Questo lavoro è stato sostenuto da NIH K08 EB003996 e la Paralyzed Veterans of America Research Foundation Grant 2573.

1. Scegli un polimero <ol><li> Scegli un polimero (per esempio, poli-L-lattico (PLLA), policaprolattone (PCL), polistirolo (PS) o di nylon) in base alle vostre specifiche (ad esempio, biodegradabile, termoplastico o cross-collegabili) e un solvente di quel polimero. Scegliere adeguati dispositivi di protezione individuale in base alla selezione. </li><li> Seleziona un substrato in base all&#39;applicazione (ad esempio, wafer di vetro, plastica, metallo o silicone). </li></ol> 2. Scegli un collettore <ol><li> Scegli la geometria del collettore in base alle specifiche. Fibre casuali possono essere raccolte su piastre fisse. Fibre allineate possono essere raccolti in rapida rotazione ruote, tamburi o aste o su piastre parallele. </li><li> Il collettore deve essere conduttivo e deve rimanere isolato dal suo asse in modo che possa essere messo a terra anche senza messa a terra oggetti adiacenti, il piano del tavolo, ecc </li></ol> 3. Approssimare la concentrazione Entanglement critico empirico 1 <ol><li> Preparare diverse concentrazioni di polimero candidati (ad esempio, 4, 10, 15, 20, 30% in peso) e scegliere una concentrazione che scorre (la soluzione dovrebbe essere un liquido viscoso, ma non un gel) per iniziare. </li><li> Sistemare il dispositivo di electrospinning 2,3,4,5 (vedi Figura 1) <ol><li> Caricare la pompa a siringa e impostare la velocità della pompa in modo che ogni goccia di soluzione cancellato dalla punta viene immediatamente sostituito. </li><li> Terra il collettore e la clip il filo ad alta tensione alla piastra conduttore (un piccolo quadrato di materiali conduttivi, come un foglio di alluminio attraverso i quali il sporge siringa punta). </li><li> Avviare la ruota gira. </li><li> Assicurarsi che l&#39;alimentazione è impostato a zero prima di accenderla. </li></ol></li><li> Osservare il flusso <ol><li> Rampa di tensione lentamente e guardare il tallone della soluzione sulla punta dell&#39;ago. </li><li> Regolare la tensione per ottenere un flusso lungo e costante. Se un flusso costante non è possibile ottenere, regolare la concentrazione della soluzione polimerica. Vedi Tabella 1 per un esempio. </li></ol></li></ol> 4. Risoluzione dei problemi - la corrente: <ol><li> Problemi di visualizzazione del flusso <ol><li> Utilizzare uno sfondo scuro opaco e posto una sorgente di luce unidirezionale (come una torcia elettrica) tra lo spettatore e il torrente (vedi Figura 2). </li></ol></li><li> Dripping dalla siringa <ol><li> Se la soluzione polimero è sgocciolamento verso il basso senza attrazione verso la ruota, assicurarsi che la piastra conduttore è in contatto con la punta dell&#39;ago e che il collettore è il contatto con la terra. </li><li> Se la goccia di soluzione di polimero in punta siringa è appoggiata in direzione della ruota, ma non è che formano un ruscello, aumentare la tensione. La qualità dello stream può essere regolata variando la distanza e la tensione fino a quando un flusso costante è visibile. Vedi Figura 3 per le distanze suggerite con tensioni corrispondenti per una soluzione PLLA 4% e un 8x8 piatto conduttore cm. </li></ol></li><li> Globs grande alla punta della siringa <ol><li> Quando la soluzione polimero comincia a raccogliere e ad indurire la punta di un ago swipe, il glob via con un tovagliolo di carta attaccato ad un bastone non conduttivo. </li></ol></li><li> Oscillante o &#39;agita&#39; flussi Quando il flusso si agita su e giù velocemente, abbassare la tensione o aumentare la distanza tra la punta della siringa e la ruota. Se il flusso continua ad utilizzare wag una maggiore concentrazione di polimero o aggiungere un po &#39;di solvente con una evaporazione più lenta. </li><li> Corsi brevi o discontinue Visibile flussi costante il contatto osservabile con il set ruota a una velocità di rotazione alta resa la più alta qualità di uniformità e l&#39;allineamento. Quando il flusso è breve e discontinua, aumentando la soluzione polimerica, aggiungendo più lenta evaporazione di solventi, e regolando la tensione a migliorare la durata e la stabilità del flusso. </li></ol> 5. Risoluzione dei problemi - Fiber Morfologia 6,7,8 (vedi figura 4) <ol><li> Bordatura Quando le gocce vengono scoperti nelle fibre, aumentare la soluzione polimerica e assicurarsi che la piastra conduttore è il contatto continuo con l&#39;ago e che la spazzola metallica messa a terra è in contatto continuo con la ruota. </li><li> Nastri e fibre di sanguinamento Quando le fibre si stanno formando come nastri o sono il sanguinamento insieme, utilizzare una maggiore concentrazione di polimero o un solvente con un alto tasso di evaporazione (più volatili). </li><li> Fibre svolazzo o ondulato Quando le fibre si stanno formando onde o riccioli, aumentare la velocità delle ruote o spostare la punta dell&#39;ago ulteriormente dal collettore. Inoltre, verificare che la piastra di conduttore e collezionista non sono vibranti. </li><li> Porosità 9 Se i pori sono desiderati, utilizzare un solvente evapora rapidamente. Se i pori non sono desiderati, provare ad aggiungere una piccola quantità di co-solvente che è meno volatile rispetto al solvente principali. </li><li> Allineamento 10 Quando il collettore è movzione ad un basso numero di giri oa riposo, la qualità è scarsa l&#39;allineamento. Aumentare l&#39;allineamento aumentando la velocità della ruota. </li></ol> 6. Rappresentante dei risultati: Si prega di fare riferimento alla Figura 4 per le raffigurazioni di risultati fibra tipici. <img src="/files/ftp_upload/2494/2494fig1.jpg" alt="Figura 1" /> Figura 1. Electrospinning Una configurazione tipica. Una soluzione di polimero (blu) è erogato da una pompa a siringa (arancione). Un alta tensione in corrente continua (verde) ragioni un collezionista ruota in rapida rotazione (grigio) su cui sono raccolti nanofibre allineati. Il getto polimerico tra la siringa e il collettore è costituito da un segmento costante streaming e un segmento in rapida oscillazione fustigazione. <img src="/files/ftp_upload/2494/2494fig2.jpg" alt="Figura 2" /> . Figura 2 Il getto è visibile in streaming in uscita la punta della siringa, il getto fustigazione è troppo piccolo per essere visto. Approssimare la concentrazione critica groviglio di PLLA <table border="1"><tbody><tr><td> PLLA (% peso / v) </td><td> Osservazione </td><td> Concentrazione di regolazione </td></tr><tr><td> 0,5 </td><td> Gocciolamento, nessun flusso </td><td> Aumentare </td></tr><tr><td> 2,0 </td><td> Sputare globs piccoli; alcun flusso </td><td> Increse leggermente </td></tr><tr><td> 4.0 </td><td> Flusso costante </td><td> Bene </td></tr><tr><td> 6.0 </td><td> Sputare globs grande o perline </td><td> Diminuire leggermente </td></tr><tr><td> 12,0 </td><td> Aggregazione alla punta; alcun flusso </td><td> Diminuire </td></tr></tbody></table> Tabella 1. Un esempio raffigurante il ravvicinamento della concentrazione critica di entanglement PLLA. Varie concentrazioni di polimero sono provati e il conseguente flusso getti osservati fino ad un flusso costante si ottiene. <img src="/files/ftp_upload/2494/2494fig3.jpg" alt="Figura 3" /> Figura 3. La distanza tra la punta della siringa e il collettore deve essere bilanciata con la tensione applicata per ottenere un getto costante di streaming. L&#39;eccesso di tensione applicata causa un oscillante o &#39;agita&#39; getto di forma che si traduce in meno fibre ben allineati. Quando la tensione è troppo bassa, non getto si forma e la soluzione sarà solo gocciolare dalla punta della siringa. La regione ombreggiata rappresenta viola di sopra del range di tensione in cui un getto continuo di streaming può essere ottenuto per PLLA in funzione della siringa-to-collezionista distanza. <img src="/files/ftp_upload/2494/2494fig4.jpg" alt="Figura 4" /> Figura 4. Fibre elettrofilate possono presentare una varietà di morfologie, tra cui bordare (A), nastri (B), ghirigori (C), globs poroso (D), un buon allineamento (E) e l&#39;allineamento poveri (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>

Nessun conflitto di interessi dichiarati.

Nota: La maggior parte degli esempi presentati qui affrontare electrospinning poli-L-lattico (PLLA) nanofibre. Questo è semplicemente perché PLLA è il polimero più comunemente filata nel nostro laboratorio. Tuttavia, abbiamo anche utilizzato con successo questi metodi per electrospin altri polimeri (ad esempio, PLGA, PCL, PS) e credono che le tecniche qui presentate sono facilmente applicabile alla maggior parte delle medie e soluzioni ad alto peso molecolare del polimero.

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		<th>Comment</th>
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<td>High voltage DC power supply</td>
<td>&nbsp;</td>
<td>Gamma High Voltage</td>
<td>ES40P-5W</td>
<td>&nbsp;</td>
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<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>
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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>
<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>
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<td>Various alligator clips and wires</td>
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<td>Dimethylformamide</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>AC11622-0010</td>
<td>&nbsp;</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>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>
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electrospinning fundamentals: optimizing solution and apparatus parameters

Elettrofilate ponteggi nanofibre hanno dimostrato di accelerare la maturazione, migliorare la crescita, e diretto la migrazione delle cellule in vitro. Electrospinning è un processo in cui è raccolto un jet carico polimero in un collettore a terra, una rapida rotazione risultati collettore in nanofibre allineate, mentre i collezionisti risultato stazionario in stuoie di fibre orientate in modo casuale. Il getto polimero si forma quando una carica elettrostatica applicata supera la tensione superficiale della soluzione. Vi è una concentrazione minima di un polimero dato, definito la concentrazione entanglement critica, sotto il quale un getto stabile non può essere ristabilita o se non nanofibre si forma - anche se le nanoparticelle possono essere raggiunti (electrospray). Un getto stabile ha due domini, un segmento di streaming e un segmento di frustate. Mentre il getto fustigazione è solitamente invisibile ad occhio nudo, il segmento streaming è spesso visibile in condizioni di luce adeguata. Osservando la lunghezza, lo spessore, la consistenza e il movimento del flusso è utile per prevedere l&#39;allineamento e la morfologia delle nanofibre in formazione. Un flusso breve, non uniforme, incoerente, e / o oscillante è indicativo di una serie di problemi, tra cui l&#39;allineamento delle fibre poveri, perline, schizzi, modelli e svolazzo o ondulato. Il flusso può essere ottimizzato regolando la composizione della soluzione e la configurazione degli apparati electrospinning, ottimizzando l&#39;allineamento e la morfologia delle fibre prodotte. In questo protocollo, vi presentiamo una procedura per la creazione di un apparato di base electrospinning, empiricamente approssimare la concentrazione critica groviglio di una soluzione polimerica e ottimizzando il processo di electrospinning. In aggiunta, discutiamo alcuni problemi comuni e le tecniche di risoluzione dei problemi.

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

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospinning tecniche possono creare una varietà di impalcature nanofibrous per l&#39;ingegneria tissutale o altre applicazioni. Descriviamo qui una procedura per ottimizzare i parametri della soluzione elettrofilatura e gli apparecchi di ottenere fibre con la morfologia e l&#39;allineamento desiderato. Problemi comuni e le tecniche di risoluzione dei problemi sono anche presentato.

Fondamenti electrospinning: Soluzione Ottimizzazione e parametri Apparatus

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

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Bioengineering

64.6K Views.  University of Michigan. Electrospinning tecniche possono creare una varietà di impalcature nanofibrous per l'ingegneria tissutale o altre applicazioni. Descriviamo qui una procedura per ottimizzare i parametri della soluzione elettrofilatura e gli apparecchi di ottenere fibre con la morfologia e l'allineamento desiderato. Problemi comuni e le tecniche di risoluzione dei problemi sono anche presentato.

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-strato Electrospinning di Mimic nativa Architettura arteriosa utilizzando Policaprolactone, elastina e collagene: uno studio preliminare

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

Ricerca

Bioingegneria

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.

Misura quantitativamente In situ Flussi utilizzando un autorespiratore subacqueo Velocimetry (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.

Piccole e Wide Angle X-Ray Scattering Studi di macromolecole biologiche in soluzione

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.

Electrospinning Growth Factor Releasing Microsfere in Fibrosi Ponteggi

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.

Combinazione di Microstereolithography e Electrospinning per la Produzione di Membrane Equipaggiato con nicchie per la rigenerazione della cornea

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.

Adattamento del processo Electrospinning per fornire tre ambienti unici per un Tri-livello<em&gt; In Vitro</em&gt; Modello della parete delle vie aeree

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.

Fabbricando degradabile Hydrogel idrogeli su scale di lunghezza Multiple tramite estrusione reattiva, microfluidica, auto-assemblaggio ed elettrofilatura

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.

Sciogliere elettrofilatura scrittura di scaffold tridimensionale Poly(ε-caprolactone) con morfologie controllabile per applicazioni di ingegneria del tessuto

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 risoluzione Patterning utilizzando due modalità di idrodinamiche Jet: Drop on Demand e Near-field elettrofilatura

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.

Apparato per la raccolta del tessuto microcolonne

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