The authors thank Tyra Witt, Cheri Mueske, Brant Newman and Dr. Peter J. Psaltis, MBBS, PhD for their valuable contributions. This study was financially supported by European Regional Development Fund &#8211; FNUSA-ICRC (No. CZ.1.05/1.100/02.0123), American Heart Association Scientist Development Grant (AHA #06-35185N), National Institutes of Health (T32HL007111) and The Grainger Innovation Fund &#8211; Grainger Foundation.

Todos los estudios con animales fueron aprobados por el Cuidado de Animales institucional y el Comité de Utilización (IACUC) en la Clínica Mayo. 1. Diseño y análisis de un stent de acero inoxidable 2205 <ol><li> El diseño de un stent metálico utilizando CAD <ol><li> Hacer un cilindro hueco extruido seleccionando en función de &#39;jefe extruido / base &quot;con el espesor de pared igual al espesor del puntal del stent. </li><li> Diseñar un modelo de stent en un plano de boceto diferente tangente al cilindro extruido. Hacer la anchura del patrón plano para que coincida con la circunferencia del cilindro hueco extruido. </li><li> Transfiera el patrón de diseño plano sobre el cilindro hueco utilizando la función de envoltura. </li><li> Guarde la pieza en su formato original y también en formato ACIS a exportar para FEA. </li></ol></li><li> El análisis de elementos finitos para los modelos de stent <ol><li> Importe la geometría sólida guardado en formato ACIS en el módulo de parte del software de FEA para más analyses. </li><li> Modelo 2 cilindros analíticos coaxialmente al stent en el modelador de parte del software FEA. El cilindro exterior tiene un diámetro inicial mayor que el diámetro de la endoprótesis para simular el rizador y el cilindro interior tiene un diámetro inicial de 1 mm para simular un globo para la inflación. </li><li> Doble click en la opción &#39;casos&#39; árbol del modelador de montaje para montar lo anterior dijo piezas en posiciones relativas. </li><li> Utilice el módulo de la malla del software FEA, especifique el tipo de elemento como 20-nodo de elemento hexaédrica con integración reducida, especifique el tamaño del elemento, y la malla del stent. </li><li> Especificar pares de contacto rígidas sin fricción entre el stent y los dos cilindros, respectivamente, en las &quot;propiedades de interacción&quot; del árbol del modelo. </li><li> Asigne el comportamiento tensión-deformación elasto-plástico de 2.205 de acero inoxidable para el modelo de stent. </li><li> Definir las condiciones de contorno para engarzar en primer lugar, el cilindro exterior de 1 mm, que simula la cRimping del stent. Retire el cilindro exterior para simular la relajación del stent engarzado. Expandir el cilindro interior a 3 mm para simular la expansión y finalmente, retirar el cilindro interior para simular retroceso del stent. </li><li> Definir los parámetros de simulación, incluyendo el número de procesadores y la cantidad de memoria RAM asignados en el ítem del árbol de modelo &quot;Análisis&quot; y ejecutar la simulación. </li><li> Una vez que la simulación es completa, abrir el archivo resultado (filename.odb) y post-proceso de los resultados para estudiar las deformaciones principales y iterativamente mejorar el diseño del stent para lograr una cepa principal de 20%, que es menor que el límite fallo del material . </li></ol></li></ol> 2. Fabricación de stent y pruebas para que prensa y Expansión <ol><li> Fabricación de stent <ol><li> Obtener los tubos de acero inoxidable de 2205 por la perforación del arma y la precisión de barras de molienda stock de material en un mecanizado de precisión empresa como Acción Precision Products en Pioneer,OH. </li><li> Transfiera los tubos de tierra de precisión y el diseño del modelo del stent plana a una empresa de corte stent como Laserage Technology Corporation en Waukegan, IL de corte por láser y electropulido. </li><li> Pasivar la superficie de los stents electropulido sumergiendo en un ácido fuerte (50% HCl) durante 10 min seguido de una base (10% NaHCO 3) durante otros 10 min. PRECAUCIÓN: manejar productos químicos con equipo de protección adecuado y bajo una campana de humos. Finalmente, lavar los stents con alcohol etílico y agua desionizada. Este proceso se llama decapado con ácido. </li></ol></li><li> Prueba de stent fabricado para prensar y expansión <ol><li> Unir el stent sobre un globo tríptico utilizando una mano herramienta que prensa. Sostenga el stent y el globo tríptico en la herramienta que prensa. Presione el asa para deformar radialmente el stent se doble, en el globo. </li><li> Inspeccione el stent engarzado con un microscopio para engaste uniforme y cualquier señal de fallo en la estructura debidoa la deformación plástica. </li><li> Expandir al diámetro de 3 mm diseñado mediante la presurización del globo tríptico con agua. Examine los stents expandidos para fracturas microscópicas y expansión uniforme. </li></ol></li></ol> 3. Caracterización de Stent para Campo Magnético Retenido NOTA: imán cilíndrico de 2 pulgadas de diámetro y 1 pulgada de altura se utilizó en este estudio. Los polos del imán están alineados a lo largo del eje. La densidad de superficie de flujo magnético del imán es de aproximadamente 1 T. <ol><li> Magnetizar los stents diametralmente o axialmente mediante un potente imán de neodimio. Sostenga el stent cerca del imán fuerte durante aproximadamente 1 min para la magnetización. </li><li> Sostenga el stent en una de las caras planas con su diámetro a lo largo de las líneas de campo magnético para ser magnetizados diametralmente o sostener el stent junto a la superficie cilíndrica con su eje a lo largo de las líneas de campo magnético para magnetizar axialmente. Fiel magnética Retenidod del stent se encontró que era estable durante al menos 24 horas, pero utilizar el stent tan pronto como sea posible después de la magnetización. </li><li> Montar los stents individualmente sobre mandriles de vidrio y luego montar los mandriles de vidrio en el mandril de precisión de la fijación de sondeo magnético. Microsensor sonda magnética puede ser posicionada precisamente cerca de la stent sin tocar la superficie usando el XYZ fases de montaje del dispositivo de fijación de sondeo magnética (Figura 4). </li><li> Medir la lectura de referencia del microsensor magnético lejos del stent y luego medir el campo magnético retenido en la superficie del stent mediante la colocación de la sonda utilizando las etapas XYZ de la fijación de sondeo magnético. </li></ol> 4. Estudios captura de células magnéticas <ol><li> La obtención de células, etiquetado con SPION y tinción con colorante fluorescente <ol><li> Derivar las células endoteliales excrecencia (EOC) de sangre periférica porcina como se describe en 5,7. Cultura en un matraz T-75 until aproximadamente el 80% de confluencia (5x10 6 a 8x10 6 células). </li><li> Sintetizar SPIONs como magnetita diámetro de 10 nm (Fe 3 O 4) núcleo rodeado por 50 nm de espesor de poli (láctico-co-glicólico) (PLGA) shell como se describe en el 8,9. </li><li> Incubar la EOC derivada con SPION a una concentración de 200 g / ml de medio de cultivo celular durante 16 horas a 37 ° C </li><li> Aspirar el medio de cultivo celular suavemente. Lave suavemente las células mediante la adición de 10 ml de solución salina tamponada con fosfato (PBS) al matraz, balanceo, y aspirar el PBS. </li><li> Teñir las células con tinte fluorescente (CM-Dil) para la visualización durante los experimentos. Esto se realiza según las instrucciones del fabricante mediante la adición del colorante a 10 ml de medio de cultivo celular a una concentración de 5 l / ml y la incubación con las células durante 30 min a 37 ° C. </li><li> Se lavan las células con PBS como en el paso 4.1.4 e incubar con 3 ml de solución de tripsina-EDTA 0,25% durante 5 min a 37 ° C alevantar las células del matraz. </li><li> Transferir la suspensión celular a un tubo cónico de 15 ml, con parte superior de PBS, y se centrifuga a 500 xg durante 5 min para formar un sedimento celular. </li><li> Vuelva a suspender el sedimento celular en PBS a una concentración de 1-2x10 6 células / ml y mezclar bien con la pipeta dentro y fuera del tubo cónico varias veces. </li></ol></li><li> Los estudios in vitro de células <ol><li> Diseñar y fabricar (por ejemplo, la impresión 3D) en un accesorio sencillo para sujetar el stent justo por encima de la superficie de un cubreobjetos de vidrio. </li><li> Desmagnetice un stent utilizando un degausser electromagnética o magnetizar un stent diametralmente o axialmente usando un imán fuerte neodimio. </li><li> Pipetear la EOC SPION marcado suspendido en PBS en el plato que contiene los stents de control axialmente magnetizados o diametralmente magnetizadas o no magnetizadas. Imagen de los stents con EOC suspendido en PBS inmediatamente para la fluorescencia utilizando un microscopio de fluorescencia invertida. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 5. En vivo Los estudios en animales <ol><li> La implantación del stent <ol><li> Dibuja sangre periférica de 4 cerdos Yorkshire sanos - un peso aproximado de 50 kg - 3 semanas antes de la implantación de stent, respectivamente, y la cultura EOC como se describe en 5,7. </li><li> Administrar la medicación antiplaquetaria comenzando 3 días antes de la cirugía (aspirina 325 mg y clopidogrel 75 mg al día). </li><li> En el día de la implantación del stent, anestesiar a los cerdos con intramuscular Telazol, xilazina y atropina (5 / 2-3 / 0,05 mg / kg, respectivamente) como se indica en las directrices para el cuidado de los animales y el uso institucionales aplicables. </li><li> Intubar y colocar el cerdo en la inhalación de 1-2,5% anestesia isoflurano. </li><li> Afeitarse la región del cuello ventral del cerdo y llevar a cabo el procedimiento en condiciones estériles generales. </li><li> Implante 1 magnetizado y 1 stent no magnetizado en la arteria coronaria derecha (CD) utilizando la técnica de cateterismo cardíaco normal. <ol><li> Catheterización de los animales debe ser realizado por un cardiólogo intervencionista entrenado. Acceder a la arteria carótida derecha con una vaina francés 9. </li><li> Canular la arteria coronaria diana y se inyecta un medio de contraste yodado para obtener imágenes de fluoroscopia. </li><li> Coloque un alambre guía estándar de 0,014 pulgadas coronaria en la arteria. Avance el balón y el stent usando este cable guía y desplegar el stent en un vaso de diámetro 3-3,5 mm. </li></ol></li><li> Ocluir el flujo de sangre dentro de la RCA proximal a los stents implantados usando un globo sobre el alambre y entregar aproximadamente 2x10 6 EOC autólogo marcado con SPION suspendido en 4 ml de PBS a través del catéter central durante un período de 2 min. </li><li> Restablecer el flujo sanguíneo a la RCA después de 2 min de la oclusión adicional. </li><li> Traslado al animal a la sala de recuperación y vigilar de cerca al animal hasta que se haya recuperado la conciencia. </li><li> Continuar para administrar medicamentos anti-plaquetas (aspirina 325 mg y clopidogrel 75 mg) después de la operación hasta el sacrificio. </li></ol></li><li> Stent explante y la histología <ol><li> La eutanasia a los animales 7 días después de la cirugía por primera anestesiar al animal como se ha explicado anteriormente y luego administrar por vía intravenosa una dosis letal de pentobarbital sódico (100 mg / kg) según las pautas de cuidado de animales y uso institucional aplicables. </li><li> Quirúrgicamente cosechar los segmentos arteriales con stent. Fijar las arterias explantadas en tampón de formalina al 10% durante un mínimo de 30 min. Deje las muestras en tampón de formol para su posterior análisis histológico. </li><li> Externalizar la muestra fijada a las instalaciones capaces de realizar la histología con stents metálicos. Durante este tratamiento, las muestras se incrustan en metacrilato de metilo, seccionada, y se analizaron histológicamente usando técnica de tinción de Mallory con tinción de azul de Prusia para partículas de hierro. </li></ol></li></ol>

<ol>
	<li>Garg, S., Serruys, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+stents:+current+status.">Coronary stents: current status.</a> J Am Coll Cardiol. 56, 1-42 (2010).</li><li>Austin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-eluting+stents+versus+bare-metal+stents+for+off-label+indications:+a+propensity+score-matched+outcome+study.">Drug-eluting stents versus bare-metal stents for off-label indications: a propensity score-matched outcome study.</a> Circ Cardiovasc Interv. 1 (1), 45-52 (2008).</li><li>Polyak, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+field+gradient+targeting+of+magnetic+nanoparticle-loaded+endothelial+cells+to+the+surfaces+of+steel+stents.">High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents.</a> P Natl Acad Sci USA. 105 (2), 698-703 (2008).</li><li>Tassiopoulos, A. K., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+mechanisms+of+endothelialization+of+cardiovascular+implants:+a+review+of+recent+investigative+strategies.">Angiogenic mechanisms of endothelialization of cardiovascular implants: a review of recent investigative strategies.</a> J Biomat Sci-Polym E. 11 (11), 1275-1284 (2000).</li><li>Pislaru, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+forces+enable+rapid+endothelialization+of+synthetic+vascular+grafts.">Magnetic forces enable rapid endothelialization of synthetic vascular grafts.</a> Circulation. 114, I314-I318 (2006).</li><li>Uthamaraj, S., et al. <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+validation+of+a+novel+ferromagnetic+bare+metal+stent+capable+of+capturing+and+retaining+endothelial+cells).">Design and validation of a novel ferromagnetic bare metal stent capable of capturing and retaining endothelial cells).</a> Ann Biomed Eng. 42 (12), 2416-2424 (2014).</li><li>Gulati, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+origin+and+function+of+cells+with+endothelial+phenotype+obtained+from+adult+human+blood.">Diverse origin and function of cells with endothelial phenotype obtained from adult human blood.</a> Circ Res. 93 (11), 1023-1025 (2003).</li><li>Lee, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticles+of+magnetic+ferric+oxides+encapsulated+with+poly(D,L+latide-co-glycolide)+and+their+applications+to+magnetic+resonance+imaging+contrast+agent.">Nanoparticles of magnetic ferric oxides encapsulated with poly(D,L latide-co-glycolide) and their applications to magnetic resonance imaging contrast agent.</a> J Magn Magn Mater. 272 (3 Special Issue), 2432-2433 (2004).</li><li>Lee, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+enhancement+of+iron+oxide+nanoparticles+encapsulated+with+poly(D,L-latide-co-glycolide).">Magnetic enhancement of iron oxide nanoparticles encapsulated with poly(D,L-latide-co-glycolide).</a> Colloid Surface A. (1-3), 255-251 (1016).</li><li>Forbes, Z. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+targeted+drug+delivery+to+magnetic+stents+for+therapeutic+applications.">Locally targeted drug delivery to magnetic stents for therapeutic applications.</a> Computer Architectures for Machine Perception, 2003 IEEE International Workshop on. , 1-6 (2003).</li><li>Rathel, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Stents+Retain+Nanoparticle-Bound+Antirestenotic+Drugs+Transported+by+Lipid+Microbubbles.">Magnetic Stents Retain Nanoparticle-Bound Antirestenotic Drugs Transported by Lipid Microbubbles.</a> Pharm Res-Dordr. 29 (5), 1295-1307 (2012).</li><li>Gunn, J., Cumberland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stent+coatings+and+local+drug+delivery+-+state+of+the+art.">Stent coatings and local drug delivery - state of the art.</a> Eur Heart J. 20 (23), 1693-1700 (1999).</li><li>Lu, A., Jia, G., Gao, G., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+magnetic+stent+on+coronary+restenosis+after+percutaneous+transluminal+coronary+angioplasty+in+dogs.">The effect of magnetic stent on coronary restenosis after percutaneous transluminal coronary angioplasty in dogs.</a> Chin Med J (Engl. 114 (8), 821-823 (2001).</li><li>Kempe, H., Kempe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+magnetite+nanoparticles+for+implant-assisted+magnetic+drug+targeting+in+thrombolytic+therapy.">The use of magnetite nanoparticles for implant-assisted magnetic drug targeting in thrombolytic therapy.</a> Biomaterials. 31 (36), 9499-9510 (2010).</li><li>Chorny, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+stents+with+local+delivery+of+paclitaxel-loaded+magnetic+nanoparticles+using+uniform+fields.">Targeting stents with local delivery of paclitaxel-loaded magnetic nanoparticles using uniform fields.</a> P Natl Acad Sci USA. 107 (18), 8346-8351 (2010).</li><li>Polyak, B., Friedman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+targeting+for+site-specific+drug+delivery:+applications+and+clinical+potential.">Magnetic targeting for site-specific drug delivery: applications and clinical potential.</a> Expert Opin Drug Del. 6 (1), 53-70 (2009).</li><li>Liu, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+stent+hyperthermia+for+esophageal+cancer:+an+in+vitro+investigation+in+the+ECA-109+cell+line.">Magnetic stent hyperthermia for esophageal cancer: an in vitro investigation in the ECA-109 cell line.</a> Oncol Rep. 27 (3), 791-797 (2012).</li><li>Gunn, J., Cumberland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+stent+design+influence+restenosis.">Does stent design influence restenosis.</a> Eur Heart J. 20 (14), 1009-1013 (1999).</li><li>Aviles, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+of+ferromagnetic+stents+for+implant+assisted-magnetic+drug+targeting.">In vitro study of ferromagnetic stents for implant assisted-magnetic drug targeting.</a> J Magn Magn Mater. 311 (1), 306-311 (2007).</li><li>Mardinoglu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+modelling+of+physiologically+stretched+vessel+in+magnetisable+stent+assisted+magnetic+drug+targeting+application.">Theoretical modelling of physiologically stretched vessel in magnetisable stent assisted magnetic drug targeting application.</a> J Magn Magn Mater. 323 (3-4), 324-329 (2011).</li><li>Liu, Z. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+corrosion+cracking+of+2205+duplex+stainless+steel+in+H2S-CO2+environment.">Stress corrosion cracking of 2205 duplex stainless steel in H2S-CO2 environment.</a> J Mater Sci. 44 (16), 4228-4234 (2009).</li><li>Alverez-Armas, I., Degallaix-Moreuill, 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> Duplex stainless steels. , (2009).</li><li>Tefft, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetizable+Duplex+Steel+Stents+Enable+Endothelial+Cell+Capture.">Magnetizable Duplex Steel Stents Enable Endothelial Cell Capture.</a> Ieee T Magn. 49 (1), 463-466 (2013).</li><li>Pelton, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatigue+and+durability+of+Nitinol+stents.">Fatigue and durability of Nitinol stents.</a> J Mech Behav Biomed Mater. 1 (2), 153-164 (2008).</li><li>Knowles, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finite+element+analysis+of+a+balloon-expandable+stent+and+superior+mesenteric+arterial+wall+interaction.">Finite element analysis of a balloon-expandable stent and superior mesenteric arterial wall interaction.</a> J Vasc Surg. 60 (6), 1722-1723 (2014).</li><li>Veeram Reddy, S. R., et al. <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+biodegradable+stent+applicable+for+use+in+congenital+heart+disease:+bench+testing+and+feasibility+results+in+a+rabbit+model.">A novel biodegradable stent applicable for use in congenital heart disease: bench testing and feasibility results in a rabbit model.</a> Catheter Cardiovasc Interv. 83 (3), 448-456 (2014).</li><li>Shellock, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+imaging+of+metallic+implants+and+materials:+a+compilation+of+the+literature.">MR imaging of metallic implants and materials: a compilation of the literature.</a> AJR Am J Roentgenol. 151 (4), 811-814 (1988).</li><li>Lopic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+magnetic+force+on+a+coronary+stent+in+MRI.">Quantitative determination of magnetic force on a coronary stent in MRI.</a> J Magn Reson Imaging. 37 (2), 391-397 (2013).</li></ol>

The authors declare that they have no competing financial interests.

Hemos desarrollado un stent magnética que puede funcionar como un stent de metal desnudo y puede atraer a las células endoteliales SPION marcado. En estudios previos relacionados con stents magnéticos, los investigadores han utilizado níquel stents recubiertos comerciales y bobinas o mallas hechas de materiales magnéticos debido a la falta de disponibilidad de un stent ferromagnético 5,10-14. Otros grupos también han utilizado la naturaleza paramagnética de stents de acero inoxidable 304-grado disponi...

Patients implanted with vascular stents manufactured from thrombogenic materials like stainless steel, cobalt chromium, and platinum chromium &#8211; both bare metal stents (BMS) and drug eluting stents (DES) &#8211; need anti-platelet therapy to prevent thrombus formation. BMS heal rapidly, but are subject to late stage restenosis due to incomplete healing. DES require long term anti-platelet therapy due to delayed healing. Anti-platelet therapy administered to avoid thrombosis as a result of incomplete or delayed healing leads to increased bleeding risk and may not be suitable for certain patients1,2. An ideal stent will heal completely and quickly thus avoiding long-term anti-platelet therapy and late stage restenosis. This complete healing can only be achieved if the stent is rapidly coated with a monolayer of endothelial cells after implantation. Coating the stents with biocompatible materials such as gold or other biopolymers has been shown to improve thrombo-resistance, but none of these techniques achieved ideal blood compatibility as may be possible by coating with endothelial cells3,4.
A stent can be coated with endothelial cells post implantation by attracting circulating progenitor cells. This self-seeding technique can be achieved by utilizing ligands and antibodies. But this technique is limited by the low number of circulating endothelial progenitor cells. A promising strategy is to deliver cells directly to the stent immediately following implantation during a short period of blood flow occlusion3,5. This strategy requires a technique for rapidly capturing cells and retaining them on the stent even after restoring blood flow. We have developed a technique in which a magnetic stent is used to attract and retain magnetically-labeled endothelial cells delivered post implantation. To achieve this, a functional BMS with sufficient magnetic properties to capture and retain magnetically-labeled endothelial cells is required6.
In this paper, we discuss the methods for designing, manufacturing, and testing a 2205 stainless steel stent. The stents were designed using CAD and FEA. The manufactured stents were magnetized using a neodymium magnet and the retained magnetic field was measured using a magneto-resistance microsensor probe. We then tested the stents for magnetically-labeled cell capture in a culture dish during our in-vitro experiments. Finally, the stents were tested in-vivo by implanting magnetic and non-magnetic stents in 4 pigs and histologically analyzing the stented arteries.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2205 Stainless steel</td>
			<td>Carpenter Technology Corporation</td>
			<td>N/A</td>
			<td>Round bar stock material</td>
		</tr>
		<tr>
			<td>Abaqus</td>
			<td>Dassault systems</td>
			<td>N/A</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td></td>
			<td></td>
			<td>Prescription drug.</td>
		</tr>
		<tr>
			<td>Clopidogrel</td>
			<td></td>
			<td></td>
			<td>Commercial name: Plavix. Prescription drug.</td>
		</tr>
		<tr>
			<td>CM-DiI</td>
			<td>Life Technologies</td>
			<td>V-22888</td>
			<td>Molecular Probes, Eugene, OR</td>
		</tr>
		<tr>
			<td>Endothelial growth medium-2</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand Held Crimping tool</td>
			<td>Blockwise engineering</td>
			<td>M1-RMC</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00011324</td>
			<td>CAUTION: wear proptective equipment and handle under fume hood</td>
		</tr>
		<tr>
			<td>Isoflurane anesthesia</td>
			<td>Piramal Critical Care, Inc.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00011674</td>
			<td></td>
		</tr>
		<tr>
			<td>NdFeB magnet 2&#34; Dia x 1&#34; thick</td>
			<td>Amazing magnets</td>
			<td>D1000P</td>
			<td>Axially magnetized disc magnet with poles on flat faces</td>
		</tr>
		<tr>
			<td>Over-The-Wire trifold balloon</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any commercially available OTW trifold balloon can be used</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td>Commonly known as PBS</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00003528</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Zoetis</td>
			<td></td>
			<td>Commercial Name: Sleepaway (26%), FatalPlus, Beuthanasi.&#160; Controlled substance to be ordered only by licensed veternarian</td>
		</tr>
		<tr>
			<td>SolidWorks</td>
			<td>Dassault systems</td>
			<td>N/A</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>SpinTJ-020 micro sensor</td>
			<td>MicroMagneitcs Sensible Solutions</td>
			<td>N/A</td>
			<td>Long probe STJ-020 microsensor</td>
		</tr>
		<tr>
			<td>SPION</td>
			<td>Mayo Clinic</td>
			<td>N/A</td>
			<td>Nanoparticles synthesized internally (Ref: Lee, S. J. et al. Nanoparticles of magnetic ferric oxides encapsulated with poly(D,L latide-co-glycolide) and their applications to magnetic resonance imaging contrast agent. J Magn Magn Mater 272, 2432-2433, doi:DOI 10.1016/j.jmmm.2003.12.416 (2004))</td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td></td>
			<td>Controlled substance to be ordered only by licensed veternarian</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td>Gibco, Grand Island, NY</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer Animal Health</td>
			<td></td>
			<td>Commercial name: Rompun. Controlled sunstance to be ordered only by a licensed veternarian</td>
		</tr>
	</tbody>
</table>

ferromagnetic bare metal stent for endothelial cell capture and retention

Se necesita endotelialización rápida de los stents cardiovasculares para reducir la trombosis del stent y evitar la terapia anti-plaquetaria que puede reducir el riesgo de hemorragia. La viabilidad de utilizar fuerzas magnéticas para capturar y retener células endoteliales excrecencia (EOC) marcadas con nanopartículas de óxido de hierro súper paramagnéticas (SPION) se ha demostrado previamente. Sin embargo, esta técnica requiere el desarrollo de un stent mecánicamente funcional a partir de un material magnético y biocompatible seguido de in-vitro e in-vivo de pruebas para demostrar la endotelización rápida. Hemos desarrollado un stent débilmente ferromagnética de acero inoxidable de 2.205 duplex utilizando el diseño asistido por ordenador (CAD) y su diseño se refinó aún más el uso de análisis de elementos finitos (FEA). El diseño final del stent exhibió una deformación principal por debajo del límite de fractura del material durante rizado mecánico y expansión. Cien stents fueron fabricados y un subconjunto de ellos se utilizó para ensayos mecánicos, retained mediciones del campo magnético, estudios in vitro de captura celular, y los estudios in vivo de implantación. Diez stents fueron probados para su despliegue para verificar si sostenidas que prensa y la expansión del ciclo sin fallo. Otros 10 stents se magnetizan usando un imán fuerte neodimio y se midió su campo magnético retenido. Los stents mostraron que el magnetismo retenido era suficiente para capturar marcado con SPION EOC en nuestros estudios in vitro. Captura EOC SPION marcado y la retención se verificó en modelos animales grandes implantando 1 stent magnetizado y 1 no magnetizado stent de control en cada uno de 4 cerdos. Las arterias con stent se explantaron después de 7 días y se analizaron histológicamente. Los stents débilmente magnéticas desarrolladas en este estudio fueron capaces de atraer y retener a las células endoteliales SPION marcado que pueden promover la curación rápida.

<html> Diseño de stent iterativo basado en FEA (Figura 1) mostró un stent que puede engarzado y ampliar con una cepa principal de 20%, que es menor que la deformación última 30%. Que prensa y ensayo de expansión (Figura 2) no mostraron signos de fractura. Fotos del stent deformado mostraron buen acuerdo con deformaciones FEA calculados y también imágenes de microscopía no mostraron fracturas (Figura 3). Como era de esperar de las mediciones del campo magnético r...

Watch this Scientific Journal Video about Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention at JoVE.com

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Nuestros objetivos eran para diseñar, fabricar y probar los stents ferromagnéticos para la captura de las células endoteliales. Diez stents fueron probados para la fractura y 10 más stents se ensayaron para determinar el magnetismo retenido. Finalmente, 10 stents se probaron in vitro y 8 más stents fueron implantados en 4 cerdos para mostrar la captura de células y la retención.

Ferromagnético Bare Metal stent para la captura de la célula endotelial y Retención

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

ferromagnetic-bare-metal-stent-for-endothelial-cell-capture

Research

JoVE Journal

Bioengineering

11.4K vistas.  Mayo Clinic.  Nuestros objetivos eran para diseñar, fabricar y probar los stents ferromagnéticos para la captura de las células endoteliales. Diez stents fueron probados para la fractura y 10 más stents se ensayaron para determinar el magnetismo retenido. Finalmente, 10 stents se probaron in vitro y 8 más stents fueron implantados en 4 cerdos para mostrar la captura de células y la retención. 

Video: Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. 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 surfaces5,6,7 (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 EPCs4,8. 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-coating9. 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.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autólogo de células progenitoras endoteliales-Siembra Tecnología y las pruebas de biocompatibilidad para dispositivos cardiovasculares en el modelo animal de gran

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Investigación

Bioingeniería

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

El seguimiento de los mecánica de la pared durante la implementación del stent en un buque

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

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Eléctrico de la célula-sustrato Impedancia de detección para la cuantificación de la proliferación endotelial, la función de barrera, y la motilidad

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabricación de Mecánicamente sintonizable y Bioactivos metal andamios para Aplicaciones Biomédicas

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

Co el trasplante de tejido ovárico humano con células endoteliales Ingeniería: una combinación de estrategia basado en la célula acelerada perfusión con entrega directa paracrina

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Inmovilización de personas en Caenorhabditis elegans con un Chip de microfluidos polidimetilsiloxano ultra-delgada de retención de agua

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Crecimiento de capas celulares endoteliales corneales humanas y ovinas en membranas biomateriales

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

Mitigación de la fijación de células de flujo sanguíneo a implantes metálicos a través de la inmovilización de péptidos derivados de CD47

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentación Del Crecimiento De Las Células Endoteliales En Placas De 6 Pozos En Una Coctelera Orbital Para Estudios Mecanobiológicos

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Un sistema microfisiológico para estudiar la interacción leucocitario-endotelial durante la inflamación

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...