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Myosin X is an unconventional actin-based molecular motor involved in filopodial formation, microtubule-actin filament interaction, and cell migration. Myosin X is an important component of filopodia regulation, localizing to tips of growing filopodia by an unclear targeting mechanism. The native α-helical dimerization domain of myosin X is thought to associate with antiparallel polarity of the two amino acid chains, making myosin X the only myosin that is currently considered to form antiparallel dimers. This study aims to determine if antiparallel dimerization of myosin X imparts selectivity toward actin bundles by comparing the motility of parallel and antiparallel dimers of myosin X on single and fascin-bundled actin filaments. Antiparallel myosin X dimers exhibit selective processivity on fascin-bundled actin and are only weakly processive on single actin filaments below saturating [ATP]. Artificial forced parallel dimers of myosin X are robustly processive on both single and bundled actin, exhibiting no selectivity. To determine the relationship between gating of the reaction steps and observed differences in motility, a mathematical model was developed to correlate the parameters of motility with the biochemical and mechanical kinetics of the dimer. Results from the model, constrained by experimental data, suggest that the probability of binding forward, toward the barbed end of the actin filament, is lower in antiparallel myosin X on single actin filaments compared to fascin-actin bundles and compared to constructs of myosin X with parallel dimerization.
Assuntos
Citoesqueleto de Actina/metabolismo , Miosinas/química , Miosinas/metabolismo , Multimerização Proteica , Animais , Bovinos , Cinética , Modelos Moleculares , Método de Monte Carlo , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
The pretranslocation complex of the ribosome can undergo spontaneous fluctuations of messenger RNA and transfer RNAs (tRNAs) between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classical and hybrid state tRNA positions have been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation. However, during multiple ongoing elongation cycles when both EF-G and ternary complexes are present, EF-G can bind to the pretranslocation complex much faster than the timescale of the classic-hybrid transitions. Using single-molecule fluorescence resonance energy transfer between adjacent tRNAs and between A-site tRNA and ribosomal protein L11, we found that the tRNAs do not fluctuate between the hybrid and classical states, but instead adopt a position with fluorescence resonance energy transfer efficiencies between those of the stalled classical and hybrid states.
Assuntos
Biossíntese de Proteínas , RNA de Transferência/genética , Ribossomos/genética , Ribossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fator G para Elongação de Peptídeos/metabolismo , Proteínas Ribossômicas/metabolismoRESUMO
Temporal sequences of fluorescence intensities in single-molecule experiments are often obtained from stacks of camera images. The dwell times of different macromolecular structural or functional states, correlated with characteristic fluorescence intensities, are extracted from the images and combined into dwell time distributions that are fitted by kinetic functions to extract corresponding rate constants. The frame rate of the camera limits the time resolution of the experiment and thus the fastest rate processes that can be reliably detected and quantified. However, including the influence of discrete sampling (framing) on the detected time series in the fitted model enables rate processes near to the frame rate to be reliably estimated. This influence, similar to the instrument response function in other types of instruments, such as pulsed emission decay fluorometers, is easily incorporated into the fitted model. The same concept applies to any temporal data that is low-pass filtered or decimated to improve signal to noise ratio.
Assuntos
Transferência Ressonante de Energia de Fluorescência/instrumentação , Modelos Teóricos , Cinética , Substâncias Macromoleculares/química , ProbabilidadeRESUMO
Targeted tissue ablation involving the anterior hippocampus is the standard of care for patients with drug-resistant mesial temporal lobe epilepsy. However, a substantial proportion continues to suffer from seizures even after surgery. We identified the fasciola cinereum (FC) neurons of the posterior hippocampal tail as an important seizure node in both mice and humans with epilepsy. Genetically defined FC neurons were highly active during spontaneous seizures in epileptic mice, and closed-loop optogenetic inhibition of these neurons potently reduced seizure duration. Furthermore, we specifically targeted and found the prominent involvement of FC during seizures in a cohort of six patients with epilepsy. In particular, targeted lesioning of the FC in a patient reduced the seizure burden present after ablation of anterior mesial temporal structures. Thus, the FC may be a promising interventional target in epilepsy.
Assuntos
Hipocampo , Neurônios , Animais , Hipocampo/patologia , Humanos , Camundongos , Neurônios/patologia , Epilepsia/patologia , Masculino , Optogenética , Feminino , Convulsões , Epilepsia do Lobo Temporal/fisiopatologia , Epilepsia do Lobo Temporal/patologia , AdultoRESUMO
People with paralysis express unmet needs for peer support, leisure activities, and sporting activities. Many within the general population rely on social media and massively multiplayer video games to address these needs. We developed a high-performance finger brain-computer-interface system allowing continuous control of 3 independent finger groups with 2D thumb movements. The system was tested in a human research participant over sequential trials requiring fingers to reach and hold on targets, with an average acquisition rate of 76 targets/minute and completion time of 1.58 ± 0.06 seconds. Performance compared favorably to previous animal studies, despite a 2-fold increase in the decoded degrees-of-freedom (DOF). Finger positions were then used for 4-DOF velocity control of a virtual quadcopter, demonstrating functionality over both fixed and random obstacle courses. This approach shows promise for controlling multiple-DOF end-effectors, such as robotic fingers or digital interfaces for work, entertainment, and socialization.
RESUMO
For most patients with type I diabetes, insulin therapy and glucose monitoring are sufficient to maintain glycemic control. However, hypoglycemia is a potentially lethal side effect of insulin treatment in patients who are glycemically labile or have hypoglycemia-associated autonomic failure [1]. For those patients, an alternative therapy is beta cell replacement via pancreas or islet transplantation. Pancreas transplants using cadaveric donor organs reduce insulin dependence but carry risks involved in major surgery and chronic immunosuppression. Islet transplantation, in which islets are isolated from donor pancreases and intravenously infused, require no surgery and can utilize islets isolated from pancreases unsuitable for whole organ transplantation. However, islet transplantation also requires immunosuppression, and standard steroid regimens may be toxic to beta cells [2]. The 2000 Edmonton Trial demonstrated the first long-term successful islet transplantation by using a glucocorticoid-free immunosuppressive regimen (sirolimus and tacrolimus). The Clinical Islet Transplantation (CIT) Consortium seeks to improve upon the Edmonton Protocol by using anti-thymocyte globulin (ATG) and TNFα antagonist (etanercept). The trials currently in progress, in addition to research efforts to find new sources of islet cells, reflect enormous potential for islet transplantation in treatment of type I diabetes.
Assuntos
Diabetes Mellitus Tipo 1/terapia , Transplante das Ilhotas Pancreáticas , Animais , Diabetes Mellitus Tipo 1/tratamento farmacológico , Humanos , Insulina/uso terapêutico , Transplante de PâncreasRESUMO
Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.
Assuntos
Proteínas Associadas a CRISPR , Animais , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Edição de Genes , Camundongos , RNA/metabolismoRESUMO
BACKGROUND: Precise targeting of cortical surface electrodes to epileptogenic regions defined by anatomic and electrophysiological guideposts remains a surgical challenge during implantation of responsive neurostimulation (RNS) devices. OBJECTIVE: To describe a hybrid fluoroscopic and neurophysiological technique for targeting of subdural cortical surface electrodes to anatomic regions with limited direct visualization, such as the interhemispheric fissure. METHODS: Intraoperative two-dimensional (2D) fluoroscopy was used to colocalize and align an electrode for permanent device implantation with a temporary in Situ electrode placed for extraoperative seizure mapping. Intraoperative phase reversal mapping technique was performed to distinguish primary somatosensory and motor cortex. RESULTS: We applied these techniques to optimize placement of an interhemispheric strip electrode connected to a responsive neurostimulator system for detection and treatment of seizures arising from a large perirolandic cortical malformation. Intraoperative neuromonitoring (IONM) phase reversal technique facilitated neuroanatomic mapping and electrode placement. CONCLUSION: In challenging-to-access anatomic regions, fluoroscopy and intraoperative neurophysiology can be employed to augment targeting of neuromodulation electrodes to the site of seizure onset zone or specific neurophysiological biomarkers of clinical interest while minimizing brain retraction.
Assuntos
Córtex Motor , Neurofisiologia , Eletrodos Implantados , Fluoroscopia , Humanos , Espaço SubduralRESUMO
Since ancient times we have attempted to facilitate hemostasis by application of topical agents. In the last decade, the number of different effective hemostatic agents has increased drastically. In order for the modern surgeon to successfully choose the right agent at the right time, it is essential to understand the mechanism of action, efficacy and possible adverse events as they relate to each agent. In this article we provide a comprehensive review of the most commonly used hemostatic agents, subcategorized as physical agents, absorbable agents, biologic agents, and synthetic agents. We also evaluate novel hemostatic dressings and their application in the current era. Furthermore, wholesale acquisition prices for hospitals in the United States are provided to aid in cost analysis. We conclude with an expert opinion on which agent to use under different scenarios.
Assuntos
Hemostáticos , Bandagens , Produtos Biológicos , Hemostáticos/uso terapêutico , Humanos , Guias de Prática Clínica como AssuntoRESUMO
In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100-200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100-200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.
Assuntos
Microscopia de Fluorescência , Microtecnologia/métodos , Nanoestruturas/química , Imagem Individual de Molécula , Alumínio/química , Coloides/química , Cobre/química , Cristalização , Análise de Elementos Finitos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ouro/química , Microesferas , Poliestirenos/química , PorosidadeRESUMO
The flexible, electropositive cavity of linear 1,4-diaryl-1,2,3-triazole oligomers provides a suitable host for complexation of various anions. The binding affinities for various combinations of oligomer and anion were determined by (1)H NMR titrations. Effective ionic radius is found to be a primary determinant of the relative binding interactions of various guests, with small but measurable deviations in the case of nonspherical anions. Solvent effects are significant, and the strength of the binding interaction is found to depend directly on the donor ability of the solvent. A picture emerges in which anion binding can be effectively interpreted in terms of a competition between two solvation spheres: one provided by the solvent and a second dominated by a folded cavity lined with electropositive 1,2,3-triazole CH protons. Implications for rigid macrocycles and other multivalent hosts are discussed.
Assuntos
Ânions/química , Polímeros/química , Triazóis/química , Sítios de Ligação , Compostos Macrocíclicos , Espectroscopia de Ressonância Magnética , Conformação Molecular , SolventesRESUMO
In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 µm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100-300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment.
Assuntos
Coloides/química , Nanotecnologia , Imagem Individual de Molécula/métodos , Espectrometria de Fluorescência/métodos , Alumínio/química , Vidro/química , Ouro/química , Nanoestruturas/química , Óptica e Fotônica/tendênciasRESUMO
Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the 'arginine anchor' on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA.
Assuntos
Arginina/metabolismo , Proteína Centromérica A/metabolismo , Centrômero/metabolismo , DNA/metabolismo , Nucleossomos/metabolismo , Animais , Centrômero/química , Centrômero/genética , Proteína Centromérica A/química , Proteína Centromérica A/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA/genética , Feminino , Humanos , Masculino , Camundongos , Nucleossomos/química , Nucleossomos/genética , Ligação Proteica , Domínios ProteicosRESUMO
The most promising alternatives to heart transplantation are left ventricular assist devices and artificial hearts; however, their use has been limited by thrombotic complications. To reduce these, sintered titanium (Ti) surfaces were developed, but thrombosis still occurs in approximately 7.5% of patients. We have invented a rapid-seeding technology to minimize the risk of thrombosis by rapid endothelialization of sintered Ti with human cord blood-derived endothelial cells (hCB-ECs). Human cord blood-derived endothelial cells were seeded within minutes onto sintered Ti and exposed to thrombosis-prone low fluid flow shear stresses. The hCB-ECs adhered and formed a confluent endothelial monolayer on sintered Ti. The exposure of sintered Ti to 4.4 dynes/cm for 20 hr immediately after rapid seeding resulted in approximately 70% cell adherence. The cell adherence was not significantly increased by additional ex vivo static culture of rapid-seeded sintered Ti before flow exposure. In addition, adherent hCB-ECs remained functional on sintered Ti, as indicated by flow-induced increase in nitric oxide secretion and reduction in platelet adhesion. After 15 day ex vivo static culture, the adherent hCB-ECs remained metabolically active, expressed endothelial cell functional marker thrombomodulin, and reduced platelet adhesion. In conclusion, our results demonstrate the feasibility of rapid-seeding sintered Ti with blood-derived hCB-ECs to generate a living antithrombotic surface.
Assuntos
Células Endoteliais/fisiologia , Coração Auxiliar/efeitos adversos , Sistemas Automatizados de Assistência Junto ao Leito , Trombose/prevenção & controle , Células Cultivadas , Sangue Fetal/citologia , Humanos , Adesividade Plaquetária , TitânioRESUMO
AIM: Peripheral blood-derived endothelial cells (pBD-ECs) are an attractive tool for cell therapies and tissue engineering, but have been limited by their low isolation yield. We increase pBD-EC yield via administration of the chemokine receptor type 4 antagonist AMD3100, as well as via a diluted whole blood incubation (DWBI). MATERIALS & METHODS: Porcine pBD-ECs were isolated using AMD3100 and DWBI and tested for EC markers, acetylated LDL uptake, growth kinetics, metabolic activity, flow-mediated nitric oxide production and seeded onto titanium tubes implanted into vessels of pigs. RESULTS: DWBI increased the yield of porcine pBD-ECs 6.6-fold, and AMD3100 increased the yield 4.5-fold. AMD3100-mobilized ECs were phenotypically indistinguishable from nonmobilized ECs. In porcine implants, the cells expressed endothelial nitric oxide synthase, reduced thrombin-antithrombin complex systemically and prevented thrombosis. CONCLUSION: Administration of AMD3100 and the DWBI method both increase pBD-EC yield.
Assuntos
Transplante de Células/métodos , Células Endoteliais/citologia , Engenharia Tecidual/métodos , Animais , Benzilaminas , Separação Celular , Ciclamos , Células Endoteliais/efeitos dos fármacos , Citometria de Fluxo , Compostos Heterocíclicos/administração & dosagem , Compostos Heterocíclicos/farmacologia , Modelos Animais , Reologia/efeitos dos fármacos , Estresse Mecânico , Sus scrofa , Transplante Autólogo , Veia Cava Inferior/efeitos dos fármacos , Veia Cava Inferior/fisiologiaRESUMO
The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12).
Assuntos
Técnicas Citológicas/instrumentação , Células Endoteliais/citologia , Células-Tronco/citologia , Animais , Técnicas Citológicas/métodos , Fracionamento por Campo e Fluxo/instrumentação , Fracionamento por Campo e Fluxo/métodos , Humanos , Resistência ao Cisalhamento , ViscosidadeRESUMO
Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs). By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating. After administration of 100 USP/kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope. We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface. Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.
Assuntos
Células Endoteliais/citologia , Teste de Materiais/métodos , Próteses e Implantes , Células-Tronco/citologia , Tromboembolia/etiologia , Titânio , Animais , Fenômenos Fisiológicos Cardiovasculares , Feminino , SuínosRESUMO
Titanium (Ti) is commonly utilized in many cardiovascular devices, e.g. as a component of Nitinol stents, intra- and extracorporeal mechanical circulatory assist devices, but is associated with the risk of thromboemboli formation. We propose to solve this problem by lining the Ti blood-contacting surfaces with autologous peripheral blood-derived late outgrowth endothelial progenitor cells (EPCs) after having previously demonstrated that these EPCs adhere to and grow on Ti under physiological shear stresses and functionally adapt to their environment under flow conditions ex vivo. Autologous fluorescently-labeled porcine EPCs were seeded at the point-of-care in the operating room onto Ti tubes for 30 min and implanted into the pro-thrombotic environment of the inferior vena cava of swine (n = 8). After 3 days, Ti tubes were explanted, disassembled, and the blood-contacting surface was imaged. A blinded analysis found all 4 cell-seeded implants to be free of clot, whereas 4 controls without EPCs were either entirely occluded or partially thrombosed. Pre-labeled EPCs had spread and were present on all 4 cell-seeded implants while no endothelial cells were observed on control implants. These results suggest that late outgrowth autologous EPCs represent a promising source of lining Ti implants to reduce thrombosis in vivo.
Assuntos
Prótese Vascular/efeitos adversos , Sangue , Modelos Animais de Doenças , Células Endoteliais/citologia , Células-Tronco/citologia , Trombose/prevenção & controle , Animais , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Suínos , Trombose/etiologia , TitânioRESUMO
Implantable and extracorporeal cardiovascular devices are commonly made from titanium (Ti) (e.g. Ti-coated Nitinol stents and mechanical circulatory assist devices). Endothelializing the blood-contacting Ti surfaces of these devices would provide them with an antithrombogenic coating that mimics the native lining of blood vessels and the heart. We evaluated the viability and adherence of peripheral blood-derived porcine endothelial progenitor cells (EPCs), seeded onto thin Ti layers on glass slides under static conditions and after exposure to fluid shear stresses. EPCs attached and grew to confluence on Ti in serum-free medium, without preadsorption of proteins. After attachment to Ti for 15 min, less than 5% of the cells detached at a shear stress of 100 dyne / cm(2). Confluent monolayers of EPCs on smooth Ti surfaces (Rq of 10 nm), exposed to 15 or 100 dyne/cm(2) for 48 h, aligned and elongated in the direction of flow and produced nitric oxide dependent on the level of shear stress. EPC-coated Ti surfaces had dramatically reduced platelet adhesion when compared to uncoated Ti surfaces. These results indicate that peripheral blood-derived EPCs adhere and function normally on Ti surfaces. Therefore EPCs may be used to seed cardiovascular devices prior to implantation to ameliorate platelet activation and thrombus formation.