Questo lavoro è stato condotto utilizzando le strutture del Dipartimento di Ingegneria Biomedica presso la Florida International University.

1. Setup Electrospinning Collector <ol><li> Tagliare il foglio di alluminio in un pezzo rettangolare. </li><li> Piegare il pezzo rettangolare in una striscia rettangolare, e allegarlo perpendicolare ad una piastra metallica piana con nastro (Figura 1). Nota: La dimensione del tappeto di fibre dipende dalla lunghezza e la larghezza della striscia. Pertanto, le dimensioni strip possono essere regolate come necessario. </li></ol> 2. Polimerico Solution Preparazione <ol><li> Mescolare glicerolo e acido dodecanodioico (DDA) in rapporto 1:1 molare in un becher a 120 ° C per 100 ore per ottenere polimero PGD. </li><li> Sciogliere poli (ossido di etilene) (PEO) e gelatina in 65% di etanolo, con un rapporto in peso di 1.5:3:95.5 in una provetta da 15 ml, serrare il tappo e scaldare la miscela in un forno a 60 ° C per 1 ora sotto agitazione fino ad ottenere una soluzione omogenea (soluzione Basal). </li><li> Per elettrofilatura, mescolare il polimero PGD e la soluzione basale in 04:06 rapporto di peso. Nota: concentrazione PGD ha un big effetto sul diametro delle fibre. 30% -50% per cento PGD è accettabile per la produzione di fibre di lunghezza superiore a 5 cm, con un aumento dei diametri delle fibre. </li><li> Aggiungere 0,1% riboflavina alla soluzione polimerica e mescolare bene. </li></ol> 3. Electrospinning <ol><li> Alimentare la soluzione polimerica in una siringa da 5 ml di serie con un 18 G smussati ago in acciaio inossidabile. </li><li> Inserire la siringa nella pompa a siringa. </li><li> Fissare il cavo di messa a terra di una sorgente di alimentazione ad alta tensione per la piastra metallica e il conduttore carica positiva all&#39;ago. </li><li> Regolare la distanza tra l&#39;ago e la striscia di foglio di alluminio a 15 cm. </li><li> Posizionare la pompa a siringa in un angolo di circa 15 ° con l&#39;orizzontale per prevenire l&#39;aggregazione delle fibre nella parte anteriore della striscia. </li><li> Accendere la pompa a siringa e regolare la portata della pompa di 0,6 ml / hr. </li><li> Accendere la fonte di alimentazione ad alta tensione e impostare la tensione di esercizio a 14,6 kV. </li></ol> 4.Fiber Processing <ol><li> Dopo la collezione è completa, esporre il tappeto di fibre di luce UV per 60 minuti per la reticolazione. </li><li> Trasferire il tappeto di fibre dalla striscia di foglio di alluminio per una capsula di Petri 100 mm, ed esporlo alla luce UV per altri 20 minuti per la sterilizzazione. </li><li> In un armadio biosicurezza, tagliare il tappeto di fibre in pezzi rotondi della stessa dimensione con una lama chirurgica e disporre i pezzi in una piastra da 24 pozzetti. </li><li> Per cellula di trattamento pre-semina, immergere campioni di fibre in 1 ml di tampone fosfato (PBS) e incubare a 37 ° C per una notte. </li><li> Il giorno seguente, aspirare PBS con attenzione, aggiungere 1 ml di terreno di differenziamento (DMEM/F12, N2, e FGF2) (vedi ricetta in tabella materiali) in ciascun pozzetto ed incubare a 37 ° C per 3 ore. </li><li> Aspirare il terreno di differenziamento accuratamente, aggiungere 0,2 ml di matrigel a ciascun campione fibra e incubare a 37 ° C per 30 min. Nota: laminina può essere utilizzato anche per il rivestimento di fibra. Aggiungere 0,2 ml di 20 mg / ml lamininaalla fibra e incubare a temperatura ambiente per 3 ore. </li><li> Rimuovere l&#39;eccesso di matrigel da ogni pozzetto. Lavare con 2 ml di terreno di differenziazione volta. Nota: I campioni di fibre sono pronti per la coltura cellulare. </li></ol> 5. Semina delle cellule su fibre <ol><li> Aggiungere 1 ml di Accutase alla cella piatto cultura MES e incubare per 10 min a 37 ° C. </li><li> Dopo 10 minuti, le cellule MES sono staccate dalla piastra di coltura. Aggiungere 4 ml di terreno di differenziamento alla piastra. Raccogliere le cellule MES galleggianti in una provetta da 15 ml e le cellule pipetta su e giù per rompere colonie (circa 15x). </li><li> Centrifugare a 400 xg per 5 min e risospendere le cellule in 4 ml di terreno di differenziamento. </li><li> Contare le cellule utilizzando un emocitometro. Trasferire 200 microlitri della sospensione cellulare in una provetta da 15 ml e diluire 10x con terreno di differenziamento. Trasferire 15 ml di sospensione cellulare diluita ad una camera sul emocitometro con una copertura antiscivolo in posto. Contarele celle nel centro di piazza 1 mm e le quattro piazze d&#39;angolo del emocitometro sotto il microscopio. Nota: Cell per ml = conteggio medio per quadrato x fattore di diluizione x 10 4 </li><li> Porre circa 5 x 10 4 cellule MES su ogni bene. Lentamente cadere la sospensione cellulare in mezzo ai campioni di fibre, invece di scivolare fuori al lato del pozzo, per evitare che la soluzione di drenaggio tappeto di fibre. </li><li> Aggiungere 1 ml di terreno di differenziamento in ciascun pozzetto, e mantenere la piastra in un incubatore a 37 ° C e 5% di CO 2 per consentire cellule attaccamento, crescita e differenziazione. </li><li> Aspirare il vecchio mezzo da ciascun pozzetto e sostituirlo con 1 ml di terreno di differenziamento fresco ogni altro giorno. </li></ol> 6. Vitalità cellulare <ol><li> Aspirare il vecchio mezzo da ogni pozzetto. </li><li> Mescolare 1/10 del volume di resazurina reagente fluorescenza con terreno di coltura e aggiungere 1 ml in ogni pozzetto. </li><li> Incubmangiato a 37 ° C e 5% di CO 2 per 4 ore, al riparo dalla luce diretta. </li><li> Dopo 4 ore, trasferire 100 microlitri 3x del reagente da ciascun pozzetto di una piastra da 96 pozzetti, e poi i campioni sono pronti per essere misurata mediante uno spettrofotometro a fluorescenza usando impostazioni di filtro 560EX nm/590EM. </li></ol> 7. Real Time PCR <ol><li> Dopo 14 giorni di coltura, aggiungere tampone di lisi per campioni e isolare l&#39;RNA totale da cellule sui campioni di fibre. </li><li> Utilizzare 4 pl di RNA totale per sintetizzare cDNA in 20 microlitri di reazione scale mediante trascrizione inversa. </li><li> Preparare la miscela di reazione PCR in ciascun tubo ottico: 10 microlitri Master Mix (2x), 0,2 ml di colorante, 8 microlitri di acqua priva di nucleasi, 1 ml cDNA, e 1 ml di fondo (primer utilizzati in questo studio sono elencati nella Tabella 1). </li><li> Impostare il programma PCR: a. 95 ° C 2:20 min, 1 ciclo b. 95 ° C 3 sec → 60 ° C 1 min, 40 ciclo c. 95 ° C 15 sec, 1 ciclo d. 60 ° C 1min, 1 ciclo e. 95 ° C 15 sec, 1 ciclo. Scegli il metodo comparativo C T per determinare i livelli di espressione genica relativa. </li><li> Dopo la PCR è finito, analizzare i real-time PCR risultati con il software StepOne ed esportare i risultati in Excel. </li></ol>

<ol>
	<li>Migneco, F., Huang, Y. -. C., Birla, R. K., Hollister, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+(glycerol-dodecanoate),+a+biodegradable+polyester+for+medical+devices+and+tissue+engineering+scaffolds.">Poly (glycerol-dodecanoate), a biodegradable polyester for medical devices and tissue engineering scaffolds.</a> Biomaterials. 30, 6479-6484 (2009).</li><li>Reneker, D. H., Yarin, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+jets+and+polymer+nanofibers.">Electrospinning jets and polymer nanofibers.</a> Polymer. 49, 2387-2425 (2008).</li><li>Wang, Y., Ameer, G. A., Sheppard, B. J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tough+biodegradable+elastomer.">A tough biodegradable elastomer.</a> Nature biotechnology. 20, 602-606 (2002).</li><li>Panunzi, S., De Gaetano, A., Mingrone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approximate+linear+confidence+and+curvature+of+a+kinetic+model+of+dodecanedioic+acid+in+humans.">Approximate linear confidence and curvature of a kinetic model of dodecanedioic acid in humans.</a> American Journal of Physiology-Endocrinology And Metabolism. 289, (2005).</li><li>Park, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apparatus+for+preparing+electrospun+nanofibers:+designing+an+electrospinning+process+for+nanofiber+fabrication.">Apparatus for preparing electrospun nanofibers: designing an electrospinning process for nanofiber fabrication.</a> Polymer Internationa l. 56, 1361-1366 (2007).</li><li>Barnes, C. P., Sell, S. A., Boland, E. D., Simpson, D. G., Bowlin, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanofiber+technology:+designing+the+next+generation+of+tissue+engineering+scaffolds.">Nanofiber technology: designing the next generation of tissue engineering scaffolds.</a> Advanced drug delivery reviews. 59, 1413-1433 (2007).</li><li>Li, W. -. J., Mauck, R. L., Tuan, R. S. <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+scaffolds:+production,+characterization,+and+applications+for+tissue+engineering+and+drug+delivery.">Electrospun nanofibrous scaffolds: production, characterization, and applications for tissue engineering and drug delivery.</a> Journal of Biomedical Nanotechnology. 1, 259-275 (2005).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <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+polymeric+nanofibers+for+tissue+engineering+applications:+a+review.">Electrospinning of polymeric nanofibers for tissue engineering applications: a review.</a> Tissue engineering. 12, 1197-1211 (2006).</li><li>Lim, S. H., Mao, H. -. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+scaffolds+for+stem+cell+engineering.">Electrospun scaffolds for stem cell engineering.</a> Advanced drug delivery reviews. 61, 1084-1096 (2009).</li><li>Lowery, J. L., Datta, N., Rutledge, G. C. <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+fiber+diameter,+pore+size+and+seeding+method+on+growth+of+human+dermal+fibroblasts+in+electrospun+poly+(epsilon-caprolactone)+fibrous+mats.">Effect of fiber diameter, pore size and seeding method on growth of human dermal fibroblasts in electrospun poly (epsilon-caprolactone) fibrous mats.</a> Biomaterials. 31, 491-504 (2010).</li><li>Tillman, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+stability+of+electrospun+polycaprolactone-collagen+scaffolds+in+vascular+reconstruction.">The in vivo stability of electrospun polycaprolactone-collagen scaffolds in vascular reconstruction.</a> Biomaterials. 30, 583-588 (2009).</li><li>Ju, Y. M., Choi, J. S., Atala, A., Yoo, J. J., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayered+scaffold+for+engineering+cellularized+blood+vessels.">Bilayered scaffold for engineering cellularized blood vessels.</a> Biomaterials. 31, 4313-4321 (2010).</li><li>McCullen, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+collagen+polymerization+of+layered+cell-seeded+electrospun+scaffolds+for+bone+tissue+engineering+applications.">In situ collagen polymerization of layered cell-seeded electrospun scaffolds for bone tissue engineering applications.</a> Tissue Engineering Part C: Methods. 16, 1095-1105 (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Le limitazioni di collettori semplici o la complessità di collettori che sono attualmente utilizzati per elettrospinning aumentare la restrizione di ottenere la lunghezza desiderata e dimensione del tappeto di fibre per alcune applicazioni rotanti. Inoltre, il trasferimento delle fibre dal collettore di terra alla piastra di coltura o altri substrati è una sfida 5. In questo rapporto, un collezionista di nuova concezione, realizzato semplicemente collegando una striscia di foglio di alluminio al collettore ...

Electrospinning è uno dei metodi di trattamento efficaci per produrre scaffold fibre dimensioni micro-to-nanometri. Il principio di base di elettrospinning comporta un cono di Taylor soluzione che viene tenuta in corrispondenza dell&#39;orifizio di un ago applicando alta tensione tra la punta dell&#39;ago e un collettore a terra. Quando la repulsione elettrostatica nella soluzione supera la tensione superficiale, un getto di fluido caricato viene espulso dalla punta dell&#39;ago, viaggia attraverso l&#39;aria con evaporazione del solvente, ed è infine depositato sul collettore a terra. La pompa a siringa fornisce un flusso continuo di soluzione che emerge dalla filiera e quindi più copie delle fibre elettrofilate può essere fabbricato in un breve periodo di tempo. Nel corso del lasciando la filiera per arrivare al collettore, il getto carica subirà stretching e frustate secondo un certo numero di parametri che includono la viscosità e la tensione superficiale della soluzione polimerica, i electrostatic forza nella soluzione, e l&#39;interazione del campo elettrico esterno, ecc 2. Nel processo electrospinning, un collettore funge da substrato conduttore dove si possono depositare le fibre micro-to-nanometri. In questo studio, un nuovo tipo di collettore fibra creata avere stuoie in fibra con la dimensione desiderata (lunghezza x larghezza). Tradizionalmente, un foglio di alluminio è usato come collettore ma è difficile trasferire le fibre dalla superficie piana per un altro substrato. La difficoltà di raccolta un feltro di fibre intatte da un collettore tradizionale è dovuto principalmente al fatto che le fibre elettrofilate attaccano fortemente alla superficie del collettore. Pertanto, abbiamo modificato il collettore piegando un pezzo di foglio di alluminio in una striscia rettangolare e legandolo perpendicolare ad una piastra metallica piana. Le fibre sono tese elettrofilate tutta l&#39;area tra la punta della striscia e la piastra metallica, che può essere facilmente trasferito ad un altro Substrate. L&#39;interesse per polimeri elastomerici termicamente reticolati è in rapida crescita a causa del lavoro pionieristico del gruppo di Robert Langer, che ha introdotto poli (glicerolo sebacate) (PGS), un poliestere che è analoga alla gomma vulcanizzata, nel 2002 3. Simile a PGS, abbiamo sviluppato con successo poli (glicerolo-dodecanoato) (PGD) per condensazione termica di glicerolo e acidi dodecanodioico e ha dimostrato la sua unica proprietà di memoria di forma 1. A differenza più rigido materiali sintetici poli (butirrato ossidrile) o poli (L-lattide) (moduli di Young di 250 MPa e 660 MPa rispettivamente), PGD esibisce proprietà elastomeriche come gomma, con un modulo di Young di 1,08 MPa quando la temperatura è superiore a 37 ° C, che è una stretta corrispondenza al nervo periferico in-situ (0,45 MPa). Inoltre, PGD è biodegradabile e il tempo di degradazione può essere messo a punto variando il rapporto di glicerolo e acidi dodecanodioico. L&#39;acido dodecanodioico è un sub dodici carbonioposizione con due gruppi carbossilici terminali, HOOC (CH 2) 10 COOH. Acidi bicarbossilici Anche numerati come l&#39;acido sebacico e acido dodecanodioico possono essere metabolizzati ad acetil-CoA e inserire il acidi tricarbossilici (TCA) / (acido citrico) ciclo. Il prodotto metabolico di acidi bicarbossilici, succinil-CoA, è un precursore gluconeogenetic e intermedio del ciclo di TCA 4. Così, alcuni studi hanno suggerito che possano essere utilizzate come substrato combustibile alternativo per la nutrizione enterale e parenterale, specialmente nelle condizioni patologiche. Inoltre, PGD presenta memoria di forma unica perché la sua temperatura di transizione vetrosa è 31 ° C, quindi si vede distinte proprietà meccaniche a temperatura ambiente e alla temperatura corporea. In sintesi, la PGD è biodegradabile, biocompatibile, esibendo uniche proprietà elastiche con proprietà meccaniche simili ai tessuti nervosi; quindi, è un materiale adatto per applicazioni di ingegneria del tessuto nervoso. In questo protocollo, il electrospunfibre lunghe che coprono una vasta area di deposito sono stati realizzati attraverso il collettore di nuova concezione da PGD. I ponteggi in fibra in grado di supportare la crescita delle cellule staminali pluripotenti di topo e la differenziazione.

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Glycerol</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">G7757</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Dodecanedioic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1009</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Gelatin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Poly (ehtylene oxide) (PEO)</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">182028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Riboflavin</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">132350250</td>
			<td style="width:167px;">0.10%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Mouse embryonic stem cells</td>
			<td style="width:175px;">GlobalStem</td>
			<td style="width:119px;">GSC-5002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Matrigel</td>
			<td style="width:175px;">Becton Dickinson</td>
			<td style="width:119px;">356234</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">DMEM/F12</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:119px;">SH30272.02</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">N2 supplement&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">17502048</td>
			<td style="width:167px;">1%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">FGF2</td>
			<td style="width:175px;">Stemgent</td>
			<td style="width:119px;">03-0002</td>
			<td style="width:167px;">10ng/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Accutase</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A11105-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Phosphate buffered saline (PBS)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">10010-031&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Resazurin fluorescence dye&#160;</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:119px;">62758-13-8&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">SV Total RNA Isolation System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">Z3100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoScript Reverse Transcription System</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A5000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">GoTaq&#129; qPCR Master Mix</td>
			<td style="width:175px;">Promega</td>
			<td style="width:119px;">A6001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Syringe pump&#160;</td>
			<td style="width:175px;">Fisher scientific</td>
			<td style="width:119px;">14-831-200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:268px;">High voltage power source&#160;</td>
			<td style="width:175px;">Spellman High Voltage Electronics Corporation</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">SL30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">UV light</td>
			<td style="width:175px;">Philips</td>
			<td style="width:119px;">308643</td>
			<td style="width:167px;">15W/G15T8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Synergy HT Multi-Mode Microplate Reader</td>
			<td style="width:175px;">BioTek</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">Perkin Elmer GeneAmp PCR System 9600</td>
			<td style="width:175px;">Perkin Elmer</td>
			<td style="width:119px;">8488</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:268px;">StepOne Real-time PCR System</td>
			<td style="width:175px;">Applied Biosystems</td>
			<td style="width:119px;">4376357</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

electrospun fibrous scaffolds of poly(glycerol-dodecanedioate) for engineering neural tissues from mouse embryonic stem cells

Per applicazioni di ingegneria tissutale, la preparazione di supporti biodegradabili e biocompatibili è il compito più desiderabile ma impegnativo. Tra i vari metodi di fabbricazione, elettrospinning è il più attraente uno per la sua semplicità e versatilità. Inoltre, nanofibre elettrofilate imitano la dimensione della matrice extracellulare naturale, garantendo un ulteriore sostegno per la sopravvivenza e la crescita delle cellule. Questo studio ha dimostrato la fattibilità della fabbricazione di fibre lunghe che coprono un&#39;area più grande deposito per un romanzo polimero biodegradabile e biocompatibile chiamato poli (glicerolo-dodecanoato) (PGD) 1 utilizzando un collettore di nuova concezione per electrospinning. PGD ​​presenta proprietà uniche elastici con proprietà meccaniche simili a tessuti nervosi, quindi è adatto per applicazioni di ingegneria tissutale neurali. La sintesi e la fabbricazione di set-up per la fabbricazione di materiali ponteggi fibrosi era semplice, altamente riproducibile e poco costoso. In biocompatibilitàtest, cellule derivate da cellule staminali embrionali di topo potrebbero aderire e crescere sulle fibre PGD elettrofilate. In sintesi, questo protocollo ha fornito un metodo di fabbricazione versatile per la fabbricazione di fibre PGD elettrofilate per sostenere la crescita di topo cellule staminali embrionali derivate cellule neurali lignaggio.

I componenti principali del elettrospinning sono mostrati in Figura 1. Velo di taglia grande era tipicamente ottenuta attraverso la striscia di foglio di alluminio perpendicolarmente divisoria e una piastra metallica piana. Figura 2 mostra la struttura del collettore e il tappeto di fibre elettrospinning. La larghezza e la lunghezza può essere regolata per diverse applicazioni. La lunghezza della fibra a base di polimero PGD e miscela soluzione basale è fino a 10 cm. La morfologia del...

Watch this Scientific Journal Video about Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells at JoVE.com

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

È stata riportata la sintesi e la fabbricazione di fibre lunghe elettrofilate coprono un&#39;area più grande deposito tramite un collettore di nuova concezione da un polimero biodegradabile romanzo chiamato poli (glicerolo-dodecanoato) (PGD). Le fibre sono in grado di sostenere la crescita di cellule derivate da cellule staminali pluripotenti di topo.

Elettrofilate Fibrosa Ponteggi di Poli (glicerolo-dodecanedioate) per l&#39;ingegneria neurale tessuti da cellule staminali embrionali di topo

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

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Research

JoVE Journal

Bioengineering

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

10.8K Views.  Florida International University.  È stata riportata la sintesi e la fabbricazione di fibre lunghe elettrofilate coprono un'area più grande deposito tramite un collettore di nuova concezione da un polimero biodegradabile romanzo chiamato poli (glicerolo-dodecanoato) (PGD). Le fibre sono in grado di sostenere la crescita di cellule derivate da cellule staminali pluripotenti di topo. 

Video: Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS elastomerico Ponteggi in Ingegneria tessuto arterioso

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

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Bioingegneria

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparazione del 3D fibrina ponteggi per le applicazioni delle cellule staminali Cultura

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Elaborazione Postproduzione di fibre elettrofilate per l&#39;ingegneria tissutale

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Ingegneria tessuti muscolo scheletrico di cellule murine progenitrici mioblasti e di applicazione di stimolazione elettrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Sviluppo, di espansione, e<em&gt; In vivo</em&gt; Monitoraggio delle cellule NK umane da cellule staminali embrionali umane (hESC) ed e cellule staminali pluripotenti indotte (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

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, ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&amp;#949;-caprolactone) Electrospun Yarns

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Ottimizzare Allegato di cellule staminali mesenchimali umane su Poli (ε-caprolattone) elettrofilate Filati

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

Elettrofilate nanofibre Ponteggi con gradazioni Organizzazione Fiber

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Generazione di cellule di topo Airway epiteliali ESC-derivati ​​dall&#39;uso di decellularized Lung Ponteggi

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Due metodi per decellularizzazione di tessuti vegetali per applicazioni di ingegneria del tessuto

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...