Name: wet-spinning-based molding process of gelatin for tissue regeneration
Uploaded: 2019-03-07T14:15:00
Description: Watch this Scientific Journal Video about Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration at JoVE.com

Este estudio fue apoyado por el Ministerio de la defensa nacional (MAB-105-070; MAB-106-077; MAB-107-032; MAB-107-065), el Ministerio de ciencia y tecnología (la mayoría 107-2320-B016-016), Tri-Service General Hospital, nacional de la defensa centro médico, Taiwán (TSGH-C106-046; TSGH-C106-115; TSGH-C107-041) y Hospital General Cheng Hsin y centro médico de nacional defensa cooperación (CH-NDMC-107-8).

Los tejidos de grasa obtenidos de cirugías ortopédicas como certificada por el institucional de Junta de Tri-Service General Hospital, Taipei, Taiwan, R.O.C. procedimientos con sujetos animales han sido aprobados por el Comité de cuidado del Animal a nivel nacional Centro médico de la defensa, Taiwán (ROC).
1. Moje el proceso de giro
<ol><li>
Preparación de la solución
	<ol>
<li>Disolver 5 g de polvo de gelatina en 100 mL de agua destilada doble para obtener la concen...

<ol>
	<li>Bagnall, A. M., Jones, L., Duffy, S., Riemsima, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+fixation+surgery+for+acute+traumatic+spinal+cord+injury.">Spinal fixation surgery for acute traumatic spinal cord injury.</a> Cochrane Database of Systematic Reviews. 1, 004725 (2008).</li><li>Fehlings, M. G., Perrin, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+timing+of+early+decompression+for+cervical+spinal+cord+injury:+update+with+a+review+of+recent+clinical+evidence.">The role and timing of early decompression for cervical spinal cord injury: update with a review of recent clinical evidence.</a> Injury. 36, 13-26 (2005).</li><li>Yang, L., Jones, N. R., Stoodley, M. A., Blumbergs, P. C., Brown, C. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroitin+sulphate+proteoglycans+in+the+central+nervous+system:+changes+and+synthesis+after+injury.">Chondroitin sulphate proteoglycans in the central nervous system: changes and synthesis after injury.</a> Biochemical Society Transactions. 31, 335-336 (2003).</li><li>Fawcett, J. W., Asher, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+scar+and+central+nervous+system+repair.">The glial scar and central nervous system repair.</a> Brain Research Bulletin. 49, 377-391 (1999).</li><li>Yang, Z., Mo, L., Duan, H., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chitosan/collagen+substrates+on+the+behavior+of+rat+neural+stem+cells.">Effects of chitosan/collagen substrates on the behavior of rat neural stem cells.</a> Science China Life Sciences. 53, 215-222 (2010).</li><li>Chawla, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fibrous Materials. , (1998).</li><li>Pickering, K. L., Aruan Efendy, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+recent+developments+in+natural+fibre+composites+and+their+mechanical+performance.">A review of recent developments in natural fibre composites and their mechanical performance.</a> Composites Part A-Applied Science and Manufacturing. 83, 98-112 (2016).</li><li>Lundgren, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+fibers+made+from+proteins.">Synthetic fibers made from proteins.</a> Advances in Protein Chemistry. 5, 305-351 (1954).</li><li>Radishevskii, M. B., Serkov, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+mechanism+in+wet+spinning+of+fibres.">Coagulation mechanism in wet spinning of fibres.</a> Fibre Chemistry. 37, 266-271 (2005).</li><li>Yannas, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+and+gelatin+in+the+solid+state.">Collagen and gelatin in the solid state.</a> Journal of Macromolecular Science Part C Polymer Reviews. 7, 49-106 (1972).</li><li>Baer, E., Cassidy, J. J., Hiltner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+structure+of+collagen+composite+Systems:+lessons+from+biology.">Hierarchical structure of collagen composite Systems: lessons from biology.</a> Pure and Applied Chemistry. 6, 961-973 (2009).</li><li>Harrington, W. F., Von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+collagen+and+gelatin.">The structure of collagen and gelatin.</a> Advances in Protein Chemistry. 16, 1-138 (1961).</li><li>Veis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Macromolecular Chemistry of Gelatin. , (1994).</li><li>Freyman, T. M., Yannas, I. V., Gibson, L. 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Presentamos el desarrollo de biomateriales basados en gelatina usando un simple mojado spinning técnica que puede ser aplicado en el estudio de polímeros naturales para regeneración de tejidos. Este trabajo demostró la posibilidad de fabricación de la gelatina como una fuente de proteína grandes sin la adición de otras fuentes, con el objetivo de optimizar las propiedades de la gelatina sí mismo. El desarrollo de biomateriales basados en gelatina se realizó enteramente en temperatura ambiente (22-26 ° C). Prepa...

El último desarrollo en regeneración de tejidos consiste en la aplicación de la ingeniería del tejido fino, que representa un desafío para la mejora de nuevas estrategias terapéuticas en tratamientos médicos. Por ejemplo, el limitado potencial de regeneración del sistema nervioso, lesiones o enfermedad, siguientes plantea un importante problema de salud en todo el mundo. Debido a la complejidad de los procesos patofisiológicos asociados con el sistema nervioso, el uso de autoinjerto tradicional o la aplicación de la cirugía de estabilización ha demostrado ofrecer beneficios en los resultados funcionales, pero no existe evidencia fuerte de los efectos de la fijación espinal cirugía1,2. El tejido en el área dañada es perdido y sustituido por astrocitos hypertrophically inducido3, eventual formando una densa cicatriz glial4,5. Esta matriz actúa como una barrera que bloquea la recuperación del nervio funcionan6,7 y es, así, enormemente obstaculiza la regeneración. Por lo tanto, se espera que un material de relleno adecuado espacio para prevenir la pérdida de tejido y reducir la formación de tejido conectivo asociado a cicatriz manteniendo la integridad de la zona dañada, así como al proporcionar el reemplazo directo de las células neuronales y circuito para promover la regeneración del axón.
Biomateriales poliméricos han preferido como andamios para la terapia de regeneración de tejido, basado en la regulación de la progresión de comportamiento y tejido celular o axón a través del apoyo natural matriz extracelular (ECM). El formato de la fibra se considera comúnmente como un bloque de construcción para los varios materiales, debido a su estructura unidimensional8. Las fibras pueden obtenerse generalmente por extrusión de derretimiento o mojado spinning método; sin embargo, el gran tamaño y costo de los equipos y la dificultad para llevar a cabo estos métodos son difíciles. Además, la mayoría de los trabajos relacionados con fibras de polímero se ha centrado en materiales sintéticos o compuestos. Polímeros naturales como fuente de biomateriales ofrecen mejores propiedades de biocompatibilidad para el cuerpo humano. Sin embargo obtener la alineación de las fibras de polímero natural es relativamente más difícil que de polímero sintético fuentes9. Por lo tanto, la conversión de un polímero natural como una fuente rica de proteína en las fibras del biomaterial es una estrategia importante, no sólo las fibras del biomaterial se pueden directamente aislada desde la materia prima, evitando una innecesaria transformación en monómeros, pero la fibras de proteína también tienen un buen aspecto y características favorables10.
En este sentido, se describe un método de tratamiento barato para la fabricación de fibras de polímero natural a través del concepto básico de la hilatura húmeda, que puede ser implementada en la escala de laboratorio para ingeniería de tejidos. Hilado húmedo se realiza en la extrusión y coagulación de una solución de polímero en un nonsolvent de polímero adecuado. Una solución apropiada, viscosa dopada en medio de la coagulación hace que las moléculas del polímero a disolver. A través de la transición de fase, los filamentos entonces pierden su solubilidad y se precipitan en forma de un polímero sólido fase11. Refiriéndose a este concepto, expanden el desarrollo de la gelatina en forma de tubo por un proceso de moldeo, que se considera adecuado para la aplicación de la regeneración de tejido. Además, intrínsecamente, nosotros puede también desarrollar alguna forma de material de fibras de gelatina (por ejemplo, conducto de gelatina enrollada de varias fibras de gelatina), el otro desea aplicaciones.
Gelatina, un polímero natural biodegradable, está formada de colágeno hidrolizado y desnaturalizado, incluyendo cualquier estado helicoidal triple, amorfo o semicristalino de colágeno12. Es bien sabido que el colágeno es la proteína estructural esencial en todos los tejidos conectivos de los vertebrados e invertebrados13,14, que es similar a la estructura de las proteínas de la ECM principal que induce el crecimiento del nervio y, al mismo tiempo, reemplaza una gran cantidad de glycosaminoglycan secretada durante las lesiones de la médula espinal. Por lo tanto, el uso de gelatina como una fuente sería una gran opción para cualquier vehículo médico. Además de ser una fuente barata, la gelatina también es biodegradable y cytocompatible y clínicamente probados ser un temporal defecto relleno15. Desarrollado en forma de tubo, descritas aquí las pruebas in vitro e in vivo demuestran que la gelatina tiene una excelente biocompatibilidad y conveniencia para futuros usos de la ingeniería del tejido. Cultivados con células madre adiposas humanas, tubos de gelatina mejoran diferenciación celular en las células progenitoras neurales utilizando tinción nestina positivas como un marcador de células neuronales. Además, gelatina como material de terraplenado gap, como producidos por el método establecido en el presente estudio, se espera que sea manejable y segura y de gran beneficio para ingenieros de tejido que actualmente están desarrollando polímeros naturales correlativos para el fortalecimiento del tejido estrategias de regeneración.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Solution preparation:</td>
		</tr>
		<tr>
			<td>Gelatin type B (porcine)</td>
			<td>Ferak</td>
			<td>Art. -Nr. 10733</td>
			<td>500 g vial</td>
		</tr>
		<tr>
			<td>Wet spinning process:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>Model M312</td>
			<td>Minipuls*3</td>
		</tr>
		<tr>
			<td>Plastic tube connector</td>
			<td>World Precision Instruments</td>
			<td>14011</td>
			<td>1 box</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Sterican</td>
			<td>5A06258541</td>
			<td>26Gx1/2&#34;(0.45 x 12mm)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Ferak</td>
			<td>Art. -Nr. 00010</td>
			<td>2.5 L vial</td>
		</tr>
		<tr>
			<td>Polycaprolactone CAPA 6500</td>
			<td>Perstorp</td>
			<td>24980-41-4</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>Scharlau</td>
			<td>CL03421000</td>
			<td>1 L vial</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Peripheral venous catheter (Introcan Certo)</td>
			<td>B. Braun</td>
			<td>1B03258241</td>
			<td>24Gx3/4&#34;(0.7 x 19mm)</td>
		</tr>
		<tr>
			<td colspan="4">Morphology of the gelatin tube:</td>
		</tr>
		<tr>
			<td>Ion sputter coater machine&#160;</td>
			<td>Hitachi</td>
			<td>e1010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Scanning electron microscopy</td>
			<td>Hitachi</td>
			<td>S-3000N</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cultivation of cells on the gelatin tube:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>488625</td>
			<td>100 mL vial</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>923119</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium&#160;</td>
			<td>Gibco</td>
			<td>31600-034</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Keratinocyte-SFM medium</td>
			<td>Gibco</td>
			<td>10744-019</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>T25 culture flask</td>
			<td>TPP</td>
			<td>90025</td>
			<td>VENT type</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Falcon</td>
			<td>1209938</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Immunocytochemistry:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate-buffered saline</td>
			<td>Gibco</td>
			<td>654471</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Acetic acid glacial</td>
			<td>Ferak</td>
			<td>Art. -Nr. 00697</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>NP-40 surfactant (Tergitol solution)</td>
			<td>Sigma</td>
			<td>056K0151</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000-20</td>
			<td>20 mL vial, concentrate</td>
		</tr>
		<tr>
			<td>Nestin (primary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-23927</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse-fluorescein isothiocyanate (secondary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2099</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Anaspec</td>
			<td>AS-83218</td>
			<td>5 mL vial</td>
		</tr>
		<tr>
			<td>In vivo biocompatibility test:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tiletamine+zolazepam&#160;</td>
			<td>Virbac</td>
			<td>BC91</td>
			<td>5 mL vial</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer korea</td>
			<td>KR03227</td>
			<td>10 mL vial</td>
		</tr>
		<tr>
			<td>Ketoprofen</td>
			<td>Astar</td>
			<td>1406232</td>
			<td>2 mL vial</td>
		</tr>
		<tr>
			<td>Povidone-iodine solution</td>
			<td>Everstar</td>
			<td>HA161202</td>
			<td>4 L barrel</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>China Chemical &#38; Pharmaceutical</td>
			<td>18P909</td>
			<td>1 g vial</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

wet-spinning-based molding process of gelatin for tissue regeneration

Este artículo presenta un método barato para fabricar gelatina, como un polímero natural, en fibras de monofilamento o en otras formas apropiadas. A través de la humedad método de giro, las fibras de la gelatina son producidas por extrusión suave en medio de una coagulación adecuada. Para aumentar la superficie funcional de esas fibras de gelatina y su capacidad para imitar las características de los tejidos, la gelatina puede ser moldeada en forma de tubo haciendo referencia a este concepto. Examinado por pruebas in vitro e in vivo, los tubos de gelatina demuestran un gran potencial para su aplicación en la ingeniería de tejidos. Actuando como un material de relleno adecuado brecha, gelatina de tubos pueden ser utilizados para sustituir el tejido en el área dañada (por ejemplo, en el sistema nervioso o cardiovascular), así como para promover la regeneración al proporcionar una sustitución directa de las células madre y los circuitos neuronales. Este protocolo proporciona un procedimiento detallado para la creación de un biomaterial basado en un polímero natural, y se espera que su aplicación beneficiaría enormemente el desarrollo de polímeros naturales correlativos, que ayudan a realizar estrategias de regeneración de tejido.

En este estudio, desarrollamos con éxito la gelatina en las fibras (figura 2A) y tubos (figura 2B, C) a través del concepto de hilado húmedo fácil de usar. Estos materiales a base de gelatina pueden ser utilizados como cualquier herramienta médica, dependiendo de sus formas. Teniendo en cuenta que la superficie funcional y el marco de tales materiales son más adecuados para la regeneración...

Watch this Scientific Journal Video about Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration at JoVE.com

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

Desarrollamos y describir un protocolo basado en el concepto de hilatura húmeda, para la construcción de la base de gelatina de biomateriales utilizados para la aplicación de la ingeniería de tejidos.

Proceso de moldeo basada en mojado-spinning de gelatina para la regeneración de los tejidos

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

Este protocolo mejora un concepto básico de hilado húmedo para desarrollar un material a partir de una sola tira de fibra en varias tiras que se pueden utilizar para cualquier aplicación médica preferida. Esta técnica es simple, económica y no requiere la adición de polímeros sintéticos para la personalización de los polímeros naturales en un recurso de proteína para materiales de interés. Esta técnica se puede extender al desarrollo de biomateriales para matar la terapia fría, como el tratamiento del sistema nervioso central o periférico, el reemplazo de los vasos sanguíneos o la reconstrucción directa del tracto urinario.Este método está destinado a la aplicación médica, incluyendo como una estrategia para la neuralización de tejidos o la medicina regenerativa. Esta técnica puede ser fácilmente implementada por principiantes. Tenga en cuenta que, después de varias repeticiones de moldeo, la calidad de la solución siempre debe comprobarse para mantener una formación óptima.Para formar el tubo giratorio húmedo, primero sumerja un catéter venoso periférico de 24 calibres por tres cuartos de pulgada en una solución de gelatina recién preparada durante tres segundos, seguido de una inmersión en solución de acetona recién preparada durante un minuto. Repita estos pasos 20 veces. Después de la última inmersión en acetona, deje secar el tubo moldeado durante cinco minutos a temperatura ambiente, y utilice un hemostat para transferir cuidadosamente el tubo de gelatina del catéter a una pipeta Pasteur de vidrio con una solución de diclorometano de policaprolactona al 2,5% para una incubación nocturna debajo del capó a temperatura ambiente.A la mañana siguiente, esterilizar la gelatina durante dos horas bajo luz ultravioleta antes de sumergir brevemente el tubo en 75%etanol. A continuación, lave el tubo dos veces con el medio de células madre para eliminar el etanol residual. Para cultivar el tubo de gelatina con el cultivo celular, sustituya el extremo supinato de las células madre adiposas humanas de un matraz de cultivo por un mililitro de 0,25% de Trypsin-EDTA para una incubación de 3,5 minutos a 37 grados centígrados y un 5% de dióxido de carbono.Cuando las células se hayan desprendido, detenga la reacción con un mililitro de suero bovino fetal y transfiera la suspensión celular a un tubo de microcentrífuga. Recoger las células por centrifugación y resuspender el pellet en un mililitro de medio de células madre. A continuación, coloque el tubo de gelatina en un pozo de una placa de seis pozos que contenga tres mililitros de medio de células madre.Después de contar las células, sembra cuatro veces 10 a la cuarta de las células madre adiposas humanas en el tubo para una incubación de dos semanas en la incubadora de cultivo celular. Después de confirmar el nivel adecuado de sedación por falta de respuesta a la pellizca del dedo del pie en una rata Sprague Dawley de ocho semanas de edad, afeitar el cabello de la zona del trapecio y esterilizar la piel expuesta con solución de povidona-yodo. Cubra a la rata con cortinas quirúrgicas estériles para reducir la contaminación bacteriana del sitio quirúrgico y use tijeras quirúrgicas para hacer una incisión cutánea superficial de dos centímetros de largo.Coloque el tubo de gelatina directamente en la capa entre la fascia y el músculo y utilice una sutura de nylon para cerrar la herida. A continuación, esterilizar la incisión con solución de povidona-yodo. Inducir a la rata a través de una inyección intramuscular de ketoprofeno y cefazolina y mantener al animal en condiciones asépticas durante siete días.Este concepto de hilado húmedo fácil de usar se puede utilizar para desarrollar gelatina en fibras y tubos. Las observaciones morfológicas del tubo de gelatina mediante microscopía electrónica de barrido revelan una superficie no muy lisa con un diámetro interior superior a 200 micrómetros y un espesor de aproximadamente 20 micrómetros. La tinción de un tubo de gelatina de cultivo de células madre adiposo humano para la expresión del marcador de células neurales demuestra una tinción positiva para el marcador junto con múltiples núcleos detectados que sugieren que las células pueden penetrar y adherirse al tubo de gelatina y diferenciarse en células progenitoras neuronales.La implantación del tubo de gelatina en la capa de fascia confirma su seguridad y biocompatibilidad, ya que no se observa ninguna indicación de enrojecimiento, hinchazón u otros síntomas de inflamación dentro del tejido local y el tubo está rodeado de tejido conectivo bien desarrollado. Al intentar este procedimiento de dos puntos uno, asegurar la inmersión del molde en el tiempo de recubrimiento específico en cada solución son muy importantes para construir una forma de tubo precisa. Después de este procedimiento, nuestros experimentos utilizan el modelo animal de la enfermedad humana se puede realizar para investigar con precisión el resultado de los biomateriales para la estrategia de regeneración de tejidos.Después de su desarrollo, esta técnica allanó el camino para los investigadores en el desarrollo de polímeros de naturaleza correlativa simples en cualquier tipo de material para una aplicación médica decidida. En condiciones de uso normal, se considera que la solución de diclorometano de policaprolactona presenta un riesgo ambiental mínimo, pero no olvide realizar ningún paso con este producto químico en un gabinete de bioseguridad adecuado.

wet-spinning-based-molding-process-of-gelatin-for-tissue-regeneration

Research

JoVE Journal

Bioengineering

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Expresión, aislamiento y purificación de soluble e insoluble Biotinylated Proteínas del Tejido Nervioso para regeneración

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Investigación

Bioingeniería

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Prototipado rápido y de bajo costo de los dispositivos médicos Utilizando 3D Impreso Moldes para moldeo por inyección de líquido

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

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. ...

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 ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Caracterización cualitativa de la fracción acuosa de hidrotérmico Licuefacción de algas mediante 2D cromatografía de gases con espectrometría de masas de tiempo de vuelo

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Alternando microgeles de gelatina de campo híbrido-Responsive magnéticos para Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

Un grano emparejado y arreglo de imán para moldear los micropocillos con geometrías variables cóncavos

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

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

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

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

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

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

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Patrón de la geometría de las colonias de células madre embrionarias humanas en sustratos compatibles para controlar la mecánica de nivel de tejido

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Protocolos de Bioimpresión 3D de Bioinks basados en Gelatin Methacryloyl Hydrogel

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...