Os autores são gratos a Shad Schipke Chesser e Daniel para a sua assistência com o design e fabricação do sistema e Myles Valtresa de assistência com a preparação do manuscrito.

1. Colheita e Preparação dos tecidos <ol><li> Valva aórtica devem ser coletadas de suínos adultos pesando não mais de £ 120 imediatamente após a morte. </li><li> Lavar as válvulas duas vezes com solução salina tamponada estéril de fosfato (PBS) e transporte para o laboratório em gelo. </li><li> Todas as etapas subseqüentes devem ser realizados em condições estéreis. </li><li> Assegurar que os folhetos não mostram qualquer sinal de degeneração, rasgar ou calcificação. Remover folhetos a partir da raiz da aorta, cortando 1 / 3 da distância entre o anel. </li><li> Folhetos lugar em poços individuais de uma placa de seis poços e incubar durante a noite com média de 3 ml de Dulbecco Modificado Águia suplementado com 1% de solução anti-biotic/anti-mycotic e 10% de soro fetal bovino a 37 ° C e 5% de CO 2. </li><li> Como alternativa, células isoladas podem ser semeados em placas de seis cultura e bem utilizada no dispositivo de pressão. Isolamento de células endoteliais e válvula de células intersticiais podem ser realizados conforme descrito anteriormente 13, 14. </li></ol> 2. Estudos pressão <ol><li> Um sistema de pressão feito por encomenda foi projetado para estudar os efeitos de pressão cíclica mechanobiological em tecido de válvula aórtica 15. </li><li> Faça logon no computador e abra o programa LabVIEW (Figura 1). </li><li> Calibração: <ol><li> Antes da experimentação, o sistema deve estar devidamente calibrado. </li><li> Ligue o fornecimento de energia para a placa de circuito. Esta fornece energia para as válvulas solenóides que controlam o fluxo de ar para dentro e para fora da câmara. </li><li> Verifique se o ar comprimido é conectado ao sistema e abrir o suprimento de ar à velocidade total. </li><li> Ligar o amplificador de sinal. Certifique-se a leitura da tensão é de 0,00. Ajustar conforme necessário </li><li> A interface LabVIEW tem um interruptor marcado &quot;TEST / RECORD&quot;. Verifique se a chave é definido como &quot;TEST&quot;. Clique no botão &quot;Air Supply&quot; para abrir a válvula solenóide de entrada. </li><li> Usando o regulador de pressão de gás, pressurizar a câmara com ar comprimido a um PSI. A pressão na câmara pode ser lido através do indicador de pressão digital localizado na parte traseira de placa terminal da câmara. Uma vez que a pressão tem equilibrado, gravar a leitura da tensão do amplificador de sinal. Repita o procedimento para PSI 2, 3, 4 e 5. </li><li> Construir uma curva de calibração de pressão versus tensão. A pressão deve ser convertido de PSI para mmHg. A equação do gráfico pode ser colocado no código do programa LabVIEW </li></ol></li><li> Remova a placa frontal de alumínio da câmara de pressão e pulverizar a câmara com etanol 70%. Deixe por um mínimo de 10 minutos para permitir que vapores de etanol residual a se dissipar. </li><li> Coloque a placa de seis poços contendo as amostras de folheto para a câmara e substituir a placa dianteira. Garantir a vedação é hermética, apertando as porcas localizados nas quatro hastes rosqueadas (localizado em cada canto da placa de final) com a mão. Coloque a câmara de pressão nos 37 ° C incubadora. Um diagrama esquemático da câmara de pressão é mostrado na figura 2. </li><li> A interface irá solicitar ao usuário para fornecer a quantidade de tempo que o sistema de ciclos entre entrada de ar comprimido e de saída. Estes devem ser definidos para 0.6s e 0.4s para imitar as condições diastólica e sistólica, respectivamente, a uma freqüência de 1Hz. O usuário também deve inserir um caminho de arquivo de dados. </li><li> No LabVIEW, clique em Executar e mudar o &quot;TEST / RECORD&quot; alternar para &quot;RECORD&quot;. </li><li> Certifique-se que a pressão está no nível desejado usando o gráfico na interface do LabVIEW. Pressão pode ser ajustado com o regulador de pressão de gás. </li><li> Executar o programa de período de tempo desejado. </li><li> Uma vez que a experiência é completa, clique no botão parar em LabVIEW, desligue a alimentação de ar e abrir a válvula de escape na câmara de pressão. </li><li> Remova a placa frontal da câmara e recuperar amostras de seis bem placa contendo. Amostras podem agora ser analisados ​​para a expressão do gene, a expressão da proteína, histologia, propriedades mecânicas, etc </li></ol> 3. Resultados representativos: O sistema de pressão é capaz de simular as pressões máxima transvalvar observado sob normotensos, estágios I e II condições hipertensos. No entanto, a pressão não foi capaz de imitar o gradiente de pressão sistólica, que é essencialmente zero in vivo. Freqüência é mantida em 1Hz, com um tempo de entrada de ar 0.6s e um tempo de escape de 0.4s. Pressão representante formas de onda de condições normais de pressão e elevada obtidos a partir do sistema pode ser visto na figura 3. <img src="/files/ftp_upload/3316/3316fig1.jpg" alt="Figura 1"/> Figura 1: Captura de tela da interface do LabVIEW. <img src="/files/ftp_upload/3316/3316fig2.jpg" alt="Figura 2"/> Figura 2: Desenho esquemático da câmara de pressão de vista Isometric A. da câmara de pressão; B. Vista lateral da câmara de pressão;C. Vista superior da câmara de pressão. <img src="/files/ftp_upload/3316/3316fig3.jpg" alt="Figura 3"/> Figura 3: Gráfico de simulação de pressão dentro da câmara de pressão (A) normotensos, (B) Fase I hipertensos, e (C) Estágio II condições hipertensos.

<ol>
	<li>Freeman, R. V., Otto, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+calcific+aortic+valve+disease:+pathogenesis,+disease+progression,+and+treatment+strategies.">Spectrum of calcific aortic valve disease: pathogenesis, disease progression, and treatment strategies.</a> Circulation. 111, 3316-3326 (2005).</li><li>Robicsek, F., Thubrikar, M. J., Fokin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+of+degenerative+disease+of+the+trileaflet+aortic+valve:+review+of+subject+and+presentation+of+a+new+theory.">Cause of degenerative disease of the trileaflet aortic valve: review of subject and presentation of a new theory.</a> Ann Thorac Surg. 73, 1346-1354 (2002).</li><li>Thubrikar, M. J., Aouad, J., Nolan, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+calcific+deposits+in+operatively+excised+stenotic+or+purely+regurgitant+aortic+valves+and+their+relation+to+mechanical+stress.">Patterns of calcific deposits in operatively excised stenotic or purely regurgitant aortic valves and their relation to mechanical stress.</a> Am J Cardiol. 58, 304-308 (1986).</li><li>Agno, F. S., Chinali, M., Bella, J. N., Liu, J. E., Arnett, D. K., Kitzman, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+sclerosis+is+associated+with+preclinical+cardiovascular+disease+in+hypertensive+adults:+the+Hypertension+Genetic+Epidemiology+Network+study.">Aortic valve sclerosis is associated with preclinical cardiovascular disease in hypertensive adults: the Hypertension Genetic Epidemiology Network study.</a> J Hypertens. 23, 867-8673 (2005).</li><li>Cawley, P. J., Otto, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+calcific+aortic+valve+stenosis+-+fact+or+fiction.">Prevention of calcific aortic valve stenosis - fact or fiction.</a> Annals of Medicine. 41, 100-108 (2009).</li><li>Durst, C. A., Grande-Allen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+physical+characterization+of+a+synchronous+multivalve+aortic+valve+culture+system.">Design and physical characterization of a synchronous multivalve aortic valve culture system.</a> Ann Biomed Eng. 38, 319-3125 (2010).</li><li>Engelmayr, G. C., Soletti, L., Vigmostad, S. C., Budilarto, S. G., Federspiel, W. J., Chandran, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flex-stretch-flow+bioreactor+for+the+study+of+engineered+heart+valve+tissue+mechanobiology.">A novel flex-stretch-flow bioreactor for the study of engineered heart valve tissue mechanobiology.</a> Ann Biomed Eng. 36, 700-712 (2008).</li><li>Sucosky, P., Padala, M., Elhammali, A., Balachandran, K., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+an+ex+vivo+culture+system+to+investigate+the+effects+of+shear+stress+on+cardiovascular+tissue.">Design of an ex vivo culture system to investigate the effects of shear stress on cardiovascular tissue.</a> J Biomech Eng. 130, 035001-03 (2008).</li><li>Syedain, Z. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cyclic+stretch+bioreactor+for+tissue-engineered+heart+valves.">Controlled cyclic stretch bioreactor for tissue-engineered heart valves.</a> Biomaterials. 30, 4078-4084 (2009).</li><li>Lagana, K., Moretti, M., Dubini, G., Raimondi, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+bioreactor+for+the+controlled+application+of+complex+mechanical+stimuli+for+cartilage+tissue+engineering.">A new bioreactor for the controlled application of complex mechanical stimuli for cartilage tissue engineering.</a> Proc Inst Mech Eng H. 222, 705-715 (2008).</li><li>Wartella, K. A., Wayne, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioreactor+for+biaxial+mechanical+stimulation+to+tissue+engineered+constructs.">Bioreactor for biaxial mechanical stimulation to tissue engineered constructs.</a> J Biomech Eng. 131, 044501-044501 (2009).</li><li>Wallis, M. C., Yeger, H., Cartwright, L., Shou, Z., Radisic, M., Haig, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+study+of+a+novel+urinary+bladder+bioreactor.">Feasibility study of a novel urinary bladder bioreactor.</a> Tissue Eng Part A. 14, 339-348 (2008).</li><li>Butcher, J. T., Nerem, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valvular+endothelial+cells+regulate+the+phenotype+of+interstitial+cells+in+co-culture:+effects+of+steady+shear+stress.">Valvular endothelial cells regulate the phenotype of interstitial cells in co-culture: effects of steady shear stress.</a> Tissue Eng. 12, 905-915 (2006).</li><li>Metzler, S. A., Pregonero, C. A., Butcher, J. T., Burgess, S. C., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+Strain+Regulates+Pro-Inflammatory+Protein+Expression+in+Porcine+Aortic+Valve+Endothelial+Cells.">Cyclic Strain Regulates Pro-Inflammatory Protein Expression in Porcine Aortic Valve Endothelial Cells.</a> J Heart Valve Dis. 17, 571-578 (2008).</li><li>Schipke, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Design of a cyclic pressure bioreactor for the ex vivo study of aortic heart valve mechanobiology. , (2008).</li><li>Smith, K. E., Metzler, S. A., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+strain+inhibits+acute+pro-inflammatory+gene+expression+in+aortic+valve+interstitial+cells.">Cyclic strain inhibits acute pro-inflammatory gene expression in aortic valve interstitial cells.</a> Biomechanics and Modeling in Mechanobiology. , (2009).</li><li>Warnock, J. N., Burgess, S. C., Shack, A., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+immediate-early+gene+responses+to+elevated+pressure+in+porcine+aortic+valve+interstitial+cells.">Differential immediate-early gene responses to elevated pressure in porcine aortic valve interstitial cells.</a> J Heart Valve Dis. 15, 34-41 (2006).</li><li>Brathwaite, D., Weissman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+new+onset+of+atrial+arrhythmias+following+major+noncardiothoracic+surgery+is+associated+with+increased+mortality.">The new onset of atrial arrhythmias following major noncardiothoracic surgery is associated with increased mortality.</a> Chest. 114, 462-468 (1998).</li><li>Walsh, S. R., Oates, J. E., Anderson, J. A., Blair, S. D., Makin, C. A., Walsh, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+arrhythmias+in+colorectal+surgical+patients:+incidence+and+clinical+correlates.">Postoperative arrhythmias in colorectal surgical patients: incidence and clinical correlates.</a> Colorectal Dis. 8, 212-216 (2006).</li><li>Walsh, S. R., Tang, T., Gaunt, M. E., Schneider, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+arrhythmias+after+non-cardiothoracic+surgery.">New arrhythmias after non-cardiothoracic surgery.</a> BMJ. 7, 333-333 (2006).</li><li>Walsh, S. R., Tang, T., Wijewardena, C., Yarham, S. I., Boyle, J. R., Gaunt, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+arrhythmias+in+general+surgical+patients.">Postoperative arrhythmias in general surgical patients.</a> Ann R Coll Surg Engl. 89, 91-95 (2007).</li></ol>

Não há conflitos de interesse declarados.

O sistema de pressão com sucesso expostos folhetos da válvula aórtica a pressões cíclicas que eram representativas de pressão transvalvar diastólica. No entanto, não foi capaz de imitar a pressão sistólica transvalvar, como a única pressão caiu para 40 mmHg. Pressão transvalvar é a diferença entre a pressão na aorta ascendente e do ventrículo esquerdo. Durante a diástole, quando a válvula é fechada, a diferença de pressão é 80mmHg em condições normotensos e 90 mmHg e 100 mmHg na fase I e hiperte...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> DMEM </td><td> Sigma </td><td> D5671 </td><td></td></tr><tr><td> PBS Dulbecco </td><td> Sigma </td><td> D5652 </td><td></td></tr><tr><td> Solução Anti-mycotic/antibiotic </td><td> Sigma </td><td> A5955 </td><td></td></tr><tr><td> Soro fetal bovino </td><td> ThermoScientific </td><td> SH30070 </td><td></td></tr><tr><td> Viton solenóide válvulas de diafragma </td><td> McMaster Carr </td><td> 4868K11 </td><td></td></tr><tr><td> Transdutor de pressão </td><td> Omega Engineering, Inc. </td><td> PX302-200GV </td><td></td></tr><tr><td> Condicionador de célula de carga </td><td> Encore Electronics, Inc. </td><td> 4025-101 </td><td></td></tr><tr><td> Módulo de Aquisição de Dados (DAQ) </td><td> Computing medição </td><td> PMD1608 </td><td></td></tr></tbody></table>

design of a cyclic pressure bioreactor for the ex vivo study of aortic heart valves

A valva aórtica, localizada entre o ventrículo esquerdo ea aorta, permite um fluxo unidirecional do sangue, impedindo o refluxo para o ventrículo. Folhetos da válvula aórtica são compostas de células intersticiais suspenso dentro de uma matriz extracelular (ECM) e são revestidas com uma monocamada de células endoteliais. A válvula resiste a um ambiente adverso e dinâmico e está constantemente exposto ao cisalhamento, flexão, tensão e compressão. A pesquisa mostrou lesões calcificadas em válvulas doentes ocorrer em áreas de alta resistência mecânica, como resultado do rompimento ou dano endotelial matriz intersticial 1-3. Por isso, não é surpreendente que estudos epidemiológicos têm demonstrado a pressão arterial elevada ser um factor de risco mais importante no aparecimento da doença da válvula aórtica 4. A única opção de tratamento atualmente disponíveis para a doença da válvula é a substituição cirúrgica da valva doente com uma válvula de prótese biológica ou mecânica 5. Melhor compreensão da biologia da válvula em resposta ao stress físico ajudaria a elucidar os mecanismos de patogênese da válvula. Por sua vez, isso poderia ajudar no desenvolvimento de terapias não invasivas, como a intervenção farmacêutica ou de prevenção. Biorreatores várias tenham sido previamente desenvolvidos para estudar a mechanobiology de válvulas cardíacas nativas ou engenharia 6-9. Biorreatores pulsátil também têm sido desenvolvidos para estudar uma variedade de tecidos, incluindo cartilagens 10, 11 e osso da bexiga 12. O objetivo deste trabalho foi desenvolver um sistema de pressão cíclica que poderia ser usado para elucidar a resposta biológica de folhetos da válvula aórtica a cargas de maior pressão. O sistema consistia de uma câmara de acrílico para colocar as amostras e produzir pressão cíclica, válvulas de diafragma viton solenóide para controlar o tempo do ciclo de pressão, e um computador para controle de dispositivos elétricos. A pressão foi monitorada através de um transdutor de pressão, eo sinal foi condicionado usando um condicionador de célula de carga. Um programa LabVIEW regulamentou a pressão utilizando um dispositivo analógico para bombear o ar comprimido para o sistema a uma taxa apropriada. O sistema imitou a dinâmica dos níveis de pressão transvalvar associado com a válvula aórtica, uma onda dente de serra produziu um aumento gradual na pressão, típico do gradiente de pressão transvalvar que está presente através da válvula durante a diástole, seguido por uma acentuada queda de pressão mostrando abertura da válvula em sístole. O programa permitiu que os usuários LabVIEW para controlar a magnitude ea freqüência de pressão cíclica. O sistema foi capaz de submeter amostras de tecido para condições de pressão fisiológicos e patológicos. Este dispositivo pode ser usado para aumentar a nossa compreensão de como válvulas cardíacas responder às mudanças no ambiente local mecânica.

Watch this Scientific Journal Video about Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves at JoVE.com

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Um biorreator pressão cíclica capaz de submeter o tecido de válvula cardíaca em condições de pressão fisiológicos e patológicos tem sido projetado. Um programa LabVIEW permite aos usuários controlar a pressão magnitude amplitude e frequência. Este dispositivo pode ser usado para estudar a mechanobiology do tecido cardíaco válvula ou células isoladas.

Projeto de um biorreator de pressão cíclica para o Ex Vivo Estudo de válvulas cardíacas Aórtica

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

design-cyclic-pressure-bioreactor-for-ex-vivo-study-aortic-heart

Research

Education

JoVE Core

JoVE Journal

Physics

Bioengineering

Unit 1

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Sources of Magnetic Fields

SCIENTISTS IN ACTION

15.6K visualizações.  Mississippi State University. Um biorreator pressão cíclica capaz de submeter o tecido de válvula cardíaca em condições de pressão fisiológicos e patológicos tem sido projetado. Um programa LabVIEW permite aos usuários controlar a pressão magnitude amplitude e frequência. Este dispositivo pode ser usado para estudar a mechanobiology do tecido cardíaco válvula ou células isoladas.

Vídeo: Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Patient-specific Modeling of the Heart: Estimation of Ventricular Fiber Orientations

Patient-specific simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered by the absence of in vivo imaging technology for clinically acquiring myocardial fiber orientations. The objective of this project was to develop a methodology to estimate cardiac fiber orientations from in vivo images of patient heart geometries. An accurate representation of ventricular geometry and fiber orientations was reconstructed, respectively, from high-resolution ex vivo structural magnetic resonance (MR) and diffusion tensor (DT) MR images of a normal human heart, referred to as the atlas. Ventricular geometry of a patient heart was extracted, via semiautomatic segmentation, from an in vivo computed tomography (CT) image. Using image transformation algorithms, the atlas ventricular geometry was deformed to match that of the patient. Finally, the deformation field was applied to the atlas fiber orientations to obtain an estimate of patient fiber orientations. The accuracy of the fiber estimates was assessed using six normal and three failing canine hearts. The mean absolute difference between inclination angles of acquired and estimated fiber orientations was 15.4 &deg;. Computational simulations of ventricular activation maps and pseudo-ECGs in sinus rhythm and ventricular tachycardia indicated that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.The new insights obtained from the project will pave the way for the development of patient-specific models of the heart that can aid physicians in personalized diagnosis and decisions regarding electrophysiological interventions.

A methodology to estimate ventricular fiber orientations from in vivo images of patient heart geometries for personalized modeling is described. Validation of the methodology performed using normal and failing canine hearts demonstrate that that there are no significant differences between estimated and acquired fiber orientations at a clinically observable level.

Paciente específico Modelagem do Coração: Estimativa de Orientações de fibra ventriculares

Patient-specific  simulations of heart (dys)function aimed at personalizing cardiac therapy are hampered  by the absence of in vivo imaging technology for ...

Bioengenharia

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Projeto de um biorreator Carregando Mecânica Biaxial para Engenharia de Tecidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.

Fermentors are used to increase culture yield and productivity of bioengineered cells. After screening multiple microbial or animal cell culture candidates in shake flasks, the next logical step is to increase the selected culture&#x2019;s biomass with the fermentor. This video demonstrates the setup and operation of a typical benchtop bioreactor system.

Funcionamento de um biorreator de bancada

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

Caracterização de Sistemas Complexos Usando o Projeto de Experimentos Abordagem: Transient Protein Expression em tabaco como um Estudo de Caso

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

Anatomically Realistic Neonatal Heart Model for Use in Neonatal Patient Simulators

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff practice clinical interventions such as chest compressions to ensure patient survival in the case of bradycardia or cardiac arrest. The simulators used currently are of low physical fidelity and therefore cannot provide qualitative insight into the procedure of chest compressions. The embedding of an anatomically realistic heart model in future simulators enables the detection of cardiac output generated during chest compressions; this can provide clinicians with an output parameter, which can deepen the understanding of the effect of the compressions in relation to the amount of blood flow generated. Before this monitoring can be achieved, an anatomically realistic heart model must be created containing: two atria, two ventricles, four heart valves, pulmonary veins and arteries, and systemic veins and arteries. This protocol describes the procedure for creating such a functional artificial neonatal heart model by utilizing a combination of magnetic resonance imaging (MRI), 3D printing, and casting in the form of cold injection molding. Using this method with flexible 3D printed inner molds in the injection molding process, an anatomically realistic heart model can be obtained.

This protocol describes a procedure for creating functional artificial neonatal heart models by utilizing a combination of magnetic resonance imaging, 3D printing, and injection molding. The purpose of these models is for integration into the next generation of neonatal patient simulators and as a tool for physiological and anatomical studies.

Modelo de coração Neonatal anatomicamente realista para uso em simuladores de paciente Neonatal

Neonatal patient simulators (NPS) are artificial patient surrogates used in the context of medical simulation training. Neonatologists and nursing staff ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

Microscopia de fluorescência de luz-folha para o estudo do coração murino

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Um novo modelo Ex Vivo para a avaliação de desempenho de Material de injeção submucosa endoscópica

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Biaxial Mechanical Characterizations of Atrioventricular Heart Valves

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive models, which provide a mathematical representation of the mechanical function of those structures. This presented biaxial mechanical testing protocol involves (i) tissue acquisition, (ii) the preparation of tissue specimens, (iii) biaxial mechanical testing, and (iv) postprocessing of the acquired data. First, tissue acquisition requires obtaining porcine or ovine hearts from a local Food and Drug Administration-approved abattoir for later dissection to retrieve the valve leaflets. Second, tissue preparation requires using tissue specimen cutters on the leaflet tissue to extract a clear zone for testing. Third, biaxial mechanical testing of the leaflet specimen requires the use of a commercial biaxial mechanical tester, which consists of force-controlled, displacement-controlled, and stress-relaxation testing protocols to characterize the leaflet tissue&#39;s mechanical properties. Finally, post-processing requires the use of data image correlation techniques and force and displacement readings to summarize the tissue&#39;s mechanical behaviors in response to external loading. In general, results from biaxial testing demonstrate that the leaflet tissues yield a nonlinear, anisotropic mechanical response. The presented biaxial testing procedure is advantageous to other methods since the method presented here allows for a more comprehensive characterization of the valve leaflet tissue under one unified testing scheme, as opposed to separate testing protocols on different tissue specimens. The proposed testing method has its limitations in that shear stress is potentially present in the tissue sample. However, any potential shear is presumed negligible.

This protocol involves characterizations of atrioventricular valve leaflets with force-controlled, displacement-controlled, and stress-relaxation biaxial mechanical testing procedures. Results acquired with this protocol can be used for constitutive model development to simulate the mechanical behavior of functioning valves under a finite element simulation framework.

Biaxial caracterizações mecânicas das válvulas cardíacas Atrioventricular

Extensive biaxial mechanical testing of the atrioventricular heart valve leaflets can be utilized to derive&#160;optimal parameters used in constitutive ...

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

Um dispositivo de teste de atrito-bioreator para estudo de biomecânica articular sinovial, mecanobiologia e regulação física

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an evolving reconstructive avenue for transferring multiple tissues as a composite subunit. Despite the promising nature of VCA, the long-term immunosuppressive requirements are a significant limitation due to the increased risk of malignancies, end-organ toxicity, and opportunistic infections. Tissue engineering of acellular composite scaffolds is a potential alternative in reducing the need for immunosuppression. Herein, the procurement of a rat hindlimb and its subsequent decellularization using sodium dodecyl sulfate (SDS) is described. The procurement strategy presented is based upon the common femoral artery. A machine perfusion-based bioreactor system was constructed and used for ex vivo decellularization of the hindlimb. Successful perfusion decellularization was performed, resulting in a white translucent-like appearance of the hindlimb. An intact, perfusable, vascular network throughout the hindlimb was observed. Histological analyses showed the removal of nuclear contents and the preservation of tissue architecture across all tissue compartments.

Procurement and Decellularization of Rat Hindlimbs Using an Ex Vivo Perfusion-Based Bioreactor for Vascularized Composite Allotransplantation

We describe the surgical technique and decellularization process for composite rat hindlimbs. Decellularization is conducted using low-concentration sodium dodecyl sulfate through an ex vivo machine perfusion system.

Captação e Descelularização de Membros Posteriores de Rato Usando Biorreator Ex Vivo à Base de Perfusão para Alotransplante Composto Vascularizado

Patients with severe traumatic injuries and tissue loss require complex surgical reconstruction. Vascularized composite allotransplantation (VCA) is an ...