Diese Arbeit wurde vom NIH K08 EB003996 und die gelähmten Veteranen of America Research Foundation Grants 2573 unterstützt.

1. Wählen Sie ein Polymer <ol><li> Wählen Sie ein Polymer (z. B. Poly-L-Milchsäure (PLLA), Polycaprolacton (PCL), Polystyrol (PS) oder Nylon) nach Ihren Vorgaben (zB biologisch abbaubare, thermoplastische oder vernetzbare) und einem Lösungsmittel, das Polymer. Wählen Sie eine geeignete persönliche Schutzausrüstung auf der Basis Ihrer Auswahl. </li><li> Wählen Sie ein Substrat, auf Ihre Anwendung basiert (z. B. Glas, Kunststoff, Metall oder Silizium-Wafer). </li></ol> 2. Wählen Sie eine Collector <ol><li> Wählen Sie den Kollektor Geometrie nach Ihren Vorgaben. Zufällige Fasern können auf stationären Platten gesammelt werden. Ausgerichteten Fasern kann auf schnell rotierenden Rädern, Trommeln oder Stangen oder auf parallelen Platten gesammelt werden. </li><li> Der Kollektor muss leitfähig sein und muss aus seiner axel in einer Weise isoliert bleiben, dass es ohne auch Erdung angrenzenden Objekten, die Tischplatte, etc. können geerdet werden </li></ol> 3. Angleichung der Critical Entanglement Konzentration Empirisch 1 <ol><li> Bereiten Sie mehrere Kandidaten Polymer-Konzentrationen (z. B. 4, 10, 15, 20, 30 Gew.%) und wählen Sie eine Konzentration, die (die Lösung sollte eine zähe Flüssigkeit, aber kein Gel) fließt, mit zu beginnen. </li><li> Richten Sie die Elektrospinnen Apparat 2,3,4,5 (siehe Abbildung 1) <ol><li> Laden Sie die Spritzenpumpe und stellen Sie die Drehzahl der Pumpe, so dass jede Perle der Lösung von der Spitze abgewischt wird sofort ersetzt. </li><li> Boden des Sammlers und Clip das Zündkabel an die Leiterplatte (ein kleines Quadrat aus leitfähigem Material wie Aluminiumfolie, durch die die Spitze der Spritze ragt). </li><li> Starten Sie das Rad zu drehen. </li><li> Achten Sie darauf, die Stromversorgung auf Null ist, bevor Sie ihn einstellen. </li></ol></li><li> Beobachten Sie den Strom <ol><li> Rampenspannung langsam und beobachten Sie die Perle der Lösung an der Nadelspitze. </li><li> Stellen Sie die Spannung auf eine lange und stetigen Strom zu erhalten. Wenn ein stetiger Strom nicht erreicht werden können, passen Sie die Konzentration der Polymerlösung. Siehe Tabelle 1 zeigt ein Beispiel. </li></ol></li></ol> 4. Fehlerbehebung - der Stream: <ol><li> Probleme beim Anzeigen der Strom <ol><li> Verwenden Sie eine dunkle matte Hintergrund und legen Sie eine unidirektionale Lichtquelle (wie z. B. einer Taschenlampe) zwischen dem Betrachter und dem Strom (siehe Abbildung 2). </li></ol></li><li> Dripping aus der Spritze <ol><li> Wenn die Polymerlösung gerade nach unten tropft ohne Reiz, um das Rad sicher, dass die Leiterplatte ist die Kontaktaufnahme mit der Nadelspitze und dass der Kollektor Kontakt mit Boden. </li><li> Wenn die Tropfen Polymerlösung an der Spitze der Spritze in die Richtung des Rades gelehnt ist, aber nicht bilden einen Strom, die Spannung erhöhen. Die Qualität des Streams kann durch Variation des Abstandes und die Spannung bis zu einem stetigen Strom sichtbar eingestellt werden. Siehe Abbildung 3 für empfohlene Strecken mit entsprechenden Spannungen für eine 4% PLLA-Lösung und einem 8x8 cm Leiterplatte. </li></ol></li><li> Große globs an die Spitze der Spritze <ol><li> Wenn die Polymerlösung zu sammeln und zu härten an der Spitze der Nadel, Streichen der glob weg mit einem Papiertuch an einem nicht-leitenden Stab beginnt. </li></ol></li><li> Oszillierende oder &quot;wedelt&quot; Streams Wenn der Stream wackelt schnell auf und ab, drehen Sie die Spannung erhöhen oder den Abstand zwischen der Spitze der Spritze und dem Lenkrad. Wenn der Stream weiterhin wag Verwendung einer höheren Konzentration an Polymer oder fügen Sie ein wenig Lösungsmittel mit einem langsameren Verdunstung. </li><li> Kurze oder diskontinuierliche Abwasserströme Visible stetige Ströme machen beobachtbaren Kontakt mit dem Radsatz auf einem hohen Rotationsgeschwindigkeit ergeben die höchste Qualität der Einheitlichkeit und Ausrichtung. Wenn der Strom kurz und diskontinuierlich ist, die Erhöhung der Polymer-Lösung, indem mehr langsam verdampfende Lösungsmittel, und die Anpassung der Spannung wird die Länge und Festigkeit des Baches zu verbessern. </li></ol> 5. Fehlerbehebung - Fasermorphologie 6,7,8 (siehe Abbildung 4) <ol><li> Friesen Wenn Kugeln in die Fasern entdeckt, erhöhen Sie die Polymerlösung und sicherstellen, dass die Leiterplatte macht ständigem Kontakt mit der Nadel und dem geerdeten Drahtbürste macht ständigem Kontakt mit dem Lenkrad. </li><li> Bänder und Blutungen Fasern Als Fasern werden als Bänder bilden oder zusammen Blutungen, verwenden Sie eine höhere Konzentration an Polymer oder ein Lösungsmittel mit einer höheren Rate der Verdunstung (volatiler). </li><li> Schnörkel oder wellig Fasern Wenn Fasern bilden Wellen oder Schnörkel, erhöhen Sie die Raddrehzahl oder bewegen Sie die Nadelspitze weiter vom Kollektor. Überprüfen Sie auch, dass die Leiterplatte und Sammler sind nicht vibrieren. </li><li> Porosität 9 Wenn Poren gewünscht sind, verwenden Sie eine schnell verdampfende Lösungsmittel. Wenn Poren nicht erwünscht sind, versuchen Sie, eine kleine Menge an Co-Lösungsmittel, das weniger volatil als die wichtigsten Lösungsmittel. </li><li> Alignment 10 Wenn der Kollektor movIng. bei niedriger Drehzahl oder in Ruhe, ist die Ausrichtung von schlechter Qualität. Erhöhen Sie die Ausrichtung durch die Erhöhung der Drehzahl des Rades. </li></ol> 6. Repräsentative Ergebnisse: Bitte beachten Sie 4 für Darstellungen von typischen Faser Ergebnisse Abbildung. <img src="/files/ftp_upload/2494/2494fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Ein typisches Elektrospinnen Setup. Ein Polymer-Lösung (blau) wird von einer Spritzenpumpe (orange) verzichtet. Eine Hochspannungs-Gleichstrom-Netzteil (grün) wegen einer sich schnell drehenden Rades Kollektor (grau), auf das ausgerichtet Nanofasern gesammelt werden. Das Polymer-Jet zwischen der Spritze und Kollektor besteht aus einem stabilen Streaming-Segment und einem schnell schwingenden Peitschen-Segment. <img src="/files/ftp_upload/2494/2494fig2.jpg" alt="Abbildung 2" /> . Abbildung 2: Der Streaming-Jet sichtbar Verlassen der Spitze der Spritze, die Peitschen-Jet ist zu klein, zu sehen. Durch die Annäherung der kritischen Verschränkung Konzentration von PLLA <table border="1"><tbody><tr><td> PLLA (% Gew. / v) </td><td> Beobachtung </td><td> Concentration Adjustment </td></tr><tr><td> 0,5 </td><td> Dripping, kein Strom </td><td> Erhöhen </td></tr><tr><td> 2,0 </td><td> Spitting kleine Klümpchen, kein Strom </td><td> Increse leicht </td></tr><tr><td> 4,0 </td><td> Stetigen Strom </td><td> Gut </td></tr><tr><td> 6,0 </td><td> Spitting große Kleckse oder Perlen </td><td> Leicht sinken </td></tr><tr><td> 12,0 </td><td> Verklumpung an der Spitze, kein Strom </td><td> Rückgang </td></tr></tbody></table> Tabelle 1. Ein Beispiel der Darstellung der Angleichung der kritischen Verschränkung Konzentration von PLLA. Verschiedene Polymer-Konzentrationen sind erprobt und die daraus resultierenden Streaming-Jets bis zu einem stetigen Strom beobachtet wird erhalten. <img src="/files/ftp_upload/2494/2494fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Der Abstand zwischen Spitze der Spritze und der Kollektor ist mit der angelegten Spannung ausgeglichen werden, um eine stetige Streaming-Jet erhalten. Überschüssiges angelegte Spannung bewirkt eine oszillierende oder &quot;wedelt&quot; jet, dass die Ergebnisse in weniger gut ausgerichteten Fasern zu bilden. Wenn die Spannung zu niedrig ist, wird kein Strahl bilden und die Lösung wird nur von der Spitze der Spritze tropft. Die lila schattierte Bereich oberhalb repräsentiert die Spannung Bereich, in dem eine stetige Streaming-Jet für PLLA als Funktion der Spritze-Kollektor-Strecke erhalten werden kann. <img src="/files/ftp_upload/2494/2494fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Elektrogesponnene Fasern weisen eine Vielzahl von Morphologien, darunter Perlen (A), Bänder (B), Schnörkel (C), poröse globs (D), gute Ausrichtung (E) und eine schlechte Ausrichtung (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>

Hinweis: Die Mehrzahl der hier vorgestellten Beispiele befassen sich mit Elektrospinnen Poly-L-Milchsäure (PLLA)-Nanofasern. Das liegt einfach daran PLLA ist die am häufigsten gesponnen Polymer in unserem Labor. Allerdings haben wir auch erfolgreich diese Methoden verwendet, um andere Polymere (zB PLGA, PCL, PS) electrospin und glauben, dass die hier vorgestellten Verfahren leicht anwendbar sind, die Mehrheit der mittel-bis hochmolekularen Polymerlösungen.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
<tr>
<td>Aluminum foil</td>
<td>&nbsp;</td>
<td>Reynolds</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Blunt metal tips, 23ga</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>13-850-102 </td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309585</td>
<td>&nbsp;</td>
<tr>
<td>Rotating or stationary collector</td>
<td>&nbsp;</td>
<td>Custom built</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Various alligator clips and wires</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylformamide</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC11622-0010</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC42355-0040 </td>
<td>&nbsp;</td>
<tr>
<td>PLLA</td>
<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrospinning fundamentals: optimizing solution and apparatus parameters

Elektrogesponnene Nanofaser Gerüste haben gezeigt, dass die Reifung zu beschleunigen, verbessern das Wachstum, und leiten die Migration von Zellen in vitro. Elektrospinnen ist ein Prozess, in dem ein geladenes Polymer-Jet auf einer geerdeten aufgefangen wird; einem schnell rotierenden Kollektor führt ausgerichtet Nanofasern, während stationäre Sammler Ergebnis in zufällig orientierten Fasermatten. Das Polymer-Jet wird gebildet, wenn eine angelegte elektrostatische Ladung die Oberflächenspannung der Lösung überwindet. Es gibt eine minimale Konzentration für ein bestimmtes Polymer, der sogenannten kritischen Verschränkung Konzentration, unterhalb derer eine stabile Jet nicht erreicht werden kann und keine Nanofasern bilden - auch wenn Nanopartikel können erreicht werden (Elektrospray) werden. Eine stabile Jet verfügt über zwei Domains, eine Streaming-Segment und einem Peitschen-Segment. Während die Peitschen-Jet ist in der Regel mit bloßem Auge ist die Streaming-Segment häufig unter geeigneten Lichtverhältnissen sichtbar. Die Beobachtung der Länge, Dicke, Konsistenz und die Bewegung der Strom ist nützlich, um die Ausrichtung und die Morphologie der Nanofasern gebildet vorherzusagen. Eine kurze, ungleichmäßige, inkonsistent und / oder oszillierenden Strom ist bezeichnend für eine Vielzahl von Problemen, darunter schlechte Faserausrichtung, Sicken, Spritzer und Schnörkel oder Wellenmuster. Der Strom kann durch Einstellen der Zusammensetzung der Lösung und die Konfiguration des Elektrospinnen Gerät optimiert werden, damit die Optimierung der Ausrichtung und der Morphologie der Fasern produziert. In diesem Protokoll, präsentieren wir ein Verfahren für den Aufbau eines grundlegenden Elektrospinnen Apparat, empirisch Angleichung der kritischen Verschränkung Konzentration einer Polymer-Lösung und Optimierung der Elektrospinnverfahren. Darüber hinaus diskutieren wir einige häufig auftretende Probleme und Fehlerbehebung.

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

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Elektrospinnen Techniken können eine Vielzahl von nanofibrous Gerüste für Tissue Engineering oder andere Anwendungen. Wir beschreiben hier ein Verfahren, um die Parameter des Elektrospinnen Lösung und Apparate zu optimieren, um Fasern mit den gewünschten Morphologie und Ausrichtung zu erhalten. Häufige Probleme und Techniken zur Fehlerbehebung werden ebenfalls vorgestellt.

Elektrospinnen Grundlagen: Optimierung Solution und Geräteparameter

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 Aufrufe.  University of Michigan. Elektrospinnen Techniken können eine Vielzahl von nanofibrous Gerüste für Tissue Engineering oder andere Anwendungen. Wir beschreiben hier ein Verfahren, um die Parameter des Elektrospinnen Lösung und Apparate zu optimieren, um Fasern mit den gewünschten Morphologie und Ausrichtung zu erhalten. Häufige Probleme und Techniken zur Fehlerbehebung werden ebenfalls vorgestellt.

Serie: 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-geschichteten Elektrospinnen zu Native Arterielle Architektur mit Polycaprolacton, Elastin und Collagen Mimic: A Preliminary Study

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

Forschung

Biotechnik

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.

Quantitativen Messung In situ Flows mit einem 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 ...

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.

Kleine und Wide Angle X-Ray Scattering Studium der biologischen Makromolekülen in Lösung

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.

Elektro Growth Factor Releasing Mikrosphären in Fasergerüste

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.

Kombination von Mikrostereolithographie und Elektrospinnen auf Membranen Ausgestattet mit Nischen für die Hornhautregeneration Produzieren

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.

Herstellung von abbaubaren Thermoresponsive Hydrogele auf mehreren Längenskalen über reaktive Extrusion, Mikrofluidik, Selbstmontage und Elektrospinnen

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.

Elektrospinnen Schreiben von dreidimensionalen Poly(ε-caprolactone) Gerüste mit steuerbaren Morphologien für Tissue Engineering Anwendungen zu schmelzen

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.

Hochauflösende Musterung mit zwei Modi der Electrohydrodynamic Jet: Drop-on-Demand und Nahfeld-Elektrospinnen

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.

Vorrichtung zur Ernte Gewebe 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.

Dreidimensionale echokardiographische Methode zur Visualisierung und Beurteilung spezifischer Parameter der Lungenvenen

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