Biomedical subjects

Masashige Shinkai

Publications and source records attributed to Masashige Shinkai.

13 recordsLinked to original sources

PubMed2006

Bone-like tissue formation by three-dimensional culture of MG63 osteosarcoma cells in gelatin hydrogels using calcium-enriched medium.

The aim of this study was to investigate the effect of Ca(2+) concentration in culture medium on the promotion of osteogenesis by MG63 osteoblast-like cells and to prepare bone-like tissues by supplying Ca(2+)-enriched medium to MG63 cells immobilized in three-dimensional gelatin hydrogels. Human osteosarcoma MG63 cells were cultured on tissue culture dish under various Ca(2+) concentrations to evaluate the effect of Ca(2+) concentration on calcium deposition. When Ca(2+) concentration was 8 mM, the maximum calcium deposition was obtained at day 28. Then MG63 cells were entrapped in gelatin hydrogels cross-linked by transglutaminase and cultured for 28 days, either in a standard culture medium or in medium containing 8 mM Ca(2+). Effects of Ca(2+)-enriched medium on osteoblastic phenotype of MG63 cells in gelatin hydrogels were analyzed in terms of cell number, calcium deposition content, and alkaline phosphatase (ALP) activity. The characteristics of calcified gelatin hydrogels were evaluated by x-ray diffraction (XRD), histological analysis, and scanning electron microscopy (SEM). After 28 days of culture, no significant difference in cell numbers was found between the different culture conditions. However, calcium content of gelatin hydrogels with cells cultured in Ca(2+)-enriched media was significantly higher than that of hydrogels with cells cultured in standard Ca(2+) concentration medium. After 14 days of culture, ALP activity of cells cultured in Ca(2+)-enriched media was down-regulated compared with that of cells cultured in standard Ca(2+) concentration media. XRD analysis indicated the formation of hydroxyapatite in gelatin hydrogels cultured in the Ca(2+)-enriched media at day 14, and the XRD pattern of the composite at day 21 was almost similar to that of mouse tibia. Moreover, histological analysis and SEM analysis revealed that cross-sections of hydrogels cultured in Ca(2+)-enriched media had an organic/mineral layer structure analogous to that of mouse tibia.

Alkaline Phosphatase↗

PubMed2005

Recombinant extracellular matrix-like proteins with repetitive elastin or collagen-like functional motifs.

Using overlap elongation PCR, we created repetitive DNA libraries encoding the elastin VPGVG and collagen-like GERGDRGDP sequences. From these libraries we isolated two repetitive DNA sequences, Col-5 encoding [(GERGDRGDP)(5)GER], and Ela-16 encoding [(VPGVG)(16)VPG]. Both proteins were expressed as thioredoxin fusion proteins. The resulting recombinant extracellular matrix-like proteins had the expected properties (cell adhesive ability and thermally responsive structural change) of the functional motif sequence unit used.

Amino Acid Motifs↗

PubMed2005

Medical application of functionalized magnetic nanoparticles.

Since magnetic particles have unique features, the development of a variety of medical applications has been possible. The most unique feature of magnetic particles is their reaction to a magnetic force, and this feature has been utilized in applications such as drug targeting and bioseparation including cell sorting. Recently, magnetic nanoparticles have attracted attention because of their potential as contrast agents for magnetic resonance imaging (MRI) and heating mediators for cancer therapy (hyperthermia). Magnetite cationic liposomes (MCLs), one of the groups of cationic magnetic particles, can be used as carriers to introduce magnetite nanoparticles into target cells since their positively charged surface interacts with the negatively charged cell surface; furthermore, they find applications to hyperthermic treatments. Magnetite nanoparticles conjugated with antibodies (antibody-conjugated magnetoliposomes, AMLs) are also applied to hyperthermia and have enabled tumor-specific contrast enhancement in MRI via systemic administration. Since magnetic nanoparticles are attracted to a high magnetic flux density, it is possible to manipulate cells labeled with magnetic nanoparticles using magnets; this feature has been applied in tissue engineering. Magnetic force and MCLs were used to construct multilayered cell structures and a heterotypic layered 3D coculture system. Thus, the applications of these functionalized magnetic nanoparticles with their unique features will further improve medical techniques.

Animals↗

PubMed2004

Microbial expression of proteins containing long repetitive Arg-Gly-Asp cell adhesive motifs created by overlap elongation PCR.

We developed a novel method for creating repetitive DNA libraries using overlap elongation PCR, and prepared a DNA library encoding repetitive Arg-Gly-Asp (RGD) cell adhesive motifs. We obtained various length DNAs encoding repetitive RGD from a short monomer DNA (18 bp) after a thermal cyclic reaction without a DNA template for amplification, and isolated DNAs encoding 2, 21, and 43 repeats of the RGD motif. We cloned these DNAs into a protein expression vector and overexpressed them as thioredoxin fusion proteins: RGD2, RGD21, and RGD43, respectively. The solubility of RGD43 in water was low and it formed a fibrous precipitate in water. Scanning electron microscopy revealed that RGD43 formed a branched 3D-network structure in the solid state. To evaluate the function of the cell adhesive motifs in RGD43, mouse fibroblast cells were cultivated on the RGD43 scaffold. The fibroblast cells adhered to the RGD43 scaffold and extended long filopodia.

Amino Acid Motifs↗

PubMed2004

Hyperthermia using magnetite cationic liposomes for hamster osteosarcoma.

BACKGROUND: We have developed magnetite cationic liposomes (MCLs) and applied them to local hyperthermia as a mediator. MCLs have a positive charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. In this study, the effect of hyperthermia using MCLs was examined in an in vivo study of hamster osteosarcoma. METHOD: MCLs were injected into the osteosarcoma and then subjected to an AMF. RESULTS: The tumor was heated at over 42 degrees C, but other normal tissues were not heated as much. Complete regression was observed in 100% of the treated group hamsters, whereas no regression was observed in the control group hamsters. At day 12, the average tumor volume of the treated hamsters was about 1/1000 of that of the control hamsters. In the treated hamsters, no regrowth of osteosarcomas was observed over a period of 3 months after the complete regression. CONCLUSION: These results suggest that this treatment is effective for osteosarcoma.

Journal Article↗

PubMed2004

Functional magnetic particles for medical application.

Magnetic particles for medical applications have been developed by many researchers. Since magnetic particles have unique magnetic features not present in other materials, they can be applied to special medical techniques. Separation, immunoassay, magnetic resonance imaging (MRI), drug delivery, and hyperthermia are enhanced by the use of magnetic particles. Magnetite cationic liposomes (MCLs), one of the groups of cationic magnetic particles, can be used as carriers to introduce DNA into cells since their positively charged surface associates with the negatively charged DNA. MCLs can also be used as heat mediators for cancer therapy. Magnetic particles conjugated with tumor-specific antibodies have enabled tumor-specific contrast enhancement in MRI. In addition, antibody-conjugated magnetic particles were shown to target renal cell carcinoma cells, and are applicable to the hyperthermic treatment of carcinomas. It was also found that the hyperthermic treatment using magnetic particles induced an antitumor immunity. Thus, the use of magnetic particles with their unique features will further improve medical techniques.

Animals↗

PubMed2003

Heat shock protein 70 expression induces antitumor immunity during intracellular hyperthermia using magnetite nanoparticles.

In this study we demonstrated that heat shock protein (HSP) 70 expression by hyperthermia induced antitumor immunity in the T-9 rat glioma. Our hyperthermic system using magnetic nanoparticles induced necrotic cell death that correlated with HSP70 expression. We purified the HSP70-peptide complexes from the tumor after hyperthermia to investigate whether HSP70 was involved in the antitumor immunity, and we found that in the F344 rats immunized with T-9-derived HSP70 the tumor growth of T-9 was significantly suppressed. Tumor rejection assay after hyperthermic treatment of implanted T-9 cells with incorporated magnetite cationic liposomes (MCL) was performed to investigate whether antitumor immunity was induced by release of HSP70 from the necrotic cells in the F344 rat. Tumor growth was strongly suppressed in the rats subjected to hyperthermia of implanted T-9 cells, and 50% of rats were protected from challenge with T-9 cells. Immunogenicity was enhanced when the HSP70-overexpressing T-9 cells were killed via necrosis in rats by hyperthermia, after which all rats were completely protected from challenge with T-9 cells. Our hyperthermic system produces vaccination with HSP70-peptide via necrotic tumor cell death in vivo, resulting in antitumor immunity. This phenomenon, which may be termed in situ vaccination, has important implications for the development of novel antitumor therapies.

Animals↗

PubMed2003

Anticancer effect and immune induction by hyperthermia of malignant melanoma using magnetite cationic liposomes.

The hyperthermic effect of magnetic particles was examined in an in vivo study of mouse B16 melanoma. Magnetite cationic liposomes (MCLs) have a positive surface charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. MCLs were injected into melanoma nodules, which were then subjected to an AMF. The mice were divided into four groups: group I (control), group II (hyperthermia at 43 degrees C for 30 min, once), group III (hyperthermia at 46 degrees C for 30 min, once) and group IV (hyperthermia at 46 degrees C for 30 min, twice). Complete tumour regression was observed in 90% of the mice in group IV, while no mice in groups I and II and only 40% in group III showed regression. To examine whether hyperthermia caused immune induction in B16 melanoma, in vitro cytotoxicity assays and rechallenge experiments were performed. Cytotoxic activity was observed in the spleen cells of the cured mice in group IV. In the rechallenge experiment, 66% of the cured mice rejected melanoma cells. These results suggest that hyperthermia using MCLs is an effective therapy for melanoma, since this treatment can kill the tumour cells not only by heat but also by inducing an immune response.

Animals↗

PubMed2003

Tumor regression by combined immunotherapy and hyperthermia using magnetic nanoparticles in an experimental subcutaneous murine melanoma.

Immunotherapy (IT) has become an accepted therapeutic modality. We previously reported that intracellular hyperthermia (IH) using magnetic nanoparticles induces antitumor immunity. We undertook these studies in order to study the combined effects of IT and IH on melanoma. Magnetite cationic liposomes (MCLs) have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs were injected into a B16 melanoma nodule in C57BL/6 mice, which were subjected to AMF for 30 min. The temperature at the tumor reached 43 degrees C and was maintained by controlling the magnetic field intensity. At 24 h after IH, interleukin-2 (IL-2) or granulocyte macrophage-colony stimulating factor (GM-CSF) was injected directly into the melanoma. Mice were divided into six groups: group I (control), group II (IH), group III (IL-2), group IV (GM-CSF), group V (IH + IL-2), and group VI (IH + GM-CSF). Complete regression of tumors was observed in mice of groups V and VI (75% (6/8) and 40% (4/10) of the mice, respectively), while no tumor regression was observed in mice of the other groups. This study supports the combined use of IT and IH using MCLs in patients with advanced malignancies.

Animals↗

PubMed2003

Transglutaminase-mediated gelatin matrices incorporating cell adhesion factors as a biomaterial for tissue engineering.

The goal of this work was to develop a novel biomaterial to be used for either wound dressing or as a scaffold for tissue engineering. The biodegradable hydrogels were prepared through cross-linking of gelatin with transglutaminase (TGase) in an aqueous solution. We found that the concentrations of 5 wt% gelatin and 1 unit/ml TGase were optimum for the proliferation of NIH/3T3 fibroblasts. Then, we investigated whether the cell proliferation was enhanced by incorporation of cell adhesion factors into the gelatin hydrogels. Since vitronectin and fibronectin can bind with gelatin by the action of TGase, we added these cell adhesion proteins into the gelatin hydrogels. The hydrogels incorporating these cell adhesion proteins significantly enhanced cell proliferation compared with the gelatin hydrogels without these proteins (p<0.05). Two types of synthetic Arg-Gly-Asp (RGD) peptides, RGDLLQ and RGDLLG were also added to the gelatin solution where RGDLLQ is a substrate of TGase by virtue of a glutamine (Q) residue with an epsilon-amino group and RGDLLG is not. These two RGD peptides enhanced cell proliferation, but RGDLLQ significantly enhanced the proliferation compared with RGDLLG (p<0.05). These results suggest that T-Gase-mediated incorporation of cell adhesion factors into gelatin matrices enhanced cell proliferation and this novel biomaterial is a potent tool for wound dressing or tissue engineering.

Journal Article↗

PubMed2002

Effective solitary hyperthermia treatment of malignant glioma using stick type CMC-magnetite. In vivo study.

Various kinds of hyperthermic treatment for malignant glioma had been inhibited due to both their incomplete feverish action and strict cooling effect of the brain. The author shows an effective results of hyperthermia for the treatment of malignant glioma in an in vivo study using stick type carboxymethylcellulose (CMC)-magnetite, a newly manufactured magnetite-product. A stick type CMC-magnetite, containing magnetite particles, was inserted into the T-9 glioma in the rat brain stereotactically, and the rats were exposed to an alternative magnetic field (AMF). The application time of AMF, which measured 88.9 kHz and 380 Oe, was 30 min a day. The rats were divided into three groups: three AMF applications (group I), one AMF application (group II) and no application but only injection of CMC-magnetite (control). As a result, the mean survival in days of these three groups measured 44.2+/-10.9 (group I), 17.0+/-1.5 (group II) and 14.4+/-1.5 (control). This investigation showed both significant effectiveness in attacking malignant glioma and significant prolonging of the survival time in rats. It is also a characteristic feature of the magnetite particles to spread through the tumor diffusely after three applications of AMF. This feature seemed to be one of the main factors that caused greater hyperthermic effect on glioma in this study. This method of hyperthermic treatment could be a useful strategy in the treatment of malignant glioma.

Animals↗

PubMed2002

Effect of functional magnetic particles on radiofrequency capacitive heating: an in vivo study.

Specific heating of magnetic particles in radiofrequency (RF) capacitive hyperthermia and its hyperthermic effect were investigated in an in vivo study. Magnetite cationic liposomes (MCLs) were injected into a rat tumor on the femur and 8 MHz-RF capacitive heating was applied to the rat under "mild heating" conditions. Although the input power of RF capacitive heating was low under the same power conditions, the MCLs-injected tumor was heated over 43 degrees C, whereas it was only heated to 41 degrees C in the case of the rats not injected with MCLs. A necrotic area in the tumor was observed in the heated rats. From the results of histological observation of the removed tissue, the necrotic area in the MCLs-injected tumor was wider than that in MCLs-free tumor. Complete tumor suppression was observed in 71% (5 / 7) of MCLs-injected rats, and the hyperthermic effect was greatly improved by the MCLs.

Animals↗

PubMed2002

Functional magnetic particles for medical application.

Magnetic particles for medical applications have been developed by many researchers. Since magnetic particles have unique magnetic features not present in other materials, they can be applied to special medical techniques. Separation, immunoassay, magnetic resonance imaging (MRI), drug delivery, and hyperthermia are enhanced by the use of magnetic particles. Magnetite cationic liposomes (MCLs), one of the group of cationic magnetic particles, can be used as carriers to introduce DNA into cells since their positively charged surface associates with the negatively charged DNA. They can also be used as heat mediators for cancer therapy. Magnetic particles conjugated with tumor-specific antibodies have enabled tumor-specific contrast enhancement in MRI. In addition, antibody-conjugated magnetic particles were shown to target renal cell carcinoma cells, and are applicable to the hyperthermic treatment of carcinomas. The use of magnetic particles with their unique features will further improve medical techniques.

Journal Article↗