RESUMO
In the present work, two strategies were elaborated to surface-functionalize implantable polyimide sheets. In the first methodology, cross-linkable vinyl groups were introduced on the polyimide surface using aminopropylmethacrylamide. In the second approach, a reactive succinimidyl ester was introduced on the surface of PI. Using the former approach, the aim is to apply a vinyl functionalized biopolymer coating. In the latter approach, any amine containing biopolymer can be immobilized. The foils developed were characterized in depth using a variety of characterization techniques including atomic force microscopy, static contact angle measurements, and X-ray photoelectron spectroscopy. The results indicated that both modification strategies were successful. The subcutaneous implantation in mice indicated that both modification strategies resulted in biocompatible materials, inducing only limited cellular infiltration to the surrounding tissue.
Assuntos
Materiais Biocompatíveis/química , Medicina Regenerativa , Resinas Sintéticas/química , Acrilamidas/química , Animais , Materiais Biocompatíveis/efeitos adversos , Reagentes de Ligações Cruzadas/química , Citocinas/sangue , Feminino , Implantes Experimentais/efeitos adversos , Masculino , Fenômenos Mecânicos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Força Atômica , Estrutura Molecular , Espectroscopia Fotoeletrônica , Resinas Sintéticas/efeitos adversos , Propriedades de SuperfícieRESUMO
Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzymes. For this reason, they are employed as insecticides for agricultural, gardening and indoor pest control. The biological function of the ChE enzymes is well known and has been studied since the beginning of the XXth century; in particular, acetylcholinesterase (AChE, E.C. 3.1.1.7) is an enzyme playing a key role in the modulation of neuromuscular impulse transmission. However, in the past decades, there has been increasing interest concerning its role in regulating non-neuromuscular cell-to-cell interactions mediated by electrical events, such as intracellular ion concentration changes, as the ones occurring during gamete interaction and embryonic development. An understanding of the mechanisms of the cholinergic regulation of these events can help us foresee the possible impact on environmental and human health, including gamete efficiency and possible teratogenic effects on different models, and help elucidate the extent to which OP exposure may affect human health. The chosen organophosphates were the ones mainly used in Europe: diazinon, chlorpyriphos, malathion, and phentoate, all of them belonging to the thionophosphate chemical class. This research has focused on the comparison between the effects of exposure on the developing embryos at different stages, identifying biomarkers and determining potential risk factors for sensitive subpopulations. The effects of OP oxonisation were not taken into account at this level, because embryonic responses were directly correlated to the changes of AChE activity, as determined by histochemical localisation and biochemical measurements. The identified biomarkers of effect for in vitro experiments were: cell proliferation/apoptosis as well as cell differentiation. For in vivo experiments, the endpoints were: developmental speed, size and shape of pre-gastrula embryos; developmental anomalies on neural tube, head, eye, heart. In all these events, we had evidence that the effects are mediated by ion channel activation, through the activation/inactivation of acetylcholine receptors (AChRs).
Assuntos
Colinesterases/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Compostos Organofosforados/farmacologia , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fatores de TempoRESUMO
To understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are required. Therefore, the creation of 3-dimensional (3D) cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo. Here we have characterized a stable contracting in vitro tissue model, which consists of embryonic chicken cardiomyocytes. For establishing a cell-based test system, the 3D in vitro cardiomyocyte spheres were characterized according to messenger RNA expression of special cardiac cell types and protein expression pattern of functional markers such as connexin-43. Finally, the in vitro spheroid model was used for investigating the effect of isoproterenol, a *-adrenergic receptor agonist, on the contractibility mediated by the ligand receptor interaction.
Assuntos
Miócitos Cardíacos/citologia , Esferoides Celulares/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Embrião de Galinha , Conexina 43/metabolismo , Desmina/metabolismo , Avaliação Pré-Clínica de Medicamentos , Eletrofisiologia , Isoproterenol/farmacologia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Miócitos Cardíacos/efeitos dos fármacos , RNA/isolamento & purificação , Esferoides Celulares/efeitos dos fármacosRESUMO
One major problem in cancer therapy is the immortality of tumor cells showing an active telomerase, which is responsible for the elongation of the telomeres after each cellular division and the knocking down of apoptotic suppressors. A further phenomenon occurring during cancer therapies is the problem of multicellular resistance. To develop therapeutic anticancer approaches inducing cellular apoptosis, gene-modified biological in vitro systems were established and evaluated for drug screening in a capillary system for a real-time, impedimertic monitoring. Multicellular spheroids of the human breast cancer cell line T-47D clone 11 were transfected with 1) antisense caspase-3 cDNA expression vectors for knocking down the main cell death molecule and 2) sense Bcl-xl cDNA expression vectors for overexpressing the apoptotic suppressor, resulting in more aggressive tumor models. These gene-modified tumor spheroids less sensitive for apoptosis were developed for screening drugs such as methotrexate in tumor spheroid-based biosensor systems via impedance spectroscopy. In this report, it is demonstrated that this could successfully exhibit that this real-time monitoring system with tumor spheroids positioned in a capillary system with a 4-electrode configuration is the most efficient high-content screening module for impedimetric measurements of physiological alterations during gene modification and drug application.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/química , Neoplasias da Mama/tratamento farmacológico , Modelos Biológicos , Esferoides Celulares/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias da Mama/enzimologia , Caspase 3/biossíntese , Caspase 3/genética , Inibidores de Caspase , Linhagem Celular Tumoral , Feminino , Humanos , Metotrexato/farmacologia , Esferoides Celulares/enzimologia , Proteína bcl-X/biossíntese , Proteína bcl-X/genéticaRESUMO
Newer techniques are required to identify atherosclerotic lesions that are prone to rupture. Electric impedance spectroscopy (EIS) can characterize biological tissues by measuring the electrical impedance over a frequency range. We tested a newly designed intravascular impedance catheter (IC) by measuring the impedance of different stages of atherosclerosis induced in an animal rabbit model. Six female New Zealand White rabbits were fed for 17 weeks with a 5% cholesterol-enriched diet to induce early forms of atherosclerotic plaques. All aortas were prepared from the aortic arch to the renal arteries and segments of 5-10 mm were marked by ink spots. A balloon catheter system with an integrated polyimide-based microelectrode structure was introduced into the aorta and the impedance was measured at each spot by using an impedance analyzer. The impedance was measured at frequencies of 1 kHz and 10 kHz and compared with the corresponding histomorphometric data of each aortic segment.Forty-four aortic segments without plaques and 48 segments with evolving atherosclerotic lesions could be exactly matched by the histomorphometric analysis. In normal aortic segments (P0) the change of the magnitude of impedance at 1 kHz and at 10 kHz (|Z|(1 kHz) - |Z|(10 kHz), = ICF) was 208.5 +/- 357.6 Omega. In the area of aortic segments with a plaque smaller than that of the aortic wall diameter (PI), the ICF was 137.7 +/- 192.8 Omega. (P 0 vs. P I; p = 0.52), whereas in aortic segments with plaque formations larger than the aortic wall (PII) the ICF was significantly lower -22.2 +/- 259.9 Omega. (P0 vs. PII; p = 0.002). Intravascular EIS could be successfully performed by using a newly designed microelectrode integrated onto a conventional coronary balloon catheter. In this experimental animal model atherosclerotic aortic lesions showed significantly higher ICF in comparison to the normal aortic tissue.
Assuntos
Aterosclerose/diagnóstico , Cateterismo , Impedância Elétrica , Animais , Aterosclerose/diagnóstico por imagem , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Feminino , Coelhos , Análise Espectral , UltrassonografiaRESUMO
The early development of sea urchins has been thoroughly studied since the beginning of the 20th century thanks to the particular features of the model involving cell signalling, making it easy to follow the complex cell-to-cell interactions that lead to development. In this chapter, the prominent role of cell-to-cell communication in developmental events is discussed, as well as the role of intracellular ion changes that are in turn regulated by signal molecules belonging to the cholinergic system. The results seem to indicate that the zygote stage is the most suitable to study the role of the cholinergic system, as at this stage, a calcium spike can be evoked by exposure to acetylcholine (ACh) or to muscarinic drugs, at any time before the nuclear breakdown. The described outcomes also open a path to a new way of considering biomarkers. In fact, most environmental factors have the capacity to interfere with the cholinergic system: stress, wounds, inflammation and pollution in general. In particular, this offers a way to investigate the presence in the environment and the degree of aggressiveness of neurotoxic contaminants, such as organophosphate and carbamate pesticides, largely used in European countries for many purposes, including agricultural pest control and medical treatment. These drugs exert their function by interfering with the regulation of the cholinergic system and the consequent electrical events. Thus, the sea urchin zygote could represent a reliable model to be used in biosensors with the capacity to translate the effect of neurotoxic pesticides, and generally of stress-inducing contaminants, in living cell responses, such as electrical responses.
Assuntos
Desenvolvimento Embrionário , Monitoramento Ambiental/métodos , Poluentes Ambientais/análise , Ouriços-do-Mar/fisiologia , Transdução de Sinais , Acetilcolina/metabolismo , Animais , Técnicas Biossensoriais , Comunicação Celular , Poluentes Ambientais/toxicidade , Fertilização , Modelos Biológicos , Neurotoxinas/metabolismoRESUMO
Marker free techniques are needed for the construction of efficient tissue-based test and sensor systems. In principle biological tissue can be characterized by impedance spectroscopy. In this paper we investigate by simulation how sensitive parameters of small biological tissue samples can be determined by impedance spectroscopy under optimal measurement conditions. Further, we experimentally evaluate whether the effects of different clinical relevant radio therapy variants on 3D in vitro tumor models are determinable and distinguishable by impedance spectroscopy using a tissue-based test system. The simulations demonstrate that changes in tissue parameters related to the extracellular space are determinable with a high sensitivity. The experiments show that the effect of different radiation dose levels on 3D in vitro tumor models can be determined and distinguished by using a capillary measurement system and impedance spectroscopy. These results are relevant for the development of tissue-based test and sensor systems using impedance spectroscopy to evaluate personalized therapy variants or new therapy approaches.
RESUMO
The progress in cellular engineering offers novel approaches for anti-cancer therapies. To investigate the effectiveness of potential therapies efficient screening methods are required. We propose an impedance measurement system which enables the use of multicellular spheroid models in bioelectronic screening systems either for non destructive life-time diagnostic or anti-cancer therapies. A biohybrid sensor system is created comprising gene-manipulated T47D clone 11 breast carcinoma spheroids positioned hydrodynamically in a capillary system with electrodes. A novel approach employing an antisense-5'butyrylcholinesterase expression system is probed on reaggregated tumor cells under simulated microgravity, inhibiting the gene transcription and translation of the embryonic proliferation marker butyrylcholinesterase expressed in different tumor types. Alterations in the morphology of cell aggregates e.g. apoptosis or necrosis can be detected by impedance spectroscopy monitoring the electric behavior of membranes and extracellular space with a high resolution and reproducibility. The hydrodynamic positioning of 3D in vitro cell aggregates and the short time for the measurements represent an innovative method for a synchronized multicapillary screening system. The combination of the measuring system with a bioreactor enables cyclic life time recordings of impedance spectra for monitoring the cell aggregate properties for a long period.
Assuntos
Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Adesão Celular , Contagem de Células , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , TransfecçãoRESUMO
To investigate the effectiveness of potential anticancer therapeutics or therapies, efficient screening methods are required. On the one hand, multicellular 3D aggregates (spheroids) are a powerful in vitro model for simulating the in vivo situation and on the other hand, planar electrode structures are generally highly suitable for automation and parallel testing. Here, the detection of the effect of active substances on spheroids positioned on electrodes of substrate integrated electrode arrays is exemplarily investigated. As a 3D tissue model a reaggregation system of T47D clone 11 tumor cells is used. The effect of cytotoxins (DMSO, Triton X-100) on spheroids can be detected by recording the effective impedance of planar electrodes covered by spheroids. The equivalent circuit model parameter of electrodes covered by cytotoxin treated spheroids are determined from recorded impedance spectra and compared to the parameter of electrodes covered by control spheroids as well as not covered electrodes. Spheroids on electrodes mainly influence the electrode impedance in the frequency range of 10 kHz to 1 MHz. The results are discussed in view of an optimal electrode/spheroid-interface for sensing the effects of therapeutics with high sensitivity.
Assuntos
Técnicas Biossensoriais , Análise de Sequência com Séries de Oligonucleotídeos , Algoritmos , Dimetil Sulfóxido/química , Impedância Elétrica , Eletrodos , Humanos , Modelos Teóricos , Octoxinol/química , Espectrofotometria , Células Tumorais CultivadasRESUMO
For cell biosensors and for studying neural networks using planar electrode substrates, a suitable technique for positioning single cells on electrodes was needed. We reported a new method for fast and efficient positioning of single cells on ring electrodes by controlled suction through holes. We described the microfabrication of electrode substrates with microholes and the cell positioning procedure. L929 cells and Neuro 2A cells could be positioned in parallel without cell damage.