This work was partially funded through the Richard Barber Foundation and a Thomas Rumble Fellowship (TJW).

1. agua / aceite / agua Doble Emulsión Microsphere Producción <ol><li> En primer lugar preparar 2% y 0,5% w / v de las soluciones de alcohol polivinílico (PVA) en agua desionizada. Revuelva las soluciones a 50 ° C hasta que se aclare, esto puede tardar varias horas. Preparar una solución de 2% v / v de alcohol isopropílico en agua desionizada. </li><li> Preparar una solución acuosa de proteína hidrófila deseada. La siguiente tabla proporciona ejemplos de formulaciones. </li></ol><img alt="Tabla 1" src="/files/ftp_upload/51517/51517table1highres.jpg" /> Tabla 1:. Ejemplo Proteína soluciones Las siguientes soluciones de proteínas se han encapsulado con éxito y electrospun usando este protocolo. Otras soluciones de proteínas hidrófilas se pueden usar según sea necesario. <ol start="3"><li> Coloque 40 ml de la solución de PVA al 0,5% en un tubo de centrífuga de 50 ml y reservar. </li><li> En un tubo de centrífuga de 15 ml disolver 300 mg de 65:35poli (ácido láctico-co-glicólico) (PLGA) en 3 ml de diclorometano. Un mezclador de vórtice puede ser utilizado para acelerar la disolución de PLGA. </li><li> Combinar 200 l de solución de proteína y 4 l de solución de PVA al 2%. Verter la mezcla de proteína en la solución de PLGA (paso 1.4). Las soluciones se mantendrán básicamente individualista. </li><li> Se coloca el tubo en un vaso de agua helada. Uso de un aparato de ultrasonidos varita a ~ 10 vatios (RMS), agitar la solución durante unos pocos segundos (5-10) hasta que se crea una emulsión blanca cremosa uniforme. </li><li> Se vierte la emulsión en el tubo de 50 ml que contiene 0,5% de PVA (paso 1.3). Mezcle la solución a alta velocidad en un vórtex durante ~ 20 seg. La solución desarrollará un aspecto turbio. </li><li> Se transfiere la emulsión a un vaso de 200 ml y colocar en una placa de agitación a 350 rpm durante 2 min. Añadir 50 ml de 2% de alcohol isopropílico al vaso de precipitados en la placa de agitación. Deje que la mezcla se continúa agitando durante un mínimo de 1 hora para permitir que el DCM se evapore y el PLGA a endurecerse. </li><li> Transferencia tél microesfera solución en tubos de centrífuga. </li><li> Se centrifuga a 425 g durante 3 min. Las microesferas se concentren en la parte inferior del tubo y aparecerá blanco. Retire con cuidado el sobrenadante del tubo, por encima de las microesferas, y almacenar en una botella de 500 ml. </li><li> Enjuague las microesferas con agua desionizada, llenando el tubo de tres cuartos llena y agitarlo para redistribuir las microesferas en el líquido. </li><li> Repita los pasos 1.10 y 1.11 cuatro veces. </li><li> Tras el enjuague final, separar el sobrenadante de nuevo y colocar en la botella de 500 ml con las otras muestras. Congelar las microesferas recogidos en el tubo de centrífuga, a continuación, se liofiliza durante al menos 24 h. </li><li> Visualizar las microesferas con un microscopio de luz o con un microscopio electrónico de barrido. Las microesferas no mayores de 60 micras son para electrospinning efectiva. Si las microesferas son demasiado grandes, sonicación o de vórtice tiempos más largos pueden ser requeridos en el paso 1.6 o 1.7. </li> &lt;li&gt; Guarde las microesferas secas en un -20 ° C congelador. </li><li> Opcional: Utilice un ensayo de proteínas, por las instrucciones del fabricante, para medir la cantidad de proteína en la botella de 500 ml desde el paso 30 1.10. Esto se utiliza para calcular el porcentaje de proteína encapsulada en las microesferas, restando la cantidad en la solución de lo que fue utilizado en el proceso de producción. </li></ol> NOTA: Para visualizar la ubicación de proteína en la microesfera añadir Rodamina 2 mg / ml a la solución de PLGA 31 y encapsular una proteína conjugada con FITC Figura 1 muestra un ejemplo.. 2. electrospinning con microesferas <ol><li> Antes de preparar la solución de electrospinning, crear un 0,5% w / v solución de fotoiniciador, en agua desionizada mediante la disolución a 37 ° C. Este proceso puede tardar varias horas. </li><li> Crear un 2% w / v ácido hialurónico metacrilatado (MEHA) (ver Burdick et al. Para la síntesis) 29, 3%w / v 900 kD poli (óxido de etileno) (PEO) y 0,05% w / v solución de fotoiniciador en agua desionizada. <ol><li> Calcular la cantidad correcta de MEHA y PEO para el volumen deseado. Por ejemplo, 10 ml de electrospinning solución requiere 200 mg de MEHA y 300 mg de PEO. </li><li> Disolver el PEO en agua desionizada a 90% del volumen final deseado (9 ml para este ejemplo). Esto puede tomar varias horas, una placa de agitación caliente o baño de agua a 37 ° C pueden ser utilizados para acelerar el proceso. </li><li> A continuación añadimos la MEHA y utilizar un mezclador de vórtice para agitar la solución hasta que se aclare. Esto sólo tomará unos minutos. </li><li> Finalmente añadir la solución de fotoiniciador 0,5% para llenar el volumen restante 10% (1 ml para este ejemplo). </li></ol></li><li> Añadir las microesferas a la concentración deseada de hasta 400 mg / ml. Mezclar la solución en un mezclador de vórtice hasta que las microesferas se distribuyeron uniformemente en la solución. </li><li> Transferir la solución a una jeringa y fijar un 6 pulgadas bl calibre 18aguja punta unt. </li><li> Coloque la jeringa en una bomba de jeringa y la puso para dispensar a 1,2 ml / hr. </li><li> Pegue una capa de papel de aluminio en el plato de la colecta o mandril. Esto permite una fácil limpieza y almacenamiento del andamio terminado. Un mandril giratorio se utiliza para crear fibras alineadas. Una placa plana o mandril estacionario se traducirá en fibras dispuestas al azar. </li><li> Conectar el cable de tierra de una fuente de alimentación de alto voltaje para el aparato de recogida. Conecte el cable positivo a la aguja. </li><li> Ajuste la superficie de la bomba de jeringa y la recolección de modo que haya 15 cm entre la punta de la aguja y la superficie de recogida. </li><li> Iniciar el bombeo de polímero, cuando la solución es visible en el extremo de la jeringa, encender la fuente de tensión y ajustar la tensión de 24 kV PRECAUCIÓN:. Una vez que la tensión se enciende no toque ninguna parte metálica del sistema. Carga también puede saltar distancias cortas a partir de piezas electrificadas a la piel. </li><li> Ejecute la solución hasta que dse consigue espesor andamio esired. Cuando completa apagar la fuente de tensión y la bomba de jeringa. </li><li> Retire el papel aluminio con el andamio colocado. Completadas andamios que contienen proteínas se almacenan en un congelador de -20 ° C. </li></ol> 3. Proteína Testing bioactividad <ol><li> Preparar medios de cultivo celular. Añadir 10% v / v de suero fetal bovino, 1% v / v L-glutamina, y 1% v / v penicilina-estreptomicina al medio de Eagle modificado de Dulbecco. </li><li> Seleccione cubreobjetos de vidrio que cabe totalmente en un plato bien. </li><li> Utilice 3 (trimetoxisilil) propil metacrilato para tratar los cubreobjetos tal como se describe por el fabricante. Metacrilación mejora la adherencia andamio para los cubreobjetos. </li><li> Adjuntar cubreobjetos metacrilados a la zona de recogida de la electrospinner con cinta adhesiva de doble cara antes de electrospinning. Spinning sobre los cubreobjetos facilita la manipulación y visualización. </li><li> Electrospin al espesor deseado como se describe anteriormente. </li><li> Aespués de electrospinning retire con cuidado cubreobjetos de mandril. Coloque el andamio cubreobjetos recubiertos en una cámara de nitrógeno clara y asegúrese de que todo el oxígeno se purga. </li><li> Coloque la cámara y bajo un andamio 10 mW / cm 2 365 nm de luz durante 15 min. Después de la reticulación lugar en placa de tamaño apropiado. Asegúrese de que el lado del andamio esté hacia arriba. </li><li> Coloque los andamios bajo una lámpara germicida durante 30 min para esterilizar. Si la fibronectina u otra proteína deseada se utiliza como un recubrimiento para mejorar la adhesión celular. Siga las instrucciones del fabricante de andamios abrigo. </li><li> Harvest ganglios de raíz dorsal (DRG), como se describe anteriormente por Hollenbeck 32. Se necesitará un DRG para cada andamio cubierto cubreobjetos probado. </li><li> Coloque 100-200 l de los medios de comunicación en cada andamio en la placa también. Con cuidado, coloque uno en cada DRG andamio en la gota de los medios de comunicación. Para andamios de espesor pueden ser necesarios más medios de comunicación; DRG necesita estar totalmente sumergido y no Floating. </li><li> Incubar el andamio y DRG a 37 ° C durante 4 horas para permitir que la célula se adhiera a la andamio. </li><li> Rellene los medios de comunicación para el nivel apropiado para el bien y colocar de nuevo en la incubadora. Continuar la incubación durante 4-6 días. </li><li> Tras el período de incubación retire con cuidado los medios de cada pocillo y lavar suavemente una vez con PBS. Fijar las células durante 30 min utilizando 4% w / v de paraformaldehído. </li><li> Las células de la mancha utilizando una mancha de anticuerpos para neurofilamentos. Esto permitirá la visualización del crecimiento de neuritas para la cuantificación. DAPI también se puede utilizar para ver núcleos de las células. Un ejemplo de protocolo de tinción fue descrito por Sundararaghavan y sus colegas 14. </li><li> Visualice las células utilizando un microscopio de fluorescencia. <ol><li> Colocar la placa de bien en la platina del microscopio. </li><li> Busque la masa celular utilizando los filtros de excitación y ajustes para DAPI. </li><li> Una vez que la célula es cambiar el filtro para FITC para visualizar las neuritas extendidas. Ucantar la función de puntada en el microscopio recoger y combinar tantas imágenes como sea necesario para ver toda la estructura. Repita el procedimiento para DAPI, FITC y campo claro. </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+compartment+diffusion+chamber+for+studying+axonal+chemotaxis+in+3D+collagen.">A dual compartment diffusion chamber for studying axonal chemotaxis in 3D collagen.</a> Journal of Neuroscience Methods. 215, 53-59 (2013).</li><li>Madduri, S., di Summa, P., Papalo&#239;zos, M., Kalbermatten, D., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+controlled+co-delivery+of+synergistic+neurotrophic+factors+on+early+nerve+regeneration+in+rats.">Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats.</a> Biomaterials. 31, 8402-8409 (2010).</li><li>Xu, X., 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=Polyphosphoester+microspheres+for+sustained+release+of+biologically+active+nerve+growth+factor.">Polyphosphoester microspheres for sustained release of biologically active nerve growth factor.</a> Biomaterials. 23, 3765-3772 (2002).</li><li>Yan, Q., Yin, Y., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+new+PLGL-RGD-NGF+nerve+conduits+for+promoting+peripheral+nerve+regeneration.">Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration.</a> Biomed Eng Online. 11, (2012).</li><li>Gungor-Ozkerim, P. S., Balkan, T., Kose, G. T., Sarac, A. S., Kok, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+growth+factor+loaded+microspheres+into+polymeric+electrospun+nanofibers+for+tissue+engineering+applications.">Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications.</a> J Biomed Mater Res A. , (2013).</li><li>Li, X., 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=Encapsulation+of+proteins+in+poly(L-lactide-co-caprolactone)+fibers+by+emulsion+electrospinning.">Encapsulation of proteins in poly(L-lactide-co-caprolactone) fibers by emulsion electrospinning.</a> Colloids Surf B Biointerfaces. 75, 418-424 (2010).</li><li>Wang, C. -. 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=The+effect+of+aligned+core-shell+nanofibres+delivering+NGF+on+the+promotion+of+sciatic+nerve+regeneration.">The effect of aligned core-shell nanofibres delivering NGF on the promotion of sciatic nerve regeneration.</a> J Biomater Sci Polym Ed. 23, 167-184 (2012).</li><li>Liu, J. -. J., Wang, C. -. Y., Wang, J. -. G., Ruan, H. -. J., Fan, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+regeneration+using+composite+poly(lactic+acid-caprolactone)/nerve+growth+factor+conduits+prepared+by+coaxial+electrospinning.">Peripheral nerve regeneration using composite poly(lactic acid-caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning.</a> J Biomed Mater Res A. 96, 13-20 (2011).</li></ol>

The authors have nothing to disclose.

Muchos estudios han demostrado que las células nerviosas pueden ser dirigidas por señales topográficas (alineación de la fibra) y señales químicas (factores de crecimiento) 1,2,10,11,35. Electrospinning es un método fácil para crear fibras alineadas. Los factores de crecimiento estimulan el crecimiento del nervio, pero con el fin de incluirlos en los conductos de los nervios (NC), se requiere un método para la liberación sostenida. Para crear un sistema más robusto con ambas señales, estas dos se?...

Uno de los retos actuales en la ingeniería de tejido neural es la creación de un conducto nervioso (NC) que imita la matriz extracelular, donde los nervios crecen naturalmente. La investigación ha demostrado que las células responden a varios factores en su entorno, incluyendo las señales químicas 1-3 mecánica, topográficos, adhesivo y. Uno de los principales desafíos en este campo consiste en determinar la combinación adecuada de señales y la fabricación de un sistema que puede mantener las señales durante un período prolongado para apoyar el crecimiento celular 4. Neuronas periféricas se sabe que prefieren un sustrato blando, ser dirigidos por fibras alineadas, y responden al factor de crecimiento nervioso (NGF) 5-7. CN que puede proporcionar señales químicas durante semanas se ha demostrado para proporcionar una mejor recuperación funcional más cercana a la de los aloinjertos, el estándar de oro actual para la reparación del nervio 8,9. Materiales y métodos de producción Varios se puede utilizar para producir mecánica y topográficoal cues 10-13. Señales mecánicas son inherentes al material elegido, haciendo la selección del material apropiado para la aplicación crítica 1,13. Los métodos de producción para el control de señales topográficas incluyen la separación de fases, auto-ensamblaje y electrospinning 1,13. Para aplicaciones a microescala, microfluídica, photopatterning, aguafuerte, sanguijuelas sal, o espumas de gas también se puede utilizar 14-17. Electrospinning se ha convertido en la manera más popular para diseñar sustratos fibrosos para el cultivo de tejidos, debido a su flexibilidad y facilidad de producción 13,18-23. Nanofibras electrohiladas se fabrican aplicando un alto voltaje a una solución de polímero haciendo que repeler a sí mismo y se extienden a través de una breve pausa para descargar 24. Un andamio alineados se puede crear mediante la recopilación de las fibras sobre un mandril giratorio a tierra y andamios no alineados se recogen en una placa estacionaria 25. Adhesión de señalización se puede lograr mediante el recubrimiento del ingenio andamio fibrosoh fibronectina o la conjugación de un péptido de adhesión, tales como RGD, al HA antes de electrospinning 26. Las señales químicas, tales como factores de crecimiento, son los más difíciles de mantener durante períodos prolongados, ya que necesitan una fuente de liberación controlada. Muchos sistemas se han intentado añadir liberación controlada a las redes fibrosas electrohiladas con diferentes niveles de éxito. Estos métodos incluyen electrospinning mezcla, emulsión electrospinning, electrospinning núcleo de carcasa y conjugación de la proteína 27. Además, electrospinning se realiza tradicionalmente en un disolvente volátil, que puede afectar a la viabilidad de la proteína de 28, por lo tanto mantener la bioactividad de la proteína debe ser considerado. Este enfoque aborda específicamente la combinación de señales mecánicas, topográficos, químicos y adhesivos para crear un andamio ajustable para el crecimiento de los nervios periféricos. La mecánica del andamio se controla con precisión mediante la síntesis deÁcido Hialurónico metacrilado (HA). Los sitios metacrilación se utilizan para unir fotos reticulantes reactivos. El material reticulado ya no es soluble en agua y se divide exclusivamente por las enzimas 29. La cantidad de reticulación cambia la velocidad de degradación, la mecánica y otras propiedades físicas del material. Uso de HA con 30% metacrilación, que tiene un módulo de tracción de ~ 500 Pa, crea un sustrato blando que está cerca de la mecánica nativos de tejido neural y típicamente se prefiere por las neuronas 26,29. Electrospinning en un mandril giratorio se utiliza para crear fibras alineadas para una señal topográfica. Usando electrospinning junto con microesferas proporciona señales químicas dentro del andamio durante períodos prolongados. Para apoyar microesferas de crecimiento de neuritas que contienen NGF se utilizan para crear la señal química. A diferencia de la mayoría de materiales electrospun HA es soluble en agua por lo que el NGF no se encuentra con solventes fuertes durante la producción. Para añadir una señal de adhesivo, la scaffold está revestida con fibronectina. El sistema completo contiene los cuatro tipos de señales descritos anteriormente: (mecánicas) alineados fibras suaves (topográficos) con la liberación de microesferas de NGF (química) recubiertas con fibronectina (adhesivo). Producción y pruebas de este sistema se describe en este protocolo. El proceso comienza con la producción de las microesferas con un agua-en-aceite-en-agua de doble emulsión. La emulsión se estabiliza con un tensioactivo, alcohol polivinílico (PVA). La fase acuosa interna contiene la proteína. Como se añade a la fase oleosa, que contiene el material de la cáscara PLGA disuelto en diclorometano (DCM), el tensioactivo crea una barrera entre las fases que protegen la proteína de la DCM. Esta emulsión es de otra fase dispersa en agua que contiene PVA para crear la superficie exterior de las microesferas. La emulsión estable se agita para permitir que el DCM se evapore. Después de enjuagar y liofilización que se quedan con la microesferas cont secoaining la proteína. Después de que las microesferas se completan están listos para ser electrospun en andamios. En primer lugar se prepara la solución electrospinning. La viscosidad de la solución es crítico para la formación de fibras adecuado. Las soluciones de HA pura no cumplen con este requisito; PEO se agrega como un polímero portador para permitir electrospinning. Las microesferas se añaden a la solución y electrospun resultando en un andamio fibroso con microesferas distribuidas a lo largo. Una vez que la producción se ha completado, la proteína debe ser probado para verificar su viabilidad. Para ello, una célula primaria que responde a NGF puede ser utilizado. Este protocolo utiliza ganglios de raíz dorsal (DRG) 8-10 días embriones de pollo de edad. Los haces de células se siembran en los andamios que contienen microesferas llenas de NGF los que están vacías o. Si el NGF es todavía viable debería ver un mayor crecimiento de axones en los andamios que contienen NGF. Si el NGF ya no es viable que lo haráno promover neuritas para ampliar y debe ser similar a la de control. El procedimiento exacto se describe en el presente documento se centra en el apoyo neural, sin embargo, con modificaciones sencillas en el material, el método de electrospinning, y proteínas que el sistema puede ser optimizado para diferentes tipos de células y tejidos.

<table><tbody><tr><td>DAPI</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>Irgacure 2959</td><td>BASF</td><td>24650-42-8</td><td>Protect from light</td></tr><tr><td>PEO 900 kDa</td><td>Sigma-Aldrich</td><td>189456</td><td></td></tr><tr><td>Methacryloxethyl thiocarbamoyl rhodamine B</td><td>Polysciences, Inc.</td><td>23591-100</td><td>Prepare stock solution in DMSO</td></tr><tr><td>Syringe Pump</td><td>KD Scientific</td><td>KDS100</td><td></td></tr><tr><td>Power Source</td><td>Gamma High Voltage</td><td>ES30P-5W</td><td></td></tr><tr><td>Motor</td><td>Triem Electric Motors, Inc</td><td>0132022-15</td><td>Must attach to a custom built mandrel</td></tr><tr><td>Tachometer</td><td>Network Tool Warehouse</td><td>ESI-330</td><td>Use to monitor mandrel speed</td></tr><tr><td>Omnicure UV Spot Cure System with collimating adapter</td><td>EXFO</td><td>S1000</td><td></td></tr><tr><td>Needles</td><td>Fisher Scientific</td><td>14-825-16H</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-545-81</td><td></td></tr><tr><td>Polyvinyl Alcohol</td><td>Sigma-Aldrich</td><td>P8136-250G</td><td></td></tr><tr><td>Isoporopyl Alcohol</td><td>Sigma-Aldrich</td><td>I9030-500mL</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Fisher Scientific</td><td>BP9703-100</td><td></td></tr><tr><td>BSA-FITC</td><td>Sigma-Aldrich</td><td>080M7400</td><td></td></tr><tr><td>&amp;beta;-Nerve Growth Factor (NGF)</td><td>R&amp;amp;D Systems</td><td>1156-NG</td><td></td></tr><tr><td>65:35 Poly-Lactic-Glycolic-Acid (PLGA)</td><td>Sigma-Aldrich</td><td>1001554270</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma-Aldrich</td><td>34856-2L</td><td></td></tr><tr><td>Coomassie (Bradford) Protein Assay</td><td>Thermo Scientific</td><td>1856209</td><td></td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate</td><td>Sigma-Aldrich</td><td>1001558456</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F2006</td><td></td></tr><tr><td>DMEM</td><td>Lonza</td><td>12-604F</td><td></td></tr><tr><td>FBS</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>PBS</td><td>Hyclone</td><td>SH30256.01</td><td></td></tr><tr><td>Glutamine</td><td>Fisher Scientific</td><td>G7513</td><td></td></tr><tr><td>Pen-Strep</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Alfa Aesar</td><td>A11313&amp;nbsp;</td><td></td></tr></tbody></table>

electrospinning growth factor releasing microspheres into fibrous scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

Las microesferas 50 ± 14 micras de diámetro con una proteína de encapsulación más del 85% se han producido y electrospun en andamios consistentemente. Tamaño se determinó mediante la formación de imágenes de muestras de microesferas de tres lotes de producción separadas. Las imágenes en las que capturaron en un microscopio óptico y longitudes donde mide utilizando software laboratorio comercial. Figura 1 muestra un histograma de la distribución de tamaños. Tasa de encapsulación también s...

Watch this Scientific Journal Video about Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds at JoVE.com

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Factor de Crecimiento de electrospinning Liberar microesferas en Fibrosa andamios

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

electrospinning-growth-factor-releasing-microspheres-into-fibrous

Research

JoVE Journal

Bioengineering

12.2K vistas.  Wayne State University. This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

Video: Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

The Culture of Primary Motor and Sensory Neurons in Defined Media on Electrospun Poly-L-lactide Nanofiber Scaffolds

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned to completely random. In addition, fibers can be spun from a variety of materials, including biodegradable polymers such as poly-L-lactic acid (PLLA). These characteristics make electrospun fibers suitable for a variety of scaffolding applications in tissue engineering. Our focus is on the use of aligned electrospun fibers for nerve regeneration. We have previously shown that aligned electrospun PLLA fibers direct the outgrowth of both primary sensory and motor neurons in vitro. We maintain that the use of a primary cell culture system is essential when evaluating biomaterials to model real neurons found in vivo as closely as possible. Here, we describe techniques used in our laboratory to electrospin fibrous scaffolds and culture dorsal root ganglia explants, as well as dissociated sensory and motor neurons, on electrospun scaffolds. However, the electrospinning and/or culture techniques presented here are easily adapted for use in other applications.

Aligned electrospun fibers direct the growth of neurons in vitro and are a potential component of nerve regeneration scaffolds. We describe a procedure for preparing electrospun fiber substrates and the serum-free culture of primary rat E15 sensory (DRG) and motor neurons. Visualization of neurons by immunocytochemistry is also included.

La cultura de la motora primaria y las neuronas sensoriales en los medios de comunicación definidas en Electrospun poli-L-láctico andamios nanofibras

Electrospinning is a technique for producing micro- to nano-scale fibers. Fibers can be electrospun with varying degrees of alignment, from highly aligned ...

Investigación

Neurociencias

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 ...

Bioingeniería

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease control of composition, structure, and diameter of fibers. However, despite these advantages, traditional electrospun nanofiber scaffolds come with limitations including disorganized nanofiber orientation, low porosity, small pore size, and mainly two-dimensional mats. As such, there is a great need for developing a new process for fabricating electrospun nanofiber scaffolds that can overcome the above limitations. Herein, a novel and simple method is outlined. A traditional 2D nanofiber mat is transformed into a 3D scaffold with desired thickness, gap distance, porosity, and nanotopographic cues to allow for cell seeding and proliferation through the depressurization of subcritical CO2 fluid. In addition to providing a scaffold for tissue regeneration to occur, this method also provides the opportunity to encapsulate bioactive molecules such as antimicrobial peptides for local drug delivery. The CO2 expanded nanofiber scaffolds hold great potential in tissue regeneration, wound healing, 3D tissue modeling, and topical drug delivery.

This article demonstrates the technique of expanding a traditional, two-dimension (2D) electrospun nanofiber mat into a three-dimension (3D) scaffold through the depressurization of subcritical CO2 fluid. These augmented scaffolds are 3D, closely mimic cellular nanotopographic cues, and preserve the functions of biologic molecules encapsulated within the nanofibers.

Extensión de esteras de nanofibras Electrospun de dos dimensiones en tres dimensiones andamios

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

Genética

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Electrospun fibroso Andamios de poli (glicerol-dodecanodioato) de Ingeniería Neural Los tejidos de las células madre embrionarias de ratón

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Electrospun nanofibras andamios con gradaciones en Organización de fibra

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Adaptación del Proceso Electrohilado para proporcionar tres ambientes únicos para un Tri-capa<em&gt; In Vitro</em&gt; Modelo de la pared de la vía aérea

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Fabricación superhidrófobas materiales poliméricos para aplicaciones biomédicas

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

Electrospinning andamios fibroso polímero para ingeniería de tejidos y cultivos celulares

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

Biología

Fabrication and Characterization of Griffithsin-modified Fiber Scaffolds for Prevention of Sexually Transmitted Infections

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to prevent and treat sexually transmitted infections (STIs). Moreover, many EF technologies focus on encapsulating the active agent, relative to utilizing the surface to impart biofunctionality. Here we describe a method to fabricate and surface-modify poly(lactic-co-glycolic) acid (PLGA) electrospun fibers, with the potent antiviral lectin Griffithsin (GRFT). PLGA is an FDA-approved polymer that has been widely used in drug delivery due to its outstanding chemical and biocompatible properties. GRFT is a natural, potent, and safe lectin that possesses broad activity against numerous viruses including human immunodeficiency virus type 1 (HIV-1). When combined, GRFT-modified fibers have demonstrated potent inactivation of HIV-1 in vitro. This manuscript describes the methods to fabricate and characterize GRFT-modified EFs. First, PLGA is electrospun to create a fiber scaffold. Fibers are subsequently surface-modified with GRFT using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS)chemistry. Scanning electron microscopy (SEM) was used to assess the size and morphology of surface-modified formulations. Additionally, a gp120 or hemagglutinin (HA)-based ELISA may be used to quantify the amount of GRFT conjugated to, as well as GRFT desorption from the fiber surface. This protocol can be more widely applied to fabricate fibers that are surface-modified with a variety of different proteins.

This manuscript describes the procedure to fabricate and characterize Griffithsin-modified poly(lactic-co-glycolic acid) electrospun fibers that demonstrate potent adhesive and antiviral activity against human immunodeficiency virus type 1 infection in vitro. Methods used to synthesize, surface-modify, and characterize the resulting morphology, conjugation, and desorption of Griffithsin from surface-modified fibers are described.

Fabricación y caracterización de andamios de fibra modificado Griffithsin para la prevención de infecciones de transmisión sexual

Electrospun fibers (EFs) have been widely used in a variety of therapeutic applications; however, they have only recently been applied as a technology to ...