Name: wet-spinning-based molding process of gelatin for tissue regeneration
Uploaded: 2019-03-07T14:15:00
Description: Watch this Scientific Journal Video about Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration at JoVE.com

Questo studio è stato sostenuto dal Ministero della difesa nazionale (MAB-105-070; MAB-106-077; MAB-107-032; MAB-107-065), il Ministero della scienza e della tecnologia (la maggior parte 107-2320-B016-016), Tri-Service General Hospital, la Nazionale Difesa centro medico, Taiwan (TSGH-C106-046; TSGH-C106-115; TSGH-C107-041) e Cheng-Hsin General Hospital e centro medico della difesa nazionale cooperazione (CH-NDMC-107-8).

I tessuti grassi sono stati ottenuti da interventi chirurgici ortopedici, come certificato da istituzionale Review Board di Tri-Service ospedale generale, Taipei, Taiwan, r.o.c. procedure che coinvolgono soggetti animali sono state approvate dal comitato di cura degli animali a livello nazionale Centro medico della difesa, Taiwan (R.O.C).
1. Inumidire il processo di filatura
<ol><li>
Preparazione della soluzione
	<ol>
<li>Sciogliere 5 g di gelatina in polvere in 100 mL di ac...

<ol>
	<li>Bagnall, A. M., Jones, L., Duffy, S., Riemsima, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+fixation+surgery+for+acute+traumatic+spinal+cord+injury.">Spinal fixation surgery for acute traumatic spinal cord injury.</a> Cochrane Database of Systematic Reviews. 1, 004725 (2008).</li><li>Fehlings, M. G., Perrin, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+and+timing+of+early+decompression+for+cervical+spinal+cord+injury:+update+with+a+review+of+recent+clinical+evidence.">The role and timing of early decompression for cervical spinal cord injury: update with a review of recent clinical evidence.</a> Injury. 36, 13-26 (2005).</li><li>Yang, L., Jones, N. R., Stoodley, M. A., Blumbergs, P. C., Brown, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxic+model+of+post-traumatic+syringomyelia+in+the+rat.">Excitotoxic model of post-traumatic syringomyelia in the rat.</a> Spine. 26, 1842-1849 (2001).</li><li>Rolls, A., 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=Two+faces+of+chondroitin+sulfate+proteoglycan+in+spinal+cord+repair:+a+role+in+microglia/macrophage+activation.">Two faces of chondroitin sulfate proteoglycan in spinal cord repair: a role in microglia/macrophage activation.</a> PLoS Medicine. 5, 1262-1277 (2008).</li><li>Properzi, F., Asher, R. A., Fawcett, 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=Chondroitin+sulphate+proteoglycans+in+the+central+nervous+system:+changes+and+synthesis+after+injury.">Chondroitin sulphate proteoglycans in the central nervous system: changes and synthesis after injury.</a> Biochemical Society Transactions. 31, 335-336 (2003).</li><li>Fawcett, J. W., Asher, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+scar+and+central+nervous+system+repair.">The glial scar and central nervous system repair.</a> Brain Research Bulletin. 49, 377-391 (1999).</li><li>Yang, Z., Mo, L., Duan, H., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chitosan/collagen+substrates+on+the+behavior+of+rat+neural+stem+cells.">Effects of chitosan/collagen substrates on the behavior of rat neural stem cells.</a> Science China Life Sciences. 53, 215-222 (2010).</li><li>Chawla, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fibrous Materials. , (1998).</li><li>Pickering, K. L., Aruan Efendy, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+recent+developments+in+natural+fibre+composites+and+their+mechanical+performance.">A review of recent developments in natural fibre composites and their mechanical performance.</a> Composites Part A-Applied Science and Manufacturing. 83, 98-112 (2016).</li><li>Lundgren, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+fibers+made+from+proteins.">Synthetic fibers made from proteins.</a> Advances in Protein Chemistry. 5, 305-351 (1954).</li><li>Radishevskii, M. B., Serkov, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coagulation+mechanism+in+wet+spinning+of+fibres.">Coagulation mechanism in wet spinning of fibres.</a> Fibre Chemistry. 37, 266-271 (2005).</li><li>Yannas, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+and+gelatin+in+the+solid+state.">Collagen and gelatin in the solid state.</a> Journal of Macromolecular Science Part C Polymer Reviews. 7, 49-106 (1972).</li><li>Baer, E., Cassidy, J. J., Hiltner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+structure+of+collagen+composite+Systems:+lessons+from+biology.">Hierarchical structure of collagen composite Systems: lessons from biology.</a> Pure and Applied Chemistry. 6, 961-973 (2009).</li><li>Harrington, W. F., Von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+collagen+and+gelatin.">The structure of collagen and gelatin.</a> Advances in Protein Chemistry. 16, 1-138 (1961).</li><li>Veis, A. <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 Macromolecular Chemistry of Gelatin. , (1994).</li><li>Freyman, T. M., Yannas, I. V., Gibson, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+materials+as+porous+scaffolds+for+tissue+engineering.">Cellular materials as porous scaffolds for tissue engineering.</a> Progress in Materials Science. 46, 273-282 (2001).</li><li>Michalczyk, K., Ziman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nestin+structure+and+predicted+function+in+cellular+cytoskeletal+organization.">Nestin structure and predicted function in cellular cytoskeletal organization.</a> Histology and Histopathology. 20, 665-671 (2005).</li></ol>

Abbiamo presentato lo sviluppo di biomateriali a base di gelatina utilizzando un semplice wet spinning tecnica può essere applicata nello studio dei polimeri naturali per la rigenerazione tissutale. Questo lavoro ha dimostrato la possibilità di fabbricazione di gelatina come fonte proteica grande senza l'aggiunta di altre fonti, con l'obiettivo di ottimizzare le proprietà della gelatina stessa. Lo sviluppo di biomateriali a base di gelatina è stato interamente effettuato a temperatura ambiente (22-26 ° C). Una prepa...

L'ultimo sviluppo nella rigenerazione del tessuto comporta l'applicazione dell'ingegneria tissutale, che rappresenta una sfida per il miglioramento di nuove strategie terapeutiche nei trattamenti medici. Ad esempio, il limitato potenziale di rigenerazione del sistema nervoso, seguenti lesioni o malattia, pone un problema significativo per la salute in tutto il mondo. A causa della complessità dei processi fisiopatologici associati con il sistema nervoso, l'uso di autoinnesto tradizionale o l'esecuzione di chirurgia di stabilizzazione ha dimostrato di offrire benefici in esiti funzionali, ma non ci è prova forte per gli effetti della fissazione spinale Chirurgia1,2. Il tessuto presso l'area danneggiata è perso e sostituito con hypertrophically indotto astrociti3, alla fine formano un denso cicatrice gliale4,5. Questa matrice agisce come una barriera che blocchi il recupero del nervo funzione6,7 ed è, quindi, ostacola notevolmente rigenerazione. Pertanto, si prevede un materiale di divario di riempimento adatto per prevenire la perdita di tessuto e ridurre la formazione di cicatrice-collegato del tessuto connettivo, mantenendo l'integrità della zona danneggiata, nonché offrendo la sostituzione diretta delle cellule neurali e circuiti per promuovere la rigenerazione dell'assone.
Biomateriali polimerici sono state preferite come scaffold per la terapia di rigenerazione del tessuto, sulla base del regolamento della cella o dell'assone comportamento e tessuto progressione attraverso il sostegno naturale della matrice extracellulare (ECM). Il formato della fibra è comunemente considerato come un blocco di costruzione per vari materiali, a causa della sua struttura unidimensionale8. Le fibre si ottengono generalmente dei melt estrusione o wet spinning metodo; Tuttavia, le grandi dimensioni e il costo dell'apparecchiatura e la difficoltà di eseguire questi metodi sono impegnativi. Inoltre, la maggior parte del lavoro relazionato a fibre di polimeri è stata concentrata su materiali sintetici o compositi. Polimeri naturali come fonte di biomateriale offrono migliori proprietà di biocompatibilità per il corpo umano. Tuttavia, per ottenere l'allineamento delle fibre di polimeri naturali è relativamente più difficile che di polimero sintetico fonti9. Quindi, la conversione di un polimero naturale come una ricca fonte di proteine in fibre di biomateriale è una strategia importante — non solo possono essere direttamente le fibre di biomateriale isolato dalla materia, evitando così una trasformazione inutile ai monomeri, ma la fibre di proteine hanno anche un bell'aspetto e caratteristiche favorevoli10.
A questo proposito, descriviamo un metodo di trattamento economico per la produzione di fibre di polimeri naturali attraverso il concetto di base della filatura bagnata, che può essere implementata su scala di laboratorio per l'ingegneria tissutale. Filatura bagnata avviene mediante estrusione e la coagulazione di una soluzione di polimero in un nonsolvent di polimero adatto. Una soluzione appropriata, viscosa drogata nel mezzo di coagulazione provoca le molecole di polimero a dissolversi. Attraverso la transizione di fase, i filamenti quindi perdono la loro solubilità e sono precipitati sotto forma di un polimero solido fase11. Riferendosi a questo concetto, abbiamo quindi ampliato lo sviluppo della gelatina in forma tubo tramite un processo di stampaggio, che è considerato adeguato per l'applicazione di rigenerazione del tessuto. Inoltre, intrinsecamente, possiamo anche sviluppare qualsiasi forma di materiale dalle fibre di gelatina (ad es., condotto di gelatina arrotolato da parecchie fibre di gelatina), per altre applicazioni desiderate.
Gelatina, un polimero naturale biodegradabile, è formata da collagene denaturato ed idrolizzato, compreso qualsiasi stato semicristalline, amorfo o Tripla elicoidale di collagene12. È ben noto che il collagene è la proteina strutturale essenziale in tutti i tessuti connettivi di vertebrati e invertebrati13,14, che è simile alla struttura di proteina della ECM principale che induce la crescita del nervo e, contemporaneamente, sostituisce una grande quantità di glicosaminoglicani secernuta durante lesioni del midollo spinale. Pertanto, l'utilizzo della gelatina come fonte sarebbe un'ottima scelta per qualsiasi veicolo di intervento medico. Oltre ad essere una fonte economica, la gelatina è anche biodegradabile e citocompatibili e clinicamente dimostrato di essere un temporaneo difetto filler15. Sviluppato in una forma di tubo,, test in vitro e in vivo, descritti qui dimostrano che la gelatina ha un'eccellente biocompatibilità e idoneità per future applicazioni di tessuto. Coltivate con cellule staminali adipose umane, tubi di gelatina migliorano differenziamento in cellule progenitrici neurali utilizzando la macchiatura positiva nestina come un indicatore delle cellule neurali. Inoltre, la gelatina come materiale di riempimento gap, come prodotto dal metodo stabilito in questo studio, è previsto di essere gestibile e sicuro e di grande beneficio ingegneri di tessuto che stanno attualmente sviluppando correlativi polimeri naturali per la valorizzazione del tessuto strategie di rivitalizzazione.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Solution preparation:</td>
		</tr>
		<tr>
			<td>Gelatin type B (porcine)</td>
			<td>Ferak</td>
			<td>Art. -Nr. 10733</td>
			<td>500 g vial</td>
		</tr>
		<tr>
			<td>Wet spinning process:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>Model M312</td>
			<td>Minipuls*3</td>
		</tr>
		<tr>
			<td>Plastic tube connector</td>
			<td>World Precision Instruments</td>
			<td>14011</td>
			<td>1 box</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Sterican</td>
			<td>5A06258541</td>
			<td>26Gx1/2&#34;(0.45 x 12mm)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Ferak</td>
			<td>Art. -Nr. 00010</td>
			<td>2.5 L vial</td>
		</tr>
		<tr>
			<td>Polycaprolactone CAPA 6500</td>
			<td>Perstorp</td>
			<td>24980-41-4</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>Scharlau</td>
			<td>CL03421000</td>
			<td>1 L vial</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Peripheral venous catheter (Introcan Certo)</td>
			<td>B. Braun</td>
			<td>1B03258241</td>
			<td>24Gx3/4&#34;(0.7 x 19mm)</td>
		</tr>
		<tr>
			<td colspan="4">Morphology of the gelatin tube:</td>
		</tr>
		<tr>
			<td>Ion sputter coater machine&#160;</td>
			<td>Hitachi</td>
			<td>e1010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Scanning electron microscopy</td>
			<td>Hitachi</td>
			<td>S-3000N</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cultivation of cells on the gelatin tube:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>488625</td>
			<td>100 mL vial</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>923119</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium&#160;</td>
			<td>Gibco</td>
			<td>31600-034</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Keratinocyte-SFM medium</td>
			<td>Gibco</td>
			<td>10744-019</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>T25 culture flask</td>
			<td>TPP</td>
			<td>90025</td>
			<td>VENT type</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Falcon</td>
			<td>1209938</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Immunocytochemistry:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate-buffered saline</td>
			<td>Gibco</td>
			<td>654471</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Acetic acid glacial</td>
			<td>Ferak</td>
			<td>Art. -Nr. 00697</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>NP-40 surfactant (Tergitol solution)</td>
			<td>Sigma</td>
			<td>056K0151</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000-20</td>
			<td>20 mL vial, concentrate</td>
		</tr>
		<tr>
			<td>Nestin (primary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-23927</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse-fluorescein isothiocyanate (secondary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2099</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Anaspec</td>
			<td>AS-83218</td>
			<td>5 mL vial</td>
		</tr>
		<tr>
			<td>In vivo biocompatibility test:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tiletamine+zolazepam&#160;</td>
			<td>Virbac</td>
			<td>BC91</td>
			<td>5 mL vial</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer korea</td>
			<td>KR03227</td>
			<td>10 mL vial</td>
		</tr>
		<tr>
			<td>Ketoprofen</td>
			<td>Astar</td>
			<td>1406232</td>
			<td>2 mL vial</td>
		</tr>
		<tr>
			<td>Povidone-iodine solution</td>
			<td>Everstar</td>
			<td>HA161202</td>
			<td>4 L barrel</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>China Chemical &#38; Pharmaceutical</td>
			<td>18P909</td>
			<td>1 g vial</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

wet-spinning-based molding process of gelatin for tissue regeneration

Questo articolo presenta un metodo economico per fabbricare gelatina, come un polimero naturale, in fibre monofilamento o altre forme appropriate. Attraverso sul bagnato metodo di filatura, fibre di gelatina sono prodotte per estrusione liscia in un mezzo adatto coagulazione. Per aumentare la superficie funzionale di queste fibre di gelatina e la loro capacità di imitare le caratteristiche dei tessuti, la gelatina può essere modellato in una forma di tubo facendo riferimento a questo concetto. Esaminato dai test in vitro e in vivo, i tubi di gelatina dimostrano un grande potenziale per l'applicazione nell'ingegneria tissutale. Agendo come un materiale di riempimento adatto gap, gelatina tubi possono essere utilizzati per sostituire il tessuto nella zona danneggiata (ad esempio, nel sistema nervoso o cardiovascolare), nonché per promuovere la rigenerazione fornendo una sostituzione diretta delle cellule staminali e circuiti neurali. Questo protocollo fornisce una procedura dettagliata per la creazione di un biomateriale basato su un polimero naturale, e la sua attuazione è previsto per lo sviluppo del correlativi polimeri naturali, che aiutano a realizzare strategie di rivitalizzazione del tessuto di grande beneficio.

In questo studio, abbiamo sviluppato con successo la gelatina in fibre (Figura 2A) e tubi (Figura 2B, C) attraverso il concetto di filatura bagnata user-friendly. Questi materiali a base di gelatina possono essere utilizzati come qualsiasi strumento medico, a seconda delle loro forme. Considerando che la superficie funzionale e telaio di tali materiali sono più adatti per la rigenerazione tissut...

Watch this Scientific Journal Video about Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration at JoVE.com

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

Abbiamo sviluppato e descrivere un protocollo basato sul concetto di filatura bagnata, per la realizzazione di biomateriali basati su gelatina utilizzata per l'applicazione dell'ingegneria tissutale.

Processo di stampaggio basati su bagnato-filatura di gelatina per la rigenerazione tissutale

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

Questo protocollo migliora un concetto di base di filatura a umido per sviluppare un materiale da una singola striscia di fibra in varie strisce che possono essere utilizzate per qualsiasi applicazione medica preferita. Questa tecnica è semplice, economica e non richiede l'aggiunta di polimeri sintetici per la personalizzazione dei polimeri naturali in una risorsa proteica per materiali di interesse. Questa tecnica può essere estesa allo sviluppo di biomateriali per uccidere la terapia del freddo come il trattamento del sistema nervoso centrale o periferico, la sostituzione dei vasi sanguigni o la ricostruzione diretta delle vie urinarie.Questo metodo è destinato all'applicazione medica, inclusa una strategia per la nevralizzazione dei tessuti o la medicina rigenerativa. Questa tecnica può essere facilmente implementata dai principianti. Si noti che, dopo diverse ripetizioni di stampaggio, la qualità della soluzione deve sempre essere controllata per mantenere una formazione ottimale.Per formare il tubo rotante bagnato, immergere prima un catetere venoso periferico da 24 gauge per tre quarti di pollice in una soluzione di gelatina preparata al momento per tre secondi, seguita da immersione in soluzione di acetone preparata al momento per un minuto. Ripetere questi passaggi 20 volte. Dopo l'ultima immersione in acetone, lasciare asciugare il tubo stampato per cinque minuti a temperatura ambiente e utilizzare un emostato per trasferire con cura il tubo di gelatina dal catetere in una pipetta Pasteur in vetro con soluzione di clorometano policaprolattone al 2,5% per un'incubazione notturna sotto il cofano a temperatura ambiente.La mattina seguente, sterilizzare la gelatina per due ore sotto la luce ultravioletta prima di immergere brevemente il tubo nel 75% di etanolo. Quindi lavare il tubo due volte con il mezzo delle cellule staminali per rimuovere l'etanolo residuo. Per coltivare il tubo di gelatina con la coltura cellulare, sostituire l'estremità supinata delle cellule staminali adipose umane da un pallone da coltura con un millilitro dello 0,25% di tripside-EDTA per un'incubazione di 3,5 minuti a 37 gradi Celsius e 5% di anidride carbonica.Quando le cellule si sono staccate, arrestare la reazione con un millilitro di siero bovino fetale e trasferire la sospensione cellulare in un tubo di microcentrifugo. Raccogliere le cellule mediante centrifugazione e rimosogliere il pellet in un millilitro di mezzo di cellule staminali. Quindi posizionare il tubo di gelatina in un pozzo di una piastra di sei po 'contenente tre millilitri di mezzo di cellule staminali.Dopo aver contato le cellule, seminare quattro volte 10 al quarto delle cellule staminali adipose umane nel tubo per un'incubazione di due settimane nell'incubatore di coltura cellulare. Dopo aver confermato il livello appropriato di sedazione per mancanza di risposta al dito del dito in un topo Sprague Dawley femmina di otto settimane, radere i capelli dalla zona del trapezio e sterilizzare la pelle esposta con soluzione di povidone-iodio. Coprire il topo con tende chirurgiche sterili per ridurre la contaminazione batterica del sito chirurgico e utilizzare forbici chirurgiche per fare un'incisione cutanea superficiale lunga due centimetri.Posizionare il tubo di gelatina direttamente nello strato tra la fascia e il muscolo e utilizzare una sutura di nylon per chiudere la ferita. Quindi sterilizzare l'incisione con soluzione di povidone-iodio. Indurre il ratto attraverso un'iniezione intramuscolare di chetoprofene e cefalolina e mantenere l'animale in condizioni asettiche per sette giorni.Questo concetto di filatura bagnata di facile utilizzo può essere utilizzato per sviluppare gelatina in fibre e tubi. Le osservazioni morfologiche del tubo di gelatina mediante microscopia elettronica a scansione rivelano una superficie non molto liscia con un diametro interno superiore a 200 micrometri e uno spessore di circa 20 micrometri. La colorazione di un tubo di gelatina di coltura di cellule staminali adipose umane per l'espressione marcatore di cellule neurali dimostra una colorazione positiva per il marcatore insieme a più nuclei rilevati che suggeriscono che le cellule possono penetrare e aderire al tubo di gelatina e differenziarsi in cellule progenitrici neurali.L'impianto del tubo di gelatina nello strato di fascia conferma la sua sicurezza e biocompatibilità, poiché non si osserva alcuna indicazione di arrossamento, gonfiore o altri sintomi infiammatori all'interno del tessuto locale e il tubo è circondato da tessuto connettivo ben sviluppato. Durante il tentativo di questa procedura di due punti uno, garantire l'immersione dello stampo nel tempo di rivestimento specifico in ogni soluzione è molto importante per costruire una forma precisa del tubo. Seguendo questa procedura, i nostri esperimenti utilizzano il modello animale della malattia umana può essere eseguito per indagare con precisione l'esito dei biomateriali per la strategia di rigenerazione dei tessuti.Dopo il suo sviluppo, questa tecnica ha spianato la strada ai ricercatori nello sviluppo di semplici polimeri naturali correlativi in qualsiasi tipo di materiale per un'applicazione medica decisa. In condizioni di uso normale, la soluzione di policlorometano policaprolattone è considerata un pericolo ambientale minimo, ma non dimenticare di eseguire alcun passo con questa sostanza chimica in un armadio di biosicurezza appropriato.

wet-spinning-based-molding-process-of-gelatin-for-tissue-regeneration

Research

JoVE Journal

Bioengineering

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Espressione, isolamento e purificazione di solubili e insolubili Biotinylated proteine ​​per Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Ricerca

Bioingegneria

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Rapida e basso costo Prototipazione di dispositivi medici che usano 3D Stampo Stampato per stampaggio ad iniezione Liquid

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

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

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

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Caratterizzazione qualitativa della frazione acquosa da idrotermale Liquefazione di alghe Utilizzando 2D gascromatografia con tempo di volo spettrometria di massa

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Alternando microgel ibrida Gelatina di campo magnetico-Responsive per Controlled Drug uscita

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

Una perlina accoppiato e matrice di magnete per lo stampaggio di micropozzetti con geometrie variabili concavi

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Tessutale tridimensionale allineato Astrocyte reti per ricapitolare i meccanismi dello sviluppo e facilitare la rigenerazione del sistema nervoso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Modellazione della geometria delle colonie di cellule staminali embrionali umane su substrati conformi per controllare la meccanica a livello di tessuto

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Protocolli di biostampa 3D di Gelatin Abasecryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...