Este estudo foi financiado em parte pelo Nebraska NASA Space Grant e National Science Foundation, Grant No. 0926880.

1. Preparação do Navio Latex <ol><li> Fixar as duas extremidades do recipiente de látex para conexões de mangueira farpadas, que são fixados sobre uma bancada resistente. </li><li> Medir a área de interesse em látex vaso para determinar o campo de visão. A área de interesse para um teste de stent deve ser centrado entre os conectores de mangueira farpadas e incluem aproximadamente uma polegada de cada lado do stent, a fim de observar as estirpes fora da área do stent. </li><li> Gravar a distância entre a extremidade externa de uma ligação de mangueira farpado para o local central entre os conectores, que é também o centro aproximado do vaso de látex. Traduzir a distância no cateter através da medição a partir do centro do stent para cima do cateter. Em seguida, marcar o cateter com um marcador. </li><li> Remover o vaso de látex dos conectores de mangueira farpado. </li><li> Preparar o vaso de látex por pulverização da área de interesse, com um padrão de tinta de pulverização estocástica branco e preto ou marcaçãoa área de interesse com pontos aleatórios usando marcador permanente. Para amostras de menores dimensões e de padrão estocástica mais fina é necessária. </li></ol> 2. Sistema de Teste in vitro e calibração do sistema ARAMIS <ol><li> Seleccione o painel de calibração que é ligeiramente maior do que a área de interesse medida no passo 1. </li><li> Coloque o painel de calibração entre os conectores de mangueira farpadas na área de interesse, e assegurar que a área de interesse, é bem iluminada. </li><li> Ajustar a distância entre duas câmaras, a distância a partir da amostra, ea altura da câmara com base no painel de calibração seleccionado. Cada painel de calibração é diferente, portanto, o manual do usuário ARAMIS terá que ser consultado para determinar essas distâncias. </li><li> Abra um novo projeto no ARAMIS selecionando &quot;File&quot;, depois em &quot;New Project&quot;. Em seguida clique em &quot;Sensor&quot; e selecione &quot;Calibração&quot;, então &quot;Calibration Full&quot;. </li><li> O software ARAMIS agora vai andar o usuário através dos passos para calibrar t<html> ele câmeras. </li><li> Com a abertura da lente totalmente aberta, o foco da câmera no painel de calibração, soltando o parafuso de ajuste da câmera e rotação das lentes. Uma vez focado, reapertar o parafuso de ajuste e fechar a abertura. </li><li> Tome a primeira imagem do processo de calibração. Deslocar ou rodar o painel de calibração de acordo com a demonstração no computador, até que a imagem está focada no ecrã do computador. Tome a segunda imagem. Repetir este processo para o restante das imagens de calibração. </li><li> Depois de todas as imagens de calibração são tomadas, o software de análise de imagem ARAMIS irá calcular as configurações de calibração. O processo de calibração deve ser repetido, se o desvio de calibração é maior do que 0,04. Quaisquer ajustes feitos para o foco da câmera ou a distância entre as câmeras gerará a nulidade de processo de calibração. </li><li> Remova o painel de calibração e coloque o recipiente de látex pintado de volta para os conectores de mangueira farpado. </li></ol>título &quot;&gt; 3. Pré-teste para evitar o ruído de fundo excessivo <ol><li> Determinar o número de quadros por segundo, que é desejado para o teste. Aumento quadros por segundo irá produzir resultados estirpe mais uniformes. </li><li> Ajustar a velocidade do obturador para menos de 1 quadro por segundo e de modo que nenhum vermelho é exibido na imagem. </li><li> Take 5 imagens. </li><li> Adicionar iniciar pontos sobre a série de imagem e calcular o teste. </li><li> Enquanto estiver segurando &quot;ctrl&quot;, clique no centro da amostra para observar o ruído de fundo. Se o pré-teste do ruído é superior a 75 microstrain o processo de calibração deve ser refeito. </li></ol> 4. Implantação de stent <ol><li> Selecione a quantidade de imagens desejadas para tomar durante o teste. 200 imagens será suficiente para a expansão do stent. </li><li> Gradualmente inserir o cateter para dentro do vaso de látex, e usando o indicador de marcador no cateter para guiar a inserção do stent até que atinja o local central. </li><li> Comece a tirar fotos com ARAMIS. </li><li> Para o stent balão expansível, aumentar gradualmente a pressão de balão para expandir stent até que o balão está totalmente expandido, em seguida, diminuir gradualmente a pressão do balão a zero eo balão é esvaziado e retirado juntamente com o cateter. </li><li> Para o stent auto-expansão, gradualmente remover a bainha até que o stent está totalmente expandido, em seguida, gradualmente retrair o cateter. </li></ol> 5. Análise de Imagens <ol><li> História estirpe de um ponto específico no vaso <ol><li> Criar um ponto de palco, segurando a tecla &quot;Ctrl&quot; e clicando na área de interesse. </li><li> Seleccione o tipo de estirpe que é desejada, isto é, na estirpe X, Y, XY, a estirpe maior, estirpe menor, ou estirpe Mises. </li><li> A trama, no canto inferior direito irá exibir a estirpe no ponto seleccionado ao longo da duração do teste. </li></ol></li><li> Estirpe espacial ao longo de um caminho específico do navio <ol><li> CrEATE uma linha ponto de múltiplos estágios, clicando em &quot;seções&quot; guia, em seguida, &quot;criar&quot; seção. Seleccionar uma linha paralela à imagem para o eixo X, com Y igual a zero. Isto irá criar uma série de pontos de estágio em uma linha. </li><li> Após a linha de vários estágios é criado o enredo no canto inferior irá mostrar uma série de linhas em um gráfico. Cada linha representa a estirpe menos um exemplo no tempo ao longo do comprimento da secção. </li></ol></li><li> Criando um cilindro melhor ajuste para analisar a taxa de expansão e raio do vaso <ol><li> Na barra de ferramentas superior selecione &quot;primitivas&quot;, então &quot;cilindro melhor ajuste&quot;. </li><li> Selecione uma pequena parte da imagem usando o &quot;selecionar através da superfície&quot; ferramenta na barra de ferramenta certa. </li><li> O software ARAMIS irá gerar uma de três cilindros melhor ajuste dimensional. </li><li> As imagens podem então ser reciclados através observar como o diâmetro do vaso de látex é variável. </li></ol></li><li> Avaliando a distância entredois pontos <ol><li> Sob a guia clique em &quot;análise&quot; em &quot;ponto a ponto à distância&quot;. </li><li> Seleccionar um comprimento sobre a imagem que é desejado para análise, seleccionando dois pontos. </li><li> As imagens podem então ser reciclados através de observar a mudança na distância entre os dois pontos ao longo do tempo. </li></ol></li></ol> 6. Os resultados representativos As hastes do stent expandir para o exterior da parede do vaso, as estirpes será geralmente maior em torno de localização do stent. A Figura 1 é um exemplo de mapeamento de tensão durante o processo de recuo do balão expansível stent, bem como a história estirpe principal em um ponto específico. Os pontos pretos na Figura 1 são pontos de referência, que foram utilizados pelas câmeras de alta velocidade para capturar e controlar os deslocamentos desses pontos de referência sobre a conduta. Com base no movimento gravada de pontos de referência, o software será então utilizada para calcular as tensões da conduta ou umny outro objeto alvo. Estirpe Major, também referida como a tensão máxima principal, é calculada como se segue: <img alt="Equação 1" src="/files/ftp_upload/3945/3945eq1.jpg" /> É claro que o stent implantado levou à distribuição não uniforme tensão sobre a superfície do vaso. Isto poderia ser explicado pelo carregamento de recuo a partir de extremidades-constrangido conduta de látex ea estrutura de malha de stent. Este campo estirpe corresponde à fase inicial de recuo do stent, tal como identificado pelo marcador Cruz Vermelha na imagem de baixo da Figura 1. A curva tensão-história importante de um ponto específico 10 demonstraram fases distintas de implante de stent. A expansão do balão ocorre a partir de aproximadamente 10 a 12 segundos, e de recuo do stent após a deflação do balão ocorre entre 12 e 14 segundos. <img alt="A Figura 1" src="/files/ftp_upload/3945/3945fig1.jpg" /> Figura 1. </strong&gt; Configuração experimental (topo); distribuição não uniforme tensão sobre a superfície da conduta stent (meio); a história estirpe importante no ponto 10 (parte inferior).

<ol>
	<li>Fischman, D. L., Leon, M. B., Baim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+randomized+comparison+of+coronary-stent+placement+and+balloon+angioplasty+in+the+treatment+of+coronary+artery+disease.+Stent+Restenosis+Study+Investigators.">A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. Stent Restenosis Study Investigators.</a> N. Engl. J. Med. 331, 496-501 (1994).</li><li>Abul Hasan Muhammad Bashar, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Properties+of+Various+Z-Stent+Designs:+An+Endovascular+Stent-Grafting+Perspective.">Mechanical Properties of Various Z-Stent Designs: An Endovascular Stent-Grafting Perspective.</a> Artificial Organs. 27, 714-721 (2003).</li><li>Nuutinen, J. u. h. a. -. P. e. k. k. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+and+in+vitro+degradation+of+bioabsorbable+self-expanding+braided+stents.">Mechanical properties and in vitro degradation of bioabsorbable self-expanding braided stents.</a> Journal of Biomaterials Science -- Polymer Edition. , 255-266 (2003).</li><li>C. Schulz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+stent+symmetry+and+vascular+injury+determine+experimental+restenosis.">Coronary stent symmetry and vascular injury determine experimental restenosis.</a> Heart. 83, 462-467 (2000).</li><li>Jiménez, J. M., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamically+Driven+Stent+Strut+Design.">Hemodynamically Driven Stent Strut Design.</a> Annals of Biomedical Engineering. 1483, (2009).</li><li>Johnston, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mechanical+Properties+of+Endovascular+Stents:+An+In+Vitro+Assessment.">The Mechanical Properties of Endovascular Stents: An In Vitro Assessment.</a> Cardiovascular Engineering: An International Journal. 10, 128-135 (2010).</li><li>Mejia, J. u. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+stent+strut+profile+on+shear+stress+distribution+using+statistical+moments.">Evaluation of the effect of stent strut profile on shear stress distribution using statistical moments.</a> Biomedical Engineering Online. , 1-10 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ARAMIS User Manual. , (2009).</li><li>Chesler, N. C., Thompson-Figueroa, J., Millburne, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+Mouse+Pulmonary+Biomechanics.">Measurements of Mouse Pulmonary Biomechanics.</a> Journal of Biomechanical Engineering. 126, 309-314 (2004).</li><li>de Korte, C. L., Sierevogel, M. J., Mastik, F., Strijder, C., Schaar, J. A., Velema, E., Pasterkamp, G., Serruys, P. W., van der Steen, A. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Atherosclerotic+Plaque+Components+With+Intravascular+Ultrasound+Elastography+In+Vivo+A+Yucatan+Pig+Study.">Identification of Atherosclerotic Plaque Components With Intravascular Ultrasound Elastography In Vivo A Yucatan Pig Study.</a> Circulation. 105, 1627-1630 (2002).</li></ol>

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A estéreo óptico superfície sistema de medição de tensão é utilizado para medir as estirpes locais sobre a superfície deformar para ambos os dentro e fora-de-plano movimentos sem contactar com a amostra. Este sistema usa duas câmeras de alta velocidade óptica para tirar fotos de um padrão aleatório contrastando colocar na superfície para a construção de medições precisas dos movimentos de cada ponto, com uma alta precisão de resolver tensões superficiais. Deve notar-se que...

<table border="1"><tbody><tr><td> Equipamento usado </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> ARAMIS Sistema de Câmaras de </td><td> GOM: técnicas de medição óptica </td><td></td><td></td></tr><tr><td> Palmaz STENT BILIAR transhepático Gênesis </td><td> Cordis Corporation </td><td> PG5910B </td><td> Balão expansível stent </td></tr><tr><td> Z-MED cateter de dilatação do balão </td><td> B. Braun Medical Inc. </td><td> PDZ336 </td><td> Cateter de dilatação com balão </td></tr></tbody></table>

monitoring the wall mechanics during stent deployment in a vessel

Os ensaios clínicos têm relatado taxas de reestenose diferentes para diferentes desenhos de stents 1. Especula-se que as concentrações de stent induzidas estirpe na parede arterial levar a lesão do tecido, o que inicia a restenose 2-7. Esta hipótese necessita investigações adicionais incluindo quantificações melhores de distribuição não uniforme tensão sobre a artéria após a implantação do stent. A não-contato de superfície método de medição tensão para a artéria com stent é apresentada neste trabalho. Estéreo ARAMIS óptico superfície sistema de medição de tensão utiliza duas câmaras ópticas de alta velocidade para capturar o movimento de cada ponto de referência, e resolver três estirpes dimensionais sobre a superfície deformando 8,9. Como um stent de malha é implantado dentro de um vaso de látex com um padrão aleatório contrastante pulverizado ou desenhada na sua superfície exterior, a estirpe de superfície é registada a cada instante da deformação. As distribuições de deformação calculados podem então ser utilizados para compreender o LOcal resposta lesão, validar os modelos computacionais, e formular hipóteses para posterior estudo in vivo.

Watch this Scientific Journal Video about Monitoring the Wall Mechanics During Stent Deployment in a Vessel at JoVE.com

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Stent induzidas distribuições de deformação arteriais são caracterizados utilizando um sistema óptico superfície medição da deformação. Esta técnica de visualização é usado para obter insights sobre o impacto do implante de stent no vaso hospedeiro.

Monitorização da Mecânica de parede durante a implantação de stent em um vaso

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

monitoring-the-wall-mechanics-during-stent-deployment-in-a-vessel

Research

JoVE Journal

Bioengineering

9.2K visualizações.  University of Nebraska-Lincoln. Stent induzidas distribuições de deformação arteriais são caracterizados utilizando um sistema óptico superfície medição da deformação. Esta técnica de visualização é usado para obter insights sobre o impacto do implante de stent no vaso hospedeiro.

Vídeo: Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Metodologia Elastografia por Ressonância Magnética para a Avaliação de engenharia de tecidos Crescimento Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Bioengenharia

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Citometria de massa: Protocolo para Ajuste Diário e amostras de células em execução em um citômetro de Massa CyTOF

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

A plataforma automatizada versátil para experiências de estimulação Micro escala celulares

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Investigação Experimental de Estruturas de fluxo secundário a jusante de uma falha Modelo Tipo IV Stent em uma seção de teste de 180 ° Curved Artéria

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adaptação do Processo Electrospinning para fornecer três ambientes únicos para um Tri-camadas<em&gt; In Vitro</em&gt; Modelo da parede das vias aéreas

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

Encapsulamento de células de mamífero em grânulos de alginato usando um recipiente agitado simples

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

Simulando a mecânica de lente Alojamento através de uma lente Manual esticador

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Monitoramento não destrutiva de desenvolvimento degradáveis baseados em andaime engenharia de tecidos dos vasos sanguíneos, usando a tomografia de coerência óptica

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

Uma Nanoemulsão estabilizada tripeptídeo de ácido oleico

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Padronização da geometria de colônias de células-tronco embrionárias humanas em substratos compatíveis para controlar a mecânica de nível de tecido

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...