RESUMEN
Alginate is a polysaccharide that can be crosslinked by divalent cations, such as calcium ions, to form a gel. Chemical modification is typically used to improve its cell adhesive properties for tissue engineering applications. In this study, alginates were modified with peptides containing RGD (arginine-glycine-aspartic acid) or PHSRN (proline-histidine-serine-arginine-asparagine) sequences from fibronectin to study possible additive and synergistic effects on adherent cells. Alginates modified with each peptide were mixed at different ratios to form gels containing various concentrations and spacing between the RGD and PHSRN sequences. When normal human osteoblasts (NHOsts) were cultured on or in the gels, the ratio of RGD to PHSRN was found to influence cell behaviors, especially differentiation. NHOsts cultured on gels composed of RGD- and PHSRN-modified alginates showed enhanced differentiation when the gels contained >33 % RGD-alginate, suggesting the relative distribution of the peptides and the presentation to cells are important parameters in this regulation. NHOsts cultured in gels containing both RGD- and PHSRN-alginates also demonstrated a similar enhancement tendency of calcium deposition that was dependent on the peptide ratio in the gel. However, calcium deposition was greater when cells were cultured in the gels, as compared to on the gels. These results suggest that modifying this biomaterial to more closely mimic the chemistry of natural cell adhesive proteins, (e.g., fibronectin) may be useful in developing scaffolds for bone tissue engineering and provide three-dimensional cell culture systems which more closely mimic the environment of the human body.
Asunto(s)
Alginatos/química , Matriz Extracelular/química , Osteoblastos/citología , Ingeniería de Tejidos/métodos , Materiales Biocompatibles , Células Cultivadas , Geles/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Andamios del Tejido/químicaRESUMEN
Currently, nonanimal methods of skin sensitization testing for various chemicals, biodegradable polymers, and biomaterials are being developed in the hope of eliminating the use of animals. The human cell line activation test (h-CLAT) is a skin sensitization assessment that mimics the functions of dendritic cells (DCs). DCs are specialized antigen-presenting cells, and they interact with T cells and B cells to initiate immune responses. Phenotypic changes in DCs, such as the production of CD86 and CD54 and internalization of MHC class II molecules, have become focal points of the skin sensitization test. In this study, we used h-CLAT to assess the effects of biodegradable polymers. The results showed that several biodegradable polymers increased the expression of CD54, and the relative skin sensitizing abilities of biodegradable polymers were PLLG (75 : 25) < PLLC (40 : 60) < PLGA (50 : 50) < PCG (50 : 50). These results may contribute to the creation of new guidelines for the use of biodegradable polymers in scaffolds or allergenic hazards.
RESUMEN
To facilitate research and development (R&D) and to expedite the review processes of medical devices, the Ministry of Health, Labor and Welfare (MHLW) and the Ministry of Economy, Trade and Industry (METI) founded a joint committee to establish guidance for newly emerging technology. From 2005 to 2007, two working groups held discussions on ventricular assist devices and total artificial hearts, including out-of-hospital programs, based on previous guidance documents and standards. Based on this discussion, the METI published the R&D Guidelines for innovative artificial hearts in 2007, and in 2008 the MHLW published a Notification by Director regarding the evaluation criteria for emerging technology.
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Corazón Artificial/normas , Corazón Auxiliar/normas , Animales , Seguridad de Productos para el Consumidor , Aprobación de Recursos/normas , Corazón Artificial/efectos adversos , Corazón Auxiliar/efectos adversos , Humanos , Japón , Diseño de Prótesis , Medición de RiesgoRESUMEN
A compositional gradient structure in hyaluronic acid (HA) and poly(N-isopropylacrylamide) (PIPAAm) blend film was self-organized from a homogeneous aqueous solution in a plasma-treated polystyrene dish (PTPSD), and the formation mechanisms of the gradient structure were studied by casting the same solution on PTPSD and a non-treated polystyrene dish (NTPSD) under ambient and vacuum conditions. The formation of a compositional gradient structure in HA/PIPAAm blend film was confirmed by scanning electron microscopy, energy dispersive X-ray (EDX) mapping analysis and step-scan photoacoustic Fourier transformed infrared spectroscopy (PAS-FT-IR) measurements. The EDX mapping measurements for Na element revealed that the HA component gradually decreases from the dish-side to the air-side of the film cast on PTPSD, while for the film cast on NTPSD no such obvious change was observed on the cross-section. Further studies on the films prepared on PTPSD and NPTPSD under ambient and vacuum conditions demonstrated that the hydrophilic interaction and the solvent evaporation rate were the most significant factors leading to the formation of a compositional gradient structure in the HA/PIPAAm blend system.
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Acrilamidas/química , Materiales Biocompatibles/química , Ácido Hialurónico/química , Membranas Artificiales , Polímeros/química , Resinas Acrílicas , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
We examined the effects of serum-free medium on the gene expression changes in human mesenchymal stem cells (hMSCs) during the in vitro culture using a DNA microarray analysis. In this study, we cultured hMSCs with two kinds of medium; 1) MSCGM (contain 10% fetal bovine serum) or 2) STK2 (serum-free medium developed for mesechymal stem cells multiplication), and compared hMSCs proliferation, cell morphology, and gene expression changes until 50 days culture. Expression analysis was performed with Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. hMSC proliferation was significantly higher in STK2 medium than in MSCGM medium. The cell morphology of hMSC cultured with STK2 was not significantly changed in 50 days culture. The gene expression changes in hMSCs during the in vitro culture were significantly higher in STK2 than in MSCGM. After 50 days culture, 1991 genes were significantly changed the expression levels compared with 3 days in STK2 but not MSCGM. The expressions of genes related to cell cycle, cancer, proliferation, and cell growth were significantly changed by STK2 for 50 days culture. It was also changed by STK2 that the expressions of genes related to the signaling pathways contain various growth factors, such as IGF-1, FGF, TGF-ß, EGF, proliferation, and cell cycle. These results suggest that STK2 may be useful to obtain an enough number of hMSC cells for tissue engineered medical devices in short-term, however, it should be recognized that STK2 would alter the expressions of genes related to a variety of signaling pathways in hMSC if the culture period would be extended to obtain a large number of cells.
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Medio de Cultivo Libre de Suero , Expresión Génica/genética , Células Madre Mesenquimatosas , Análisis de Secuencia por Matrices de Oligonucleótidos , Proliferación Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Transducción de Señal/genética , Factores de TiempoRESUMEN
The purpose of this study was to clarify the actual condition of 26 types of carcinogenic primary aromatic amines (PAAs) originated from azo dyes in commercial textile products in Japan. In the case of textiles made of various fibers of various colors, the fibers were separated by color and analyzed. A total of 86 textile products (117 samples) were analyzed. Twenty-one kinds of PAAs were detected in the samples and almost all the PAAs were detected at low concentrations. However, the concentrations of benzidine, 3,3'-dimethoxybenzidine, and 2,4-diaminotoluene (56-440 µg g⻹) in placemats made of cotton were found to exceed EU regulation limits of 30 µg g⻹. Although such placemats do not always come into contact with the user's skin, it is thought that they should be handled more carefully. Finally, 7 products (8 samples) contained PAAs at concentrations that exceeded the regulation limits. Two sample preparation methods (with and without solvent extraction) were performed on the same sample in order to compare the PAAs in samples in which it is difficult to separate the component materials, such as a cotton fabric that contained polyester fibers. In a comparison of the results obtained from the two methods, it was observed that the concentrations and/or kinds of PAAs detected in the samples were different. It was therefore thought that textile products that present this particular challenge should be analyzed by both methods.
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Aminas/análisis , Compuestos Azo/análisis , Colorantes/análisis , Hidrocarburos Aromáticos/análisis , Textiles/análisis , JapónRESUMEN
Neurotoxicities of dibutyltin (DBT), tin(II) octylate (OT), poly-L-lactides (PLLA, molecular weight [MW]=5000, PLLA 5000), PLLA without tin (MW=3000, PLLA 3000), PLLA with a large amount (590 ppm) of tin (S3), poly(glycolic acid-co-epsilon-caprolactone) oligomer (MW=6200, PGC oligomer), and poly(L-lactic acid-co-glycolic acid-co-epsilon-caprolactone) oligomer (MW=6400, PLGC oligomer) related to artificial dura mater were examined using the murine astrocyte cell line, CRL-2534. The indices were cell viability, glutamate concentration in the cell supernatant, and cell proliferation. Lower cell viability was observed among cells exposed to 0.5 microM DBT or 10 microg/ml of S3. There were no differences in cell viability of astrocytes exposed to OT, PLLA 5000, PLLA 3000, PGC oligomer, or PLGC oligomer. Mean glutamate concentration in the supernatant of cells exposed to 0.25 muM DBT was higher than that of the control after 2 h incubation. Lower mean concentration of glutamate in the supernatant of cells exposed to 5 microg/ml of S3 was observed after 2 h incubation. Cells exposed to 50 microg/ml of PGC oligomer had a higher mean concentration of glutamate in the supernatant. OT only inhibited cell proliferation at 100 microM. Proliferation of cells exposed to 0.25 microM or 0.5 microM DBT was inhibited, as was that of cells exposed to 100 microM OT, 50 microg/ml PLLA 5000, 50 microg/ml PLLA 3000, and 5 microg/ml S3, 5 d and 7 d after exposure. Although DBT does not reach levels that induced neurotoxicity in artificial dura mater, these results suggest that DBT is neurotoxic and PLLA toxicity increases with the increase in tin concentration.
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Astrocitos/efectos de los fármacos , Bioprótesis , Supervivencia Celular/efectos de los fármacos , Duramadre , Compuestos Orgánicos de Estaño/toxicidad , Poliésteres/toxicidad , Compuestos de Estaño/toxicidad , Implantes Absorbibles , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glutamatos/farmacología , Ratones , Compuestos Orgánicos de Estaño/química , Poliésteres/químicaRESUMEN
The international standard ISO 8124-3: 1997 "Safety of toys -Part 3: Migration of certain elements" and "Interim Enforcement Policy for Children's Metal Jewelry Containing Lead- 2/3/2005" by the U.S. Consumer Product Safety Commission (CPSC) to control the amount of eluted lead from metal accessories cannot be simply compared, because the acid extraction methods and the limit values are different from each other. Therefore acid extraction tests based on the ISO standard and the CPSC policy were conducted and the amounts of eluted lead from small metal products were compared between both tests. There was less eluted lead in the ISO method than in the CPSC method. Moreover, the amount of eluted lead in the ISO method did not even reach that of the first elution in the CPSC method. It became clear that the acid extraction test of the ISO standard was not as good as that of the CPSC policy, because of the difference in test conditions. In 16 small metal products, seven products were unsuitable for the ISO standard and 14 products were unsuitable for the CPSC policy; however, all these products were originally inapplicable to the ISO standard and the CPSC policy. The calculation grounds of the limit values were also different between the ISO standard and the CPSC policy. The standardization of an acid extraction test that simulates the lead elution to gastric juice is required so as to prevent adverse health effects in children due to their accidental ingestion of small metal products containing lead.
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Seguridad de Productos para el Consumidor/normas , Electroforesis Capilar/métodos , Joyas/análisis , Plomo/análisis , Metales/análisis , Juego e Implementos de Juego , Espectrometría por Rayos X/métodos , Ácido ClorhídricoRESUMEN
Fullerenes are condensed ring aromatic compounds with extended pi systems; they have unique cage structures. Current studies suggest that several fullerene derivatives have neuroprotective effects, and it is expected that fullerenes will be useful in drug delivery system and novel medical devices targeting the brain. However, little is known about the effects of fullerenes and its derivative on brain function. We examined the effect of fullerene(OH)24 on the central nervous system in this study. In a V79 colony assay, the IC50 of fullerene(OH)24 was 1.74 microg/ml. In an MTT assay, fullerene(OH)24 reduced proliferation of normal human astrocytes obviously. In an vivo study, 0.25 mg/kg(-1) of fullerene(OH)24 was injected into the lateral ventricle of rat brains. The intracerebral injection of fullerene(OH)24 remarkably decreased body weight and locomotor behavior of rats on day 1, but drastically increased locomotor behavior on day 7. The intracerebral injection of fullerene(OH)24 changed the monoamine concentration greatly on day 1 and slightly on day 30 after the injection. These results suggest that intracerebral injection of fullerene(OH)24 had strong and acute effects on the central nervous system, but that the effects were not permanent. In conclusion, we suggest that fullerene's derivative, fullerene(OH)24 had toxic effects on brain cells and that intracerebral injection of fullerene(OH)24 had acute harmful effects on brain monoamines neurotransmission and locomotor activity.
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Monoaminas Biogénicas/metabolismo , Química Encefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Fulerenos/farmacología , Actividad Motora/efectos de los fármacos , Neurotransmisores/metabolismo , Análisis de Varianza , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Conducta Animal/efectos de los fármacos , Encéfalo/citología , Encéfalo/metabolismo , Células CHO , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Humanos , Hidroxilación , Inyecciones Intraventriculares , Masculino , Ratas , Ratas Wistar , SolubilidadRESUMEN
A surface of biomaterials is known to affect the behavior of cells after their adhesion on the surface, indicating that surface characteristics of biomaterials play an important role in cell adhesion, proliferation, and differentiation. To assess the effects of functional groups on biomaterial surface, normal human osteoblasts (NHOsts) were cultured on surfaces coated with self-assembled monolayers (SAMs) containing various functional groups, and the adhesion, proliferation, differentiation, and gap junctional intercellular communication (GJIC) of the NHOsts were investigated. In the case of SAM with terminal methyl groups (hydrophobic surface), NHOst adhesion and proliferation was less prevalent. In contrast, NHOsts were adhered well on SAMs with hydroxyl, carboxyl, amino, phosphate, and sulfate group, which are relatively hydrophilic, their proliferation and differentiation level were dependent on the type of functional groups. Especially, when they were cultured on either SAMs with phosphate or sulfate group, both their alkaline phosphate activity and the calcium deposition by them were enhanced more than those cultured on a collagen-coated dish. More interestingly, GJIC of NHOsts, which has been reported to play a role in cell differentiation as well as homeostasis of cells, were not significantly different among the SAM surfaces tested. These suggest that a specific functional group on a material surface can regulate NHOst adhesion, proliferation, and differentiation via cell-functional group interaction without influencing their homeostasis.
Asunto(s)
Materiales Biocompatibles/química , Adhesión Celular , Diferenciación Celular , Proliferación Celular , Osteoblastos/fisiología , Materiales Biocompatibles/metabolismo , Comunicación Celular , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Conexina 43/genética , Conexina 43/metabolismo , Uniones Comunicantes/metabolismo , Homeostasis , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales , Estructura Molecular , Osteoblastos/citología , Propiedades de SuperficieRESUMEN
The use of tributyltin (TBT) and triphenyltin (TPT) in some household products are prohibited by "Act on the Control of Household Products Containing Harmful Substances" in Japan. In this study, methods for determination of TBT and TPT in water soluble paints and adhesives were developed by GC-MS. These compounds in paints and adhesives, which were mainly composed of vinyl acetate, urethane and acryl resins, and chloroprene rubber, were firstly extracted with HCl-acetone, and then extracted with hexane. On the other hand, the adhesive composed of natural rubber was firstly dispersed in water before acidification. The organotins were extracted with hexane from this solution and then these compounds were extracted with acetonitrile from hexane extract. These extracts were purified by a florisil cartridge column after ethyl-derivation with sodium tetraethylborate, and analyzed by GC-MS. The quantifications using deuterated compound of both organotins as surrogate standard were conducted, and good results were obtained. The recoveries were 81 to 118% and the coefficients of variation were 0.83 to 4.3% (TBT and TPT added; 5 microg/g). The method quantification limits were 0.0090 to 0.025 microg/g, which were lower than those of an official method. These methods were applied to monobutyltin (MBT), dibutyltin (DBT), monophenyltin (MPT), and diphenyltin (DPT). DBT and DPT in paints and adhesives were quantified, except for DPT in natural rubber. These methods were applied to commercial products. DBT was detected at low concentrations (t.r.-0.19 microg/g) in some paint samples, while TBT and TPT were not detected in all samples.
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Cromatografía de Gases y Espectrometría de Masas/métodos , Compuestos Orgánicos de Estaño/análisis , Pintura/análisis , Agua , Compuestos Orgánicos de Estaño/aislamiento & purificación , Solubilidad , Compuestos de Trialquiltina/análisis , Compuestos de Trialquiltina/aislamiento & purificaciónRESUMEN
To evaluate the role of particle size in cytotoxicity tests of nanomaterials (NMs), we exposed Chinese hamster cells to polystyrene (PS) spheres with defined diameters ranging from 0.1 to 9.2 microm. We found that the 4.45-microm PS particles were most cytotoxic while sizes 0.1 and 0.2 microm showed no cytotoxicity up to 1000 microg/ml. In the chromosome aberration test, the 4.45-microm PS particles induced polyploidy in a mass concentration-dependent manner in 24- and 48-h treatments. The 5.26-microm PS particles induced polyploidy only at 1000 microg/ml for 48 h. Next, we performed the cytotoxicity test with as-grown single walled carbon nanohorns (NHas). These were suspended in DMSO and then transferred into the culture medium followed by sonication. Six suspensions differently sonicated showed the same apparent toxicity, although the total particle size distributions differed. However, the sizes of NHas particles predicted to be most toxic from the experiments with PS particles, i.e. 1.01-4.47 microm constituted 40-60% of all particles in all six suspensions. The results suggest that the cytotoxicity of NMs in suspension depends on specific sizes of aggregates and therefore suspensions should be checked with regard to particle size distributions in assays of toxic effects. The uptake of particles into cells was confirmed by confocal microscopy.
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Bioensayo/métodos , Supervivencia Celular/efectos de los fármacos , Nanoestructuras/administración & dosificación , Nanoestructuras/ultraestructura , Pruebas de Toxicidad/métodos , Animales , Células CHO , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Tamaño de la PartículaRESUMEN
To apply human mesenchymal stem cells (hMSC) to regenerative medicines, it is necessary to multiply hMSC in vitro in a short period. In addition, it is desirable that the medium which is used for the hMSC multiplication is not supplemented with the serum, because the addition of the serum has risks of infection. In this study, we cultured hMSC with three kinds of medium used for multiplying hMSC (DMEM, MSCGM, STK2) and compared hMSC proliferation in each medium. As a result, it was confirmed that hMSC proliferation was significantly higher in STK2 medium which is a novel serum-free medium developed for hMSC multiplication. Moreover, we compared the hMSC proliferation in these media under the environment that assumed bone reproduction. When we cultured hMSC in each medium with hydroxyapatite (HAp), the proliferative inhibition by HAp depended on the additive amount, and the degree of the proliferative inhibition was different among the media but the lowest inhibitory effect was observed in STK2 medium.
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Proliferación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Durapatita/farmacología , Células Madre Mesenquimatosas/citología , Células Cultivadas , Depresión Química , Relación Dosis-Respuesta a Droga , Humanos , Estimulación QuímicaRESUMEN
The purpose of tumorigenicity testing, as applied not only to cell substrates used for viral vaccine manufacture but also stem cells used for cell-based therapy, is to discriminate between cells that have the capacity to form tumors and cells that do not. Therefore, tumorigenicity testing is essential in assessing the safety of these biological materials. Recently developed NOD/Shi-scid IL2Rg(null) (NOG) mice have been shown to be superior to NOD/Shi-scid (SCID) mice for xenotransplantation of both normal and cancerous cells. To select a suitable mouse strain as a xenogenic host for tumorigenicity testing, we compared the susceptibility of NOG (T, B, and NK cell-defective), SCID (T and B cell-defective), and the traditionally used nude (T cell-defective) mice to tumor formation from xenotransplanted HeLa S3 cells. When 10(4) HeLa S3 cells were subcutaneously inoculated into the flanks of these mice, the tumor incidence on day 22 was 10/10 (100%) in NOG, 2/10 (20%) in SCID, and 0/10 (0%) in nude mice. The subcutaneous tumors formed reproducibly and semiquantitatively in a dose-dependent manner. Unexpectedly, half of the NOG mice (5/10) that had been inoculated with a mere 10(1) HeLa S3 cells formed progressively growing subcutaneous tumors on day 78. We confirmed that the engrafted tumors originated from inoculated HeLa S3 cells by immunohistochemical staining with anti-HLA antibodies. These data suggest that NOG mice may be the best choice as a suitable strain for testing tumorigenicity.
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Predisposición Genética a la Enfermedad , Trasplante de Neoplasias , Animales , Pruebas de Carcinogenicidad/métodos , Femenino , Células HeLa/trasplante , Humanos , Masculino , Ratones , Ratones Endogámicos NOD/genética , Ratones Desnudos/genética , Ratones SCID/genética , Neoplasias Experimentales/patología , Tejido Subcutáneo/patología , Factores de Tiempo , Trasplante HeterólogoRESUMEN
Fullerenes are a family of carbon allotropes, molecules composed entirely of carbon. Fullerenes have been developed in various forms and functions and are expected to be used for novel medical materials targeting on brain. Information on cytotoxicity of fullerenes on brain function, however, is few; thus we examined the effect of fullerenes on the brain astrocytes in this study. We used fullerene [60], hydroxylated-fullerene [60], carboxylated-fullerene [60], dimalonilated-fullerene [60], carboxylated-carbon nanotube and amino-carbon nanotube. At first, we examined cytotoxicity of fullerenes by V79 colony assay. Fullerenes inhibited the cell growth in a concentration-dependent manner, but 50 percent growth inhibition concentrations were different among fullerene derivatives, which we used. Cytotoxicity of carbon nanotubes was stronger than that of fullerenes. Secondly, we performed the microtiter tetrazolium assay of normal human astrocytes and measured the effects of fullerenes on cell activity. Fullerenes and carbon nanotubes decreased mitochondrial activity. In addition to this, it was observed that fullerenes and nanotubes adhered to cells. These results suggest that fullerenes and carbon nanotubes have cytotoxicity and the effects are different from each other due to their side chain and steric forms. We expected that fullerenes and carbon nanotubes gave physical stress to cells and caused cytotoxicity. In conclusion, it was suggested that safety evaluation is needed for fullerenes and carbon nanotubes individually.
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Astrocitos/efectos de los fármacos , Encéfalo/citología , Fulerenos/toxicidad , Nanotubos de Carbono/toxicidad , Animales , Astrocitos/citología , División Celular/efectos de los fármacos , Células Cultivadas , Cricetinae , Relación Dosis-Respuesta a Droga , Fulerenos/química , Humanos , Nanotubos de Carbono/químicaRESUMEN
Astrocyte proliferation is strictly controlled during development and in the adult nervous system. In this study, we examined the role of sulfated hyaluronan (SHya) in the proliferation and differentiation of normal human astrocytes (NHAs). Cells were cultured with different concentrations of SHya for 7 days, and the number of viable cells and the presence of neural cell-specific genes were determined to assess their proliferation and development, respectively. With SHya, cell proliferation increased nonsignificantly. Furthermore, remarkable enhancing action by SHya on connexin-26, -32, and -43 gene expressions were observed during the culture of NHAs. It has been suggested that a fraction of NHAs have neural precursor activity that gives rise to astrocytes themselves, oligodendrocytes, and neurons. Our results clearly demonstrated that the expression of specific genes for neural precursor cells, astrocytes, neurons, and oligodendrocytes was significantly increased to 50 mug/mL in SHya-treated cultures when compared with that of the control culture. These findings suggest that SHya plays an important role in the proliferation and differentiation of NHAs and in the production of a novel material for tissue engineering.
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Astrocitos/citología , Conexinas/genética , Ácido Hialurónico/farmacología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Conexina 26 , Conexina 43/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ingeniería de Tejidos/métodos , Proteína beta1 de Unión ComunicanteRESUMEN
We investigated mRNA expression of c-myc and chromosome aberrations at the c-myc locus in the same passage number of human mesenchymal stem cells (hMSCs). To understand the sensitivity of mRNA expression and the induction of chromosome aberrations, we first tested them in hMSC and cancer cell lines (HeLa S3, HOS, and OUMS-27). The c-myc mRNA expressions in HeLa S3 and OUMS-27 were significantly higher than those in hMSC, but then those in HOS were not. On the other hand, c-myc aberrant cells detected by fluorescence in situ hybridization in HeLa S3, HOS, and OUMS-27 were significantly higher than that in hMSC. Both analyses were performed in hMSCs derived from five donors for the culture period of 50 days. In hMSCs from one donor, the frequency of c-myc aberrant cells significantly increased at 20 and 50 days respectively, and each mRNA expressions had a tendency to increase, but there is no significant change among 3, 20 and 50 days. In hMSCs from the others, both endpoints did not change for 50 days. For safe use of somatic stem cells in the regenerative medicine, the investigation of characteristic change of them during the in vitro culture is important. In the present study, we showed the mRNA expressions and chromosome aberrations of hMSCs in in vitro culture as the first step for establishing of safety evaluation of tissue engineered medical devices using normal hMSCs.
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Aberraciones Cromosómicas , Expresión Génica , Genes myc/genética , Células Madre Mesenquimatosas , Línea Celular Tumoral , Células Cultivadas , Humanos , ARN Mensajero , Medicina Regenerativa , Factores de Tiempo , Ingeniería de TejidosRESUMEN
Fullerenes are condensed ring aromatic compounds with extended pi systems and unique cage structures. Fullerenes are used for medical devices such as carbon nanotubes because they are very flexible and suitable for drug delivery systems. Recently, fullerene derivatives and tube-shaped materials have been used for neuroregeneration studies, and we expect that fullerenes and carbon nanotubes have potential uses as materials in novel medical devices targeting the brain. However, little information on the effects of fullerenes on brain function is available; thus, we examined the effects of [60]fullerene (C60) on the central nervous system in this study. In a V79 cell colony Asia, the IC50 of C60 was 1620 microg/ml. In an in vivo study, 0.25 mg/kg B.W. of C60 was injected into the lateral brain ventricle or abdominal cavity of rats. The intracerebral injection of C60 increased the locomotor behavior of the rats on days 1 and 30 after the injection. The intraperitoneal injection of C60 did not change the locomotor behavior of rats acutely, but it was decreased on day 30. The intracerebral injection of C60 affected monoamine concentrations in the rat brain. In particular, serotonin turnover rates were increased in the hypothalamus, cerebral cortex, striatum, and hippocampus, and dopamine turnover rates were increased in the hypothalamus, cerebral cortex, and striatum. The intraperitoneal injection of C60 decreased only the dopamine turnover rate in the hippocampus. These results suggest that intracerebral injection of C60 had different effects on the central nervous system than intraperitoneal injection. In conclusion, it was suggested that fullerene did not cross the blood-brain barrier. The intracerebral injection of C60 affected neurotransmission in the brain widely, and the monoamine dysbolism might be related to changes in locomotor activity.
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Encéfalo/efectos de los fármacos , Fulerenos/farmacología , Animales , Encéfalo/fisiología , Línea Celular , Cricetinae , Cricetulus , Dopamina/metabolismo , Fulerenos/administración & dosificación , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Masculino , Microinyecciones , Actividad Motora/efectos de los fármacos , Nanotubos de Carbono , Ratas , Ratas Wistar , Serotonina/metabolismo , Transmisión SinápticaRESUMEN
Human mesenchymal stem cells (hMSCs) are able to self-replicate and differentiate into a variety of cell types including osteoblasts, chondrocytes, adipocytes, endothelial cells, and muscle cells. It was reported that fibroblast growth factor-2 (FGF-2) increased the growth rate and multidifferentiation potentials of hMSCs. In this study, we investigated the genes involved in the promotion of osteogenic and chondrogenic differentiation potentials of hMSCs in the presence of FGF-2. hMSCs were maintained in the medium with FGF-2. hMSCs were harvested for the study of osteogenic or chondrogenic differentiation potential after 15 days' culture. To investigate osteogenic differentiation, the protein levels of alkaline phosphatase (ALP) and the mRNA expression levels of osteocalcin were measured after the induction of osteogenic differentiation. Moreover, the investigation for chondrogenic differentiation was performed by measuring the mRNA expression levels of type II and type X collagens after the induction of chondrogenic differentiation. The expression levels of ALP, type II collagen, and type X collagen of hMSCs cultured with FGF-2 were significantly higher than control. These results suggested that FGF-2 increased osteogenic and chondrogenic differentiation potentials of hMSCs. Furthermore, microarray analysis was performed after 15 days' culture in the medium with FGF-2. We found that the overall insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) signaling pathways were inactivated by FGF-2. These results suggested that the inactivation of IGF-I and TGF-beta signaling promotes osteogenic and chondrogenic differentiation potential of hMSCs in the presence of FGF-2.
RESUMEN
We demonstrated the effect of synthesized sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan, and basic fibroblast growth factor 2 (FGF-2) on normal human astrocytes (NHA) activity and its morphological transformation in vitro study. Astrocyte is a kind of glial cell and stellated astrocyte (activating astrocyte) supports axons network, neurons survival and synaptic plasticity. Treatment of SHya hardly affected NHA proliferation. However combination treatment of SHya and FGF-2 increased NHA proliferation. Treatment of SHya promoted transformation of normal astrocyte into a stella morphology (stellation) and combination treatment of SHya and FGF-2 promoted stellation than that of SHya only. Treatment of SHya increased glial fibrillary acidic protein (GFAP), nestin mRNA and GFAP protein expression in the stellated NHA. The cell-cell adhesion of NHA increased by treatment of SHya. Treatment of SHya increased heparin-binding trophic factors FGF-2, midkine, and some other trophic factors mRNA level in the NHA. These results suggested that the treatment of SHya promoted NHA activity due to enhancing neurotrophins production and the morphological transformation of NHA and the effect of SHya on astrocytes partly involved FGF-2 activity. These findings indicate that SHya may be involved in the astrocyte activity and support neurons survivals.