Este trabajo fue apoyado por subvenciones del Instituto Nacional de Corazón y Pulmón (R01 HL086582) y el Fondo de Células Madre de Ciencias de Nueva York (NYSTEM, Contrato # &#160; C024316) a STA y DDS ilustraciones utilizadas en vídeo JoVe fueron completados por Juan Nyquist; Ilustrador médico de la Universidad Estatal de Nueva York en Buffalo.

NOTA: Este protocolo ha sido aprobado por el Comité de Cuidado y Uso de Animales de la Universidad Estatal de Nueva York en Buffalo. Preparación 1. Pre-quirúrgico <ol><li> Utilice ovejas (cross Dorset, hembra, de aproximadamente 1-3 años de edad con un peso de 40-60 kg) para el siguiente estudio. Administrar ciclosporina A (200 mg / día), aspirina (975 mg / día), y Coumadin (20-30 mg / día) por vía oral, comenzando 3 días antes de la cirugía y continuar durante la duración de todos los estudios. </li><li> Asegúrese de que la oveja ha ayunado 12 h antes de la cirugía (alimento normal = 1,8 kg y 0,4 kg de heno de granos). </li><li> Esterilizar todos los suministros quirúrgicos utilizando un autoclave de vapor a 121 ° C y 15 psi durante 30 min. </li><li> Coloque los siguientes elementos en una hora desecador paraformaldehído 48 antes de la cirugía: sondas de 4 mm de flujo Doppler, 1 mm cristales ultrasónicos, permanentes catéteres arteriales, tubos de extensión y 42 &quot;Tygon Tubería. NOTA: Una tabla de equipo médico pertinente necesaria para lacirugía y para la esterilización se enumeran en la Tabla 1. Pre-colocación de la instrumentación en el tubo Tygon en este paso le ayudará a ahorrar tiempo durante la cirugía. <ol><li> Etiquete cada extremo de las sondas, catéteres y cristales como la izquierda y la derecha, en su caso. </li></ol></li></ol> 2. Operación Quirúrgica <ol><li> Inducir ovejas para la anestesia con diazepam (0,5 mg / kg) y ketamina (4 mg / kg) por vía intravenosa (IV). Alternativamente, utilizar Telazol (4 mg / kg) IV. <ol><li> Realizar intubación orotraqueal con un tubo endotraqueal diámetro 8,5-10,0 mm interno esposado 7. </li><li> Administrar anestesia inhalatoria a través de un circuito de reinhalación con un ventilador de las mareas volumen (7-10 ml / kg) o un ventilador de presión regulada (15-20 cm H 2 O). Use un vaporizador de precisión para administrar isoflurano o sevoflurano en una cantidad apropiada (3% -4% inicialmente) para llegar a un medio a lo profundo del escenario anestésico quirúrgico. La concentración alveolar mínima paraovejas es de 1,4% o 1,9%, respectivamente 8. </li><li> Evaluar la profundidad anestésica mediante la observación de la respuesta motora a los estímulos, reflejo palpebral, posición de los ojos, y la frecuencia cardíaca. De la saturación de oxígeno en la sangre (95% -100%) utilizando la oximetría de pulso, concentración de CO 2 (45-55 mmHg) en los gases espiratorios utilizando capnografía y mantener la temperatura corporal durante el procedimiento (38,5-39,5 ° C) usando una manta de calentamiento auto regulado. </li></ol></li><li> Afeitado lana de todo el cuello de la oveja, y más de un vena cefálica, utilizando una cuchilla # 40 en máquinas de cortar estándar. Preparar la piel de ambos sitios para la cirugía utilizando un 70% de alcohol isopropílico y 7,5% matorrales betadine gasa saturada. Comienza con una gasa alcohol para facilitar la extracción de aceites de la piel. Alternar entre gasas betadine y alcohol gasa tres veces. </li><li> Coloque ovejas en la mesa de operaciones en decúbito dorsal en la parte superior de una manta térmica. Pasar una sonda orogástrica de tamaño medio para permitir la expulsión pasiva de los contenidos estomacales. Extender elcuello y de oveja utilizar amortiguación de apoyo, según sea necesario para mantener la colocación. <ol><li> Realice una limpieza aséptica final utilizando 7,5% betadine gasa embebida y deje reposar durante 5 minutos antes de la cirugía. </li></ol></li><li> Administrar líquidos por vía intravenosa (solución de lactato de Ringer o solución salina 0,9%) a 10 ml / kg / hora a través de un angiocath colocado en la vena cefálica. Administrar antibióticos intraoperatorios y analgesia: Penicilina G procaína 6.600 U / kg por vía intramuscular (IM), gentamicina 1,6 mg / kg IM, y buprenorfina 0,005-0,01 mg IV o IM. </li><li> Hacer una incisión cm ~ 12 longitudinalmente sobre el cuello de la línea media ventral usando electro cauterio. Aislar arterias carótidas izquierda y derecha (~ 6 cm) mediante la eliminación de tejido conectivo usando una técnica de disección roma. Ate y cauterizar microvasos de ramificación de las arterias carótidas para minimizar la hemorragia. </li><li> Mantener la esterilidad mediante la utilización (a no estéril) enfermera quirúrgica para ayudar con la madriguera todos (sonda de flujo cableado y tubos, alambres de cristal ultrasónicosy el tubo de catéter) en la capa subcutánea de la piel. Use un trocar romo, que sale a través de la incisión preparada quirúrgicamente en el cuello dorsolateral. <ol><li> Alcanzar bajo el paño estéril y gire la cabeza de las ovejas para que el lado del cuello puede ser visualizado bajo la sábana. </li><li> Utilice una pinza hemostática curva 8 cm de túnel a través del espacio subcutáneo entre la incisión en el cuello en la línea media ventral y el lado del cuello. Abra y cierre la pinza hemostática para diseccionar sin rodeos un espacio para la tubería ~ 1.5 cm de ancho. Las puntas de la pinza hemostática deben residir a medio camino entre la cabeza y los hombros, a unos 10 cm caudal a la oreja derecha o izquierda. Gire la pinza hemostática para que las puntas están apuntando hacia la piel superficial. </li><li> Alcance bajo el paño estéril y hacer una incisión de 1,5 cm a través de la piel, sobre las puntas de la pinza hemostática con una cuchilla # 11 estéril. Visualice la punta de la pinza hemostática para confirmar una salida clara a través de la piel. </li><li> Pase el tubo de Tygon que contiene unall cableado y la tubería a través del túnel subcutáneo. Mantenga los cables y los tubos por encima del campo estéril. </li><li> Llegar debajo de la cortina para retirar el tubo de Tygon exterior del cuello, exponiendo el cableado implantado, ya que deja el cuello de la oveja. Tire de las líneas individuales para minimizar la holgura en el espacio subcutáneo. Deje suficiente distancia para fijar correctamente la instrumentación de la arteria. </li></ol></li><li> Coloque 4 mm sondas de flujo Doppler en ambas arterias carótidas y alcanzar una lectura inicial (Figura 1). Administrar 100 U / kg de heparina IV 30 min antes de la sujeción de la arteria. </li><li> Continuar la administración de heparina a 100 U / kg / hr hasta el final de la cirugía. Sujete la arteria carótida utilizando no trituración pinzas vasculares y extirpar una porción (aproximadamente 4 cm de longitud). La velocidad de flujo de la carótida contralateral aumentará 50% -100% para mantener el flujo de sangre al cerebro. NOTA: Es posible limitar el estiramiento longitudinal por retroceso del vaso nativo mediante la eliminaciónsegmentos más cortos que están sustituyendo y / o estirar la arteria nativa con vascular abrazaderas para acortar brecha hasta que se complete el procedimiento de anastomosis completo. Esto ayudará a limitar la tensión en las suturas de retención individuales y el injerto implantado. </li><li> Suturar la TEV en su lugar con puntos interrumpidos simples con 7-0 prolina ETHALLOY doble sutura monofilamento armado. Si es necesario, aplique relajantes musculares lisas vasculares como la papaverina (15 mg / ml) o nicardipina (1,25 mg / ml) por vía tópica a la vasculatura nativa con el fin de evitar la vasoconstricción lo que dificultaría la sutura de la anastomosis. NOTA: Comience colocando suturas con aproximadamente 1 mm de distancia. Esto puede variar mucho de un caso a otro. La composición y el espesor de TEV afectarán a la distancia efectiva entre las suturas. A medida que el espesor del tejido nativo o TEV disminuye, puede ser necesario colocar las suturas más juntos. <ol><li> Primero ancla cuatro puntos de la TEV a la arteria nativa mediante la colocación de dos s opuestastitches en ambos extremos proximal y distal (Figura 2 -Di). Mantenga cada ancla enseñado usando pinzas hemostáticas. NOTA: descripciones proximal y distal están en referencia a la dirección del flujo de sangre a través del papel. </li><li> Añadir 5-6 suturas más en el lado superficial de ambos los extremos proximal y distal para comenzar la anastomosis. (Figura 2 -Pr). Simultáneamente gire el vascular abrazaderas 180 grados. </li><li> Vuelva a establecer la tensión en las suturas de anclaje. Añadir adicional (5 a 6) suturas interrumpidas a extremos proximal y distal en el lado girada de la TEV. </li></ol></li><li> Una vez que el TEV se sutura firmemente en su lugar, gire de nuevo a la posición original y retire el vascular abrazaderas de uno a la vez, pinza distal primero. Un ligero sangrado en los sitios de anastomosis es común. Esto puede resolver de forma natural después de varios minutos de liberación de la abrazadera y reclamping o exigir la colocación de suturas adicionales. Coloque la sonda de flujo Doppler (Figura 3-FL) De nuevo en proximal de la arteria nativa de flujo de sangre que entra en el TEV y la velocidad de flujo del monitor. NOTA: Espere que los caudales de la carótida izquierda y la derecha se equilibren después de aproximadamente 15 min. Si la velocidad de flujo en la arteria carótida con implantado TEV cae de manera constante, es posible que el TEV es la coagulación. Otras anormalidades posibles respecto al flujo pueden ser atribuidos a la constricción de la proximal arterias nativas o distal a la TEV. Si esto ocurre, el uso de relajante del músculo liso vascular adicional puede ser aplicado, y el vaso nativo debería volver a un tono basal después de 30-60 min tras el cierre de tejido sobre el sitio del injerto. <ol><li> Si lo desea, extirpar la arteria carótida contralateral y se sutura en su lugar como un control &quot;Sham&quot;. Esto es más clínicamente relevante que dejando la arteria carótida derecha sola y única fijación de la sonda de flujo, cristales ultrasónicos, y el catéter. Si se quiere un simulacro de control, realice esto antes de ir al paso 2.11. </li></ol></li><li> Sutura 1 mm cristales ultrasónicos (Figura 3 -CR1 CR2) a los lados de la VET oponerse utilizando 7-0 Proline. Hilo de sutura a través de la cabeza de cristal ultrasónico y cosa sólo a la capa superficial de la TEV. </li><li> Cateterización de la arteria mediante un modificado 18 G catéter con un bolsito de teflón tejido (Figura 3- Ca y Figura 4A). Coloque el catéter distal a la TEV en tejido arterial nativo. <ol><li> Suturar la tapeta de la pared arterial con 5/0 Ethibond para controlar el sangrado. Utilice ciclohexanona para adherir el tubo capilar al catéter que ha sido purgado con solución salina. Utilice la tubería como una línea de extensión. </li><li> Utilice un adaptador Stub 20 G Luer con un tapón de inyección SurFlo para sellar el extremo exteriorizado de tubo (Figura 4B). Para mantener la permeabilidad del catéter, obtener el volumen de cebado de la línea y lavarlo con 10 ml de solución salina y luego 5.000 U / ml de la inyección de heparina sódica cada 2-3 días. </li></ol></li><li> Registre la distancia entre la sonda de flujo y los cristales ultrasónicos, así como la distancia entre la sonda de flujo y el catéter. Esto permitirá a la velocidad de la onda de pulso que se calcula en relación con el software. Si no se necesitan estos cálculos, no implante de un catéter. </li><li> Obtener una lectura intraoperatoria si se desea asegurar todo el material implantado es funcional (véase la sección 3). </li><li> Asegure las líneas y cables implantados a la cercana musculatura utilizando 2/0 seda y una aguja cónica (Figura 3). <ol><li> Coloque la sonda de flujo vascular cable paralelo al buque, con el caudal de la sonda y el cable que se extiende cranealmente y, a continuación, hacer una &quot;U-Turn&quot; hacia la musculatura lateral. Fije el cable a la musculatura adyacente, usando seda 2-0 en una aguja cónica en dos lugares, de manera que la sonda de alambre o de flujo no es capaz de colocar cualquier cepa en el buque. Asegúrese suturas estén ajustadas, pero no apriete demasiado y estrangular musculatura (Figura 3). </li><li> Suturar la línea de cables de cristal y arterial catéter a la musculatura lateral, lo que permite ~ 1,5 cm de holgura, similar a los pasos anteriores para asegurar sonda de flujo (Figura 3). </li><li> Grupo todos los cables y líneas juntas y anclarlos a la musculatura justo antes de salir a través de túnel subcutáneo, similar a los pasos anteriores. </li></ol></li><li> Cierre la zona quirúrgica con una sutura de Vicryl 2-0 en capas utilizando un patrón de sutura que se ejecuta en el salpicadero y subdérmico, corriendo colchón puntadas en la piel (salpicadero, la aguja no cortante, la piel, el corte de la aguja). Cerrar la incisión 1,5 cm en el cuello dorsal alrededor de los cables y líneas exteriorizados usando Vicryl 2/0 y una aguja de corte. </li><li> Coloque los cables de sonda de flujo, líneas de catéteres y alambres de cristal de ultrasonidos en una bolsa (10 cm x 10 cm) que se sutura firmemente a la piel de las ovejas (Figura 5 - después de la recuperación). </li><li> Poco a poco dejar de depender de las ovejas de la anestesiay el ventilador volumen corriente y luego retirar la intubación a las ovejas cuando se reanuda la respiración espontánea. Retire la angiocath inserta en la vena cefálica y el vendaje de ser necesario. </li><li> Vendar el cuello usando un ungüento antibiótico triple en las incisiones, una almohadilla Telfa, gasa rodillo tensor y Elasticon. </li><li> Administrar analgesia postoperatoria: flunixina meglumina 2,2 mg / kg IM una vez durante la recuperación, luego 1.1 mg / kg IM una vez al día durante dos días, buprenorfina 0,005-0,01 mg IV o IM dos veces al día durante un día. </li></ol> 3. En Monitoreo Vivo <ol><li> Coloque las ovejas en el carro móvil para garantizar la seguridad adecuada. Esto permite a las ovejas para mantener la calma y consciente, sin comprometer el hardware. Es posible que necesite para aclimatarse ovejas para la compra 2 o 3 veces durante 30 minutos antes de obtener grabaciones de instrumentación. </li><li> Retire todos los cables y las líneas de la bolsa y conectarse a los dispositivos de vigilancia. Conecte la sonda de flujo a un medidor de flujo, los cristales de 1 mm de ultrasonidos conectado a TRBCaja -USB y catéteres líneas a transductores de presión. Un diagrama de flujo de esta configuración se proporciona (Figura 6). </li><li> Calibrar sondas de flujo y transductores de presión antes de la adquisición de datos. NOTA: Debido a la variabilidad potencial entre las versiones de software y las diferencias en los equipos utilizados, calibraciones y ajustes variarán de un caso a otro. </li><li> Utilizar un osciloscopio para afinar la medición de cristal Sonometrics, de acuerdo con el protocolo del fabricante. </li><li> Registrar los datos utilizando software informático (Figura 7). Rastros en la mitad superior de la figura 7 en el color blanco corresponde a la TEV implantado, mientras trazas en la mitad inferior de color rojo corresponde al Sham / nativos. Tanto para el TEV y Sham la velocidad de flujo (ml / min), la presión arterial de la sangre (mm Hg) y el diámetro (mm) se registran en vivo. </li><li> Registro durante al menos 1 min sin perturbaciones. Exportar estos datos para un análisis más detallado. Después de la grabación, DISCONNect todos los cables y lugar de nuevo en la bolsa se sutura en el cuello de la oveja. </li></ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+vascular+tissue-engineering.">Animal models for vascular tissue-engineering.</a> Current Opinion in Biotechnology. 24, 916-925 (2013).</li><li>Liang, M. -. S., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+fibrin-binding+TGF-&#946;1+for+sustained+signaling+and+contractile+function+of+MSC+based+vascular+constructs.">Engineering fibrin-binding TGF-&#946;1 for sustained signaling and contractile function of MSC based vascular constructs.</a> Biomaterials. 32, 8684-8693 (2011).</li><li>Han, J., Liu, J. Y., Swartz, D. D., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+functional+effects+of+organismal+ageing+on+smooth+muscle+cells+derived+from+bone+marrow+mesenchymal+stem+cells.">Molecular and functional effects of organismal ageing on smooth muscle cells derived from bone marrow mesenchymal stem cells.</a> Cardiovascular Research. 87, 147-155 (2010).</li></ol>

There are no disclosures to report.

El propósito de este informe es proporcionar un procedimiento fiable y reproducible para VETs implantes de interés en la arteria carótida ovina. Las arterias carótidas nativas de los animales utilizados en este modelo fueron 0,5-0,75 mm de espesor y 4.5-5 mm de diámetro exterior. La técnica quirúrgica descrita aquí ha sido un éxito para la implantación VETs de geometrías variables que miden 0,25 a 1 mm de espesor, de 4-5 mm de diámetro exterior y 4 cm de longitud, con gran éxito la prueba efectiva de hasta ...

El enfoque principal para el desarrollo de TEV ha sido la de proporcionar un sustituto para el reemplazo de injerto autólogo cuando los vasos autólogos no están disponibles y limitar los donantes vista morbilidad. Por ejemplo, el número de cirugías de revascularización coronaria por año ha superado los 350.000 en los EE.UU., y la fuente ideal de injertos adecuados sigue siendo la arteria mamaria interna izquierda, arteria descendente anterior coronaria y la vena safena 1. Dado que muchas personas que sufren de enfermedades vasculares pueden no tener las arterias y venas adecuadas para la sustitución de injerto autólogo, el desarrollo de TEVs por lo tanto se ha convertido en un intenso campo de la investigación durante décadas 1-6. Mientras que la ingeniería y optimización de nuevos VETs han sufrido muchos avances, informes sobre las técnicas quirúrgicas empleadas para implantar las VETs mismos no ha sido un tema de tanta discusión intensa. Más bien, los protocolos relativos a la implantación de TEVs en modelos animales se dejaron en gran partehasta investigar los investigadores. La siguiente manuscrito muestra cómo implantar TEVs mediante la utilización de un enfoque anastomosis de extremo a extremo. Este procedimiento se ha optimizado mediante el uso de un patrón de sutura de la anastomosis específica, la estabilización de técnica de sutura, la optimización de la tensión longitudinal y la adición de la instrumentación de monitoreo in vivo. Este método se contrasta con algunas de las muchas variaciones que se han utilizado previamente. Además, este procedimiento se describe cómo adquirir parámetros tales como la presión arterial, la TEV diámetro / cumplimiento y el caudal a través de la TEV después de la cirugía hasta la explantación. Esta recogida de datos proporciona un análisis indispensable de la TEV mientras está en el proceso de remodelación.

<table border="0" cellpadding="0" cellspacing="0" width="1505">
	<tbody>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:335px;">Equipment</td>
			<td dir="LTR" style="width:208px;">Manufacturer</td>
			<td dir="LTR" style="width:311px;">Serial/Catalog #</td>
			<td dir="LTR" style="width:363px;">Notes</td>
		</tr>
		<tr height="56">
			<td dir="LTR" height="94" rowspan="2" style="height:94px;width:335px;">Pressure Transducer</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Becton Dickinson</td>
			<td dir="LTR" rowspan="2" style="width:311px;">P23XL-1</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each artery) 
			&#160;Used with water-filled diaphragm domes</td>
		</tr>
		<tr height="172">
			<td dir="LTR" height="172" style="height:172px;width:335px;">Amplifier and transducer box</td>
			<td dir="LTR" style="width:208px;">Gould</td>
			<td dir="LTR" style="width:311px;">5900 Signal Conditioner Cage</td>
			<td dir="LTR" style="width:291px;">1 
			Two transducers and amplifiers should be included in cage. While this specific unit may be discontinued, other commercially available pressure transducers with a BNC/analog output will communicate with the Sonometrics equipment</td>
		</tr>
		<tr height="121">
			<td dir="LTR" height="121" style="height:121px;width:335px;">T403 Console with TS420 perivascular flowmeter module(x2)&#160;</td>
			<td dir="LTR" style="width:208px;">Transonic Systems</td>
			<td dir="LTR" style="width:311px;">T403 module and TS420 (x2)</td>
			<td dir="LTR" style="width:291px;">1 
			Flow probes measuring flow through each of the carotid arteries will connect to each of the TS420 units.&#160;&#160;</td>
		</tr>
		<tr height="126">
			<td dir="LTR" height="126" style="height:126px;width:335px;">Digital ultrasonic measurement unit</td>
			<td dir="LTR" style="width:208px;">Sonometrics</td>
			<td dir="LTR" style="width:311px;">TR-USB</td>
			<td dir="LTR" style="width:291px;">1</td>
		</tr>
		<tr height="48">
			<td dir="LTR" height="48" style="height:48px;width:335px;">Flow Probe Precision S-Series 4mm</td>
			<td dir="LTR" rowspan="4" style="width:208px;">Transonic Systems Inc.</td>
			<td dir="LTR" rowspan="4" style="width:311px;">MC4PSS-LS-WC100-CM4B-GA</td>
			<td dir="LTR" rowspan="4" style="width:291px;">2</td>
			<td dir="LTR" rowspan="4" style="width:363px;"></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="49" rowspan="2" style="height:49px;width:335px;">1mm&#160; Sonometrics Crystals</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Sonometrics Systems</td>
			<td dir="LTR" rowspan="2" style="width:311px;">1R-38S-20-NC-SH</td>
			<td dir="LTR" style="width:291px;">&#160;2-4 (2 for each artery)</td>
		</tr>
		<tr height="81">
			<td dir="LTR" height="134" rowspan="2" style="height:134px;width:335px;">Catheter for implantation</td>
			<td dir="LTR" style="width:208px;">BD (Becton Dickinson)</td>
			<td dir="LTR" rowspan="2" style="width:311px;">&#160;381447</td>
			<td dir="LTR" style="width:291px;">&#160;1+ (1 for each artery) 
			Catheter is cut and secured to microbore tubing, stylette is utilized for insertion&#160;</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="73" rowspan="2" style="height:73px;width:335px;">Tygon Microbore Tubing</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Norton Performance Plastics</td>
			<td dir="LTR" style="width:311px;">(AAQ04127) Formulation S-54-HL</td>
			<td dir="LTR" style="width:291px;">NA (cut to length for an extension set)</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="49" rowspan="2" style="height:49px;width:335px;">Luer Stub Adapter</td>
			<td dir="LTR" style="width:208px;">BD (Becton Dickinson)</td>
			<td dir="LTR" style="width:311px;">427564 (20 gauge)</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each arterial catheter)</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="82" rowspan="2" style="height:82px;width:335px;">Surflo Injection Plug</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Terumo</td>
			<td dir="LTR" rowspan="2" style="width:311px;">SR-IP2</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each arterial catheter)</td>
		</tr>
		<tr height="39">
			<td dir="LTR" height="128" rowspan="2" style="height:128px;width:335px;">Meadox</td>
			<td dir="LTR" rowspan="2" style="width:208px;">PTFE (Teflon) Felt</td>
			<td dir="LTR" rowspan="2" style="width:311px;">19306</td>
			<td dir="LTR" style="width:291px;">NA (cut to size) 
			The PTFE felt used in our studies was discontinued. However, comparable companies such as &#8220;Surgical Mesh&#8221; offer products which are equivalent.&#160;</td>
		</tr>
	</tbody>
</table>

surgical technique for the implantation of tissue engineered vascular grafts and subsequent in vivo monitoring

El desarrollo de la ingeniería de tejidos vasculares (TEV) es avanzado por la capacidad de VETs implantes rutinaria y eficaz (4-5 mm de diámetro) en un modelo animal de gran tamaño. Un protocolo paso a paso para la colocación de inter-posicional de la evaluación digital de TEV y en tiempo real de la TEV y las arterias carótidas nativas se describe aquí. En vivo monitoreo es posible gracias a la implantación de sondas de flujo, catéteres y cristales ultrasónicos (capaz de registro de los cambios dinámicos de diámetro de TEVs implantados y arterias carótidas nativos) en el momento de la cirugía. Una vez implantados, los investigadores pueden calcular los patrones de flujo sanguíneo arterial, presión arterial invasiva y diámetro de la arteria produciendo parámetros como la velocidad de la onda de pulso, índice de aumento, las presiones de pulso y el cumplimiento. La adquisición de datos se lleva a cabo utilizando un único programa de ordenador para el análisis de toda la duración del experimento. Tales datos invaluable da una idea de remodelación de la matriz TEV, su resemblance / a controles simulados nativos y el rendimiento general del TEV en vivo.

Más de 30 ovejas han sido sometidos a la técnica quirúrgica se describe en este informe para la implantación de TEV (en prensa) 9. Una tabla que resume las más recientes operaciones de ovejas después de optimización de protocolos se muestran en la Tabla 2. Todas las ovejas recuperados después de la implantación TEV sin complicaciones potencialmente mortales. En algunos animales, la fibrosis se observó en la arteria nativa cerca de la punta del catéter arterial. No se ha observado u...

Watch this Scientific Journal Video about Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring at JoVE.com

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

Un protocolo de paso a paso para la colocación inter-posicional de ingeniería tisular buques (TEV) en la arteria carótida de una oveja mediante anastomosis de extremo a extremo y la evaluación digital en tiempo real en vivo hasta el sacrificio de animales.

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 ...

surgical-technique-for-implantation-tissue-engineered-vascular-grafts

Research

JoVE Journal

Bioengineering

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

11.6K vistas.  State University of New York Buffalo School of Medicine.  Un protocolo de paso a paso para la colocación inter-posicional de ingeniería tisular buques (TEV) en la arteria carótida de una oveja mediante anastomosis de extremo a extremo y la evaluación digital en tiempo real en vivo hasta el sacrificio de animales. 

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

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Bioluminiscencia de imágenes para la evaluación de la respuesta inmune después de la implantación de tejido cardiaco de Ingeniería (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Investigación

Bioingeniería

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

La ingeniería biológica basada en los injertos vasculares El uso de un biorreactor pulsátil

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sin contacto, libre de marca Seguimiento de células y matriz extracelular utilizando espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

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 ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Atímicos Rata Modelo de Evaluación de Ingeniería de ligamento cruzado anterior injertos

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

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 ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

El escalado de Engineered injertos vasculares Usando 3D Impreso guías y el método de anillo de apilamiento

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

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 ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.