weights and compositions of PID chains were equivalent except for terminal groups, micelles having the outermost surface phenyl groups exhibited drastically lower phase transition temperature shifts, especially for micelles with low molecular weight PID chains.

Amines↗

PubMedDate not supplied

Immobilization of cell-adhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest.

We have developed temperature-responsive cell culture surfaces to harvest intact cell sheets for tissue-engineering applications. Both cost and safety issues (e.g., prions, bovine spongiform encephalopathy) are compelling reasons to avoid use of animal-derived materials, including serum, in such culture. In the present study, synthetic cell-adhesive peptides are immobilized onto temperature-responsive polymer-grafted surfaces, and cell adhesion and detachment under serum-free conditions were examined. The temperature-responsive polymer poly(N-isopropylacrylamide) (PI-PAAm) was functionalized by copolymerization with a reactive comonomer having both a carboxyl group and an isopropylacrylamide group. These copolymers were covalently grafted onto tissue culture-grade polystyrene dishes. Synthetic cell-adhesive peptides were then immobilized onto these surfaces via carboxyl groups. Bovine aortic endothelial cells both adhered and spread on these surfaces even under serum-free conditions at 37 degrees C, similar to those in 10% serum-supplemented culture. Spread cells promptly detached from the surfaces on lowering culture temperatures below the lower critical solution temperature of the polymer, 32 degrees C. These surfaces would be useful for serumfree culture for tissue-engineering applications.

Adsorption↗

PubMedDate not supplied

Novel method to examine hepatocyte-specific gene expression in a functional coculture system.

To mimic native tissue function, coculture systems are an extremely useful model. In many cases, differentiated functions can be maintained only through the interactions of various cell types. Therefore, methods for examining the interactions between cocultured cells are necessary. The assessment of cell-to-cell cross-talk at the level of gene expression is one such method to examine interactions between different cell types. However, it is generally difficult to determine the gene expression of specific cell types in coculture without first separating cell populations. To overcome these obstacles, we have established a novel method to determine gene expression levels of a targeted cell population in coculture, using species-specific primers. With this approach, we were able to determine hepatocyte-specific gene expression of Fao cells (a rat hepatocyte cell line) in culture with human umbilical vein endothelial cells (HUVECs). Expression of both albumin and apolipoprotein A-I (apoA-I) increased time dependently for 10 days and maintained significantly higher expression in the coculture system as compared with isolated Fao cells. This indicates that hepatocyte function increased gradually in our coculture system and could be maintained long-term, suggesting that the construction of mature cell-to-cell communication between the two cell lines required a considerable amount of time. The expression of HNF-4 and HNF-1alpha, which are liver-enriched transcription factors, did not differ between the monolayer and cocultured Fao cells, suggesting that expression of HNF-4 and HNF-1alpha was not responsible for the increased expression albumin and apoA-I. Our findings suggest that this novel method for the detection of gene expression of targeted cell populations can be a useful tool in determining the molecular mechanisms that regulate communication between different cell types.

Animals↗

PubMedDate not supplied

Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model.

Conventional periodontal regeneration methods remain insufficient to attain complete and reliable clinical regeneration of periodontal tissues. We have developed a new method of cell transplantation using cell sheet engineering and have applied it to this problem. The purpose of this study was to investigate the characteristics of human periodontal ligament (HPDL) cell sheets retrieved from culture on unique temperature-responsive culture dishes, and to examine whether these cell sheets can regenerate periodontal tissues. The HPDL cell sheets were examined histologically and biochemically, and also were transplanted into a mesial dehiscence model in athymic rats. HPDL cells were harvested from culture dishes as a contiguous cell sheet with abundant extracellular matrix and retained intact integrins that are susceptible to trypsin-EDTA treatment. In the animal study, periodontal ligament-like tissues that include an acellular cementum-like layer and fibrils anchoring into this layer were identified in all the athymic rats transplanted with HPDL cell sheets. This fibril anchoring highly resembles native periodontal ligament fibers; such regeneration was not observed in nontransplanted controls. These results suggest that this technique, based on the concept of cell sheet engineering, can be useful for periodontal tissue regeneration.

Acrylic Resins↗