Name: implantation of electrospun vascular grafts with optimized structure in a rat model
Uploaded: 2018-06-27T13:00:00.000+00:00
Duration: 8 min 46 s
Description: Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

Questo lavoro è stato sostenuto finanziariamente da progetti NSFC (81522023, 81530059, 91639113, 81772000, 81371699 e 81401534).

L'uso di animali da esperimento è stato approvato dal animale esperimenti etico Comitato di Nankai University e svolte in conformità con la guida per la cura e l'uso di animali da laboratorio.
1. fabbricazione di innesti elettrofilate PCL
Nota: Nel presente documento, una tecnica di elettrofilatura è stata utilizzata per fabbricare gli innesti vascolari.
<ol><li>Preparare soluzioni PCL di 25 wt % e 10% in peso, sciogliendo PCL in una miscela di metanolo e cloroformio, rispettivamente, (rapporto volume 1:5), a temperatura ambiente (TA) per 12 h.</li>
	<li>Caricare la soluzione PCL in una siringa di vetro da 10 mL.</li>
	<li>Posizionare la siringa con un ago 21G.</li>
	<li>Posizionare il mandrino in acciaio inox (2 mm di diametro, 25 cm di lunghezza) lo strumento di raccolta.</li>
	<li>Per gli innesti più spessa-fibra, utilizzare la soluzione PCL del 25% in peso, la distanza di lavoro di 17 cm dalla punta dell'ago al collezionista, portata di 8 mL/h e la tensione di 11 kV come i parametri di elettrofilatura. Per gli innesti di diluente-fibra, utilizzare la soluzione PCL di 10% in peso, distanza di lavoro di 20 cm dalla punta dell'ago al collezionista, una portata di 2 mL/h e la tensione di 18 kV come i parametri di elettrofilatura.</li>
	<li>Assicurare che gli innesti ottenuti siano immessi nel vuoto durante la notte per rimuovere il solvente residuo. Sterilizzare tutti gli strumenti prima della procedura e mantenere asettiche.</li>
	<li>Prima dell'impianto, è necessario disinfettare gli innesti di immergendole in 10 mL di etanolo al 75% per 30 min e poi esporli ai raggi UV durante la notte.</li>
	<li>Misure di dimensione del poro e fibra: calcolare il diametro medio della fibra utilizzando software ImageJ basato sulla scansione immagini di microscopia elettronica (SEM).</li>
	<li>Prove meccaniche dei ponteggi:<ol>
<li>Tagliare i ponteggi tubolari in sezioni di 3 mm di lunghezza con una lama di rasoio. Misurare lo spessore degli scaffold utilizzando un micrometro.</li>
		<li>Posizionare i ponteggi tubolari su una macchina con una capacità di carico di 100 N. prove di trazione</li>
		<li>Fissare i ponteggi con un 1 mm di distanza inter-morsetto e tirare longitudinalmente ad un tasso di 10 mm/min fino alla rottura. Misurare la resistenza alla trazione e allungamento a rottura. Calcolare il modulo di Young della regione lineare iniziale (fino al 5% di deformazione) della curva sforzo-deformazione.</li>
	</ol>
</li>
</ol>2. ratto Aorta addominale l'impianto modello
Nota: Tutti i materiali e gli strumenti utilizzati nella chirurgia sono sterili. Durante l'intervento, assicurarsi che l'operatore indossa una maschera di garze e guanti sterili per evitare le infezioni. Garantire che la temperatura ambiente è mantenuta in 27-30 ° C per mantenere la temperatura del corpo animale. Seguire le linee guida locali IACUC per quanto riguarda l'analgesia.
<ol><li>Utilizzare i ratti maschii di Sprague Dawley pesano 240-270 g come destinatari dell'innesto vascolare. Garantire che il ratto ha digiunato 24 h prima dell'intervento chirurgico. Lo scopo del digiuno ratti per 24 h è a vuoto le feci nel tratto intestinale, sufficientemente, quindi allargare orizzonte dell'operatore.</li>
	<li>Sul retro del collo del ratto di afferrare e tenere la testa verso il basso, inserire l'ago di springe nella cavità addominale dell'addome più basso. Indurre il ratto per anestesia con cloralio idrato (330 mg/kg) tramite un'iniezione intraperitoneale.</li>
	<li>Confermare amputate adeguata facendo in modo che il ratto ha rilassato i muscoli e la respirazione costante. Posto il ratto sotto il microscopio operatorio in posizione supina.</li>
	<li>Applicare unguento oftalmico IFP petrolato sugli occhi per prevenire la secchezza mentre sotto anestesia. Amministrare l'anticoagulazione (100 UI/kg) con soluzione fisiologica eparinizzata (50 UI/mL) di iniezione della vena della coda prima della chirurgia.</li>
	<li>Radersi la pelliccia nella parete addominale anteriore con una lama di rasoio e pulire la pelle con soluzione di iodio e alcool medico soluzione.</li>
	<li>Eseguire un'incisione laparotomia del midline con forbici chirurgiche e garantire che l'incisione è circa 4-5 cm di lunghezza e quindi esporre la cavità addominale.</li>
	<li>Ritrarre e avvolgere l'intestino con una garza inumidita con soluzione fisiologica preferenzialmente.</li>
	<li>La dissezione dell'aorta addominale con attenzione.</li>
	<li>Identificare e legare tutti i piccoli rami utilizzando suture in nylon monofilamento di 9-0.</li>
	<li>Morsetto della sezione isolata (fino a 1 cm di lunghezza) dell'aorta utilizzando due pinze vascolari. L'aorta può rimanere bloccato per 20-30 min.</li>
	<li>Transetto dell'aorta addominale tra due morsetti utilizzando micro-forbici per creare luoghi anastomotic.</li>
	<li>Lavare le due estremità dell'aorta con soluzione fisiologica eparinata (50 UI/mL) per rimuovere i residui di sangue.</li>
	<li>Staccare il adventitia utilizzando micro-forbici.</li>
	<li>Anastomotizzare l'innesto con diametro interno di 2 mm e 1 cm di lunghezza per aorta addominale del ratto con un modello di figura di otto sutura con punti di sutura di 9-0 monofilamento in nylon.</li>
	<li>In primo luogo, costruire quattro anastomosi secondo la sequenza di 9, 3, 12 e 6 posizioni sul lato prossimale, quindi anastomotizzare i bordi tagliati in 4 punti di sutura tra due punti di sutura. Dopo aver terminato la sutura prossimale, suturare il lato distale con lo stesso metodo. Nota: Ogni punto è necessario affinché che il lato nativo è leggermente incorporato nell'innesto.</li>
	<li>Rimuovere il morsetto distale per permettere al sangue di fluire nell'innesto, quindi rimuovere il morsetto prossimale.</li>
	<li>Premere le estremità della sutura per fermare l'emorragia utilizzando un batuffolo di cotone sterile o una piccola garza-spugna. Premere per circa 3 minuti, finché l'emostasi.</li>
	<li>Restituire l'intestino nella cavità addominale.</li>
	<li>Sciacquare la cavità addominale utilizzando soluzione fisiologica tiepida con gentamicina (320 U/mL).</li>
	<li>Cucire la parete addominale utilizzando una sutura in Nylon 3-0 nello strato del muscolo e la pelle, rispettivamente.</li>
	<li>Luogo il ratto in una gabbia pulita ed asciutta e mettere un rilievo di riscaldamento sotto la gabbia per mantenere la temperatura corporea degli animali; quindi attendere che il topo a recuperare dall'anestesia. Partecipare all'animale fino a quando ha riacquistato coscienza sufficiente per mantenere decubito sternale.</li>
	<li>Dopo esso riprende conoscenza, mettere il ratto in una gabbia singola con cibo e acqua. Applicare iodio sulla ferita per prevenire l'infezione dopo l'intervento chirurgico. Tornare il ratto la compagnia di altri animali fino a quando non viene completamente ripristinata.</li>
	<li>Eutanasia ratti secondo le linee guida istituzionali a intervalli di tempo predeterminati.</li>
</ol>

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolds+in+vascular+regeneration:+current+status.">Scaffolds in vascular regeneration: current status.</a> Vasc Health Risk Manag. 11, 79-91 (2015).</li><li>Pektok, E., 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=Degradation+and+healing+characteristics+of+small-diameter+poly+(e-caprolactone)+vascular+grafts+in+the+rat+systemic+arterial+circulation.">Degradation and healing characteristics of small-diameter poly (e-caprolactone) vascular grafts in the rat systemic arterial circulation.</a> Circulation. 118 (24), 2563-2570 (2008).</li><li>Innocente, F., 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=Paclitaxel-eluting+biodegradable+synthetic+vascular+prostheses:+a+step+towards+reduction+of+neointima+formation?.">Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?.</a> Circulation. 120 (11 Suppl), S37-S45 (2009).</li><li>de Valence, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+bilayered+vascular+grafts+for+surgical+applicability+and+tissue+regeneration.">Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration.</a> Acta Biomater. 8 (11), 3914-3920 (2012).</li><li>Assmann, 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=Acceleration+of+autologous+in+vivo+recellularization+of+decellularized+aortic+conduits+by+fibronectin+surface+coating.">Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating.</a> Biomaterials. 34 (25), 6015-6026 (2013).</li><li>Hasan, 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=Electrospun+scaffolds+for+tissue+engineering+of+vascular+grafts.">Electrospun scaffolds for tissue engineering of vascular grafts.</a> Acta Biomater. 10 (1), 11-25 (2014).</li><li>Baker, B. 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=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29 (15), 2348-2358 (2008).</li><li>Wang, K., 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=Creation+of+macropores+in+electrospun+silk+fibroin+scaffolds+using+sacrificial+PEO-microparticles+to+enhance+cellular+infiltration.">Creation of macropores in electrospun silk fibroin scaffolds using sacrificial PEO-microparticles to enhance cellular infiltration.</a> Journal of Biomedical Materials Research Part A. 101 (12), 3474-3481 (2013).</li><li>Lee, B. L. 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=Femtosecond+laser+ablation+enhances+cell+infiltration+into+three-dimensional+electrospun+scaffolds.">Femtosecond laser ablation enhances cell infiltration into three-dimensional electrospun scaffolds.</a> Acta Biomaterialia. 8 (7), 2648-2658 (2012).</li><li>Rnjak-Kovacina, J., Weiss, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+the+pore+size+of+electrospun+scaffolds.">Increasing the pore size of electrospun scaffolds.</a> Tissue Eng Part B Rev. 17 (5), 365-372 (2011).</li><li>Zhong, S., Zhang, Y., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+large+pores+in+electrospun+nanofibrous+scaffolds+for+cellular+infiltration:+a+review.">Fabrication of large pores in electrospun nanofibrous scaffolds for cellular infiltration: a review.</a> Tissue Eng Part B Rev. 18 (2), 77-87 (2012).</li><li>Pham, Q. P., Sharma, U., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+poly(epsilon-caprolactone)+microfiber+and+multilayer+nanofiber/microfiber+scaffolds:+characterization+of+scaffolds+and+measurement+of+cellular+infiltration.">Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.</a> Biomacromolecules. 7 (10), 2796-2805 (2006).</li><li>Rnjak-Kovacina, J., 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=Tailoring+the+porosity+and+pore+size+of+electrospun+synthetic+human+elastin+scaffolds+for+dermal+tissue+engineering.">Tailoring the porosity and pore size of electrospun synthetic human elastin scaffolds for dermal tissue engineering.</a> Biomaterials. 32 (28), 6729-6736 (2011).</li><li>Wang, Z., 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+thick+fibers+and+large+pores+of+electrospun+poly(epsilon-caprolactone)+vascular+grafts+on+macrophage+polarization+and+arterial+regeneration.">The effect of thick fibers and large pores of electrospun poly(epsilon-caprolactone) vascular grafts on macrophage polarization and arterial regeneration.</a> Biomaterials. 35 (22), 5700-5710 (2014).</li><li>Hutcheson, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genesis+and+growth+of+extracellular-vesicle-derived+microcalcification+in+atherosclerotic+plaques.">Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques.</a> Nat Mater. 15 (3), 335-343 (2016).</li><li>Tara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-organized+neointima+of+large-pore+poly(L-lactic+acid)+vascular+graft+coated+with+poly(L-lactic-co-epsilon-caprolactone)+prevents+calcific+deposition+compared+to+small-pore+electrospun+poly(L-lactic+acid)+graft+in+a+mouse+aortic+implantation+model.">Well-organized neointima of large-pore poly(L-lactic acid) vascular graft coated with poly(L-lactic-co-epsilon-caprolactone) prevents calcific deposition compared to small-pore electrospun poly(L-lactic acid) graft in a mouse aortic implantation model.</a> Atherosclerosis. 237 (2), 684-691 (2014).</li></ol>

Gli autori non hanno nessun conflitto di interessi finanziario.

L'infiltrazione delle cellule è fondamentale per la rigenerazione e rimodellamento dell'innesto vascolare in vivo16. L'infiltrazione delle cellule limitato è spesso legato ai pori relativamente piccoli dell'innesto che ostacolano la migrazione delle cellule nella parete dell'innesto. Per risolvere questa difficoltà, abbiamo sviluppato un metodo modificato per preparare con grande poro struttura gli innesti vascolari elettrofilate PCL. In dettaglio, il formato del poro aumentato con l'a...

Gli innesti vascolari fatti da polimeri sintetici sono ampiamente utilizzati in clinica per la terapia delle malattie cardiovascolari (CPV). Purtroppo, nel caso di innesti vascolari di piccolo diametro (D < 6 mm) non sono presenti prodotti di successo disponibile dovuto la pervietà bassa innescata dalla velocità del flusso sanguigno ridotto, che spesso porta a trombosi, iperplasia intimale e altri le complicazioni1.
Ingegneria tissutale fornisce una strategia alternativa per realizzare evidenza a lungo termine e l'omeostasi basato su una rigenerazione vascolare impalcatura-guidata e ricostruzione. In dettaglio, l'innesto vascolare, come un modello tridimensionale, potrebbe fornire supporto meccanico e strutturale durante la rigenerazione del tessuto vascolare e influenza funzioni cellulari, tra cui l'adesione delle cellule, la migrazione, la proliferazione, e secrezione di matrice extracellulare2. Fino ad ora, sono stati valutati vari polimeri sintetici per applicazioni in ingegneria del tessuto vascolare. Tra questi polimeri, poly(ε-caprolactone) (PCL) è stato intensamente studiato a causa delle cellule di buona compatibilità e lento degrado che vanno da parecchi mesi a due anni3. Nel ratto dell'aorta modello4,5,6, innesti vascolari PCL elaborati da elettrofilatura espone eccellente integrità strutturale e la pervietà, l'invasione delle cellule come pure continuamente aumentato e neovascolarizzazione nella parete dell'innesto per fino a 6 mesi. Tuttavia, rimodellamento tissutale negativi, tra cui la regressione delle cellule e vasi capillari e calcificazione, inoltre sono stati osservati timepoints più a lungo, fino a 18 mesi.
Cellularization dell'innesto vascolare è un fattore chiave per determinare la rigenerazione tissutale e rimodellamento7. Elettrofilatura, come una tecnica versatile, è stato ampiamente impiegato per la preparazione di innesti vascolari con struttura fibrosa nano8. Purtroppo, la struttura di poro relativamente piccolo conduce spesso ad infiltrazione cellulare insufficiente nell'innesto vascolare elettrofilate, che limita la rigenerazione successiva del tessuto. Per risolvere questo problema, diverse tecniche sono state tentate per aumentare la dimensione dei pori e la porosità totale, tra cui il sale/polimero lisciviazione9,10, modificazione dell'apparato collettore, post-trattamento di radiazione laser11 , ecc. Infatti, la struttura degli innesti elettrofilate (tra cui il diametro della fibra, la dimensione dei pori e porosità) è collegato strettamente al trattamento condizioni12,13. Durante elettrofilatura, il diametro della fibra può essere facilmente controllato modificando i parametri, quali la concentrazione della soluzione polimerica, portata, tensione, ecc. 14 , 15, e di conseguenza, la dimensione dei pori e la porosità sono stati migliorati di conseguenza.
Recentemente abbiamo segnalato un innesto di elettrofilate PCL modificato con struttura macroporosa (fibre con diametro di 5-7 µm e pori di 30-40 µm). L'impianto in vivo mediante la sostituzione dell'aorta addominale del ratto ha mostrato alto tasso di pervietà, come pure buona rigenerazione endotelizzazione e muscolo liscio a 3 mesi post-intervento chirurgico16. Ancora più importante, nessun rimodellamento del tessuto negativo compreso regressione calcificazione e cella potrebbe essere osservato anche dopo un anno di impianto.

<table><tbody><tr><td>Poly(&amp;epsilon;-caprolactone) (PCL) pellets (M&lt;sub&gt;n&lt;/sub&gt;=80,000)</td><td>Sigma</td><td>704067</td><td></td></tr><tr><td>Methanol</td><td>Tianjin Chemical Reagent Company</td><td>1060</td><td></td></tr><tr><td>Alcohol</td><td>Tianjin Chemical Reagent Company</td><td>1083</td><td></td></tr><tr><td>Chloroform</td><td>Tianjin Chemical Reagent Company</td><td>A1007</td><td></td></tr><tr><td>Sucrose</td><td>Tianjin Fengchuan Company</td><td>2296</td><td></td></tr><tr><td>Triton X-100</td><td>Alfa Aesar</td><td>A16046</td><td></td></tr><tr><td>Sprague Dawley rats</td><td>Laboratory Animal Center of the Academy of Military Medical Sciences</td><td></td><td></td></tr><tr><td>Normal saline</td><td>Hebei Tiancheng Pharmaceutical company</td><td></td><td></td></tr><tr><td>Chloral hydrate</td><td>Tianjin Ruijinte chemical company</td><td>2223</td><td></td></tr><tr><td>Heparin sodium Injection</td><td>Tianjin Biochem Pharmaceutical company</td><td></td><td></td></tr><tr><td>Gentamycin Sulfate Injection</td><td>Jiangsu Lianshui Pharmaceutical company</td><td></td><td></td></tr><tr><td>Mouse anti-&amp;alpha;-SMA primary antibody</td><td>Abcam</td><td>ab7817</td><td></td></tr><tr><td>Mouse anti-smooth MYH primary antibody</td><td>Abcam</td><td>ab683</td><td></td></tr><tr><td>Rabbit polyclonal anti-rat elastin antibody</td><td>Abcam</td><td>ab23748</td><td></td></tr><tr><td>Rabbit anti-von Willebrand factor primary antibody</td><td>Abcam</td><td>ab6994</td><td></td></tr><tr><td>Goat anti-mouse IgG (Alexa Fluor 488)</td><td>Invitrogen</td><td>ab150117</td><td></td></tr><tr><td>Goat anti-rabbit IgG (Alexa Fluor 488)</td><td>Invitrogen</td><td>ab150077</td><td></td></tr><tr><td>5% normal goat serum</td><td>Zhongshan Golden bridge</td><td>ZLI9022</td><td></td></tr><tr><td>Hematoxylin and eosin (H&amp;amp;E)</td><td>Beijing leagene biotech</td><td>DH0006</td><td></td></tr><tr><td>Masson&#039;s trichrome</td><td>Beijing leagene biotech</td><td>DC0032</td><td></td></tr><tr><td>Verhoeff-van Gieson (VVG)</td><td>Beijing leagene biotech</td><td>DC0059</td><td></td></tr><tr><td>Von Kossa</td><td>Beijing leagene biotech</td><td>DS0003</td><td></td></tr><tr><td>Surgical sutures needles with thread,3-0 silk</td><td>Shanghai Jinhuan medical supplies company</td><td>G3002b</td><td></td></tr><tr><td>Surgical sutures needles with thread,9-0 silk</td><td>Shanghai Jinhuan medical supplies company</td><td>H901</td><td></td></tr></tbody></table>

implantation of electrospun vascular grafts with optimized structure in a rat model

Qui, presentiamo un protocollo per fabbricare macroporosa innesto vascolare di PCL e descrivere un protocollo di valutazione utilizzando un modello del ratto di sostituzione dell'aorta addominale. Gli innesti vascolari elettrofilate possiedono spesso relativamente piccoli pori, che limitare l'infiltrazione delle cellule in innesti e ostacolano la rigenerazione e rimodellamento delle neo-arterie. In questo studio, gli innesti vascolari PCL con fibre più spesse (5-6 µm) e più grandi pori (~ 30 µm) sono stati realizzati utilizzando una tecnica di lavorazione modificate. Le prestazioni a lungo termine dell'innesto è stata valutata tramite impianto in un modello di ratto dell'aorta addominale. Analisi di ultrasuono hanno mostrato che gli innesti rimasero brevetti senza aneurisma o stenosi che si verificano anche dopo 12 mesi dall'impianto. Struttura macroporosa migliorato il ingrowth delle cellule e così promosso tessuto rigenerato a 3 mesi. Ancora più importante, non c'era nessun segno di rimodellamento negativi, come la calcificazione all'interno della parete dell'innesto dopo 12 mesi. Pertanto, con modificate macroporosa elaborazione gli innesti vascolari elettrofilate PCL tenere potenziali per essere un sostituto dell'arteria per l'impianto a lungo termine.

Gli innesti PCL explanted a 3 mesi e 12 mesi postoperatorio e analizzati mediante tecniche istologiche standard per ematossilina ed eosina (H & E), Masson tricromica, Verhoeff-van Gieson (LCA), Von Kossa e immunofluorescenza che macchia per α-SMA, MYH, sindrome di Raynaud e l'elastina. Le immagini istologiche sono state prese utilizzando un microscopio dritto, e le immagini di immunofluorescenza sono state prese utilizzando un microscopio ﬂuorescenti.
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Watch this Scientific Journal Video about Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model at JoVE.com

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Impianto degli innesti vascolari elettrofilate con struttura ottimizzata in un modello del ratto

Qui, presentiamo un metodo di elettrofilatura modificate per fabbricare gli innesti vascolari PCL con fibre spesse ed i grandi pori e descrivere un protocollo per valutare l'efficacia in vivo in un modello del ratto di sostituzione dell'aorta addominale.

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

implantation-electrospun-vascular-grafts-with-optimized-structure-rat

Research

JoVE Journal

Biology

7.9K Views.  Nankai University. Qui, presentiamo un metodo di elettrofilatura modificate per fabbricare gli innesti vascolari PCL con fibre spesse ed i grandi pori e descrivere un protocollo per valutare l'efficacia in vivo in un modello del ratto di sostituzione dell'aorta addominale.

Video: Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

La formazione di una sofisticata tecnica microchirurgica: Interposizione della vena giugulare esterna innesto nella carotide comune in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Ricerca

Medicina

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

L&#39;impianto di ingegneria tessutale nel Cuore Rat

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Biologia

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

Tecnica chirurgica per l&#39;impianto di ingegneria tessutale vascolari Innesti e successive<em&gt; In Vivo</em&gt; Monitoraggio

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Bioingegneria

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generazione e innesto delle navi tissutale in un modello di topo

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

Atimico Rat modello per la valutazione di Engineered legamento crociato anteriore Innesti

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

L&#39;impianto di Vena cava inferiore Interposizione Graft in modello murino

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

Il arterovenosa (AV) loop in un modello di Piccoli Animali per studiare Angiogenesi e ingegneria dei tessuti vascolarizzati

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Innesto ingegnerizzato per la ricostruzione esofagea circonferenza nei ratti

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

Vena interposizione Modello: un modello adatto per studiare Bypass Graft pervietà

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

Impianto degli innesti vascolari elettrofilate con struttura ottimizzata in un modello del ratto

Riepilogo

Esplora Altri Video

Capitoli in questo video

La formazione di una sofisticata tecnica microchirurgica: Interposizione della vena giugulare esterna innesto nella carotide comune in Rats

L'impianto di ingegneria tessutale nel Cuore Rat

Tecnica chirurgica per l'impianto di ingegneria tessutale vascolari Innesti e successive

Generazione e innesto delle navi tissutale in un modello di topo

Atimico Rat modello per la valutazione di Engineered legamento crociato anteriore Innesti

L'impianto di Vena cava inferiore Interposizione Graft in modello murino

Il arterovenosa (AV) loop in un modello di Piccoli Animali per studiare Angiogenesi e ingegneria dei tessuti vascolarizzati

Innesto ingegnerizzato per la ricostruzione esofagea circonferenza nei ratti

Vena interposizione Modello: un modello adatto per studiare Bypass Graft pervietà

Impianto di innesto vascolare di ingegneria tissutale nell'arteria carotide di topo tramite tecnica della cuffia