This work was partially funded through the Richard Barber Foundation and a Thomas Rumble Fellowship (TJW).

1. Acqua / Olio / Acqua Doppia Emulsione microsfere di produzione <ol><li> Prima preparare 2% e 0,5% w / v soluzioni di alcoli polivinilici (PVA) in acqua deionizzata. Mescolare le soluzioni a 50 ° C fino a chiaro, questo può richiedere diverse ore. Preparare una soluzione al 2% v / v alcool isopropilico in acqua deionizzata. </li><li> Preparare una soluzione acquosa di proteine ​​idrofila desiderato. La tabella seguente fornisce esempi di formulazioni. </li></ol><img alt="Tabella 1" src="/files/ftp_upload/51517/51517table1highres.jpg" /> Tabella 1:. Esempio proteine ​​Solutions Le seguenti soluzioni di proteine ​​sono stati inseriti con successo e elettrofilate utilizzando questo protocollo. Altre soluzioni proteiche idrofili possono essere utilizzati come necessario. <ol start="3"><li> Mettere 40 ml di soluzione di PVA 0,5% in una provetta da centrifuga da 50 ml e mettere da parte. </li><li> In una provetta da centrifuga 15 ml sciogliere 300 mg di 65:35poli (acido lattico-co-glicolico) (PLGA) in 3 ml di diclorometano. Un miscelatore a vortice può essere utilizzato per accelerare PLGA dissoluzione. </li><li> Unire 200 ml di soluzione proteica e 4 ml di soluzione di PVA 2%. Versare la miscela proteica nella soluzione PLGA (punto 1.4). Le soluzioni rimarranno per lo più separati. </li><li> Posizionare il tubo in un bicchiere di acqua ghiacciata. Utilizzando un sonicatore bacchetta a ~ 10 Watt (RMS), agitare la soluzione per alcuni secondi (5-10) fino a una crema uniforme emulsione bianco è creato. </li><li> Versare l&#39;emulsione nella provetta da 50 ml contenente 0,5% di PVA (passo 1.3). Mescolare la soluzione ad alta velocità su un vortex per ~ 20 sec. La soluzione svilupperà un aspetto torbido. </li><li> Trasferire l&#39;emulsione in un becher da 200 ml e posto su una piastra di agitazione a 350 rpm per 2 min. Aggiungere 50 ml di 2% di alcool isopropilico per il bicchiere sul piatto mescolare. Lasciare il composto per continuare a mescolare per un minimo di 1 ora per consentire al DCM evaporare e il PLGA per indurire. </li><li> Trasferimento tegli microsfere soluzione in provette da centrifuga. </li><li> Centrifugare a 425 xg per 3 min. Le microsfere si raccoglieranno nella parte inferiore del tubo e appaiono bianco. Rimuovere con attenzione il surnatante dal tubo, sopra le microsfere, e conservare in una bottiglia da 500 ml. </li><li> Sciacquare le microsfere con acqua deionizzata riempiendo il tubo di tre quarti pieno e scuotendola di ridistribuire le microsfere nel liquido. </li><li> Ripetere i punti 1.10 e 1.11 quattro volte. </li><li> Dopo l&#39;ultimo risciacquo, rimuovere il supernatante di nuovo e posto in bottiglia da 500 ml con altri campioni. Congelare le microsfere raccolti nella provetta da centrifuga, poi liofilizzare per almeno 24 ore. </li><li> Visualizza le microsfere al microscopio ottico o con un microscopio elettronico a scansione. Microsfere non più grandi di 60 micron sono per electrospinning efficaci. Se microsfere sono troppo grandi, sonicating o vortexare tempi più lunghi possono essere richiesti al punto 1.6 o 1.7. </li> &lt;li&gt; Conservare le microsfere secche in un -20 ° C freezer. </li><li> Opzionale: Utilizzare un saggio di proteine, per istruzioni del produttore, per testare la quantità di proteine ​​in bottiglia da 500 ml dal punto 1.10 30. Questo è utilizzato per calcolare la percentuale di proteine ​​incapsulato in microsfere, sottraendo la quantità di soluzione da ciò che è stato utilizzato nel processo di produzione. </li></ol> Nota: Per visualizzare la posizione proteina nel microsfere aggiungere Rodamina 2 mg / ml per la soluzione di PLGA 31 e incapsulare una proteina coniugato con FITC Figura 1 mostra un esempio.. 2 Electrospinning con microsfere <ol><li> Prima di preparare la soluzione elettrospinning, creare un 0,5% w / v soluzione di fotoiniziatore, in acqua deionizzata sciogliendo a 37 ° C. Questo processo può richiedere diverse ore. </li><li> Creare un 2% w / v metacrilato acido ialuronico (Meha) (vedi Burdick et al. Per la sintesi) il 29, 3%w / v 900 kD poli (ossido di etilene) (PEO) e 0.05% w / v soluzione fotoiniziatore in acqua deionizzata. <ol><li> Calcolare la quantità corretta di Meha e PEO per il volume desiderato. Per esempio, 10 ml di soluzione richiede elettrospinning 200 mg di Meha e 300 mg di PEO. </li><li> Sciogliere il PEO in acqua deionizzata al 90% del volume finale desiderato (9 ml per questo esempio). Questo può richiedere diverse ore, un piatto mescolare riscaldato o bagnomaria a 37 ° C possono essere utilizzati per accelerare il processo. </li><li> Successivamente aggiungere il Meha e utilizzare un miscelatore vortex per mescolare la soluzione fino a chiaro. Questo richiederà solo pochi minuti. </li><li> Infine aggiungere la soluzione fotoiniziatore 0,5% ad occupare il restante 10% del volume (1 ml per questo esempio). </li></ol></li><li> Aggiungere microsfere alla concentrazione desiderata fino a 400 mg / ml. Miscelare la soluzione su un vortex fino a quando le microsfere sono distribuiti uniformemente nella soluzione. </li><li> Trasferire la soluzione in una siringa e attaccare un 6 pollici bl 18 gaugeunt ago punta. </li><li> Posizionare la siringa in una pompa a siringa e impostarlo per dispensare a 1,2 ml / h. </li><li> Nastro uno strato di foglio di alluminio sul piatto di raccolta o mandrino. Questo permette una facile pulizia e conservazione del patibolo finito. Un mandrino rotante viene utilizzato per creare fibre allineate. Una lastra piana o mandrino fermo si tradurrà in fibre disposte in modo casuale. </li><li> Collegare il filo di terra da una fonte di alimentazione ad alta tensione per l&#39;apparato di raccolta. Collegare il conduttore positivo al ago. </li><li> Regolare la superficie della pompa siringa e la raccolta in modo che vi è di 15 cm tra la punta dell&#39;ago e la superficie di raccolta. </li><li> Avviare il pompaggio polimero, quando la soluzione è visibile alla fine della siringa, attivare il generatore di tensione e impostare la tensione di 24 kV. ATTENZIONE: Quando la tensione è acceso non toccare alcuna parte metallica del sistema. Carica può anche saltare brevi distanze dalle parti elettrificate per la pelle. </li><li> Eseguire la soluzione fino a dspessore impalcatura esired è raggiunto. Al termine spegnere fonte di tensione e pompa a siringa. </li><li> Rimuovere la pellicola con ponteggio allegato. Ponteggi completate contenenti proteine ​​vengono memorizzati in una -20 ° C freezer. </li></ol> 3. proteine ​​Bioattività Testing <ol><li> Preparare delle cellule di coltura. Aggiungere 10% v / v siero fetale bovino, 1% v / v L-glutammina, e 1% v / v penicillina-streptomicina per mezzo di Eagle modificato di Dulbecco. </li><li> Selezionare i vetrini che si adattano completamente in una piastra ben. </li><li> Utilizzare 3 (Trimethoxysilyl) propil metacrilato per trattare i coprioggetti come descritto dal produttore. Methacrylation migliora patibolo aderenza ai coprioggetti. </li><li> Fissare coprioggetti metacrilato alla zona di raccolta del electrospinner con nastro biadesivo rimovibile prima elettrospinning. Spinning sui coprioggetti facilita la gestione e la visualizzazione. </li><li> Electrospin di spessore desiderato come descritto sopra. </li><li> Laopo Electrospinning rimuovere con attenzione coprioggetto da mandrino. Posizionare il patibolo vetrini rivestiti in una camera di azoto chiaro e assicurarsi che tutto l&#39;ossigeno viene eliminato. </li><li> Posizionare la camera e il patibolo sotto di 10 mW / cm 2 365 nm luce per 15 min. Dopo la reticolazione posto in pozzetti di dimensioni appropriate. Assicurarsi che il lato impalcatura è rivolto verso l&#39;alto. </li><li> Luogo ponteggi sotto una lampada germicida per 30 minuti per sterilizzare. Se fibronectina desiderato o altra proteina viene utilizzato come rivestimento per migliorare l&#39;adesione cellulare. Seguire le istruzioni del produttore per ponteggi cappotto. </li><li> Raccolta dei gangli dorsali (DRG) come precedentemente descritto da Hollenbeck 32. Sarà necessario One DRG per ogni ponteggio coperto coprioggetto testato. </li><li> Mettere 100-200 ml di media su ogni patibolo nella piastra ben. Posizionare con cura un DRG su ogni patibolo nel droplet media. Per ponteggio di spessore possono essere necessari più mezzi di comunicazione; DRG devono essere completamente sommerse e non FLOATing. </li><li> Incubare l&#39;impalcatura e DRG a 37 ° C per 4 ore per permettere alla cellula di aderire al ponteggio. </li><li> Riempire i media a livello appropriato per il bene e mettere di nuovo in incubatrice. Continuare l&#39;incubazione per 4-6 giorni. </li><li> Dopo il periodo di incubazione, rimuovere con attenzione i media di ogni bene e lavare delicatamente una volta con PBS. Fissare le cellule per 30 minuti con 4% w / v paraformaldeide. </li><li> Cellule macchia con una macchia anticorpo per neurofilamenti. Questo permetterà la visualizzazione di crescita dei neuriti per la quantificazione. DAPI può anche essere utilizzato per visualizzare nuclei delle cellule. Un esempio di protocollo di colorazione è stato descritto da Sundararaghavan e colleghi 14. </li><li> Visualizza le cellule utilizzando un microscopio a fluorescenza. <ol><li> Posizionare pozzetti sul palco del microscopio. </li><li> Individuare la massa cellulare utilizzando le impostazioni del filtro e di eccitazione per DAPI. </li><li> Una volta che la cellula è passare il filtro FITC per visualizzare le neuriti estesi. Ucantare la funzione di punto sul microscopio raccogliere e combinare le immagini che necessario per vedere l&#39;intera struttura. Ripetere l&#39;operazione per DAPI, FITC e campo chiaro. </li></ol></li></ol>

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	<li>Wrobel, M. R., Sundararaghavan, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+migration+in+neural+tissue+engineering.">Directed migration in neural tissue engineering.</a> Tissue Eng Part B Rev. , (2013).</li><li>Schmidt, C. E., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering:+strategies+for+repair+and+regeneration.">Neural tissue engineering: strategies for repair and regeneration.</a> Annual Review of Biomedical Engineering. 5, 293-347 (2003).</li><li>Madduri, S., di Summa, P., Papaloizos, M., Kalbermatten, D., Gander, B. <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+controlled+co-delivery+of+synergistic+neurotrophic+factors+on+early+nerve+regeneration+in+rats.">Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats.</a> Biomaterials. 31, 8402-8409 (2010).</li><li>Madduri, S., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+delivery+systems+and+repair+strategies+for+damaged+peripheral+nerves.">Growth factor delivery systems and repair strategies for damaged peripheral nerves.</a> J Control Release. 161, 274-282 (2012).</li><li>Madigan, N. N., McMahon, S., O'Brien, T., Yaszemski, M. J., Windebank, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+tissue+engineering+and+novel+therapeutic+approaches+to+axonal+regeneration+following+spinal+cord+injury+using+polymer+scaffolds.">Current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury using polymer scaffolds.</a> Respir Physiol Neurobiol. 169, 183-199 (2009).</li><li>Sundararaghavan, H. G., Monteiro, G. A., Firestein, B. L., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+growth+in+3D+collagen+gels+with+gradients+of+mechanical+properties.">Neurite growth in 3D collagen gels with gradients of mechanical properties.</a> Biotechnol. Bioeng. 102, 632-643 (2009).</li><li>Hudson, T. W., Evans, G. R., Schmidt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+strategies+for+peripheral+nerve+repair.">Engineering strategies for peripheral nerve repair.</a> Clin Plast Surg. 26, 617-628 (1999).</li><li>Kokai, L. E., Bourbeau, D., Weber, D., McAtee, J., Marra, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+growth+factor+delivery+promotes+axonal+regeneration+in+long+gap+peripheral+nerve+repair.">Sustained growth factor delivery promotes axonal regeneration in long gap peripheral nerve repair.</a> Tissue Eng Part A. 17, 1263-1275 (2011).</li><li>Bronzino, J. D., Peterson, 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=.">.</a> The Biomedical Engineering Handbook, Third Edition - 3 Volume Set: Tissue Engineering and Artificial Organs. , (2006).</li><li>Bell, J. H. A., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+nerve+guides:+materials,+fabrication,+growth+factors,+and+cell+delivery.">Next generation nerve guides: materials, fabrication, growth factors, and cell delivery.</a> Tissue Eng Part B Rev. 18, 116-128 (2012).</li><li>Ruiter, G. C. W., Malessy, M. J. A., Yaszemski, M. J., Windebank, A. J., Spinner, 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=Designing+ideal+conduits+for+peripheral+nerve+repair.">Designing ideal conduits for peripheral nerve repair.</a> Neurosurgical focus. 26, (2009).</li><li>Olakowska, E., Woszczycka-Korczy&#324;ska, I., J&#281;drzejowska-Szypu&#322;ka, H., Lewin-Kowalik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nanotubes+and+nanofibres+in+nerve+repair.+A+review.">Application of nanotubes and nanofibres in nerve repair. A review.</a> Folia Neuropathol. 48, 231-237 (2010).</li><li>Gunn, J., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyblend+nanofibers+for+biomedical+applications:+perspectives+and+challenges.">Polyblend nanofibers for biomedical applications: perspectives and challenges.</a> Trends Biotechnol. 28, 189-197 (2010).</li><li>Sundararaghavan, H. G., Masand, S. N., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+generation+of+haptotactic+gradients+through+3D+collagen+gels+for+enhanced+neurite+growth.">Microfluidic generation of haptotactic gradients through 3D collagen gels for enhanced neurite growth.</a> Journal of Neurotrauma. 28, 2377-2387 (2011).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multiscale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multiscale and photopatterned porosity.</a> Macromol Biosci. 10, 265-270 (2010).</li><li>Edalat, F., Sheu, I., Manoucheri, S., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Material+strategies+for+creating+artificial+cell-instructive+niches.">Material strategies for creating artificial cell-instructive niches.</a> Current Opinion in Biotechnology. 23, 820-825 (2012).</li><li>Annabi, N., 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=Synthesis+of+highly+porous+crosslinked+elastin+hydrogels+and+their+interaction+with+fibroblasts+in+vitro.">Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro.</a> Biomaterials. 30, 4550-4557 (2009).</li><li>Casta&#241;o, O., Eltohamy, M., Kim, H. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+technology+in+tissue+regeneration.">Electrospinning technology in tissue regeneration.</a> Methods Mol. Biol. 811, 127-140 (2012).</li><li>Chew, S. Y., Wen, J., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+release+of+proteins+from+electrospun+biodegradable+fibers.">Sustained release of proteins from electrospun biodegradable fibers.</a> Biomacromolecules. 6, 2017-2024 (2005).</li><li>Han, D., Gouma, P. -. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+bioscaffolds+that+mimic+the+topology+of+extracellular+matrix.">Electrospun bioscaffolds that mimic the topology of extracellular matrix.</a> Nanomedicine. 2, 37-41 (2006).</li><li>Prabhakaran, M. P., 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=Electrospun+biocomposite+nanofibrous+scaffolds+for+neural+tissue+engineering.">Electrospun biocomposite nanofibrous scaffolds for neural tissue engineering.</a> Tissue Eng Part A. 14, 1787-1797 (2008).</li><li>Xie, J., MacEwan, M. R., Schwartz, A. G., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibers+for+neural+tissue+engineering.">Electrospun nanofibers for neural tissue engineering.</a> Nanoscale. 2, 35-44 (2010).</li><li>Yao, L., O'Brien, N., Windebank, A., Pandit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orienting+neurite+growth+in+electrospun+fibrous+neural+conduits.">Orienting neurite growth in electrospun fibrous neural conduits.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. 90, 483-491 (2009).</li><li>Sill, T. J., von Recum, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Ifkovits, J. L., Sundararaghavan, H. G., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+fibrous+polymer+scaffolds+for+tissue+engineering+and+cell+culture.">Electrospinning fibrous polymer scaffolds for tissue engineering and cell culture.</a> Journal of Visualized Experiments: JoVE. , (2009).</li><li>Sundararaghavan, H. G., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+with+depth+in+electrospun+fibrous+scaffolds+for+directed+cell+behavior.">Gradients with depth in electrospun fibrous scaffolds for directed cell behavior.</a> Biomacromolecules. 12, 2344-2350 (2011).</li><li>Meinel, A. J., Germershaus, O., Luhmann, T., Merkle, H. P., Meinel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+matrices+for+localized+drug+delivery:+current+technologies+and+selected+biomedical+applications.">Electrospun matrices for localized drug delivery: current technologies and selected biomedical applications.</a> Eur J Pharm Biopharm. 81, 1-13 (2012).</li><li>Ji, 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=Bioactive+electrospun+scaffolds+delivering+growth+factors+and+genes+for+tissue+engineering+applications.">Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications.</a> Pharm. Res. 28, 1259-1272 (2011).</li><li>Burdick, J. A., Chung, C., Jia, X., Randolph, M. A., 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=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>P&#233;an, J. M., 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=Optimization+of+HSA+and+NGF+encapsulation+yields+in+PLGA+microparticles.">Optimization of HSA and NGF encapsulation yields in PLGA microparticles.</a> International Journal of Pharmaceutics. 166, 105-115 (1998).</li><li>Cartiera, M. S., Johnson, K. M., Rajendran, V., Caplan, M. J., Saltzman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Uptake+and+Intracellular+Fate+of+PLGA+Nanoparticles+in+Epithelial+Cells.">The Uptake and Intracellular Fate of PLGA Nanoparticles in Epithelial Cells.</a> Biomaterials. 30, 2790-2798 (2009).</li><li>Hollenbeck, P. J., Bamburg, J. 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> Neurons: Methods and Applications for the Cell Biologist. , (2003).</li><li>Boer, R., 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=Rat+sciatic+nerve+repair+with+a+poly-lactic-co-glycolic+acid+scaffold+and+nerve+growth+factor+releasing+microspheres.">Rat sciatic nerve repair with a poly-lactic-co-glycolic acid scaffold and nerve growth factor releasing microspheres.</a> Microsurgery. 31, 293-302 (2011).</li><li>Pujic, Z., Goodhill, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+compartment+diffusion+chamber+for+studying+axonal+chemotaxis+in+3D+collagen.">A dual compartment diffusion chamber for studying axonal chemotaxis in 3D collagen.</a> Journal of Neuroscience Methods. 215, 53-59 (2013).</li><li>Madduri, S., di Summa, P., Papalo&#239;zos, M., Kalbermatten, D., Gander, B. <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+controlled+co-delivery+of+synergistic+neurotrophic+factors+on+early+nerve+regeneration+in+rats.">Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats.</a> Biomaterials. 31, 8402-8409 (2010).</li><li>Xu, X., 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=Polyphosphoester+microspheres+for+sustained+release+of+biologically+active+nerve+growth+factor.">Polyphosphoester microspheres for sustained release of biologically active nerve growth factor.</a> Biomaterials. 23, 3765-3772 (2002).</li><li>Yan, Q., Yin, Y., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+new+PLGL-RGD-NGF+nerve+conduits+for+promoting+peripheral+nerve+regeneration.">Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration.</a> Biomed Eng Online. 11, (2012).</li><li>Gungor-Ozkerim, P. S., Balkan, T., Kose, G. T., Sarac, A. S., Kok, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+growth+factor+loaded+microspheres+into+polymeric+electrospun+nanofibers+for+tissue+engineering+applications.">Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications.</a> J Biomed Mater Res A. , (2013).</li><li>Li, X., 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=Encapsulation+of+proteins+in+poly(L-lactide-co-caprolactone)+fibers+by+emulsion+electrospinning.">Encapsulation of proteins in poly(L-lactide-co-caprolactone) fibers by emulsion electrospinning.</a> Colloids Surf B Biointerfaces. 75, 418-424 (2010).</li><li>Wang, C. -. Y., 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+effect+of+aligned+core-shell+nanofibres+delivering+NGF+on+the+promotion+of+sciatic+nerve+regeneration.">The effect of aligned core-shell nanofibres delivering NGF on the promotion of sciatic nerve regeneration.</a> J Biomater Sci Polym Ed. 23, 167-184 (2012).</li><li>Liu, J. -. J., Wang, C. -. Y., Wang, J. -. G., Ruan, H. -. J., Fan, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+regeneration+using+composite+poly(lactic+acid-caprolactone)/nerve+growth+factor+conduits+prepared+by+coaxial+electrospinning.">Peripheral nerve regeneration using composite poly(lactic acid-caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning.</a> J Biomed Mater Res A. 96, 13-20 (2011).</li></ol>

The authors have nothing to disclose.

Molti studi hanno dimostrato che le cellule nervose possono essere diretti da stimoli topografici (allineamento della fibra) e segnali chimici (fattori di crescita) 1,2,10,11,35. Electrospinning è un metodo facile per creare fibre allineate. I fattori di crescita favoriscono la crescita dei nervi, ma al fine di includerli in condotti nervose (NC), è necessario un metodo per rilascio prolungato. Per creare un sistema più robusto con entrambi i segnali, questi due segnali devono essere combinati. Diversi met...

Una delle sfide in corso in ingegneria dei tessuti neurali è la creazione di un condotto del nervo (NC) che imita la matrice extracellulare, dove i nervi crescono naturalmente. La ricerca ha dimostrato che le cellule rispondono a diversi fattori nel loro ambiente, tra cui meccanica, topografico, adesivi e segnali chimici 1-3. Una delle sfide principali in questo settore sta determinando la combinazione appropriata di segnali e fabbricando un sistema in grado di mantenere spunti per un lungo periodo per sostenere la crescita delle cellule 4. Neuroni periferici sono noti a preferire un substrato morbido, diretto da fibre allineate e rispondere agli fattore di crescita nervoso (NGF) 5-7. NC che può fornire segnali chimici per settimane hanno dimostrato di fornire una migliore recupero funzionale più vicina a quella di allotrapianti, il gold standard attuale per la riparazione dei nervi 8,9. Materiali e metodi di produzione Diversi possono essere utilizzati per produrre meccanica e topograficoAl spunti 10-13. Spunti meccanici sono inerenti al materiale scelto, rendendo la selezione del materiale appropriato per l&#39;applicazione critica 1,13. I metodi di produzione per il controllo spunti topografiche includono la separazione di fase, auto-assemblaggio e elettrospinning 1,13. Per le applicazioni microscala, microfluidica, photopatterning, incisione, liscivie sale, o schiume a gas può essere utilizzato anche 14-17. Electrospinning è emerso come il modo più popolare per progettare substrati fibrosi per coltura di tessuti grazie alla sua flessibilità e facilità di produzione 13,18-23. Nanofibre elettrofilate sono fabbricati applicando una tensione elevata ad una soluzione polimerica inducendolo a respingere se stessa e si estendono attraverso una breve distanza per scaricare 24. Un ponteggio allineato può essere creato raccogliendo le fibre su un mandrino rotante a terra e ponteggi non allineati sono raccolti su una piastra fissa 25. Segnalazione di adesione può essere ottenuto rivestendo il ponteggio spirito fibrosoh fibronectina o coniugare un peptide adesione, come RGD, per la prima HA elettrospinning 26. Segnali chimici, come i fattori di crescita, sono i più difficili da mantenere per lunghi periodi perché hanno bisogno di una fonte per il rilascio controllato. Molti sistemi sono stati tentato di aggiungere rilascio controllato a reti fibrosi elettrofilate con vari livelli di successo. Questi metodi comprendono miscela elettrofilatura, emulsione elettrofilatura, core shell elettrofilatura e proteine ​​coniugazione 27. Inoltre, elettrofilatura è tradizionalmente fatto in un solvente volatile, che può influenzare la vitalità della proteina 28, quindi mantenendo bioattività delle proteine ​​deve essere considerato. Questo approccio affronta specificamente che combina,, segnali chimici e adesivi topografici meccaniche per creare un&#39;impalcatura sintonizzabile per la crescita dei nervi periferici. Meccanica impalcatura è controllata proprio dalla sintesimetacrilato acido ialuronico (HA). I siti methacrylation vengono utilizzati per collegare foto reticolanti reattivi. Il materiale reticolato non è solubile in acqua ed è suddiviso esclusivamente dagli enzimi 29. La quantità di reticolazione cambia la velocità di degradazione, meccanica ed altre proprietà fisiche del materiale. Utilizzo di HA con il 30% methacrylation, che ha un modulo di elasticità di ~ 500 Pa, crea un substrato morbido che è vicino alla meccanica nativi di tessuto neurale ed è in genere preferito dai neuroni 26,29. Electrospinning su un mandrino rotante viene utilizzato per creare fibre allineate per una stecca topografica. Utilizzando elettrofilatura con microsfere fornisce segnali chimici all&#39;interno del ponteggio per periodi prolungati. Per supportare microsfere di crescita dei neuriti contenenti NGF sono utilizzati per creare il segnale chimico. Diversamente dalla maggior parte dei materiali elettrofilate HA è solubile in acqua in modo che il NGF non incontra solventi aggressivi durante la produzione. Per aggiungere un segnale di adesivo, la scaffold è rivestito con fibronectina. Il sistema completo contiene tutti e quattro i tipi di segnali sopra descritti: morbide (meccanici) allineati (topografiche) fibre con NGF rilascio microsfere (chimica) rivestito con fibronectina (adesivo). Produzione e collaudo di questo sistema è descritto in questo protocollo. Il processo inizia con la produzione delle microsfere con acqua-in-olio-in-acqua doppia emulsione. L&#39;emulsione è stabilizzato con un tensioattivo, alcool polivinilico (PVA). La fase acquosa interna contiene la proteina. Come è aggiunto alla fase olio, contenente il materiale delle coperture PLGA disciolto in diclorometano (DCM), il tensioattivo crea una barriera tra le fasi che proteggono la proteina dal DCM. Questa emulsione è più dispersa in un&#39;altra fase acquosa contenente PVA per creare la superficie esterna delle microsfere. L&#39;emulsione stabile viene agitata per consentire al DCM evaporare. Dopo il risciacquo e liofilizzazione si sono lasciati con la microsfere cont seccoaining proteina. Dopo le microsfere sono completati sono pronti per essere elettrofilate in ponteggi. Per prima cosa preparate la soluzione electrospinning. La viscosità della soluzione è fondamentale per la formazione di fibre corretta. Soluzioni di puro HA non soddisfano questo requisito; PEO viene aggiunto come un polimero portante per consentire electrospinning. Le microsfere vengono aggiunti alla soluzione e elettrofilate conseguente un&#39;impalcatura fibrosa con microsfere distribuiti. Una volta che la produzione è completa, la proteina deve essere testato per verificarne la fattibilità. A tale scopo, una cellula primaria che risponde a NGF può essere utilizzato. Questo protocollo utilizza dei gangli dorsali (DRG) 8-10 giorni antichi embrioni di pollo. I fasci di cellule vengono seminate su scaffold contenenti microsfere riempite di NGF o quelli che sono vuoti. Se il NGF è ancora vitale si dovrebbe vedere una maggiore crescita dei neuriti sui ponteggi contenenti NGF. Se il NGF non è più praticabile lo faràNon promuovere neuriti per estendere e dovrebbe apparire simile al controllo. La procedura esatta qui descritta è focalizzata sul sostegno neurale, però, con semplici modifiche al materiale, metodo elettrospinning e proteine ​​del sistema può essere ottimizzato per i vari tipi di cellule e tessuti.

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DAPI</td>
			<td style="width:208px;">Invitrogen</td>
			<td style="width:139px;">D1306</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Irgacure 2959</td>
			<td style="width:208px;">BASF</td>
			<td style="width:139px;">24650-42-8</td>
			<td style="width:291px;">Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PEO 900 kDa</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">189456</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Methacryloxethyl thiocarbamoyl rhodamine B</td>
			<td style="width:208px;">Polysciences, Inc.</td>
			<td style="width:139px;">23591-100</td>
			<td style="width:291px;">Prepare stock solution in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe Pump</td>
			<td style="width:208px;">KD Scientific</td>
			<td style="width:139px;">KDS100</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Power Source</td>
			<td style="width:208px;">Gamma High Voltage</td>
			<td style="width:139px;">ES30P-5W</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Motor</td>
			<td style="width:208px;">Triem Electric Motors, Inc</td>
			<td style="width:139px;">0132022-15</td>
			<td style="width:291px;">Must attach to a custom built mandrel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tachometer</td>
			<td style="width:208px;">Network Tool Warehouse</td>
			<td style="width:139px;">ESI-330</td>
			<td style="width:291px;">Use to monitor mandrel speed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Omnicure UV Spot Cure System with collimating adapter</td>
			<td style="width:208px;">EXFO</td>
			<td style="width:139px;">S1000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Needles</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">14-825-16H</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coverslips</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">12-545-81</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Polyvinyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Isoporopyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">I9030-500mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">BP9703-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">BSA-FITC</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">080M7400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">&#946;-Nerve Growth Factor (NGF)</td>
			<td style="width:208px;">R&#38;D Systems</td>
			<td style="width:139px;">1156-NG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">65:35 Poly-Lactic-Glycolic-Acid (PLGA)</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001554270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dichloromethane</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">34856-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:208px;">Thermo Scientific</td>
			<td align="right" style="width:139px;">1856209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">3-(Trimethoxysilyl)propyl methacrylate</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001558456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fibronectin</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM</td>
			<td style="width:208px;">Lonza</td>
			<td style="width:139px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">FBS</td>
			<td style="width:208px;">Atlanta Biologicals</td>
			<td style="width:139px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PBS</td>
			<td style="width:208px;">Hyclone</td>
			<td style="width:139px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glutamine</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pen-Strep</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Paraformaldehyde</td>
			<td style="width:208px;">Alfa Aesar</td>
			<td style="width:139px;">A11313&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Microsfere 50 ± 14 micron di diametro, con un incapsulamento di proteine ​​oltre l&#39;85% sono stati costantemente prodotto e elettrofilate in ponteggi. Dimensione è stata determinata mediante l&#39;imaging campioni di microsfere da tre lotti di produzione separate. Le immagini catturate in cui su un microscopio ottico e lunghezze dove misurata utilizzando software laboratorio commerciale. Figura 1 mostra un istogramma della distribuzione dimensionale. Tasso di incapsulamento è stato testato anc...

Watch this Scientific Journal Video about Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds at JoVE.com

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

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, ...

electrospinning-growth-factor-releasing-microspheres-into-fibrous

Research

JoVE Journal

Bioengineering

12.2K Views.  Wayne State University. 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.

Video: Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

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. ...

Ricerca

Bioingegneria

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 ...

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

Axon crescita Stretch: Il meccanotrasduzione di crescita neuronale

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

Consegna dei geni e trasfezione in cellule umane di cancro al pancreas con Epidermal Growth Factor Receptor mirati Nanovectors Progettato a base di gelatina

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

In situ carico di compressione e Correlativo imaging non invasiva del Bone-parodontale legamento-dente Fibrosa comune

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

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

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

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

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

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 ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

Studiare la Stechiometria di Epidermal Growth Factor Receptor in cellule intatte con correlativa Microscopia

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

Polielettrolita complesso per eparina vincolante fattore di crescita Osteogenica dominio di consegna

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Preparazione e caratterizzazione di novel nanoparticelle che imitano HDL per l&#39;incapsulamento del fattore di crescita del nervo

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

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 ...