This work was supported by The British Heart Foundation and The Oak Foundation.

Realice todos los experimentos con animales de acuerdo a los protocolos aprobados por el Comité Institucional de Uso y Cuidado de Animales de Laboratorio. 1. Preparación de Medios de Cultivo <ol><li> Hacer medios de cultivo para la línea celular de fibroblastos humanos CCL-153: F-12K mediana, suero bovino fetal al 10% (FBS) y 100 U / ml de penicilina y estreptomicina. </li><li> Hacer reprogramación de medios para la generación de PIPSC: medio de Eagle modificado de Knockout Dulbecco (DMEM) que contiene 20% suero sustitución Knockout, 0,1 mM β-mercaptoetanol, 0,1 mM Medio Esencial Mínimo (MEM) de ácidos no esencial Amino, 10 ng / factor de crecimiento de fibroblastos básico ml 2 (bFGF-2) y 100 U / ml de penicilina y estreptomicina. Añadir bFGF-2 frescos cada vez antes de usar los medios de comunicación. </li><li> Hacer Diferenciación Media (DM) para inducir células del músculo liso (SMC) Diferenciación: α-MEM medios que contienen 10% de FBS, Factor de Crecimiento 0,1 mM β-mercaptoetanol, 100 U / penicilina y estreptomicina y 25 ml ng / ml derivado de las plaquetas(PDGF-ββ). </li><li> Hacer endotelial Diferenciación Media (EC-DM) para inducir células endoteliales (CE) Diferenciación: medios que contienen 50 ng de crecimiento endotelial medios de comunicación-2 (EGM-2) / ml factor de crecimiento vascular endotelial (VEGF) y 100 U / ml de penicilina y estreptomicina. </li></ol> 2. Reprogramación fibroblastos humanos en células madre pluripotentes inducidas (parcialmente PIPSC) <ol><li> Cultura línea celular de fibroblastos humanos CCL-153 en 0.04% de la solución de gelatina frascos revestidos en medio de cultivo. </li><li> Células Passage cada 3 días en una proporción de 1: 6. Utilice las células antes del paso 9 para una óptima eficiencia de la generación PIPSC. Las células están listas para la transfección al llegar a 80 - 90% de confluencia. </li><li> Linealizar pCAG2LMKOSimO plásmido policistrónico que contiene 4 factores de reprogramación octamer vinculante factor de transcripción 4 (OCT4), Sox2, factor de Kruppel-like 4 (KLF4) y C-MYC (pCAG-OSKM). Digerir 5 g de plásmido con 5 unidades de enzima de restricción PvuI durante 3 horas a 37 ° C. Purificarel plásmido con un kit comercial según el protocolo del fabricante. </li><li> Transfectar 2 x 10 6 fibroblastos humanos con 4 g de plásmido pCAG-OSKM linealizado mediante electroporación con el kit de fibroblastos dérmicos humanos (NHDF) nucleofection según el protocolo del fabricante. Sembrar suavemente las células transfectadas en pre 0,04% de gelatina recubierta matraz T25 que contiene 5 ml de pre-calentado reprogramación de medios. </li><li> Después de 24 horas, cambio de medio con suplementos de 25 mg / ml de neomicina para seleccionar una población pura de células transfectadas. </li><li> Cambio de medio cada dos días hasta el día 4. Nota: Los fibroblastos humanos se convierten en PIPSC después de 4 días de reprogramación. </li></ol> 3. Diferenciación de PIPSC en células endoteliales y musculares lisas <ol><li> PIPSC Seed en platos de colágeno IV pre-recubiertos que contienen DM durante 4 días para inducir la diferenciación en las SMC. Cambio de medio cada dos días hasta el día 4. </li><li> PIPSC Semilla de colágeno IV pre-recubierto platos containing endotelial-DM durante 6 días para inducir la diferenciación en las células endoteliales. Cambie medio cada dos días hasta el día 6. </li></ol> 4. Descelularizado Aorta Preparación del injerto Nota: Todas las soluciones y equipos deben ser estériles. <ol><li> Sacrificar el ratón por dislocación cervical y fijar el ratón en una posición supina bajo microscopio de disección. </li><li> Corte a través del esternón y alrededor de la caja torácica para abrir la cavidad torácica. Quite el corazón, los pulmones y el esófago para exponer la aorta. Retire con cuidado la grasa peri-aorta con fórceps. </li><li> Cortar la aorta desde el extremo anterior con unas tijeras y coloque un lado del extremo con fórceps romos. Separe la aorta de la columna de la columna vertebral por detrás mediante disección roma. Cierre con cuidado todas las arterias de ramificación de la aorta por la ligadura con un electrocoagulador bipolar y el corte desde el otro extremo de la ligadura con unas tijeras. </li><li> Utilice una jeringa de 5 ml para limpiar el lumen de la aorta con 3 ml de solución salina que contiene 100 U de heparina para prevenir la formación de coágulos de sangre. </li><li> Cortar el extremo posterior de la aorta antes se ramifica en el renal. Mantener la integridad de la aorta es muy importante durante todo el procedimiento. </li><li> Enjuague el lumen de la aorta con 5 ml de dodecilsulfato de sodio (SDS) solución de 0,075% diluido en tampón fosfato salino (PBS). </li><li> Remojar la aorta torácica en solución de SDS al 0,075% en una placa de Petri de 10 cm. Coloque la placa de Petri en un agitador orbital durante 2 horas a 150 rpm a temperatura ambiente. </li><li> Enjuague el lumen de la aorta con 5 ml de PBS. </li><li> Remoje la aorta en PBS en una placa de 10 cm de Petri. Coloque la placa de Petri en el agitador orbital durante 2 horas a 150 rpm a temperatura ambiente. Actualizar PBS cada 20 minutos. </li><li> Enjuague el lumen de la aorta con 5 ml de PBS. </li><li> Mantenga injerto aorta descelularizado en PBS a 4 ° C durante un máximo de una semana. </li></ol> 5. Dual Siembra de PIPSC y biorreactor acondicionado Nota: Todas solución des y el equipo deben ser estériles. Realizar todas las operaciones en una campana de cultivo de tejidos. <ol><li> Montar el circuito de flujo de biorreactor, como se ilustra en la Figura 1. Conectar cámara de incubación, depósito de medio, la bomba peristáltica y la cámara de cumplimiento de la tubería. Volumen mínimo de medio necesario para la circulación es de 40 ml. </li><li> Pre-condición del recipiente descelularizado con medio de cultivo empapando la aorta en un medio de DM en un plato de 10 cm Petri. Coloque la placa de Petri en el agitador orbital durante 1 hora a 50 rpm a temperatura ambiente. </li><li> Introduzca 1 cm tubos de nylon de longitud (OD 0,9 mm, ID 0,75 mm) en ambos extremos de la vasija descelularizado bajo el microscopio. Ate el recipiente y los tubos con 8-0 suturas de seda. </li><li> Montar el injerto de aorta descelularizado en la cámara de incubación mediante la conexión de los tubos de nylon con los puertos de entrada y salida (1/32 &quot;) de tubos de la pared de la cámara. Mantener cámara de incubación con 5 ml de medio cada vez. </li><li> Trypsinize PIPSC añadiendo precalentadotripsina para cubrir la placa de cultivo y el rock suavemente el plato 10 veces. Deseche la tripsina y dejar el plato en la incubadora durante 2 - 3 minutos. Añadir medio de cultivo pre-calentado en el plato y mezclar el medio con las células. </li><li> Contar el número de células usando un hemocitómetro. Centrifugar dos alícuotas de suspensiones celulares que contienen 5 x 10 5 células cada uno durante 5 min a 300 x g. Aspirar el sobrenadante por completo. </li><li> Después de aspirar el sobrenadante por completo, resuspender 1 pellet de las pepitas sedimentos celulares en 50 l de DM. Inyectar esta suspensión de células en la luz del vaso descelularizados. </li><li> A continuación, volver a suspender el sedimento celular otra pips en 100 l de Matrigel. Pipetear con cuidado la mezcla sobre el injerto de aorta descelularizado. </li><li> Espere de 10 - 15 min hasta que la mezcla se convierte en un estado similar a un gel uniformemente y se envuelve alrededor de la superficie externa del vaso. Llene la cámara de incubación con DM. </li><li> Configuración de lugar en una incubadora de CO2 al 5% todo el biorreactor a 37 ° C. Girar manualmente el injerto descelularizado 90 ° alrededor del eje longitudinal cada media hora en el primero 2 hr. </li><li> Mantenga la cultura estática durante 12 horas para permitir la adhesión celular. </li><li> Entregar DM a través del lumen por la bomba peristáltica para inducir la diferenciación de células musculares lisas. Iniciar la velocidad de flujo inicial en 5 ml / min y aumento escalonado a 20 ml / min durante 24 hr. </li><li> Mantener la tasa de flujo del medio circulante a 20 ml / min durante 24 horas. </li><li> Detener el flujo del medio y mover el ajuste de la incubadora biorreactor. </li><li> Re-semilla del lumen del injerto con PIPSC. Trypsinize, recuento y centrifugar 1 x 10 6 PIPSC como en los pasos 5.5 a 5.6. Resuspender las células en 50 l de medio EGM-2 que contienen 50 ng / ml de VEGF. Inyectar la mezcla de células en el lumen del injerto. </li><li> Cambiar el medio en la cámara de incubación y toda la circulación en EGM-2 medio que contiene 50 ng de VEGF / ml para inducir la diferenciación endotelial. </li><li> Mueva retrasando a th el biorreactore incubadora. Gire manualmente el injerto 90 ° cada 30 minutos en las primeras 2 horas y luego mantener una cultura estática durante 12 horas. </li><li> Iniciar la circulación de flujo de 5 ml / min y aumento escalonado a 35 ml / min. Mantener el caudal a 35 ml / min durante 5 días. Cambiar circulante y medio cámara de cada dos días. </li><li> Se recoge el injerto de ingeniería para su posterior injerto de ratón. </li></ol> 6. El injerto de doble cabeza de serie PIPSC injerto en ratones <ol><li> Uso NOD.CB17-Prkdc scid / NcrCrl ratones macho como receptores de injertos buque. Asegúrese siempre de que los ratones son alrededor de 10 semanas de edad, pesan aproximadamente 25 - 35 g, y se encuentran en buenas condiciones de salud. </li><li> Anestesie ratón receptor mediante la administración de una combinación de Hypnorm (25 mg / kg) y Hypnovel (25 mg / kg) por vía intraperitoneal. Aplicar vaselina pomada oftálmica en los ojos para evitar la sequedad mientras que bajo anestesia. Confirme anestesia eficiente, garantizando que los ratones tienen los músculos relajados y están respirando constantemente. <ol><li> Fijar el ratón en una posición supina con su cuello afeitó y extendido. Administrar sulfato de atropina (1,7 mg / kg) en combinación con los anestésicos para asegurar que las vías respiratorias del ratón sigue siendo clara. </li></ol></li><li> Preparar una incisión en la línea media de la mandíbula al esternón del ratón. Bajo un microscopio de disección con 5 a 10 veces la amplificación, levantar las glándulas salivales derecha lateralmente y eliminar el músculo esternocleido- derecho a exponer la arteria carótida común derecha. </li><li> Retire con cuidado los tejidos unidos a movilizar a la arteria carótida común derecha desde el extremo distal hacia el extremo proximal. Ligar la porción media de la arteria carótida común dos veces con una sutura de seda 8-0 y disecar entre los dos lazos. </li><li> Pasar a través de un manguito de tubo de nylon autoclavable en cada extremo del vaso y fijar cada extremo con pinzas microhemostat. </li><li> Quitar el lazo de sutura en un extremo y aplicar una gota de heparina (100 U / ml). Inmediatamente después, invertir el extremo distal del ésimoe arteria para cubrir todo el cuerpo del manguito y fijar el recipiente invertido con una sutura de seda 8-0 en el manguito. <ol><li> Repetir el mismo procedimiento para la otra porción de la arteria. Arteria Lavar con solución salina para eliminar los coágulos de sangre. </li></ol></li><li> Implantar un injerto de vaso descelularizado entre dos extremos de la arteria carótida por deslizamiento los extremos de los vasos de la descelularizados a través de los puños de la arteria y la fijación con suturas de seda 8-0. Eliminar pinzas vasculares de cualquiera de los extremos antes de evaluar la pulsación del injerto. </li><li> Coloque la glándula salival de vuelta a su posición original. Cierre de la piel en la ubicación quirúrgica con una sutura interrumpida usando una sutura de poliglactina 6-0. </li><li> Consistentemente observar los signos vitales de ratón después de que se completa el procedimiento, hasta que se reanude la conciencia. Sacrificar los ratones receptores por dislocación y de los vasos de la cosecha injertos cervicales ya sea 24 horas o 3 semanas más tarde para su posterior análisis. </li></ol>

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Los autores no tienen conflictos de intereses financieros.

El protocolo actual indica un sonido, la estrategia rápido, simple, eficiente y reproducible en la que los vasos de ingeniería tisular funcionales se pueden generar utilizando PIPSC a partir de fibroblastos humanos. Esta técnica representa una herramienta valiosa para la medicina regenerativa, la ingeniería de tejidos y terapia celular específica del paciente potencialmente en el futuro cercano. Los pasos críticos para garantizar la eficacia del protocolo incluyen la preparación de PIPSC, preparación de injertos...

La construcción de conductos vasculares es una estrategia fundamental para la reparación quirúrgica de los vasos dañados y heridos resultantes de enfermedades cardiovasculares. Hasta la fecha, los materiales utilizados en la cirugía de injerto biocompatible incluyen polímeros sintéticos (politetrafluoroetileno [teflón], politetrafluoroetileno expandido [ePTFE; Gore-Tex] o tereftalato de polietileno [Dacron]), aloinjertos, tejido autólogo (pericardio o vena safena) y xenoinjertos 1. Mientras que los injertos artificiales (por ejemplo, Gore-Tex y Dacron) son los más utilizados, estos materiales probablemente causan numerosas complicaciones a corto y largo plazo que incluyen la estenosis, la deposición de calcio, trombo-embolia y las infecciones. Aunque los pacientes con injertos biológicos presentes con una disminución de eventos tromboembólicos, todavía se encuentran con limitaciones, como el fracaso del injerto secundaria y durabilidad acortada debido a la degradación de la calcificación 2. Por lo tanto, a pesar de las mejoras significativas en t quirúrgicaechniques largo de los años, los investigadores y los médicos todavía están cargados con la necesidad de identificar el conducto ideal para enfermedades vasculares. Más recientemente, el campo de la investigación de la ingeniería de tejido vascular ha generado un concepto en el que las células se incorporan en estructuras biodegradables, con el objetivo de crear un entorno biomimético que personifica un recipiente funcional para injerto realizado con éxito 1. Fundamentalmente, el éxito de las construcciones vasculares depende de tres componentes esenciales; las células que componen el andamio, es decir, una capa interna de células endoteliales y una capa de células del músculo liso, un andamio que contiene la matriz extracelular apropiado para proporcionar propiedades mecánicas comparables a la vasculatura nativa, y la señalización molecular / celular que se requiere para iniciar / regulador reparación. La permeabilidad del injerto a largo plazo y el desarrollo sostenido de los neo-tejidos son altamente dependientes de la siembra de células eficaz de andamios, THereby haciendo que la decisión del tipo de célula de importancia crítica. Varios informes demuestran el uso de endoteliales maduras y las células musculares lisas de diferentes fuentes para desarrollar conductos de pequeño diámetro de 3-6. Aunque prometedores, la falta de vasos autólogos suficiente para obtener endoteliales maduras y las células musculares lisas siguen siendo una carga considerable. Más recientemente, las células madre a partir de diversas fuentes han sido explotados para aplicaciones de ingeniería de tejidos vasculares. Células mononucleares de hecho, las células madre pluripotentes una variedad de tipos de células madre, incluyendo células madre embrionarias 7, inducida (CMPI) 8,9, PIPSC 10,11, derivadas de la médula ósea 12, células madre mesenquimales 13, las células progenitoras endoteliales y la pared del vaso adulto -derivado células madre antígeno-1 (Sca-1) + células madre / progenitoras 14,15 todos se han demostrado que son capaces de diferenciarse en células endoteliales ya sea funcional o de músculo liso en respuesta a los medios de comunicación definidos ycondiciones de cultivo. Además, la capacidad ilimitada de auto renovación de las células madre a tomar mejores candidatos a diferencia endoteliales maduras y las células musculares lisas que sólo se puede dividir por un número finito de veces antes de someterse a la detención del crecimiento y la senescencia. La selección del material de andamio para generar tejido éxito recipiente diseñado para el injerto depende de varios factores tales como la biocompatibilidad, propiedades biomecánicas, y la velocidad de biodegradación. Fundamentalmente, los materiales utilizados para crear andamios para los injertos deben ser biodegradables y no se montarán las respuestas inmunes receptores innecesario. Además, debe abarcar una porosidad adecuada y la microestructura para la fijación celular y la supervivencia posterior. Hasta la fecha, los materiales más comunes usados ​​para andamios en la ingeniería de tejidos vascular incluyen polímeros de ácido poliglicólico, ácido poliláctico, y poli ε-caprolactona 16. Más recientemente, los materiales biológicos descelularizados tienenTambién ha aplicado con cierto éxito. Varios laboratorios han demostrado que la siembra de los vasos porcina humana descelularizado, canino o con células autólogas proporciona un injerto biológico que resistió la coagulación y la hiperplasia intimal 17-19. Otras estrategias de ingeniería de tejidos vascular incluyen injertos vasculares extracelulares basado proteínas de matriz por ejemplo, células de siembra en gel de fibrina 13 y generando láminas de células sin el apoyo del andamio 20, 21. El protocolo actual demuestra la diferenciación de PIPSC humano en endotelial funcional y células musculares lisas, la generación de un biorreactor que consiste en un andamio recipiente descelularizado para albergar células vasculares derivadas de PIPSC funcionales, y el injerto de los vasos de ingeniería tisular en la inmunodeficiencia combinada severa (SCID ) ratones. PIPSC son un tipo de célula óptima a utilizar para la ingeniería de tejidos de injertos de vasos ya que estas células no forman tumores en ratones o plantean ético yallo respuestas inmunes. Por otra parte, hemos demostrado que la estrategia para la generación de células musculares Pipa-células endoteliales y Pipa-lisas es eficiente y reproducible 10,11. A partir de entonces, se diseñó un recipiente de descelularizado para la siembra de las células vasculares derivadas de PIPSC para imitar las proteínas de la matriz que existe dentro de un vaso nativo, mejorando así la supervivencia de injerto y la eficacia. Además, la descelularización de los vasos antes de la siembra PIPSC previene la aparición de las respuestas inflamatorias montadas por tipos de células inmunes, tales como macrófagos. Más importante aún, este protocolo no sólo representan una metodología para generar prometiendo conductos vasculares para la traducción a los seres humanos, sino que también proporciona medios valiosos para el estudio y la comprensión de los mecanismos moleculares que regulan la regeneración del tejido vascular a través de modelos de ratón.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Human Fibroblasts CCL-153</td>
			<td >ATCC</td>
			<td >CCL-153</td>
			<td>Prenatal human embryonic fibroblasts</td>
		</tr>
		<tr >
			<td >ATCC F-12K Medium (Kaighn&#39;s Modification of Ham&#39;s F-12 Medium)</td>
			<td >ATCC</td>
			<td >30-2004</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ATCC</td>
			<td >30-2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout DMEM medium optimized for embryonic stem cells</td>
			<td >Life technologies (Gibco)</td>
			<td >12660-012</td>
			<td></td>
		</tr>
		<tr >
			<td >Knockout Serum Replacement</td>
			<td >Life technologies (Invitrogen)</td>
			<td >10828-028</td>
			<td></td>
		</tr>
		<tr >
			<td >Human Basic FGF-2&#160;</td>
			<td >Miltenyi Biotech</td>
			<td >130-093-837</td>
			<td></td>
		</tr>
		<tr >
			<td >alpha-MEM medium</td>
			<td >Life technologies (Invitrogen)</td>
			<td >32571093</td>
			<td></td>
		</tr>
		<tr >
			<td >Human PDGF</td>
			<td >R&#38;D System</td>
			<td >120-HD-001</td>
			<td></td>
		</tr>
		<tr >
			<td >Gelatin Solution 2%</td>
			<td >Sigma</td>
			<td >G1393</td>
			<td></td>
		</tr>
		<tr >
			<td >Plasmid 20866: pCAG2LMKOSimO (SOX2, OCT4, KLF4, C-MYC)</td>
			<td >Addgene</td>
			<td >20866</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;PvuI Restriction Enzyme</td>
			<td >New England Biolabs</td>
			<td >RO150S</td>
			<td></td>
		</tr>
		<tr >
			<td >SureClean Plus</td>
			<td >Bioline</td>
			<td >BIO-37047</td>
			<td></td>
		</tr>
		<tr >
			<td >Nucelofection Kit (NHDF Kit)</td>
			<td >LONZA</td>
			<td >VPD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Neomycin</td>
			<td >SIGMA</td>
			<td >G418</td>
			<td>Selection of&#160;</td>
		</tr>
		<tr >
			<td >KL 1500 LCD, Illumination for Stereo Microscopy</td>
			<td >SCHOTT</td>
			<td >KL 1500 LCD</td>
			<td>Cold light illumination for stereo microscopy</td>
		</tr>
		<tr >
			<td >Nikon Zoom Steromicroscope SMZ800</td>
			<td >Nikon</td>
			<td >SMZ800</td>
			<td></td>
		</tr>
		<tr >
			<td >Heparin sodium salt</td>
			<td >Sigma</td>
			<td >H3393</td>
			<td></td>
		</tr>
		<tr >
			<td >10% SDS Stock Solution Molecular Biology Reagent</td>
			<td >Severn Biotech</td>
			<td >CAS 151-21-3</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td >Sigma</td>
			<td >D8537</td>
			<td></td>
		</tr>
		<tr >
			<td >Matrigel (10mg/ml)</td>
			<td >BD</td>
			<td >A6661</td>
			<td></td>
		</tr>
		<tr >
			<td >Shaker IKA Vibrax with Shaking platform VX 7&#160;</td>
			<td >Jepson Bolton&#39;s, Janke&#38;Kunkel</td>
			<td >S32-102</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S Digital Pump Drive</td>
			<td >Cole-Parmer</td>
			<td >WZ-07523-80</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S 6-channel, 6-roller cartridge pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex L/S large cartridges for pump head</td>
			<td >Cole-Parmer</td>
			<td >EW-07519-75</td>
			<td></td>
		</tr>
		<tr >
			<td >Masterflex platinum-cured silicone pump tubing, L/S 14, 25 ft</td>
			<td >Cole-Parmer&#160;</td>
			<td >WZ-96410-14</td>
			<td >Tubing goes through the peristaltic pump</td>
		</tr>
		<tr >
			<td >0.5mm ID, 0.8 mm OD Silicone Tubing</td>
			<td >SILEX</td>
			<td >N/A</td>
			<td>Tubings connect incubation chamber, media reservoir and compliance chamber&#160;</td>
		</tr>
		<tr >
			<td >Fitting Reducer 0.5 to 1.6, natural Polypropyline</td>
			<td >Ibidi</td>
			<td >10829</td>
			<td>Adapter connect above two types of tubings</td>
		</tr>
		<tr >
			<td >1/32&#34; Tubing, ID 0.01&#34; (250&#181;m) Material: PEEK</td>
			<td >LabSmith</td>
			<td >T-132-010P</td>
			<td>Tubing through the incubation chamber wall which connects the graft with outside tubing</td>
		</tr>
		<tr >
			<td >One-Piece Fittings&#160;</td>
			<td >LabSmith</td>
			<td >T-132-100</td>
			<td>Fix the above tubings through the incubation chamber wall</td>
		</tr>
		<tr >
			<td >Nylon tubes (OD 0.9mm, ID 0.75mm)&#160;</td>
			<td >Smiths Medical</td>
			<td >N/A</td>
			<td>Tubings insert into two ends of the aorta graft</td>
		</tr>
		<tr >
			<td >NOD.CB17-Prkdcscid/NcrCrl mouse</td>
			<td >Charles River</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 8-0&#160; silk</td>
			<td >ETHICON</td>
			<td >W819</td>
			<td ></td>
		</tr>
		<tr >
			<td >Hypnorm</td>
			<td >Vetapharm</td>
			<td >Vm21757/4000</td>
			<td >Neuroleptanalgesic for use in mice</td>
		</tr>
		<tr >
			<td >Hypnovel (Midazolam)</td>
			<td >Roche</td>
			<td >59467-70-8</td>
			<td >Induction of anaesthesia</td>
		</tr>
		<tr >
			<td >Dissecting microscope</td>
			<td >Carl Zeiss</td>
			<td >Stemi 2000</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nylon Tubing</td>
			<td >Portex LTD</td>
			<td >800/200/100/200</td>
			<td >0.65 mm in diameter and 1 mm in length; to make artery cuff</td>
		</tr>
		<tr >
			<td >Electrocoagulator</td>
			<td >Martin&#160;</td>
			<td >SN 54.131</td>
			<td >Ligation of artery branches on aorta</td>
		</tr>
		<tr >
			<td >Bipolar micro hemostat forceps</td>
			<td >Martin</td>
			<td >80-91-12-04</td>
			<td >Fixation of vessel ends</td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFX-7</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >JFL-3dZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Vessel Dilator</td>
			<td >S&#38;T</td>
			<td >D-5aZ</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mini applier&#160;</td>
			<td >AESCULAP</td>
			<td >FE572K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Micro hemostats clips</td>
			<td >AESCULAP</td>
			<td >FE720K</td>
			<td ></td>
		</tr>
		<tr >
			<td >Surgical sutures, 6-0 VICRYL</td>
			<td >ETHICON</td>
			<td >V489</td>
			<td ></td>
		</tr>
	</tbody>
</table>

generation and grafting of tissue-engineered vessels in a mouse model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

La generación exitosa de PIPSC fue confirmado 4 días después nucleofecting fibroblastos humanos con un plásmido linealizado pCAG2LMKOSimO llevar 4 factores de transcripción, Oct4, SOX2, KLF4 y c-myc (OSKM). PIPSC muestra un fenotipo notablemente distinta en comparación con fibroblastos (Figura 2A) y expresó los 4 factores de reprogramación en el mRNA (Figura 2B) y los niveles de proteína (Figura 2C) 10. La eficacia de un injerto vascular a base de PI...

Watch this Scientific Journal Video about Generation and Grafting of Tissue-engineered Vessels in a Mouse Model at JoVE.com

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

A continuación, presentamos un protocolo para generar ingeniería tisular injertos de vasos que son funcionales para el injerto en ratones por doble siembra de células madre pluripotentes inducidas parcialmente (PIPSC) - deriva células musculares lisas y PIPSC - células endoteliales derivadas de un biorreactor andamio buque descelularizado.

Generación y Acrecentamiento de buques de ingeniería de tejidos en un modelo de ratón

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

generation-and-grafting-of-tissue-engineered-vessels-in-a-mouse-model

Research

JoVE Journal

Bioengineering

11.9K vistas.  King's College London BHF Centre.  A continuación, presentamos un protocolo para generar ingeniería tisular injertos de vasos que son funcionales para el injerto en ratones por doble siembra de células madre pluripotentes inducidas parcialmente (PIPSC) - deriva células musculares lisas y PIPSC - células endoteliales derivadas de un biorreactor andamio buque descelularizado. 

Video: Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protocolo para la evaluación relativa hidrodinámico de Tri-lámina polimérica Válvulas

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Investigación

Bioingeniería

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

Preparación de Membranas Light responden por un injerto superficial combinado y proceso de modificación posterior

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Técnica quirúrgica para la implantación de la ingeniería tisular injertos vasculares y posterior<em&gt; En Vivo</em&gt; Monitoreo

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Producción, Caracterización y posibles usos de un Humano esofágica mucosas Modelo ingeniería tisular 3D

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

Un Tejido Combinada 3D Diseñado<em&gt; In Vitro</em&gt; /<em&gt; In silico</em&gt; Modelo de tumor de pulmón para la predicción de la eficacia de drogas en Fondos Específicos mutacional

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Aplicaciones de la<em&gt; En Vivo</em&gt; Pruebas funcionales de la rata tibial anterior para la evaluación de la ingeniería tisular de reparación del músculo esquelético

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Ingeniería tisular tridimensional alineado astrositos redes para recapitular los mecanismos del desarrollo y facilitar la regeneración del sistema nervioso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Una nueva técnica de sutura de tenorrafia con injerto de colágeno de ingeniería tisular para reparar defectos grandes del tendón

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Siembra e implantación de un injerto traqueal tejido-dirigida biosintético en un modelo murino

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...