RESUMEN
Cell spheroid culture can recapitulate the tissue microstructure and cellular responses in vivo. While there is a strong need to understand the modes of toxic action using the spheroid culture method, existing preparation techniques suffer from low efficiency and high cost. Herein, we developed a metal stamp containing hundreds of protrusions for batch bulk preparation of cell spheroids in each well of the culture plates. The agarose matrix imprinted by the stamp can form an array of hemispherical pits, which facilitated the fabrication of hundreds of uniformly sized rat hepatocyte spheroids in each well. Chlorpromazine (CPZ) was used as a model drug to investigate the mechanism for drug induced cholestasis (DIC) by agarose-stamping method. Hepatocyte spheroids showed a more sensitive detection of hepatotoxicity compared to 2D and Matrigel-based culture systems. Cell spheroids were also collected for staining of cholestatic protein and showed a CPZ-concentration-dependent decrease of bile acid efflux related proteins (BSEP and MRP2) and tight junction (ZO-1). In addition, the stamping system successfully delineated the DIC mechanism by CPZ that may be associated with the phosphorylation of MYPT1 and MLC2, two central proteins in the Rho-associated protein kinase pathway (ROCK), which were significantly attenuated by ROCK inhibitors. Our results demonstrated a large-scale fabrication of cell spheroids by the agarose-stamping method, with promising benefits for exploring the mechanisms for drug hepatotoxic responses.
Asunto(s)
Colestasis , Esferoides Celulares , Ratas , Animales , Sefarosa/toxicidad , Sefarosa/metabolismo , Esferoides Celulares/metabolismo , Hepatocitos/metabolismo , Células Cultivadas , Colestasis/inducido químicamente , Colestasis/metabolismoRESUMEN
Marine polysaccharides or oligosaccharides have potential to promote wound healing due to their biocompatibility and physicochemical properties. However, microbial infection delays wound healing process, and novel antimicrobial wound dressings are urgently needed. Here, agarose oligosaccharides (AGO) obtained from marine red algae were used as a reducing and stabilizer for green synthesis of silver nanoparticles (AgNPs), and further successfully connected with odorranain A (OA), one of antimicrobial peptides (AMPs), to obtain a novel composite nanomaterial (AGO-AgNPs-OA). Transmission electron microscopy (TEM) and Malvern particle size analyzer showed that AGO-AgNPs-OA was spherical or elliptic with average size of about 100 nm. Circular dichroism (CD) spectroscopy showed that AGO-AgNPs stabilized the α-helical structure of OA. AGO-AgNPs-OA showed stronger anti-bacterial activities than AGO-AgNPs, and had good biocompatibility and significant promoting effect on wound healing. Our data suggest that AMPs conjugated marine oligosaccharides and AgNPs may be effective and safe antibacterial materials for wound therapy.
Asunto(s)
Antibacterianos/uso terapéutico , Antifúngicos/uso terapéutico , Vendajes , Nanopartículas del Metal/uso terapéutico , Sefarosa/química , Cicatrización de Heridas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/toxicidad , Antifúngicos/química , Antifúngicos/toxicidad , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Péptidos Catiónicos Antimicrobianos/toxicidad , Bacterias/efectos de los fármacos , Candida albicans/efectos de los fármacos , Línea Celular Tumoral , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Nanopartículas del Metal/química , Nanopartículas del Metal/toxicidad , Pruebas de Sensibilidad Microbiana , Oligosacáridos/síntesis química , Oligosacáridos/química , Oligosacáridos/toxicidad , Ratas Sprague-Dawley , Rhodophyta/química , Sefarosa/síntesis química , Sefarosa/toxicidad , Plata/química , Plata/uso terapéutico , Plata/toxicidad , Piel/efectos de los fármacosRESUMEN
Conventional agaroses with high gelling temperature are limited to apply to the field of drug delivery. In this study, ß-cyclodextrin (ßCD) functionalized agarose (CFA) with low gelling temperature was successfully prepared from ethylenediamine-functionalized agarose using mono-succinyl ßCD. The gelling temperature of CFA dramatically decreased to 26.7⯰C from 65⯰C and the melting temperature declined from 95⯰C to 66.1⯰C. Upon drug loading, CFA can be used at 30⯰C because of its low gelling temperature compared to agarose. CFA gel could be used both for bovine serum albumin as a full release, and for the doxorubicin (DOX) for sustained release, via inclusion complexation of ßCD. Furthermore, cytotoxicity tests revealed that CFA was noncytotoxic. DOX in the CFA gel could retain the anti-cancer activity. Newly synthesized CFA with low gelling temperature offer a new means for the development of hydrogel-based delivery systems for a variety of therapeutic drugs.
Asunto(s)
Portadores de Fármacos/química , Hidrogeles/química , Sefarosa/química , beta-Ciclodextrinas/química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Bovinos , Doxorrubicina/química , Doxorrubicina/farmacología , Portadores de Fármacos/síntesis química , Portadores de Fármacos/toxicidad , Liberación de Fármacos , Células HEK293 , Células HeLa , Humanos , Hidrogeles/síntesis química , Hidrogeles/toxicidad , Sefarosa/síntesis química , Sefarosa/toxicidad , Albúmina Sérica Bovina/química , Temperatura de Transición , beta-Ciclodextrinas/síntesis química , beta-Ciclodextrinas/toxicidadRESUMEN
Bone loss associated with skeletal trauma or metabolic diseases often requires bone grafting. In such situations, a biomaterial is necessary for migrated cells to produce new tissue. In this study, agarose-chitosan was implanted in the femoral condyle of New Zealand White rabbits that were divided into three groups: Group I was used as control; Groups II and III were used as implanted tissue with agarose-chitosan and presenting empty defects, respectively. This study evaluated the agarose-chitosan biocompatibility by determining the in vivo genotoxicity, oxidative stress balance that correlated with the hardness mechanical property. Moreover, the histopathological and quantitative elements analyzed by using inductively coupled plasma optical emission spectrometry were determined. After 30 days of implantation, the in vivo analysis of genotoxicity showed that agarose-chitosan did not induce chromosome aberration or micronucleus damage. A significant decrease in thiobarbituric and acid-reactive substance was observed after agarose-chitosan implantation in the bone tissue. Superoxide dismutase, catalase and glutathione peroxidase were significantly enhanced in agarose-chitosan-treated group compared with that of control group. A negative correlation coefficient of the mechanical property with malonyldialdehyde level was detected (R = -0.998). The histological study exhibited a significantly increased angiogenesis and newly formed tissue. No presence of inflammatory process, necrotic or fibrous tissue was detected. Major and trace elements such as Ca, P, Zn, Mg and Fe were increased significantly in the newly formed bone. These findings show that agarose-chitosan biomaterial implantation might be effective for treating trauma and bone regeneration.
Asunto(s)
Regeneración Ósea/efectos de los fármacos , Sustitutos de Huesos , Quitosano , Sefarosa , Ingeniería de Tejidos/métodos , Animales , Fenómenos Biofísicos , Células de la Médula Ósea , Sustitutos de Huesos/química , Sustitutos de Huesos/farmacología , Sustitutos de Huesos/toxicidad , Células Cultivadas , Quitosano/química , Quitosano/farmacología , Quitosano/toxicidad , Daño del ADN/efectos de los fármacos , Fémur/citología , Fémur/efectos de los fármacos , Fémur/metabolismo , Dureza , Ensayo de Materiales , Pruebas de Mutagenicidad , Estrés Oxidativo/efectos de los fármacos , Conejos , Sefarosa/química , Sefarosa/farmacología , Sefarosa/toxicidadRESUMEN
Hybrid agarose hydrogels embedded with pH-responsive diblock copolymers micelles were developed to achieve functional hydrogels capable of stimulus-triggered drug release. Specifically, a well-defined poly(ethylene oxide) (PEO)-based diblock copolymer, PEO-b-poly(2-(N,N-diisopropylamino)ethyl methacrylate) (PEO(113)-b-PDPAEMA(31), where the subscripts represent the degrees of polymerization of two blocks), was synthesized by atom transfer radical polymerization. PDPAEMA is a pH-responsive polymer with a pKa value of 6.3. The PEO(113)-b-PDPAEMA(31) micelles were formed by a solvent-switching method, and their pH-dependent dissociation behavior was investigated by dynamic light scattering and fluorescence spectroscopy. Both studies indicated that the micelles were completely disassembled at pH = 6.40. The biocompatibility of PEO(113)-b-PDPAEMA(31) micelles was demonstrated by in vitro primary cortical neural culture. Hybrid agarose hydrogels were made by cooling 1.0 wt % agarose solutions that contained various amounts of PEO(113)-b-PDPAEMA(31) micelles at either 2 or 4 °C. Rheological measurements showed that the mechanical properties of gels were not significantly adversely affected by the incorporation of diblock copolymer micelles with a concentration as high as 5.0 mg/g. Using Nile Red as a model hydrophobic drug, its incorporation into the core of diblock copolymer micelles was demonstrated. Characterized by fluorescent spectroscopy, the release of Nile Red from the hybrid hydrogel was shown to be controllable by pH due to the responsiveness of the block copolymer micelles. Based on the prominent use of agarose gels as scaffolds for cell transplantation for neural repair, the hybrid hydrogels embedded with stimuli-responsive block copolymer micelles could allow the controlled delivery of hydrophobic neuroprotective agents to improve survival of transplanted cells in tune with signals from the surrounding pathological environment.
Asunto(s)
Preparaciones de Acción Retardada/química , Polietilenglicoles/química , Ácidos Polimetacrílicos/química , Sefarosa/química , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Preparaciones de Acción Retardada/toxicidad , Colorantes Fluorescentes/química , Hidrogeles/química , Hidrogeles/toxicidad , Concentración de Iones de Hidrógeno , Cinética , Ensayo de Materiales , Micelas , Neuronas/efectos de los fármacos , Neuronas/fisiología , Oxazinas/química , Polietilenglicoles/toxicidad , Ácidos Polimetacrílicos/toxicidad , Sefarosa/toxicidad , Andamios del Tejido/química , ViscosidadRESUMEN
In order to improve bioactivity of agarose, we modified agarose by carboxylation and grafting dopamine. Under alkaline condition, carboxylated agarose was prepared using 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) oxidation system by oxidizing C(6) hydroxyl on D-galactose ring into carboxyl group, and the maximum value of the degree of carboxylation reached 30%. With the increase of the amount of oxidant, the molecular weight of the carboxylated agarose decreased to 4 kDa by gel permeation chromatography (GPC) measure. Carboxylated agarose reacted with dopamine through EDC condensation reaction to obtain agarose grafting dopamine (Ag-g-DA), and the grafting rate of dopamine was determined to be 9.3% by UV spectroscopy at 280 nm. The structures of these modified agaroses were determined by FT-IR and (13)C NMR. Both carboxylated agarose and Ag-g-DA showed no cytotoxicity and promoted cell-adhesiveness.
Asunto(s)
Ácidos Carboxílicos/química , Dopamina/química , Sefarosa/química , Sefarosa/farmacología , Células 3T3 , Animales , Carbodiimidas/química , Adhesión Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Óxidos N-Cíclicos/química , Dimetilaminas/química , Ratones , Oxidación-Reducción , Sefarosa/toxicidadRESUMEN
Agarose hydrogels containing aminopropyl triethoxy silane (APTS) have been prepared and evaluated as scaffolds for adhesion and proliferation of human mesenchymal stem cells (hMSCs). The preparation of the hydrogels involved the conventional melting of agarose in water followed by addition of APTS as functional group carrier. The resulting hydrogel supports have been studied by Fourier transformed infrared spectroscopy in order to get an insight into the hybrid molecular structure. X-ray photoelectron spectroscopy has been used for the analysis of the surface chemical composition of the hydrogels. It is deduced from these data that the resulting hybrid structure presents two phases with a clear tendency toward APTS surface segregation. Moreover, the observation of the desiccated hydrogel surfaces by atomic force microscopy shows that the films acquire a filament-mesh structure for increasing APTS content, while the pure agarose supports exhibit a granular structure. As a result of such a structure, the hydrogel surfaces show a hydrophobic behavior, as determined by water contact angle measurements. The biocompatibility of such platforms is supported by adhesion-proliferation assays performed with hMSCs. It is concluded that although adhesion is lower on APTS rich scaffolds, the proliferation rate on these surfaces is higher so that total number of proliferating cells does not significantly depend on APTS content in the hydrogels.
Asunto(s)
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/toxicidad , Ensayo de Materiales , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Sefarosa/toxicidad , Silanos/toxicidad , Adhesión Celular , Técnicas de Cultivo de Célula , Humanos , Propilaminas , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The hippocampus receives substantial input from the medial septum/diagonal band of broca (MS/DB) via the fibria-fornix (FF). Projections from the MS/DB innervate hippocampal interneurons that express alpha7 nicotinic receptors and regulate excitation in principal cell populations. In the present report we used stereotaxic surgery, whole-cell patch clamping, and immunohistochemical techniques to evaluate the effects of FF and MS/DB lesions on alpha7 nicotinic receptors in stratum radiatum interneurons. Focal somatic application of ACh (1 mM) evoked methyllycaconitine (MLA)-sensitive currents that were markedly reduced following aspirative lesions of the FF. Reductions in current amplitudes were prevented or restored to levels not significantly different from controls following in vivo treatment with the alpha7-selective agonist GTS-21, and GTS-21 treatment did not change current amplitudes measured in tissue from unlesioned animals. MS/DB injections of the selective cholinergic neurotoxin 192 IgG-saporin did not affect alpha7 receptor currents, although MS/DB ChAT and hippocampal AChE immunolabeling were significantly reduced. In contrast, kainic acid lesions of the MS/DB, potentially more selective for GABAergic projection neurons, produced significant reductions in current amplitudes. These findings are the first to show functional changes in alpha7 receptors following hippocampal denervation and suggest that MS/DB hippocampal innervation regulates functional aspects of hippocampal alpha7 receptors. The results confirm hippocampal alpha7 nicotinic receptors as viable therapeutic targets in diseases that involve degradation of the septohippocampal pathway and may indicate that GABAergic MS/DB hippocampal input plays a more substantial role in the regulation of alpha7 nicotinic receptor function than MS/DB hippocampal cholinergic input.
Asunto(s)
Hipocampo/citología , Interneuronas/fisiología , Receptores Nicotínicos/fisiología , Tabique del Cerebro/fisiología , Acetil-CoA C-Acetiltransferasa/metabolismo , Acetilcolina/farmacología , Aconitina/análogos & derivados , Aconitina/farmacocinética , Animales , Animales Recién Nacidos , Compuestos de Bencilideno/farmacología , Colina O-Acetiltransferasa/metabolismo , Estimulación Eléctrica/métodos , Agonistas de Aminoácidos Excitadores/toxicidad , Lateralidad Funcional/fisiología , Inmunohistoquímica/métodos , Técnicas In Vitro , Interneuronas/efectos de los fármacos , Ácido Kaínico/toxicidad , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacocinética , Técnicas de Placa-Clamp/métodos , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Sefarosa/análogos & derivados , Sefarosa/toxicidad , Tabique del Cerebro/lesiones , Tabique del Cerebro/metabolismo , Tabique del Cerebro/patología , Tritio/farmacocinética , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
BACKGROUND: Animal models are indispensable in order to investigate the mechanism of various diseases and to explore the counter measures for those disease states. Although there are several animal models of ischemic heart diseases, surgical interventions required to create myocardial ischemia sometimes give rise to a problem in the yield of model. This study describes a new technique for inducing myocardial ischemia in rats. METHODS AND RESULTS: A 0.014-inch guidewire was introduced via the carotid artery and selectively advanced into the coronary arteries under fluoroscopy. Transmural myocardial ischemia was confirmed by ST-segment elevation and by the appearance of left ventricular wall motion abnormalities on the echocardiogram. Reversibility of the wire-induced myocardial ischemia was demonstrated by complete resolution of both ST-segment elevation and wall motion abnormalities after removing the wire. CONCLUSION: Wire-induced myocardial ischemia was reproducible and is less invasive than conventional ischemic models in rats. This method is a powerful and useful tool for the investigation of ischemic heart disease in small animals.
Asunto(s)
Modelos Animales , Isquemia Miocárdica/etiología , Animales , Cateterismo Cardíaco/instrumentación , Enfermedad Coronaria/etiología , Electrocardiografía , Embolia/etiología , Masculino , Microesferas , Isquemia Miocárdica/diagnóstico por imagen , Isquemia Miocárdica/fisiopatología , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Sefarosa/administración & dosificación , Sefarosa/toxicidad , UltrasonografíaRESUMEN
Alveolar type II cells produce pulmonary surfactant and serve as the stem cell of the alveolar epithelium by proliferating and transforming into type I cells. The study of the differentiated function and proliferative capacity of type II cells in response to injury in vivo has been hindered by the complexity of the systemic response to injury. In vitro studies have in turn been limited by the impaired proliferative potential and loss of markers of differentiation in isolated type II cells maintained in culture. We describe an in vitro system in which type II cells proliferate spontaneously and simultaneously maintain differentiated characteristics. Other investigators have maintained slices of adult lung in culture after agarose infusion for up to 9 wk. To further develop this model for the study of epithelial cell differentiation and proliferation, we assessed epithelial differentiation, proliferative capacity, and regulation of cell-specific gene expression in slice explants of agarose-infused rat lungs. We prepared 1-mm-thick explants and maintained them in culture for up to 2 wk. Maintenance of differentiation was confirmed morphologically by light and electron microscopy, by the accumulation of epithelial cell-specific surfactant proteins, and by phospholipid analysis. Proliferative capacity was assessed by measuring [3H]thymidine incorporation in alveolar and small airway cells at baseline and in response to growth stimuli. Type II cell proliferation was inhibited in a dose-dependent manner by glucocorticoids. Glucocorticoids regulated RNA levels in explants in a manner similar to that seen in vivo and in fetal lung explants. The alveolar epithelium in adult lung slice explants maintains differentiated function and the ability to proliferate, thereby providing a useful system for the study of distal airway and alveolar cell homeostasis and response to injury.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Fosfolípidos/metabolismo , Proteolípidos/metabolismo , Alveolos Pulmonares/citología , Surfactantes Pulmonares/metabolismo , Animales , Autorradiografía , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , División Celular/genética , División Celular/fisiología , Células Cultivadas , Técnicas de Cultivo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/genética , Inmunohistoquímica , Microscopía Electrónica , Microscopía de Contraste de Fase , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/ultraestructura , Proteínas Asociadas a Surfactante Pulmonar , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sefarosa/toxicidadRESUMEN
Microlatex beads of homogenous size were made by polymerization of a mixture of acrylamide/bisacrylamide dispersed in a microemulsion. The microlatex was aggregated by dilution of the microemulsion in acrylamide solutions. The aggregates were then coagulated by polymerization at the interfaces of agarose beads circulating in a capillary tube containing paraffin oil. Biocompatibility was tested on isolated pituitary cells microencapsulated by this procedure.