Questo lavoro è stato sostenuto da finanziamenti provenienti NHMRC e Stafford Fox Medical Foundation. Gli autori riconoscono l&#39;assistenza chirurgica di Sue McKay, Liliana Pepe, Anna Deftereos e Amanda Rixon del Medical Sperimentale e l&#39;Unità Chirurgia, Ospedale di St. Vincent, Melbourne. Il supporto è fornito anche dal Dipartimento del Governo dello Stato del Victoria di innovazione, l&#39;industria e il Programma delle infrastrutture di supporto operativi per lo sviluppo regionale.

I protocolli descritti qui sono stati approvati dal Comitato Etico Animal Hospital di Melbourne di San Vincenzo, in Australia, e sono state condotte in stretta aderenza alle linee guida Australian National sanità e ricerca medica del Consiglio. NOTA: due protocolli della camera sono descritti di seguito. I due modelli diversi e loro disegni camera specifici sono illustrati in Figura 1. Sezione (1) è realizzato in policarbonato (per il modello camera di ciclo di ratto artero). È cilindrico con diametro interno 13 mm e altezza 4 mm. Una finestra a un certo punto nel muro consente l&#39;accesso senza ostacoli per il peduncolo. Nel secondo modello (per flusso continuo ratto modello camera peduncolo), la camera è realizzata poliacrilici e è rettangolare (10 x dimensioni 8 x 4 mm 3 interne). Ha due aperture 1,5 mm sui lati opposti per accogliere l&#39;arteria femorale e la vena quanto trasgrediscono la camera. 1. Rat artero-Loop Modello Camera (One Chamber per animale) NOTA: Prima di iniziare l&#39;intervento chirurgico, assicurarsi che tutti gli strumenti siano stati correttamente sterilizzati. Allo stesso modo, assicurano il resto strumenti su asciugamani sterili e sono ad una distanza ragionevole dal campo operatorio per evitare la contaminazione durante la procedura. <ol><li> Preparazione del Animal per la chirurgia <ol><li> Utilizzare ratti peso di almeno 250 g per le grandi dimensioni delle navi per la realizzazione del ciclo artero. </li><li> Anestetizzare l&#39;animale con il 4% inalazione isoflurano. Corroborare un&#39;adeguata profondità dell&#39;anestesia valutando refrattarietà alla punta-pizzico. Dopo l&#39;anestesia, tenere l&#39;animale adeguatamente anestetizzato durante tutta la procedura con il 2% isoflurano. </li><li> Posto l&#39;animale in posizione supina su un blocco riscaldante e applicare il lubrificante sterile per gli occhi per prevenire l&#39;essiccamento durante l&#39;intervento chirurgico. </li><li> L&#39;utilizzo di un rasoio elettrico, la barba sia inguini e rimuovere i capelli con un pezzo di garza umida. </li><li> Preparare i siti chirurgici con clorexidina/ Soluzione di etanolo al 70% e drappeggio l&#39;animale con teli sterili. Somministrare una singola dose di Carprofen (5 mg / kg, per via sottocutanea) come analgesico. </li></ol></li><li> Raccolta di vena femorale Graft <ol><li> Utilizzando una lama # 15, fare un lungo 4 cm incisione cutanea sulla all&#39;inguine sinistro parallelo al legamento inguinale. Ciò espone il cuscinetto adiposo inguinale. </li><li> Tagliare il cuscinetto adiposo circonferenziale con le forbici lasciandola attaccata al suo peduncolo vascolare sulla base dei vasi epigastrici. </li><li> Utilizzando le forbici micro, liberare le aderenze del tessuto connettivo vaporosi tra la parete addominale e vasi femorali sottostanti. </li><li> Posizionare un divaricatore sulla parete addominale e tirare mediale. Ciò espone il legamento inguinale e tutta la lunghezza dei vasi femorali. </li><li> Utilizzando micro pinze e forbici curve sezionare la vena epigastrica e isolarlo dal suo grasso circostante tirando e taglio con delicatezza. Questa vena agisce come un tether nel costruire il ciclo. </li><li> usinG Micro pinze e micro forbici curve aprire la guaina perivascolare che contiene i vasi femorali e dei nervi tutta la strada dal legamento inguinale alla sua distale biforcazione al ramo epigastrica. </li><li> Utilizzando micro pinze, prendere la vena femorale per la sua avventizia e delicatamente separarlo dai tessuti circostanti e di accompagnamento delle arterie. Fare questo con una pinza micro e curve micro forbici rotonde punte tirando il tessuto a parte e taglio attraverso di essa. NOTA: Non afferrare tutto lo spessore della parete venosa come questo potrebbe causare traumi alla intima che lo rende incline alla trombosi. </li><li> rami laterali legare trovato durante la dissezione con 10/0 sutura in nylon o coagulano con un coagulatore bipolare. </li><li> Con la vena femorale completamente libero, legare la sua estremità prossimale e distale con 4/0 punti di sutura in seta. Assicurarsi di ottenere una vena innesto di almeno 10 mm di lunghezza e comprende circa 0.5 cm Lunghezza del ramo epigastrica per essere usato come un tether corda ragazzoper tenere l&#39;anello aperto nella camera. </li><li> Utilizzando pinze micro e micro forbici diritte, tagliare l&#39;avventizia dalla estremità del trapianto tirando e tagliando delicatamente. Questo può essere fatto anche in seguito, prima di anastomosi microchirurgiche. </li><li> Lavare l&#39;innesto della vena con soluzione fisiologica eparinizzata (10 U / ml di eparina) e lasciare riposare nella soluzione. Chiudere la ferita con funzionamento continuo 4/0 sutura di seta più due o tre ulteriori semplici punti interrotti. </li></ol></li><li> Creazione di arterovenose Loop e l&#39;impianto della Camera <ol><li> Ripetere i passaggi da 1.2.1 a 1.2.4 in esattamente allo stesso modo sull&#39;arto controlaterale. </li><li> Utilizzando micro pinze, sezionare e isolare sia l&#39;arteria epigastrica e la vena formare il cuscinetto adiposo circostante. Per fare ciò, tirando delicatamente il tessuto lontano da vasi </li><li> Utilizzando micro pinze, prendere l&#39;arteria femorale dal suo avventizia e liberarla dai tessuti circostanti. Fare questo con una pinza micro e curvo mic rotonda punteforbici ro tirando il tessuto a parte e taglio attraverso di essa. Legare o coagulare i suoi rami laterali. </li><li> Legare l&#39;arteria femorale e la vena distale alla nascita dei vasi epigastrici utilizzano 4/0 di seta di sutura. </li><li> Posizionare un morsetto prossimalmente su ciascuna arteria femorale e la vena. Utilizzando un forte dritto micro forbice, fare una trasversale pulito tagliato in ogni distale nave alla nascita dei rami epigastrico. Posizionare un contrasto di fondo di plastica sterile sotto i vasi. </li><li> Lavare le navi energicamente con abbondante soluzione salina eparinizzata finché tutto il sangue viene rimosso dal lume. </li><li> Portare l&#39;innesto della vena nel campo operatorio e rimuovere eventuali avventizia esubero dalle estremità delle navi di cui al punto 1.2.10, se necessario. </li><li> Eseguire entrambe le anastomosi microchirurgiche con 10/0 sutura in nylon. Anastomose l&#39;estremità prossimale della vena graft alla vena femorale e l&#39;estremità distale all&#39;arteria femorale. Questo permetterà al sangue di fluire fROM arterioso al lato venoso senza resistenza dalle valvole all&#39;interno del trapianto vena. NOTA: Assicurarsi che i vasi femorali e della vena trapianto resto nella loro posizione naturale, senza colpi di scena. </li><li> Controllare eventuali perdite in entrambi i siti anastomosi. Risolvere piccole perdite, che sembrano non pulsante il sangue che esce dal sito anastomotico, mettendo un piccolo pezzo di grasso sulla parte superiore e delicatamente compressione per 5-10 min. Grandi perdite pulsanti che rapidamente inondano l&#39;intero campo avranno bisogno di punti di sutura aggiuntivi. </li><li> Controllare la pervietà del ciclo artero-venosa. occlusione Gentle dell&#39;arteria femorale dovrebbe rendere termoretraibile mentre la stessa nella vena femorale dovrebbe engorge esso. </li><li> Posizionare la base della camera di ingegneria dei tessuti sotto il ciclo arterovenosa con quest&#39;ultimo riposo nella sua posizione naturale senza torsioni o piegature. </li><li> Fissare la base della camera al legamento inguinale e sottostante fascia muscolare con 6/0 punti di sutura in nylon. </li><li> Mettere il coperchio sopra ilbase in modo che i vasi femorali entrano nella camera attraverso una tacca (finestra nel lato della camera). Quando si chiude il coperchio, assicurarsi che cattura i rami epigastrico, tra la base e il coperchio della camera, che agiscono come pastoie di tenere l&#39;anello artero-venosa in posizione. </li><li> Chiudere la ferita con funzionamento continuo 4/0 sutura di seta più due o tre ulteriori semplici punti interrotti. </li><li> Lasciare che l&#39;animale per recuperare da anestesia su un blocco di riscaldamento. </li><li> Non lasciare l&#39;animale incustodito fino a quando non ha ripreso conoscenza sufficiente a mantenere decubito sternale. Allo stesso modo, non restituiscono un animale che ha subito un intervento chirurgico per la compagnia di altri animali fino alla completa guarigione. 24 ore più tardi, somministrare un&#39;altra dose singola di Carprofen (5 mg / kg, per via sottocutanea) come analgesico. </li><li> Trattare la ferita con attualità pomata antibiotica per 5 giorni. Se la ferita è aperta, anestetizzare l&#39;animale come al punto 1.1.2 tramite 1.1.5 e chiudere la ferita come in 1.3.14. Monitorare la salute di animali al giorno. Eutanasia l&#39;animale con una dose letale di iniezione lethabarb intraperitoneale (163 mg / kg a 0,25 ml per 23 ago G) se l&#39;animale mostra più di segni moderati di inacitivity, scarso appetito, perdita di peso e perdita di colore. </li></ol></li></ol> 2. flusso continuo Pedicle Camera (due camere per animale) <ol><li> Preparazione del Animal per la chirurgia <ol><li> Ripetere i passaggi 1.1.1 tramite 1.1.4. Due camere possono essere impiantati in entrambe le regioni all&#39;inguine di un singolo topo. </li></ol></li><li> Isolamento di vasi femorali e inserimento di sezione <ol><li> Ripetere i passaggi 1.2.1 tramite 1.2.8. </li><li> Con entrambi arteria e vena completamente liberata tessuti circostanti e loro rami ligato, portare la camera nel campo operatorio. </li><li> Mettere ciascuno dei vasi femorali intatte sulla corrispondente feritoia della base della camera assicurandosi che non vi siano torsioni o curvature. </li><li> Chiudere la camera da attaching il coperchio alla base. Chiudere la ferita utilizzando funzionamento continuo 4/0 sutura seta più di due o tre ulteriori semplici punti interrotti e permettere all&#39;animale di recuperare come precedentemente descritto. </li></ol></li></ol> 3. Raccolta delle Camere e la lavorazione dei tessuti <ol><li> Una volta punti di tempo dell&#39;esperimento (4-6 settimane post-impianto) vengono raggiunti, anestetizzare l&#39;animale come al punto 1.1.2 e 1.1.3 ripetere i passaggi attraverso 1.1.5. </li><li> Aprire la ferita con una lama # 15 e tagliare i tessuti con le forbici finché la camera è completamente esposto. </li><li> Esporre i vasi femorali prossimale al costrutto e di prova per la pervietà vascolare: occlude delicatamente il vaso con due microforceps, poi il latte il sangue in una direzione distale e, infine, rilasciare il forcipe prossimali. Se la nave si riempie di sangue nuovo, ciò conferma la pervietà. Legare i vasi femorali prossimalmente nel caso del ciclo artero ed entrambi prossimale e distale nel caso di the flow-through configurazione e rimuovere le camere con il tessuto contenente in blocco. </li><li> Alla fine dell&#39;esperimento, eutanasia l&#39;animale con una dose letale di iniezione lethobarb intraperitoneale (163 mg / kg in 0,25 ml per 23 ago G). </li><li> Fissare tessuti in paraformaldeide 4% a temperatura ambiente per 24 ore. Tessuti dividere in più sezioni trasversali (1-2 mm di spessore) e incorporare in cera di paraffina o composto ottimale temperatura di taglio per le sezioni di paraffina (5 micron) o sezioni congelate (10 micron), rispettivamente. 3,4,8,17,22, 24 </li><li> Eseguire routine di colorazione istologica come ematossilina ed eosina esaminare la morfologia generale dei tessuti. Eseguire la colorazione immunoistochimica con anticorpi specifici per identificare il tipo cellulare di interesse, 3,4,8,17,22,24,29 per T immunostaining esempio, la troponina cardiaca per cardiomiociti. </li></ol>

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K., Cassell, O. C., Kelly, J., Abberton, K. M., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+vascularized+organoid+using+skeletal+muscle+as+the+inductive+source.">Generation of a vascularized organoid using skeletal muscle as the inductive source.</a> FASEB J. 19 (11), 1570-1572 (2005).</li><li>Lim, S. Y., Hern&#225;ndez, D., Dusting, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+vascularized+heart+tissue+from+stem+cells.">Growing vascularized heart tissue from stem cells.</a> J Cardiovasc Pharmacol. 62 (2), 122-129 (2013).</li><li>Poon, C. 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=Preparation+of+an+adipogenic+hydrogel+from+subcutaneous+adipose+tissue.">Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.</a> Acta Biomater. 9 (3), 5609-5620 (2013).</li><li>Dilley, R. J., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularisation+to+improve+translational+potential+of+tissue+engineering+systems+for+cardiac+repair.">Vascularisation to improve translational potential of tissue engineering systems for cardiac repair.</a> Int J Biochem Cell Biol. 56, 38-46 (2014).</li><li>Lesman, A., Koffler, J., Atlas, R., Blinder, Y. J., Kam, Z., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vessel-like+networks+within+multicellular+fibrin-based+constructs.">Engineering vessel-like networks within multicellular fibrin-based constructs.</a> Biomaterials. 32 (31), 7856-7869 (2011).</li><li>Hussey, A. 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=Seeding+of+pancreatic+islets+into+prevascularized+tissue+engineering+chambers.">Seeding of pancreatic islets into prevascularized tissue engineering chambers.</a> Tissue Eng Part A. 15 (12), 3823-3833 (2009).</li><li>Chen, X., Aledia, A. S., Popson, S. A., Him, L., Hughes, C. C., George, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+anastomosis+of+endothelial+progenitor+cell-derived+vessels+with+host+vasculature+is+promoted+by+a+high+density+of+cotransplanted+fibroblasts.">Rapid anastomosis of endothelial progenitor cell-derived vessels with host vasculature is promoted by a high density of cotransplanted fibroblasts.</a> Tissue Eng Part A. 16 (2), 585-594 (2010).</li><li>Lin, R. Z., Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor-2+facilitates+rapid+anastomosis+formation+between+bioengineered+human+vascular+networks+and+living+vasculature.">Fibroblast growth factor-2 facilitates rapid anastomosis formation between bioengineered human vascular networks and living vasculature.</a> Methods. 56 (3), 440-451 (2012).</li><li>Dolderer, J. H., 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=Spontaneous+large+volume+adipose+tissue+generation+from+a+vascularized+pedicled+fat+flap+inside+a+chamber+space.">Spontaneous large volume adipose tissue generation from a vascularized pedicled fat flap inside a chamber space.</a> Tissue Eng. 13 (4), 673-681 (2007).</li><li>Wei, F. C., Lin Tay, S. K., Neligan, P. C., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+techniques+of+microvascular+surgery.">Principles and techniques of microvascular surgery.</a> Plastic Surgery. Volume 1. , 587-620 (2013).</li><li>Sekine, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+fabrication+of+functional+three-dimensional+tissues+with+perfusable+blood+vessels.">In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels.</a> Nat.Comm. 4 (1399), 1-10 (2013).</li><li>Lim, S. Y., Sivakumaran, P., Crombie, D. E., Dusting, G. J., P&#233;bay, A., Dilley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trichostatin+A+enhances+differentiation+of+human+induced+pluripotent+stem+cells+to+cardiogenic+cells+for+cardiac+tissue+engineering.">Trichostatin A enhances differentiation of human induced pluripotent stem cells to cardiogenic cells for cardiac tissue engineering.</a> Stem Cells Transl Med. 2 (9), 715-725 (2013).</li><li>Lim, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+tissue+engineering+chamber+supports+human+induced+pluripotent+stem+cell+survival+and+rapid+differentiation.">In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.</a> Biochem Biophys Res Commun. 422 (1), 75-79 (2012).</li><li>Piao, Y., Hung, S. S., Lim, S. Y., Wong, R. C., Ko, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+integration-free+human+induced+pluripotent+stem+cells+from+keratinocytes+by+simple+transfection+of+episomal+vectors.">Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.</a> Stem Cells Transl Med. 3 (7), 787-791 (2014).</li></ol>

Gli autori dichiarano interessi in gioco.

Ingegneria della microcircolazione è attualmente in fase di studio essenzialmente attraverso due approcci. La prima comporta lo sviluppo di una rete vascolare altamente interconnessa all&#39;interno del costrutto in vitro in modo che quando impiantato, capillari dal letto vascolare ospitante collegano con quelli del trapiantato costruire attraverso un processo chiamato inosculation, garantendo così la consegna di nutrienti non solo la periferia ma anche al nucleo. 21,32,33 Questo è chiamato pre-va...

Fabbricazione del tessuto vascolarizzato funzionale utilizzando un approccio di ingegneria dei tessuti è un paradigma emergente nel campo della medicina rigenerativa. 1,2 Molti approcci di ingegnerizzare tessuti nuovi e sani per la sostituzione di tessuto danneggiato o organi difettosi sono stati sviluppati, 3-6 sperimentalmente in modelli animali di piccole dimensioni con promettente potenziale clinico. 7,8 Tuttavia, vascolarizzazione rimane una delle grandi sfide per l&#39;ingegneria dei tessuti che limitano il potenziale per crescere tessuti di dimensioni clinicamente rilevante. 9 Gli attuali approcci per vascolarizzano tessuto seguire sia una via estrinseca in cui i nuovi vasi crescono da letto vascolare destinatario e invadono tutto il tessuto impiantato costruisce 10 o un percorso di vascolarizzazione intrinseca dove il sistema vascolare cresce e si espande in sintonia con il tessuto di recente sviluppo. 11 L&#39;approccio estrinseca coinvolge tradizionalmente le cellule di semina su una impalcaturain vitro e impiantare il costrutto completo nella animale vivente con l&#39;aspettativa che i nutrienti, in precedenza forniti da terreni di coltura, sarà acquistato dalla circolazione. 12,13 Il concetto è semplice come crescita interna vascolare è troppo lento e solo gli impianti molto sottili (&lt; di spessore 1-2 mm) rimane valida. Fornire sostanze nutritive e ossigeno per mezzo di una vascolarizzazione sufficiente e rapida è al centro di tutti i tentativi riusciti a crescere sostituti tissutale più complesse e più grandi, come ossa, muscoli, grasso e gli organi solidi. 14,15 vascolarizzazione intrinseca offre il potenziale per costrutti più grandi per lo sviluppo dalla crescita di tessuto progressiva compatibile con la sua espansione apporto di sangue. Un disegno è l&#39;impianto in vivo in una camera di un peduncolo vascolare con o senza un ponteggio seminato cellule. 5,6 Questo ha aperto la strada a nuove procedure per la generazione di tessuti intrinsecamente vascolarizzati spessi. 16,17 &lt;/ P&gt; Più recentemente, sono state sviluppate strategie di pre-vascolarizzano innesti di tessuto, prima dell&#39;impianto. Queste reti dei vasi sanguigni incorporate hanno lo scopo di inosculate con vasi di accoglienza presso l&#39;impianto consentono la rapida fornitura di un apporto di sangue completo per migliorare la sopravvivenza di tutte le parti di un innesto di tessuto spesso trapiantato. 18 Siamo stati pionieri di un vascolarizzato vivo modello in ingegneria dei tessuti nei piccoli animali che coinvolge una via sottocutanea impiantato semirigido camera chiusa contenente un peduncolo vascolare perfusione e biomateriali cellulari contenenti. La camera crea un ambiente ischemico che stimola sprouting angiogenico dai vasi impiantati. 3 Il peduncolo vascolare può essere un ciclo arterovenosa ricostruite o un&#39;arteria flusso continuo intatta e vena. 3-6,19 This vascolari germogli peduncolari un funzionamento ed estesa artero rete -capillary-venosa che collega sia l&#39;arteeriole e venoso si conclude con il peduncolo vascolare. 3,20, inoltre, la circostante camera di supporto cava protegge il tessuto in via di sviluppo da potenzialmente deformare forze meccaniche e prolunga l&#39;unità ischemico per migliorare la vascolarizzazione. 3,21,22 Se il peduncolo nave è semplicemente impiantato in tessuto normale e non all&#39;interno dello spazio protetto della camera, germinazione angiogenico cessa lungo la stessa timeline come una ferita normale e nessun nuovo tessuto si accumulano intorno al peduncolo. Gli investigatori hanno utilizzato questa configurazione in vivo per produrre costrutti di tessuto tridimensionali funzionali vascolarizzati con vascolarizzazione di sostegno e di dimensioni clinicamente rilevante. 4,23 Inoltre, i costrutti di tessuto vascolarizzate ingegnerizzati con il suo peduncolo vascolare intatto possono essere raccolti per il successivo trapianto presso il sito della lesione . 24,25 Uno scenario più clinicamente fattibile sarebbe la creazione della camera presso il sito definitivo per la ricostruzione sale come il petto. Così, questa de novo tessuto approccio ingegneristico potrebbe avere potenziale clinico di fornire una nuova fonte di tessuto bersaglio funzionale per la chirurgia ricostruttiva. 26-28 Il seguente protocollo fornirà una guida generale per costruire un vascolarizzati camera di ingegneria tissutale in vivo nel ratto, che potrebbe essere adattato in diversi modelli animali e impiegato per esaminare i processi complicati dell&#39;angiogenesi, produzione della matrice, e la migrazione e la differenziazione cellulare.

<table border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">1 15 Blade Scalpel</td>
			<td style="width:187px;">Braun</td>
			<td style="width:163px;">BB515</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Toothed Adson Forceps</td>
			<td>Braun</td>
			<td>BD527R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Needle Holder</td>
			<td>Braun</td>
			<td>BM201R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Bipolar Coagulator&#160;</td>
			<td>Braun</td>
			<td>US335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Needle Holder B-15-8.3</td>
			<td>S &#38; T</td>
			<td>00763</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Dilator Forceps D-5a.2</td>
			<td>S &#38; T</td>
			<td>00125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Jeweler&#39;s Forceps JF-5</td>
			<td>S &#38; T</td>
			<td>00108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Straight SAS-11</td>
			<td>S &#38; T</td>
			<td>00098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Curved SDC-11</td>
			<td>S &#38; T</td>
			<td>00090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 Single Clamps B-3</td>
			<td>S &#38; T</td>
			<td>00400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 10/0 nylon suture</td>
			<td>S &#38; T</td>
			<td>03199</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 6/0 nylon suture</td>
			<td>Braun</td>
			<td>G2095469</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 4/0 Silk Sutures</td>
			<td>Braun</td>
			<td>C0760145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xilocaine 1%</td>
			<td>Dealmed</td>
			<td>150733</td>
			<td>10 mg/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin Sodium</td>
			<td>Dealmed</td>
			<td>272301</td>
			<td>5000 UI / ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ringer Lactate</td>
			<td>Baxter</td>
			<td>JB2323</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 dome-shaped tissue engineering chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 flow-through chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin I, Griffonia Simplicifolia&#160;</td>
			<td>Vector Laboratories</td>
			<td>B-1105</td>
			<td>1.67 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Troponin T antibody</td>
			<td>Abcam</td>
			<td>Ab8295</td>
			<td>4 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human-specific Ku80 antibody</td>
			<td>Abcam</td>
			<td>Ab80592</td>
			<td style="width:176px;">0.06 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Desmin antibody</td>
			<td>Dako</td>
			<td>M0760</td>
			<td style="width:176px;">2.55 &#956;g/mL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Tracker CM-DiI dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C-7000</td>
			<td>3 mg/106 cells</td>
		</tr>
	</tbody>
</table>

tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber

Nella chirurgia ricostruttiva, vi è una necessità clinica di un&#39;alternativa ai metodi attuali di ricostruzione autologo che sono complessi, costosi e commercio un difetto per un altro. L&#39;ingegneria dei tessuti mantiene la promessa di affrontare questa crescente domanda. Tuttavia, la maggior parte delle strategie di ingegneria tissutale non riescono a generare sostituti stabili e funzionali dei tessuti a causa della scarsa vascolarizzazione. Questo documento si concentra su una ingegneria dei tessuti modello in vivo della camera di vascolarizzazione intrinseca in cui un&#39;arteria perfusione e una vena o come un ciclo artero o di una configurazione peduncolo a flusso continuo è diretto all&#39;interno di una camera cava protetta. In questo sistema camerale basato germinazione angiogenica verifica dai vasi arterovenose e questo sistema attrae migrazione cellulare endogena ischemica e infiammatoria motore che riempie progressivamente spazio della camera con tessuto fibro-vascolare. cellula esogena / matrice impianto al momento della costruzione della camera migliora sur cellularevivenza e determina la specificità dei tessuti ingegnerizzati che si sviluppano. I nostri studi hanno dimostrato che questo modello camera può generare con successo diversi tessuti come grasso, muscolo cardiaco, fegato e altri. Tuttavia, sono necessarie modifiche e perfezionamenti per garantire tessuto bersaglio formazione è coerente e riproducibile. Questo articolo descrive un protocollo standardizzato per la realizzazione di due differenti modelli a camera ingegneria tissutale vascolarizzate in vivo.

La creazione microchirurgico di camere ingegneria tissutale è stata eseguita come descritto nel protocollo di cui sopra. I tessuti generati all&#39;interno delle camere possono essere esaminate istologicamente come descritto nel passaggio protocollo 3. Vari tipi di tessuto sono stati progettati con successo utilizzando la camera vascolarizzata in vivo (Figura 2). Questi includono tessuto cardiaco con cardiomiociti neonatali di ratto (Figura 2A),</strong...

Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingegneria dei tessuti da parte intrinseca Vascolarizzazione in un<em&gt; In Vivo</em&gt; Camera ingegneria dei tessuti

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

tissue-engineering-intrinsic-vascularization-an-vivo-tissue

Research

JoVE Journal

Bioengineering

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

8.8K Views.  St Vincent's Institute of Medical Research. This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling. 

Video: Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

Fabbricazione di derivazione biologica Materiali iniettabili per l&#39;ingegneria tissutale miocardica

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Ricerca

Bioingegneria

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

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

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

PGS elastomerico Ponteggi in Ingegneria tessuto arterioso

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

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

L&#39;ingegneria dei tessuti del colon in un modello murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capillare Forza litografia per Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingegneria dei tessuti di un Umano 3D<em&gt; In vitro</em&gt; Sistema Tumore test

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

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. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparazione di Thermoresponsive Nanostrutturati Superfici Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

L&#39;isolamento di Murine adiposo derivate dal tessuto microvascolare Frammenti come vascolarizzazione Unità per Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Ingegneria dei tessuti da parte intrinseca Vascolarizzazione in un

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Capitoli in questo video

Fabbricazione di derivazione biologica Materiali iniettabili per l'ingegneria tissutale miocardica

PGS elastomerico Ponteggi in Ingegneria tessuto arterioso

L'ingegneria dei tessuti del colon in un modello murino

Capillare Forza litografia per Cardiac Tissue Engineering

Ingegneria dei tessuti di un Umano 3D

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

Ingegneria 3D Cellularized collagene Gel per Vascolare Rigenerazione Tissutale

Microambienti sandwich di sfruttare Cellulari / Materiale Interazioni

Preparazione di Thermoresponsive Nanostrutturati Superfici Tissue Engineering

L'isolamento di Murine adiposo derivate dal tessuto microvascolare Frammenti come vascolarizzazione Unità per Tissue Engineering