Este trabajo fue apoyado por subvenciones del NHMRC y Fundación Médica Stafford Fox. Los autores agradecen la asistencia quirúrgica de Sue McKay, Liliana Pepe, Anna Deftereos y Amanda Rixon de la Medicina Experimental y Unidad Quirúrgica, Hospital de San Vicente, de Melbourne. También se presta apoyo por el Departamento de Innovación, Industria del Gobierno del Estado de Victoria y el Programa de Apoyo a la Infraestructura Operacional de Desarrollo Regional.

Los protocolos descritos aquí han sido aprobados por el Comité de Ética Animal del Hospital St. Vincent de Melbourne, Australia, y se llevaron a cabo bajo una estricta adherencia a las directrices del Consejo de Investigación Médica y Salud Nacional de Australia. NOTA: Dos protocolos de la cámara se describen a continuación. Los dos modelos diferentes y sus diseños de cámara específicos se ilustran en la Figura 1. Cámara (1) está hecha de policarbonato (para el modelo de cámara bucle arteriovenosa rata). Su forma es cilíndrica con un diámetro 13 mm interno y altura 4 mm. Una ventana en un punto en la pared permite el acceso libre del pedículo. En el segundo modelo (por flujo a través del pedículo rata modelo de cámara), la cámara está hecha de acrílico y es rectangular (10 x dimensiones de 8 x 4 mm 3 internos). Tiene dos aberturas de 1,5 mm en los lados opuestos para acomodar la arteria femoral y la vena, ya que traspasan la cámara. 1. Rata arteriovenosa Loop Cámara Modelo (Una Chamber por animal) NOTA: Antes de iniciar la cirugía, asegúrese de que todos los instrumentos han sido esterilizados adecuadamente. Del mismo modo, garantizar la instrumentos resto en las toallas estériles y están a una distancia razonable del campo quirúrgico para evitar la contaminación durante el procedimiento. <ol><li> Preparación del animal para la cirugía <ol><li> Utilice las ratas con un peso mínimo de 250 gramos por su gran tamaño de los vasos para la creación del asa arteriovenosa. </li><li> Anestesiar al animal con 4% de inhalación de isoflurano. Corroborar la adecuada profundidad de la anestesia mediante la evaluación de la falta de respuesta a los pies-pellizco. Después de la anestesia, mantener al animal anestesiado adecuadamente durante todo el procedimiento con isoflurano al 2%. </li><li> Colocar el animal en posición supina sobre una almohadilla de calentamiento y aplicar lubricante estéril para los ojos para evitar la desecación durante la cirugía. </li><li> El uso de una máquina de afeitar eléctrica, afeitarse ambas ingles y eliminar el vello con un trozo de gasa húmeda. </li><li> Prepara los sitios quirúrgicos con clorhexidina/ Solución de etanol al 70% y la caída del animal con toallas estériles. Administrar una sola dosis de carprofeno (5 mg / kg, vía subcutánea) como analgésico. </li></ol></li><li> Cosecha de la vena femoral del injerto <ol><li> Utilizando una cuchilla # 15, hacer una incisión en la piel de 4 cm de largo en la ingle izquierda paralelo al ligamento inguinal. Esto expone la almohadilla de grasa inguinal. </li><li> Corte a través de la almohadilla de grasa circunferencialmente con unas tijeras dejando lo saque de su pedículo vascular basado en los vasos epigástricos. </li><li> Usando tijeras micro, liberar las adherencias del tejido conectivo membranosas entre la pared abdominal y vasos femorales subyacentes. </li><li> Coloque un retractor en la pared abdominal y tire en sentido medial. Esto expone el ligamento inguinal y toda la longitud de los vasos femorales. </li><li> El uso de micro fórceps y tijeras curvas diseccionar la vena epigástrica y aislarlo de la grasa que rodea y tirando suavemente de corte. Esta vena actúa como una correa de sujeción en la construcción de bucle. </li><li> using micro pinzas y tijeras curvadas micro abren la vaina perivascular que contiene los vasos femorales y los nervios de todo el camino desde el ligamento inguinal hasta su bifurcación distal a la rama epigástrica. </li><li> El uso de micro fórceps, recoger la vena femoral por su adventicia y suavemente separarlo de los tejidos circundantes y la arteria que acompaña. Hacer esto con micro fórceps y tijeras curvadas micro puntas redondas por tirar del tejido aparte y cortar a través de él. NOTA: Nunca agarrar todo el espesor de la pared de la vena ya que esto podría causar un trauma a la íntima por lo que es propenso a la trombosis. </li><li> Se ligan las ramas laterales encontraron durante la disección con sutura de nylon 10/0 o coagulan con un coagulador bipolar. </li><li> Con la vena femoral completamente gratis, ligar sus extremos proximal y distal con suturas de seda 4/0. Asegúrese de obtener un injerto de vena de al menos 10 mm de longitud e incluye aproximadamente 0,5 cm de longitud de la rama epigástrica para ser utilizado como una correa de sujeción tipo cuerdapara mantener el circuito abierto en la cámara. </li><li> El uso de micro pinzas y tijeras micro rectas, recortar la adventicia de los extremos del injerto tirando suavemente y corte. Esto también se puede hacer más tarde, antes de anastomosis microquirúrgicas. </li><li> Enjuague bien el injerto de vena con solución salina heparinizada (10 U / ml de heparina) y se deja reposar en la solución. Cerrar la herida con un funcionamiento continuo 4/0 sutura de seda más dos o tres puntos interrumpidos simples adicionales. </li></ol></li><li> Creación de arteriovenosa Loop y la implantación de la Cámara <ol><li> Repita los pasos 1.2.1 a 1.2.4 de la misma manera exacta en la extremidad contralateral. </li><li> El uso de micro fórceps, diseccionar y aislar tanto la arteria y la vena epigástrica formar la capa de grasa que rodea. Para ello, tirando suavemente el tejido lejos de los vasos </li><li> El uso de micro fórceps, recoger la arteria femoral por su adventicia y liberarla de los tejidos circundantes. Hacer esto con micro fórceps y micro-redonda en punta curvadatijeras ro tirando el tejido aparte y cortar a través de él. Ligar o coagular sus ramas laterales. </li><li> Ligar la arteria femoral y la vena distal a la aparición de los vasos epigástricos utilizando sutura de seda 4/0. </li><li> Colocar una sola pinza proximal en cada uno de la arteria femoral y la vena. Usando una tijera afilada micro recta, transversal hacer un corte limpio en cada vaso distal a la aparición de las ramas epigástricos. Colocar un fondo de contraste de plástico estéril debajo de los buques. </li><li> Enjuague los vasos vigorosamente con cantidades generosas de solución salina heparinizada hasta que toda la sangre se retira del lumen. </li><li> Llevar el injerto de vena en el campo operatorio y eliminar cualquier adventicia redundante de los extremos de los buques como en el paso 1.2.10, si es necesario. </li><li> Realizar ambas anastomosis microquirúrgica con sutura de nylon 10/0. Anastomosan el extremo proximal del injerto de vena a la vena femoral y el extremo distal a la arteria femoral. Esto permitirá que la sangre fluya fesde la arterial al lado venoso sin resistencia por parte de las válvulas dentro de la vena del injerto. NOTA: Asegúrese de que los vasos femorales y el injerto de vena reposo en su posición natural sin ningún tipo de giros. </li><li> Compruebe si hay fugas en ambos sitios de anastomosis. Resolver las fugas pequeñas, que parecen no pulsante de la sangre que sale del lugar de la anastomosis, mediante la colocación de un pequeño trozo de grasa en la parte superior y suavemente comprimir durante 5-10 minutos. Grandes fugas pulsantes que inundan rápidamente todo el campo necesitarán puntos adicionales. </li><li> Comprobar la permeabilidad del asa arteriovenosa. oclusión suave de la arteria femoral debe hacerlo encoge mientras que el mismo en la vena femoral debe engorge ella. </li><li> Colocar la base de la cámara de la ingeniería de tejidos bajo el asa arteriovenosa con este último en reposo en su posición natural, sin giros o torceduras. </li><li> Fije la base de la cámara al ligamento inguinal y la fascia muscular subyacente con suturas de nylon 6/0. </li><li> Coloque la tapa sobre labase de manera que los vasos femorales entran en la cámara a través de una muesca (ventana en el lado de la cámara). Al cerrar la tapa, asegúrese de que las capturas las ramas epigástricos, entre la base de la cámara y la tapa, que actúan como correas de sujeción para sujetar el asa arteriovenosa en su posición. </li><li> Cerrar la herida con un funcionamiento continuo 4/0 sutura de seda más dos o tres puntos interrumpidos simples adicionales. </li><li> Deje que el animal se recupere de la anestesia en un cojín de calentamiento. </li><li> No dejar al animal sin vigilancia hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. Del mismo modo, no devuelva un animal que ha sido sometido a una cirugía para la compañía de otros animales hasta que esté completamente recuperado. 24 horas más tarde, administrar otra dosis única de carprofeno (5 mg / kg, vía subcutánea) como analgésico. </li><li> Tratar la herida con un ungüento antibiótico tópico durante 5 días. Si se abre la herida, anestesiar al animal como en el paso 1.1.2 a través de 1.1.5 y cerrar la herida como en 1.30.14. Controlar la salud de los animales todos los días. La eutanasia del animal usando una dosis letal de la inyección intraperitoneal lethabarb (163 mg / kg en 0,25 ml por 23 agujas G) si el animal muestra más de un signos moderados de inacitivity, falta de apetito, pérdida de peso y pérdida de color. </li></ol></li></ol> 2. El flujo a través de la Cámara de pedículo (dos cámaras por animal) <ol><li> Preparación del animal para la cirugía <ol><li> Repita los pasos 1.1.1 a 1.1.4. Dos cámaras se pueden implantar en las dos regiones de la ingle de una sola rata. </li></ol></li><li> Aislamiento de vasos femorales y la inserción de la Cámara <ol><li> Repita los pasos 1.2.1 a través de 1.2.8. </li><li> Con ambos arteria y la vena completamente liberado de los tejidos circundantes y sus ramas se ligó, llevar la cámara en el campo operatorio. </li><li> Coloque cada uno de los vasos femorales intactas en la correspondiente ranura de asegurarse de que no hay torceduras o dobleces de la base de la cámara. </li><li> Cerrar la cámara por attaching de la tapa a la base. Cierre de la herida utilizando un funcionamiento continuo 4/0 sutura de seda más dos o tres puntos de sutura interrumpidas simples adicionales y permitir que el animal se recupere como se describió previamente. </li></ol></li></ol> 3. Cosecha de Cámaras y procesamiento de tejido <ol><li> Una vez que se alcanzan los puntos de tiempo del experimento (4-6 semanas después de la implantación), anestesiar al animal como en el paso 1.1.2 y repetir los pasos 1.1.3 a través de 1.1.5. </li><li> Abra la herida usando una cuchilla # 15 y cortar a través de los tejidos con tijeras hasta que la cámara está completamente expuesto. </li><li> Exponer los vasos femorales proximales a la construcción y prueba de la permeabilidad vascular: ocluye suavemente el recipiente con dos microforceps, a continuación, la leche de la sangre en una dirección distal y finalmente liberar la pinza proximal. Si el recipiente se llena de sangre de nuevo, esto confirma la permeabilidad. Ligar los vasos femorales proximalmente en el caso del bucle arteriovenosa y ambos proximal y distalmente en el caso de THe flujo a través de la configuración, y quitar las cámaras con el tejido que contiene en bloque. </li><li> Al final del experimento, la eutanasia del animal usando una dosis letal de inyección lethobarb intraperitoneal (163 mg / kg en 0,25 ml por 23 aguja G). </li><li> Fijar los tejidos en paraformaldehído al 4% a temperatura ambiente durante 24 hr. Divida a los tejidos múltiples secciones transversales (1-2 mm de espesor) y embed de cera de parafina o compuesto de temperatura óptima de corte para las secciones de parafina (5 micras) o secciones congeladas (10 micras), respectivamente. 3,4,8,17,22, 24 </li><li> Realizar la tinción histológica de rutina, tales como hematoxilina y eosina para examinar la morfología general de los tejidos. Realizar la tinción inmunohistoquímica con un anticuerpo específico para identificar el tipo de célula de interés, por ejemplo 3,4,8,17,22,24,29 troponina T cardiaca inmunotinción de los cardiomiocitos. </li></ol>

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K., Cassell, O. C., Kelly, J., Abberton, K. M., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+vascularized+organoid+using+skeletal+muscle+as+the+inductive+source.">Generation of a vascularized organoid using skeletal muscle as the inductive source.</a> FASEB J. 19 (11), 1570-1572 (2005).</li><li>Lim, S. Y., Hern&#225;ndez, D., Dusting, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+vascularized+heart+tissue+from+stem+cells.">Growing vascularized heart tissue from stem cells.</a> J Cardiovasc Pharmacol. 62 (2), 122-129 (2013).</li><li>Poon, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+an+adipogenic+hydrogel+from+subcutaneous+adipose+tissue.">Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.</a> Acta Biomater. 9 (3), 5609-5620 (2013).</li><li>Dilley, R. J., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularisation+to+improve+translational+potential+of+tissue+engineering+systems+for+cardiac+repair.">Vascularisation to improve translational potential of tissue engineering systems for cardiac repair.</a> Int J Biochem Cell Biol. 56, 38-46 (2014).</li><li>Lesman, A., Koffler, J., Atlas, R., Blinder, Y. J., Kam, Z., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vessel-like+networks+within+multicellular+fibrin-based+constructs.">Engineering vessel-like networks within multicellular fibrin-based constructs.</a> Biomaterials. 32 (31), 7856-7869 (2011).</li><li>Hussey, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeding+of+pancreatic+islets+into+prevascularized+tissue+engineering+chambers.">Seeding of pancreatic islets into prevascularized tissue engineering chambers.</a> Tissue Eng Part A. 15 (12), 3823-3833 (2009).</li><li>Chen, X., Aledia, A. S., Popson, S. A., Him, L., Hughes, C. C., George, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+anastomosis+of+endothelial+progenitor+cell-derived+vessels+with+host+vasculature+is+promoted+by+a+high+density+of+cotransplanted+fibroblasts.">Rapid anastomosis of endothelial progenitor cell-derived vessels with host vasculature is promoted by a high density of cotransplanted fibroblasts.</a> Tissue Eng Part A. 16 (2), 585-594 (2010).</li><li>Lin, R. Z., Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor-2+facilitates+rapid+anastomosis+formation+between+bioengineered+human+vascular+networks+and+living+vasculature.">Fibroblast growth factor-2 facilitates rapid anastomosis formation between bioengineered human vascular networks and living vasculature.</a> Methods. 56 (3), 440-451 (2012).</li><li>Dolderer, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+large+volume+adipose+tissue+generation+from+a+vascularized+pedicled+fat+flap+inside+a+chamber+space.">Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space.</a> Tissue Eng. 13 (4), 673-681 (2007).</li><li>Wei, F. C., Lin Tay, S. K., Neligan, P. C., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+techniques+of+microvascular+surgery.">Principles and techniques of microvascular surgery.</a> Plastic Surgery. Volume 1. , 587-620 (2013).</li><li>Sekine, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fabrication+of+functional+three-dimensional+tissues+with+perfusable+blood+vessels.">In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels.</a> Nat.Comm. 4 (1399), 1-10 (2013).</li><li>Lim, S. Y., Sivakumaran, P., Crombie, D. E., Dusting, G. J., P&#233;bay, A., Dilley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichostatin+A+enhances+differentiation+of+human+induced+pluripotent+stem+cells+to+cardiogenic+cells+for+cardiac+tissue+engineering.">Trichostatin A enhances differentiation of human induced pluripotent stem cells to cardiogenic cells for cardiac tissue engineering.</a> Stem Cells Transl Med. 2 (9), 715-725 (2013).</li><li>Lim, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+tissue+engineering+chamber+supports+human+induced+pluripotent+stem+cell+survival+and+rapid+differentiation.">In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.</a> Biochem Biophys Res Commun. 422 (1), 75-79 (2012).</li><li>Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+integration-free+human+induced+pluripotent+stem+cells+from+keratinocytes+by+simple+transfection+of+episomal+vectors.">Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.</a> Stem Cells Transl Med. 3 (7), 787-791 (2014).</li></ol>

Los autores declaran no tener conflictos de intereses.

Ingeniería de la microcirculación está siendo investigado actualmente fundamentalmente a través de dos enfoques. El primero implica el desarrollo de una red vascular altamente interconectada dentro de la construcción in vitro de manera que cuando se implanta, los capilares de la cama vascular anfitrión se conectan con los de la trasplantado construir a través de un proceso llamado inosculation, asegurando así el suministro de nutrientes no sólo a la periferia pero también para el núcleo. 21,32,3...

La fabricación de tejido vascularizado funcional utilizando un enfoque de la ingeniería de tejidos es un paradigma emergente en la medicina regenerativa. 1,2 Muchos enfoques para diseñar tejido nuevo y sano para el reemplazo de tejido lesionado o órganos defectuosos se han desarrollado de forma experimental, 3-6 en pequeños modelos animales con potencial clínico prometedor. 7,8 Sin embargo, la vascularización sigue siendo uno de los grandes desafíos para la ingeniería de tejidos, limitando su potencial para crecer tejidos de tamaño clínicamente relevante. 9 Los enfoques actuales para vascularizar tejidos seguir ya sea una vía extrínseca donde los nuevos vasos crecen desde el lecho vascular receptor e invaden todo el tejido implantado construye 10 o una vía vascularización intrínseca donde la vasculatura crece y se expande al unísono con el tejido de reciente desarrollo. 11 El enfoque extrínseca tradicionalmente involucra células de siembra en un andamioin vitro y la implantación de la construcción completa en el animal vivo con la expectativa de que los nutrientes, suministradas previamente por los medios de cultivo, se obtiene de la circulación. 12,13 El concepto es simple como crecimiento vascular es demasiado lento y sólo implantes muy delgadas (&lt; 1-2 mm de espesor) seguirá siendo viable. El aporte de nutrientes y oxígeno a través de una vascularización suficiente y rápida está en el corazón de todos los intentos exitosos para crecer sustitutos de ingeniería tisular más complejos y de mayor tamaño, como los huesos, los músculos, la grasa y los órganos sólidos. 14,15 vascularización intrínseca ofrece la posibilidad de construcciones más grandes a desarrollar por el crecimiento tisular progresiva acorde con la expansión de su suministro de sangre. Un diseño es la implantación in vivo en una cámara de un pedículo vascular con o sin un andamio sembrado celular. 5,6 Esto ha allanado el camino a nuevos procedimientos para la generación de tejidos vascularizados intrínsecamente más gruesas. 16,17 &lt;/ P&gt; Más recientemente, se han desarrollado estrategias para pre-vascularizan injertos de tejido, antes de la implantación. Estas redes de vasos sanguíneos incorporados están dirigidos a inosculate con vasos de acogida en la implantación que permitan la rápida provisión de un suministro de sangre completa para mejorar la supervivencia de todas las partes de un injerto de tejido grueso trasplantado. 18 Fuimos pioneros en un modelo in vivo vascularizado ingeniería de tejidos en animales pequeños, que involucra una cámara cerrada semi-rígido implantado subcutáneamente que contiene un pedículo vascular perfundido y biomateriales que contienen células. La cámara crea un ambiente isquémico que estimula la germinación de los vasos angiogénicos implantados. 3 El pedículo vascular puede ser o bien un bucle arteriovenosa reconstruida o una arteria de flujo a través intacta y la vena. 3-6,19 Este vasculares brotes pediculares un funcionamiento y una amplia arterio -capillary red venosa que une en el arteeriole y venoso termina con el pedículo vascular. 3,20 Además, la cámara de soporte hueco que rodea protege el tejido en desarrollo de la deformación potencialmente fuerzas mecánicas y prolonga la unidad isquémico para mejorar la vascularización. 3,21,22 Si el pedículo recipiente simplemente se implanta en tejido normal y no dentro del espacio protegido de la cámara, el brote angiogénico cesa a lo largo de la misma línea de tiempo como una herida normal y no hay nuevos tejidos se acumulan alrededor del pedículo. Los investigadores han utilizado esta configuración in vivo para producir construcciones de tejido vascularizado funcionales tridimensionales con vasculatura de apoyo y de tamaño clínicamente relevante. 4,23 Por otra parte, las construcciones de tejido vascularizado de ingeniería con su pedículo vascular intacto se pueden cosechar para posterior trasplante en el sitio de la lesión 24,25. Un escenario más clínicamente factible sería la creación de la cámara en el lugar definitivo para la reconstrucción sUCH como el de mama. Por lo tanto, este enfoque de novo la ingeniería de tejidos podría tener potencial clínico para proporcionar una nueva fuente de tejido diana funcional para la cirugía reconstructiva. 26-28 El siguiente protocolo proporcionará una guía general para la construcción de una cámara de la ingeniería de tejidos vascularizado in vivo en la rata, que podría adaptarse en diferentes modelos animales y se emplea para examinar los procesos complejos de la angiogénesis, la producción de la matriz, y la migración celular y la diferenciación.

<table border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">1 15 Blade Scalpel</td>
			<td style="width:187px;">Braun</td>
			<td style="width:163px;">BB515</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Toothed Adson Forceps</td>
			<td>Braun</td>
			<td>BD527R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Needle Holder</td>
			<td>Braun</td>
			<td>BM201R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Bipolar Coagulator&#160;</td>
			<td>Braun</td>
			<td>US335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Needle Holder B-15-8.3</td>
			<td>S &#38; T</td>
			<td>00763</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Dilator Forceps D-5a.2</td>
			<td>S &#38; T</td>
			<td>00125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Jeweler&#39;s Forceps JF-5</td>
			<td>S &#38; T</td>
			<td>00108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Straight SAS-11</td>
			<td>S &#38; T</td>
			<td>00098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Curved SDC-11</td>
			<td>S &#38; T</td>
			<td>00090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 Single Clamps B-3</td>
			<td>S &#38; T</td>
			<td>00400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 10/0 nylon suture</td>
			<td>S &#38; T</td>
			<td>03199</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 6/0 nylon suture</td>
			<td>Braun</td>
			<td>G2095469</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 4/0 Silk Sutures</td>
			<td>Braun</td>
			<td>C0760145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xilocaine 1%</td>
			<td>Dealmed</td>
			<td>150733</td>
			<td>10 mg/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin Sodium</td>
			<td>Dealmed</td>
			<td>272301</td>
			<td>5000 UI / ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ringer Lactate</td>
			<td>Baxter</td>
			<td>JB2323</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 dome-shaped tissue engineering chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 flow-through chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin I, Griffonia Simplicifolia&#160;</td>
			<td>Vector Laboratories</td>
			<td>B-1105</td>
			<td>1.67 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Troponin T antibody</td>
			<td>Abcam</td>
			<td>Ab8295</td>
			<td>4 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human-specific Ku80 antibody</td>
			<td>Abcam</td>
			<td>Ab80592</td>
			<td style="width:176px;">0.06 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Desmin antibody</td>
			<td>Dako</td>
			<td>M0760</td>
			<td style="width:176px;">2.55 &#956;g/mL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Tracker CM-DiI dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C-7000</td>
			<td>3 mg/106 cells</td>
		</tr>
	</tbody>
</table>

tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber

En la cirugía reconstructiva, existe una necesidad clínica de una alternativa a los métodos actuales de reconstrucción autóloga que son complejos, costosos y el comercio un defecto para otro. La ingeniería de tejidos mantiene la promesa para hacer frente a esta creciente demanda. Sin embargo, la mayoría de las estrategias de ingeniería de tejidos no logran generar sustitutos de tejidos estables y funcionales debido a la mala vascularización. Este documento se centra en un modelo de cámara de la ingeniería de tejidos in vivo de la vascularización intrínseca de una arteria y una vena perfundido ya sea como un asa arteriovenosa o una configuración de flujo a través del pedículo se dirige dentro de una cámara hueca protegida. En este sistema basado en cámara de germinación angiogénico se produce a partir de los vasos arteriovenosas y este sistema atrae la migración celular endógeno isquémica e inflamatoria impulsado que llena gradualmente el espacio de la cámara con el tejido fibro-vascular. celular exógena / implantación de la matriz en el momento de la construcción de la cámara aumenta sur celularsupervivencia y determina la especificidad de los tejidos de ingeniería que se desarrollan. Nuestros estudios han demostrado que este modelo de cámara puede generar con éxito diferentes tejidos tales como la grasa, el músculo cardíaco, el hígado y otros. Sin embargo, se requieren modificaciones y mejoras para garantizar la formación de tejido diana es consistente y reproducible. En este artículo se describe un protocolo estandarizado para la fabricación de dos modelos de cámara ingeniería de tejidos vascularizados diferentes in vivo.

Se realizó la creación microquirúrgico de cámaras de ingeniería de tejidos como se describe en el protocolo anterior. Los tejidos generados dentro de las cámaras pueden ser examinados histológicamente como describen en el paso protocolo 3. Varios tipos de tejidos han sido diseñados con éxito utilizando la cámara de vascularizado in vivo (Figura 2). Estos incluyen el tejido cardíaco con los cardiomiocitos neonatales de rata (Figura 2a),</strong...

Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingeniería de tejidos por intrínseca Vascularización en una<em&gt; En Vivo</em&gt; Cámara Ingeniería de Tejidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

tissue-engineering-intrinsic-vascularization-an-vivo-tissue

Research

JoVE Journal

Bioengineering

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

8.8K vistas.  St Vincent's Institute of Medical Research. This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling. 

Video: Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomérico andamios en ingeniería de tejidos arteriales

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Investigación

Bioingeniería

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

La encapsulación de los cardiomiocitos en un hidrogel de fibrina para ingeniería de tejidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproducción de procesamiento de las Fibras electrospun de Ingeniería de Tejidos

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Ingeniería de tejidos del intestino en un modelo murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Fuerza Litografía Cardiaca de Ingeniería de Tejidos

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingeniería de tejidos de un humano 3D<em&gt; In vitro</em&gt; Sistema de Prueba de Tumores

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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 ...

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. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microentornos-Sandwich como para aprovechar Celulares / Materiales Interacciones

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

Aislamiento de murino tejido adiposo derivado de microvascular Fragmentos como Unidades Vascularización para ingeniería de tejidos

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...