Ce travail a été soutenu par le NIH et le K08 EB003996 Paralyzed Veterans of America Foundation subvention de recherche 2573.

1. Choisir un polymère <ol><li> Choisissez un polymère (par exemple, la poly-L-lactique (PLLA), polycaprolactone (PCL), polystyrène (PS) ou nylon) en fonction de votre cahier des charges (par exemple, biodégradable, thermoplastique ou réticulable) et un solvant de ce polymère. Choisissez un équipement de protection individuelle en fonction de votre sélection. </li><li> Sélectionner un substrat à base de votre application (par exemple, plaquette de verre, plastique, métal ou de silicium). </li></ol> 2. Choisir un Collector <ol><li> Choisissez la géométrie collecteur en fonction de votre cahier des charges. Fibres aléatoires peuvent être collectées sur plaques fixes. Fibres alignées peuvent être recueillies sur la rotation rapide des roues, tambours ou barres, ou sur des plaques parallèles. </li><li> Le collecteur doit être conducteur et doit rester isolée de son axel de telle manière qu&#39;il peut être mis à la terre sans également mise à la terre les objets adjacents, les dessus de table, etc </li></ol> 3. Approximative de la concentration critique Entanglement Empiriquement 1 <ol><li> Préparez plusieurs concentrations en polymère candidat (par exemple, 4, 10, 15, 20, 30% en poids) et de choisir une concentration des flux (la solution doit être un liquide visqueux, mais pas un gel) pour commencer. </li><li> Mettre en place l&#39;appareil électrofilage 2,3,4,5 (voir Figure 1) <ol><li> Chargez la pompe seringue et régler la vitesse de la pompe de telle sorte que toute goutte de solution rayé de la pointe est immédiatement remplacé. </li><li> Terrain le collecteur et le clip le fil haute tension à la plaque de conducteur (un petit carré de matériau conducteur tel que du papier d&#39;aluminium à travers laquelle l&#39;extrémité de la seringue dépasse). </li><li> Démarrez le rouet. </li><li> Assurez-vous que l&#39;alimentation est mis à zéro avant de l&#39;allumer. </li></ol></li><li> Observez le flux <ol><li> Rampe de tension lentement et regardez le talon de la solution à la pointe de l&#39;aiguille. </li><li> Régler la tension pour obtenir un flux long et régulier. Si un flux régulier ne peut être obtenue, ajuster la concentration de la solution polymère. Voir tableau 1 pour un exemple. </li></ol></li></ol> 4. Dépannage - Stream: <ol><li> Problèmes avec le flux <ol><li> Utilisez un fond foncé mat et le lieu d&#39;une source lumineuse unidirectionnelle (comme une lampe de poche) entre le spectateur et le flux (voir figure 2). </li></ol></li><li> Dripping de l&#39;extrémité de la seringue <ol><li> Si la solution de polymère est dégoulinant vers le bas sans attrait à la roue, assurez-vous que la plaque de conducteur est en contact avec la pointe de l&#39;aiguille et que le collecteur est en contact avec le sol. </li><li> Si la goutte de solution de polymère à l&#39;extrémité de la seringue est penché dans la direction de la roue, mais ne forme pas un ruisseau, augmenter la tension. La qualité des cours d&#39;eau peut être ajustée en faisant varier la distance et la tension jusqu&#39;à ce qu&#39;un flux constant est visible. Voir Figure 3 pour les distances proposées avec des tensions correspondant à une solution PLLA 4% et une plaque de conducteur de 8x8 cm. </li></ol></li><li> Globs Large à l&#39;extrémité de la seringue <ol><li> Lorsque la solution de polymère commence à recueillir et à durcir à la pointe de l&#39;aiguille, glisser le glob repartir avec une serviette en papier attaché à un bâton non-conducteur. </li></ol></li><li> Oscillant ou «remue» ruisseaux Lorsque le flux est remuant de haut en bas rapidement, baissez la tension ou augmenter la distance entre le bout de la seringue et la roue. Si le flux continue à remuer utiliser une concentration plus élevée de polymère ou d&#39;ajouter un peu de solvant avec une évaporation plus lente. </li><li> Filières courtes ou discontinues Visible flux réguliers en contact observables avec le mobile à une vitesse de rotation élevée de rendement le plus élevé de qualité de l&#39;uniformité et l&#39;alignement. Lorsque le flux est court et discontinu, l&#39;augmentation de la solution de polymère, en ajoutant plus lente évaporation de solvants, et en ajustant la tension sera d&#39;améliorer la durée et la constance du flux. </li></ol> 5. Dépannage - morphologie de fibres 6,7,8 (voir figure 4) <ol><li> Perlage Lorsque les perles sont découverts dans les fibres, l&#39;augmentation de la solution de polymère et assurez-vous que la plaque de conducteur est en contact continu avec l&#39;aiguille et que la brosse métallique à la terre est en contact continu avec la roue. </li><li> Rubans et fibres saignements Lorsque les fibres se forment comme des rubans ou des saignements sont ensemble, utiliser une concentration plus élevée de polymère ou un solvant avec un taux plus élevé d&#39;évaporation (plus volatils). </li><li> Fibres fioriture ou ondulés Lorsque les fibres forment des vagues ou de fioritures, d&#39;augmenter la vitesse de la roue ou de déplacer la pointe de l&#39;aiguille d&#39;autres à partir du collecteur. Aussi, vérifiez que la plaque conductrice et le collecteur sont pas vibrer. </li><li> Porosité 9 Si les pores sont souhaitées, utiliser un solvant qui s&#39;évapore rapidement. Si les pores ne sont pas souhaitées, essayez d&#39;ajouter une petite quantité de co-solvant qui est moins volatil que le solvant majeur. </li><li> Alignement 10 Lorsque le collecteur est movING à bas régime ou au repos, la qualité de l&#39;alignement est pauvre. Augmenter l&#39;alignement en augmentant la vitesse de la roue. </li></ol> 6. Les résultats représentatifs: S&#39;il vous plaît se référer à la figure 4 pour les représentations des résultats de fibre typique. <img src="/files/ftp_upload/2494/2494fig1.jpg" alt="Figure 1" /> Figure 1. Une configuration typique de l&#39;électrofilage. Une solution de polymère (en bleu) est distribué à partir d&#39;une seringue électrique (orange). Une alimentation haute tension en courant continu (vert) les motifs d&#39;un collecteur de roue en rotation rapide (en gris) sur lequel nanofibres alignées sont collectées. Le jet de polymère entre la seringue et le collecteur est constitué d&#39;un segment stable de streaming et un segment oscillant rapidement à fouetter. <img src="/files/ftp_upload/2494/2494fig2.jpg" alt="Figure 2" /> . Figure 2 Le jet continu est visible sortant du bout de la seringue; le jet à fouetter est trop petit pour être vu. Approximation de la concentration critique de l&#39;enchevêtrement PLLA <table border="1"><tbody><tr><td> PLLA (% p / v) </td><td> Observation </td><td> Ajustement de la concentration </td></tr><tr><td> 0.5 </td><td> Dripping; aucun flux </td><td> Augmenter </td></tr><tr><td> 2.0 </td><td> Cracher Globs petite, pas de flux </td><td> INCRESE légèrement </td></tr><tr><td> 4,0 </td><td> Flux régulier </td><td> Bonne </td></tr><tr><td> 6,0 </td><td> Cracher Globs grandes ou des perles </td><td> Diminuer légèrement </td></tr><tr><td> 12,0 </td><td> Agglomérante à la pointe, pas de flux </td><td> Diminuer </td></tr></tbody></table> Tableau 1. Un exemple illustrant le rapprochement de la concentration critique de l&#39;enchevêtrement PLLA. Différentes concentrations en polymère sont jugés et les jets résultant de streaming observées jusqu&#39;à un flux constant est obtenu. <img src="/files/ftp_upload/2494/2494fig3.jpg" alt="Figure 3" /> Figure 3. La distance entre l&#39;extrémité de la seringue et le collecteur doit être équilibrée avec la tension appliquée pour obtenir un jet constant de streaming. L&#39;excès de tension appliquée provoque un jet d&#39;oscillation ou de «remue» pour former que des résultats en moins de fibres bien alignées. Lorsque la tension est trop basse, pas de jet se forme et la solution ne goutte à goutte du bout de la seringue. La région de mauve grisé ci-dessus représente la plage de tension sur lequel un jet constant de diffusion peut être obtenue pour PLLA en fonction de la seringue à collecteur à distance. <img src="/files/ftp_upload/2494/2494fig4.jpg" alt="Figure 4" /> Figure 4. Fibres électrofilées peuvent présenter une variété de morphologies, y compris les perles (A), rubans (B), fioritures (C), Globs poreux (D), un bon alignement (E) et un mauvais alignement (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 majorité des exemples présentés ici portent électrofilage de poly-L-lactique (PLLA) nanofibres. C&#39;est tout simplement parce PLLA est le polymère le plus couramment filé dans notre laboratoire. Toutefois, nous avons également utilisé avec succès ces méthodes pour electrospin autres polymères (par exemple, PLGA, PCL, PS) et de croire que les techniques présentées ici sont facilement applicables à la majorité de la mi-à haut poids moléculaire des solutions de polymères.

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		<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>
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<td>Polypropylene syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309585</td>
<td>&nbsp;</td>
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<td>Rotating or stationary collector</td>
<td>&nbsp;</td>
<td>Custom built</td>
<td>&nbsp;</td>
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<td>Various alligator clips and wires</td>
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<td>&nbsp;</td>
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<td>Dimethylformamide</td>
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<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC42355-0040 </td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Boehringer Ingelheim</td>
<td>Resomer L210</td>
<td>&nbsp;</td>
<tr>
<td>PLGA 85:15</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43471</td>
<td>&nbsp;</td>
<tr>
<td>Carbon tape</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13073-1</td>
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electrospinning fundamentals: optimizing solution and apparatus parameters

Électrofilées échafaudages de nanofibres ont été montré pour accélérer la maturation, améliorer la croissance, et de diriger la migration des cellules in vitro. Électrofilage est un processus dans lequel un jet polymère chargé est collectée sur un collecteur de terre, une rotation rapide des résultats collectionneur en nanofibres alignées alors entraîner l&#39;arrêt des collectionneurs de mats de fibres orientées de façon aléatoire. Le jet de polymère est formé quand une charge électrostatique appliqué surmonte la tension superficielle de la solution. Il ya une concentration minimale pour un polymère donné, appelée la concentration d&#39;enchevêtrement critique en dessous duquel un jet stable ne peut être atteint et aucune nanofibres formeront - bien que les nanoparticules peuvent être atteints (électrospray). Un jet stable a deux domaines, un segment de streaming et un segment de fouetter. Alors que le jet à fouetter est généralement invisible à l&#39;œil nu, le segment de streaming est souvent visible dans les conditions d&#39;éclairage approprié. Observer la longueur, l&#39;épaisseur, la consistance et le mouvement du flux est utile pour prédire l&#39;alignement et la morphologie des nanofibres formé. Un ruisseau court, non uniforme, incohérentes et / ou oscillante est indicative d&#39;une variété de problèmes, y compris l&#39;alignement des fibres pauvres, le perlage, les éclaboussures, et les modèles curlicue ou ondulés. Le flux peut être optimisé en ajustant la composition de la solution et la configuration de l&#39;appareil électrofilage, optimisant ainsi l&#39;alignement et la morphologie des fibres produites. Dans ce protocole, nous présentons une procédure pour la mise en place d&#39;un appareil électrofilage de base, de façon empirique se rapprochant de la concentration d&#39;enchevêtrement critique d&#39;une solution de polymère et l&#39;optimisation du processus électrofilage. De plus, nous discutons de certains problèmes communs et les techniques de dépannage.

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

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Électrofilage techniques peuvent créer une variété d&#39;échafaudages nanofibrous pour l&#39;ingénierie tissulaire ou d&#39;autres applications. Nous décrivons ici une procédure d&#39;optimiser les paramètres de la solution électrofilage et appareils pour obtenir des fibres à la morphologie et l&#39;alignement désiré. Les problèmes courants et les techniques de dépannage sont également présentés.

Fondements Électrofilage: Solution Optimisation et paramètres Appareillage

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 vues.  University of Michigan. Électrofilage techniques peuvent créer une variété d'échafaudages nanofibrous pour l'ingénierie tissulaire ou d'autres applications. Nous décrivons ici une procédure d'optimiser les paramètres de la solution électrofilage et appareils pour obtenir des fibres à la morphologie et l'alignement désiré. Les problèmes courants et les techniques de dépannage sont également présentés.

Vidéo: 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-couches électrofilage pour imiter l&#39;architecture native artérielle à l&#39;aide polycaprolactone, l&#39;élastine et du collagène: une étude préliminaire

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

Recherche

Bio-ingénierie

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.

Mesurer quantitativement In situ Flux aide d&#39;un appareil autonome à la vélocimétrie subaquatique (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.

Les petites et Wide Angle études diffraction des rayons X de macromolécules biologiques en solution

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.

Électrofilage facteur de croissance libération de microsphères en fibre échafaudages

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.

Combinaison de Microstéréolithographie et Électrofilage à produire des membranes Equipé Niches pour la régénération de la cornée

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

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.

Adapter le processus Électrofilage de fournir trois environnements uniques pour un Tri-couches<em&gt; In Vitro</em&gt; Modèle du mur Airway

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

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.

Fabrication des Thermoresponsive Hydrogels dégradable sur plusieurs échelles de longueur par Extrusion réactive, microfluidique, auto-assemblage et électrofilage

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.

Faire fondre électrofilage écrit d’échafaudages Poly(ε-caprolactone) en trois dimensions avec des Morphologies contrôlables pour les Applications de génie tissulaire

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.

Haute résolution en utilisant deux Modes de Jet électrohydrodynamique le Patterning : Drop on Demand et contigu électrofilage

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.

Appareil pour la récolte de tissus Microcolumns

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