Este trabajo fue apoyado por el NIH K08 EB003996 y los Veteranos Paralizados de América Investigación Beca de la Fundación 2573.

1. Elegir un polímero <ol><li> Elegir un polímero (por ejemplo, poli-L-láctico (PLLA), policaprolactona (PCL), poliestireno (PS) o nylon) con base en las especificaciones (por ejemplo, biodegradable, termoplástico o cruz enlazables) y un disolvente de que el polímero. Elegir el equipo adecuado de protección personal sobre la base de su selección. </li><li> Seleccione un sustrato a base de su aplicación (por ejemplo, obleas de vidrio, plástico, metal o el silicio). </li></ol> 2. Elegir un colector <ol><li> Elija la geometría colector basado en sus especificaciones. Fibras al azar se pueden recoger en las placas fijas. Fibras alineadas se pueden recoger en rápida rotación de las ruedas, tambores o barras, o en placas paralelas. </li><li> El colector debe ser conductora y debe permanecer aislado de su eje de tal manera que pueda ser conectado a tierra, sin tierra también los objetos adyacentes, la superficie de la mesa, etc </li></ol> 3. Aproximar la concentración entrelazamiento crítico Empíricamente un <ol><li> Prepare varias concentraciones de polímeros candidatos (por ejemplo, 4, 10, 15, 20, 30% en peso) y elija una concentración que los flujos (la solución debe ser un líquido viscoso, pero no un gel) para empezar. </li><li> La instalación del equipo electrospinning 2,3,4,5 (ver Figura 1) <ol><li> Cargar la bomba de jeringa y fijar la velocidad de la bomba de manera que cualquier gota de solución de borrado de la punta es reemplazada inmediatamente. </li><li> Baja del colector y el clip de alambre de alta tensión a la placa de conductor (un pequeño cuadrado de material conductor, como papel de aluminio a través del cual sobresale la punta de la jeringa). </li><li> Inicie el giro de la rueda. </li><li> Asegúrese de que la fuente de alimentación se ajusta a cero antes de encenderlo. </li></ol></li><li> Observe el flujo de <ol><li> Rampa de tensión lentamente y ver la gota de la solución en la punta de la aguja. </li><li> Ajustar la tensión para obtener una larga y constante. Si un flujo constante no se puede obtener, ajustar la concentración de la solución de polímero. Ver Tabla 1 para un ejemplo. </li></ol></li></ol> 4. Resolución de problemas - la corriente: <ol><li> Problemas para ver el flujo de <ol><li> Use un fondo oscuro mate y colocar una fuente de luz unidireccional (como una linterna) entre el espectador y la corriente (ver Figura 2). </li></ol></li><li> Goteo de la punta de la jeringa <ol><li> Si la solución de polímero es que gotea hacia abajo sin ningún atractivo a la rueda, asegúrese de que la placa de conductor hace contacto con la punta de la aguja y que el colector está haciendo contacto con el suelo. </li><li> Si la gota de solución de polímero en la punta de la jeringa se inclina en la dirección de la rueda, pero no es la formación de una corriente, aumentar la tensión. La calidad de la corriente se puede ajustar mediante la variación de la distancia y la tensión hasta que un flujo constante es visible. Ver Figura 3 para distancias sugeridas con voltajes correspondientes a una solución PLLA 4% y una placa de conductor de 8x8 cm. </li></ol></li><li> Globos grande en la punta de la jeringa <ol><li> Cuando la solución de polímero comienza a recoger y se endurecen en la punta de la aguja, mueva la globalización con una toalla de papel pegado a un palo no conductor. </li></ol></li><li> Oscilante o corrientes de agua &#39;moviendo&#39; Cuando la corriente está moviendo arriba y abajo rápidamente, bajar la tensión o aumentar la distancia entre la punta de la jeringa y la rueda. Si la corriente sigue a menear utilizar una mayor concentración de polímero o añadir un poco de disolvente con una evaporación más lenta. </li><li> Programas cortos o discontinuos Visible corrientes constantes toma de contacto observable con el conjunto de la rueda a una velocidad de rotación de alto rendimiento de la más alta calidad de la uniformidad y la alineación. Cuando la corriente se corta y discontinua, el aumento de la solución de polímero, la adición de más solvente de evaporación lenta, y el ajuste de la tensión mejorará la longitud y la firmeza de la corriente. </li></ol> 5. Resolución de problemas - Morfología de fibra 6,7,8 (ver Figura 4) <ol><li> Cuentas Cuando cuentas que se descubren en las fibras, aumento de la solución de polímero y asegúrese de que la placa de conductor es el contacto continuo con la aguja y que el cepillo de alambre a tierra es el contacto continuo con la rueda. </li><li> Cintas y fibras de sangrado Cuando las fibras se forman como cintas o sangrado en conjunto, utilice una mayor concentración de polímero o un disolvente con una mayor tasa de evaporación (más volátiles). </li><li> Fibras floritura u ondulado Cuando las fibras se forman las olas o volutas, aumentar la velocidad de las ruedas o mover la punta de la aguja más lejos de la colección. Además, verifique que la placa de conductor y el colector no están vibrando. </li><li> Porosidad 9 Si los poros son lo desea, use un solvente de evaporación rápida. Si los poros no son deseados, pruebe a añadir una pequeña cantidad de co-disolvente, que es menos volátil que el principal solvente. </li><li> Alineación 10 Cuando el colector se movción a un régimen bajo o en reposo, la calidad de la alineación es deficiente. Aumentar la alineación por el aumento de la velocidad de la rueda. </li></ol> 6. Los resultados representativos: Por favor refiérase a la Figura 4 para las representaciones de los resultados de fibra típica. <img src="/files/ftp_upload/2494/2494fig1.jpg" alt="Figura 1" /> Figura 1. Una configuración típica de electrospinning. Una solución de polímero (azul) se dispensa de una bomba de jeringa (naranja). Un alto voltaje de corriente directa (verde) un colector de motivos de la rueda que gira rápidamente (en gris) en los que se recogen nanofibras alineados. El chorro de polímero entre la jeringa y el colector consta de un segmento de constante flujo y un segmento de rápido oscilante azotes. <img src="/files/ftp_upload/2494/2494fig2.jpg" alt="Figura 2" /> . Figura 2 El chorro de streaming se puede ver salir de la punta de la jeringa, el chorro de azotes es demasiado pequeño para ser visto. La aproximación de la concentración crítica de enredo PLLA <table border="1"><tbody><tr><td> PLLA (% p / v) </td><td> Observación </td><td> Ajuste de la concentración </td></tr><tr><td> 0.5 </td><td> Goteo, sin corriente </td><td> Aumento </td></tr><tr><td> 2.0 </td><td> Escupiendo globos pequeños, sin corriente </td><td> INCRESE ligeramente </td></tr><tr><td> 4.0 </td><td> Flujo constante </td><td> Bueno </td></tr><tr><td> 6.0 </td><td> Escupiendo globos grandes o cuentas </td><td> Disminuyen ligeramente </td></tr><tr><td> 12.0 </td><td> Formación de grumos en la punta, sin corriente </td><td> Disminución </td></tr></tbody></table> Tabla 1. Un ejemplo que muestra la aproximación de la concentración crítica de PLLA enredo. Diferentes concentraciones de polímeros son juzgados y los chorros resultantes de transmisión observada hasta una corriente constante se obtiene. <img src="/files/ftp_upload/2494/2494fig3.jpg" alt="Figura 3" /> Figura 3. La distancia entre la punta de la jeringa y el colector debe estar equilibrada con la tensión aplicada para obtener un chorro constante de streaming. Exceso de voltaje aplicado causa una oscilación o &quot;menear&quot; chorro de forma que los resultados en menos fibras bien alineados. Cuando la tensión es demasiado baja, no se forma de chorro y la única solución por goteo de la punta de la jeringa. La región sombreada en color morado de arriba representa el rango de tensión sobre el cual puede ser un chorro constante flujo obtenidos para PLLA en función de la distancia jeringa-colector. <img src="/files/ftp_upload/2494/2494fig4.jpg" alt="Figura 4" /> Figura 4. Fibras Electrospun puede exhibir una variedad de morfologías, incluyendo cuentas (A), cintas (B), arabescos (C), globos poroso (D), una buena alineación (E) y una mala alineación (F).

<ol>
	<li>Shenoy, S. L., Bates, W. D., Frisch, H. L., Wnek, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+chain+entanglements+on+fiber+formation+during+electrospinning+of+polymer+solutions:+good+solvent,+non-specific+polymer-polymer+interaction+limit.">Role of chain entanglements on fiber formation during electrospinning of polymer solutions: good solvent, non-specific polymer-polymer interaction limit.</a> Polymer. 46, 3372-3384 (2005).</li><li>Gertz, C. C., Leach, M. K., Birrel, L. K., Martin, D. C., Feldman, E. L., Corey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+neuritogenesis+and+maturation+of+primary+spinal+motor+neurons+in+response+to+nanofibers.">Accelerated neuritogenesis and maturation of primary spinal motor neurons in response to nanofibers.</a> Dev. Neurobiol. 70, 589-603 (2010).</li><li>Lin, D. Y., Johnson, M. A., Vohden, R. A., Chen, D., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+nanofiber+morphologies+using+modulated+electrospinning+for+biomedical+applications.">Tailored nanofiber morphologies using modulated electrospinning for biomedical applications.</a> Mat. Res. Soc. Symp. Proc. 736, D3.8.1-D3.8.6 (2003).</li><li>Corey, J. M., Gertz, C. C., Wang, B. S., Birrell, L. K., Johnson, S. L., Martin, D. C., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+electrospun+PLLA+nanofiber+scaffolds+compatible+with+serum-free+growth+of+primary+motor+and+sensory+neurons.">The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of primary motor and sensory neurons.</a> Acta. Biomater. 4, 863-875 (2008).</li><li>Corey, J. M., Lin, D. Y., Mycek, K. B., Chen, Q., Samuel, S., Feldman, E. L., Martin, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+electrospun+nanofibers+specify+the+direction+of+dorsal+root+ganglia+neurite+growth.">Aligned electrospun nanofibers specify the direction of dorsal root ganglia neurite growth.</a> J. Biomed. Mater. Res. A. 83, 636-645 (2007).</li><li>Tan, S. -. H., Kotaki, M., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+parameter+study+for+ultra-fine+fiber+fabrication+via+electrospinning+process.">Systematic parameter study for ultra-fine fiber fabrication via electrospinning process.</a> Polymer. 46, 6128-6134 (2005).</li><li>Yang, F., Murugan, R., Wang, S., Ramakrishna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+of+nano/micro+scale+poly(L-lactic+acid)+aligned+fibers+and+their+potential+in+neural+tissue+engineering.">Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering.</a> Biomaterials. 26, 2603-2610 (2005).</li><li>Li, W., Laurencin, C. T., Caterson, E. J., Tuan, R. S., Ko, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibrous+structure:+A+novel+scaffold+for+tissue+engineering.">Electrospun nanofibrous structure: A novel scaffold for tissue engineering.</a> J. Biomed. Mater. Res. A. 60, 613-621 (2002).</li><li>Kim, C. H., Jung, Y. H., Kim, H. Y., Lee, D. R. <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+collector+temperature+on+the+porous+structure+of+electrospun+fibers.">Effect of collector temperature on the porous structure of electrospun fibers.</a> Macromol. Res. 14, 59-65 (2006).</li><li>Wang, H. B., Mullins, M. E., Cregg, J. M., Hurtado, A., Oudega, M., Trombley, M. T., Gilbert, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+highly+aligned+electrospun+poly-L-lactic+acid+fibers+for+nerve+regeneration+applications.">Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications.</a> J. Neural Eng. 6, (2009).</li></ol>

Nota: La mayoría de los ejemplos que aquí se presenta frente a electrospinning poli-L-láctico (PLLA) nanofibras. Esto es simplemente porque PLLA es el polímero más común giró en nuestro laboratorio. Sin embargo, también hemos utilizado con éxito estos métodos para electrospin otros polímeros (por ejemplo, PLGA, PCL, PS) y creemos que las técnicas presentadas aquí son fácilmente aplicables a la mayoría de mediano y alto peso molecular de soluciones de polímeros.

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>High voltage DC power supply</td>
<td>&nbsp;</td>
<td>Gamma High Voltage</td>
<td>ES40P-5W</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pump</td>
<td>&nbsp;</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>&nbsp;</td>
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<td>Aluminum foil</td>
<td>&nbsp;</td>
<td>Reynolds</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Blunt metal tips, 23ga</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-850-102 </td>
<td>&nbsp;</td>
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<td>Polypropylene syringe</td>
<td>&nbsp;</td>
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<td>309585</td>
<td>&nbsp;</td>
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<td>Rotating or stationary collector</td>
<td>&nbsp;</td>
<td>Custom built</td>
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<td>Various alligator clips and wires</td>
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<td>Dimethylformamide</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Fisher</td>
<td>AC42355-0040 </td>
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<td>&nbsp;</td>
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<td>Resomer L210</td>
<td>&nbsp;</td>
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<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
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<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
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electrospinning fundamentals: optimizing solution and apparatus parameters

Electrospun andamios nanofibras han demostrado que acelera la maduración, mejorar el crecimiento, y dirigir la migración de las células in vitro. Electrospinning es un proceso en el que se recoge una carga chorro de polímero en un colector de tierra, una rápida rotación resultados colector en nanofibras alineados mientras que el resultado colectores fijos en esteras de fibra orientadas al azar. El chorro de polímero se forma cuando una carga electrostática aplicada supera la tensión superficial de la solución. Hay una concentración mínima de un polímero dado, la denominada concentración crítica enredo, por debajo del cual un chorro estable, no se puede lograr y no nanofibras se forman - a pesar de las nanopartículas se puede lograr (electrospray). Un avión de estabilidad tiene dos dominios, un segmento de transmisión y un segmento de azotes. Mientras que el chorro de azotes suele ser invisible a simple vista, el segmento de transmisión es a menudo visible bajo condiciones de iluminación adecuadas. La observación de la longitud, grosor, consistencia y el movimiento de la corriente es útil para predecir la alineación y la morfología de las nanofibras se forman. Una corriente de corto, no uniforme, inconsistentes, y / u oscilante es indicativo de una variedad de problemas, incluyendo la alineación de la fibra pobres, puntillas, salpicando, y los patrones de floritura u ondulado. La corriente se puede optimizar mediante el ajuste de la composición de la solución y la configuración del aparato electrospinning, optimizando así la alineación y la morfología de las fibras que se producen. En este protocolo, se presenta un procedimiento para la creación de un aparato de electrospinning básicos, empíricamente, la aproximación de la concentración de enredo crítico de una solución de polímero y la optimización del proceso de electrospinning. Además, se describen algunos problemas comunes y las técnicas de resolución de problemas.

Watch this Scientific Journal Video about Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters at JoVE.com

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospinning técnicas pueden crear una variedad de andamios nanofibras para ingeniería de tejidos u otras aplicaciones. Se describe aquí un procedimiento para optimizar los parámetros de la solución de electrospinning y aparatos para la producción de fibras con la morfología y la alineación que desee. Problemas comunes y las técnicas de resolución de problemas también se presentan.

Fundamentos electrospinning: Solución de Optimización de los parámetros y aparatos

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

electrospinning-fundamentals-optimizing-solution-apparatus

Research

JoVE Journal

Bioengineering

64.6K vistas.  University of Michigan. Electrospinning técnicas pueden crear una variedad de andamios nanofibras para ingeniería de tejidos u otras aplicaciones. Se describe aquí un procedimiento para optimizar los parámetros de la solución de electrospinning y aparatos para la producción de fibras con la morfología y la alineación que desee. Problemas comunes y las técnicas de resolución de problemas también se presentan.

Video: Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery under pulsatile deformations. The goal of this study was to mimic the structure of native artery by fabricating a multi-layered electrospun conduit composed of poly(caprolactone) (PCL) with the addition of elastin and collagen with blends of 45-45-10, 55-35-10, and 65-25-10 PCL-ELAS-COL to demonstrate mechanical properties indicative of native arterial tissue, while remaining conducive to tissue regeneration. Whole grafts and individual layers were analyzed using uniaxial tensile testing, dynamic compliance, suture retention, and burst strength. Compliance results revealed that changes to the middle/medial layer changed overall graft behavior with whole graft compliance values ranging from 0.8 - 2.8 % / 100 mmHg, while uniaxial results demonstrated an average modulus range of 2.0 - 11.8 MPa. Both modulus and compliance data displayed values within the range of native artery. Mathematical modeling was implemented to show how changes in layer stiffness affect the overall circumferential wall stress, and as a design aid to achieve the best mechanical combination of materials. Overall, the results indicated that a graft can be designed to mimic a tri-layered structure by altering layer properties.

The aim of this study was to mimic the native three layered architecture of the arterial wall. To accomplish this, electrospinning was employed with the use of a 3-1 (input-output) nozzle and blends of polycaprolactone, elastin, and collagen.

Tri-capa electrospinning que imita la arquitectura nativa arterial con policaprolactona, elastina, colágeno y: un estudio preliminar

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

Investigación

Bioingeniería

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as oceanography, ecology, biology, and fluid mechanics. Field measurements introduce practical challenges such as environmental conditions, animal availability, and the need for field-compatible measurement techniques. To avoid these challenges, scientists typically use controlled laboratory environments to study animal-fluid interactions. However, it is reasonable to question whether one can extrapolate natural behavior (i.e., that which occurs in the field) from laboratory measurements. Therefore, in situ quantitative flow measurements are needed to accurately describe animal swimming in their natural environment.
We designed a self-contained, portable device that operates independent of any connection to the surface, and can provide quantitative measurements of the flow field surrounding an animal. This apparatus, a self-contained underwater velocimetry apparatus (SCUVA), can be operated by a single scuba diver in depths up to 40 m. Due to the added complexity inherent of field conditions, additional considerations and preparation are required when compared to laboratory measurements. These considerations include, but are not limited to, operator motion, predicting position of swimming targets, available natural suspended particulate, and orientation of SCUVA relative to the flow of interest. The following protocol is intended to address these common field challenges and to maximize measurement success.

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA

This protocol provides instructions on how to use a self-contained underwater velocimetry apparatus (SCUVA), which is designed for quantification of in situ animal-generated flows. In addition, this protocol addresses challenges posed by field conditions, and includes operator motion, predicting position of animals, and orientation of SCUVA.

La medición cuantitativa de In situ Flujos de utilizar un aparato autónomo Velocimetry Subacuático (SCUVA)

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as ...

Small and Wide Angle X-Ray Scattering Studies of Biological Macromolecules in Solution

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique where X-rays are elastically scattered by an inhomogeneous sample in the nm-range at small angles (typically 0.1 - 5&deg;) and wide angles (typically &gt; 5&deg;). This technique provides information about the shape, size, and distribution of macromolecules, characteristic distances of partially ordered materials, pore sizes, and surface-to-volume ratio. Small Angle X-ray Scattering (SAXS) is capable of delivering structural information of macromolecules between 1 and 200 nm, whereas Wide Angle X-ray Scattering (WAXS) can resolve even smaller Bragg spacing of samples between 0.33 nm and 0.49 nm based on the specific system setup and detector. The spacing is determined from Bragg's law and is dependent on the wavelength and incident angle.
In a SWAXS experiment, the materials can be solid or liquid and may contain solid, liquid or gaseous domains (so-called particles) of the same or another material in any combination. SWAXS applications are very broad and include colloids of all types: metals, composites, cement, oil, polymers, plastics, proteins, foods, and pharmaceuticals. For solid samples, the thickness is limited to approximately 5 mm.
Usage of a lab-based SWAXS instrument is detailed in this paper. With the available software (e.g., GNOM-ATSAS 2.3 package by D. Svergun EMBL-Hamburg and EasySWAXS software) for the SWAXS system, an experiment can be conducted to determine certain parameters of interest for the given sample. One example of a biological macromolecule experiment is the analysis of 2 wt% lysozyme in a water-based aqueous buffer which can be chosen and prepared through numerous methods. The preparation of the sample follows the guidelines below in the Preparation of the Sample section. Through SWAXS experimentation, important structural parameters of lysozyme, e.g. the radius of gyration, can be analyzed.

The demonstration of the small and wide angle X-ray scattering (SWAXS) procedure has become instrumental in the study of biological macromolecules. Through the use of the instrumentation and procedures of specific angle methods and preparation, the experimental data from the SWAXS displays the atomic and nano-scale characterization of macromolecules.

Pequeñas y Wide Angle X-Ray Estudios de dispersión de macromoléculas biológicas en disolución

In this paper, Small and Wide Angle X-ray Scattering (SWAXS) analysis of macromolecules is demonstrated through experimentation. SWAXS is a technique ...

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.

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, ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Combinación de Microstereolithography y electrospinning para Fabricación de membranas Equipado con nichos para la regeneración de la córnea

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

Fabricación de hidrogeles degradables de interpenetrantes en múltiples escalas de la longitud mediante extrusión reactiva, microfluídica, uno mismo-Asamblea y Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

Escritura de Electrospinning de andamios Poly(ε-caprolactone) tridimensional con morfologías controlables para aplicaciones de ingeniería de tejidos del derretimiento

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

High-resolution Patterning Using Two Modes of Electrohydrodynamic Jet: Drop on Demand and Near-field Electrospinning

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning tool. EHD printing uses a fluidic supplier to maintain the extruded meniscus by pushing the ink out of the nozzle tip. The electric field is then used to pull the meniscus down to the substrate to produce high-resolution patterns. Two modes of EHD printing have been used for fine patterning: continuous near-field electrospinning (NFES) and dot-based drop-on-demand (DOD) EHD printing. According to the printing modes, the requirements for the printing equipment and ink viscosity will differ. Even though two different modes can be implemented with a single EHD printer, the realization methods significantly differ in terms of ink, fluidic system, and driving voltage. Consequently, without a proper understanding of the jetting requirements and limitations, it is difficult to obtain the desired results. The purpose of this paper is to present a guideline so that inexperienced researchers can reduce the trial and error efforts to use the EHD jet for their specific research and development purposes. To demonstrate the fine-patterning implementation, we use Ag nanoparticle ink for the conductive patterning in the protocol. In addition, we also present the generalized printing guidelines that can be used for other types of ink for various fine-patterning applications.

Here, we present a protocol to produce high-resolution conductive patterns using electrohydrodynamic (EHD) jet printing. The protocol includes two modes of EHD jet printing: the continuous near-field electrospinning (NFES) and the dot-based drop-on-demand (DOD) EHD printing.

Alta resolución patrones usando dos modos de electrohidrodinámicos Jet: caída en la demanda y Electrospinning de campo cercano

Electrohydrodynamic (EHD) jet printing has drawn attention in various fields because it can be used as a high-resolution and low-cost direct patterning ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

Aparato para la recolección de tejido Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Three-Dimensional Echocardiographic Method for the Visualization and Assessment of Specific Parameters of the Pulmonary Veins

The dimensions of the pulmonary veins are important parameters when planning pulmonary vein isolation (PVI), especially with the cryoballoon ablation technique. Acknowledging the dimensions and anatomical variations of the pulmonary veins (PVs) may improve the outcome of the intervention. Conventional 2D transoesophageal echocardiography can only provide limited data about the dimensions of the PVs; however, 3D echocardiography can further evaluate relevant diameters and areas of the PVs, as well as their spatial relationship to surrounding structures. In previous literature data, parameters influencing the success rate of PVI have already been identified. These are the left lateral ridge, the intervenous ridge, the ostial area of the PVs and the ovality index of the ostium. Proper imaging of the PVs by 3D echocardiography is a technically challenging method. One crucial step is the collection of images. Three individual transducer positions are necessary to visualize the important structures; these are the left lateral ridge, the ostium of the PVs and the intervenous ridge of the left and right PVs. Next, 3D images are acquired and saved as digital loops. These datasets are cropped, which result in the en face views displaying spatial relationships. This step can also be employed to determine the anatomical variations of the PVs. Finally, multiplanar reconstructions are created to measure each individual parameter of the PVs.
Optimal quality and orientation of the acquired images are paramount for the appropriate assessment of PV anatomy. In the present work, we examined the 3D visibility of the PVs and the suitability of the above method in 80 patients. The aim was to provide a detailed outline of the essential steps and potential pitfalls of PV visualization and assessment with 3D echocardiography.

The dimensions of the pulmonary veins (PV) are important parameters when planning pulmonary vein isolation. 2D transoesophageal echocardiography can only provide limited data about the PVs; however, 3D echocardiography can evaluate relevant diameters and areas of the PVs, as well as their spatial relationship to surrounding structures.

Método ecocardiográfico tridimensional para la visualización y evaluación de parámetros específicos de las venas pulmonares

The dimensions of the pulmonary veins are important parameters when planning pulmonary vein isolation (PVI), especially with the cryoballoon ablation ...