Name: tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber
Uploaded: 2016-05-30T15:00:00.000+00:00
Duration: 9 min 55 s
Description: Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

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><tbody><tr><td>1 15 Blade Scalpel</td><td>Braun</td><td>BB515</td><td></td></tr><tr><td>1 Toothed Adson Forceps</td><td>Braun</td><td>BD527R</td><td></td></tr><tr><td>1 Needle Holder</td><td>Braun</td><td>BM201R</td><td></td></tr><tr><td>1 Bipolar Coagulator&amp;nbsp;</td><td>Braun</td><td>US335</td><td></td></tr><tr><td>1 Micro Needle Holder B-15-8.3</td><td>S &amp;amp; T</td><td>00763</td><td></td></tr><tr><td>1 Micro Dilator Forceps D-5a.2</td><td>S &amp;amp; T</td><td>00125</td><td></td></tr><tr><td>1 Micro Jeweler&#039;s Forceps JF-5</td><td>S &amp;amp; T</td><td>00108</td><td></td></tr><tr><td>1 Micro Scissors - Straight SAS-11</td><td>S &amp;amp; T</td><td>00098</td><td></td></tr><tr><td>1 Micro Scissors - Curved SDC-11</td><td>S &amp;amp; T</td><td>00090</td><td></td></tr><tr><td>2 Single Clamps B-3</td><td>S &amp;amp; T</td><td>00400</td><td></td></tr><tr><td>2 10/0 nylon suture</td><td>S &amp;amp; T</td><td>03199</td><td></td></tr><tr><td>1 6/0 nylon suture</td><td>Braun</td><td>G2095469</td><td></td></tr><tr><td>2 4/0 Silk Sutures</td><td>Braun</td><td>C0760145</td><td></td></tr><tr><td>Xilocaine 1%</td><td>Dealmed</td><td>150733</td><td>10 mg/ml</td></tr><tr><td>Heparin Sodium</td><td>Dealmed</td><td>272301</td><td>5,000 UI/ml</td></tr><tr><td>Ringer Lactate</td><td>Baxter</td><td>JB2323</td><td>500 ml</td></tr><tr><td>1 dome-shaped tissue engineering chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>1 flow-through chamber</td><td>custom made</td><td></td><td></td></tr><tr><td>Lectin I, Griffonia Simplicifolia&amp;nbsp;</td><td>Vector Laboratories</td><td>B-1105</td><td>1.67 &amp;mu;g/ml</td></tr><tr><td>Troponin T antibody</td><td>Abcam</td><td>Ab8295</td><td>4 &amp;mu;g/ml</td></tr><tr><td>Human-specific Ku80 antibody</td><td>Abcam</td><td>Ab80592</td><td>0.06 &amp;mu;g/ml</td></tr><tr><td>Desmin antibody</td><td>Dako</td><td>M0760</td><td>2.55 &amp;mu;g/ml</td></tr><tr><td>Cell Tracker CM-DiI dye</td><td>Thermo Fisher Scientific</td><td>C-7000</td><td>3 mg/10&lt;sup&gt;6&lt;/sup&gt; cells</td></tr><tr><td>Lethabarb (sodium pentobarbitone)</td><td><a href="www.virbac.com.au" target="_blank"><img src="/img/new_window.png" /> Virbac Animal Health</a></td><td>LETHA450</td><td>325 mg/ml</td></tr><tr><td>Heat pad flexible</td><td><a href="http://www.redzoneheating.com.au" target="_blank"><img src="/img/new_window.png" /> Redzone Heating</a></td><td>RZ/Medium</td><td></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

Universidad de Cartagena UDEC

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

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 ...

Investigación

Bioingeniería

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 ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Una piel llena de defectos modelo para evaluar la vascularización de Biomateriales<em&gt; En Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

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

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

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

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

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

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. ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning y Montaje de Microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

El asa arteriovenosa (AV) en un modelo de pequeños animales para estudiar la angiogénesis e Ingeniería tejido vascularizado

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Vascularizado de Bioprinting de microfluidos para la ingeniería de tejidos y organitas

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bioingeniería Humanos redes microvasculares en ratones inmunodeficientes

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Biología

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Andamios de impresión de núcleo/concha para la ingeniería de tejidos de estructuras tubulares

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Ingeniería de tejidos por intrínseca Vascularización en una

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