Questo lavoro è stato sostenuto da sovvenzioni dal Cuore e Polmone Istituto Nazionale (R01 HL086582) e la Stem Cell Science Fund di New York (NYSTEM, Contract # &#160; C024316) per STA e DDS illustrazioni utilizzate in video JoVE sono state completate da John Nyquist; Medical Illustrator dalla State University di New York a Buffalo.

NOTA: Questo protocollo è stato approvato dalla cura e l&#39;uso degli animali Comitato presso la State University di New York a Buffalo. Preparazione 1. Pre-chirurgica <ol><li> Utilizzare pecore (Dorset croce, femmina, circa 1-3 anni con un peso di 40-60 kg) per il seguente studio. Somministrare ciclosporina A (200 mg / die), aspirina (975 mg / die), e coumadin (20-30 mg / die) per via orale, iniziando 3 giorni prima chirurgia e continua per tutta la durata di tutti gli studi. </li><li> Assicurarsi che la pecora ha digiunato 12 ore prima dell&#39;intervento chirurgico (alimentazione normale = 1,8 kg e 0,4 kg di fieno grano). </li><li> Sterilizzare tutte le forniture chirurgiche in autoclave a vapore a 121 ° C e 15 psi per 30 min. </li><li> Posizionare le seguenti voci di un paraformaldeide essiccatore 48 ore prima dell&#39;intervento chirurgico: 4 mm sonde flusso Doppler, 1 millimetro cristalli ultrasuoni, cateteri arteriosi, tubi di estensione, e 42 &quot;Tygon Tubing. NOTA: Una tabella di attrezzature mediche, necessarie perchirurgia e per la sterilizzazione sono elencati nella Tabella 1. Pre-posizionamento della strumentazione nel tubo Tygon in questo passaggio aiuterà risparmiare tempo durante l&#39;intervento chirurgico. <ol><li> Etichettare ogni estremità delle sonde, cateteri e cristalli come destra e sinistra, se applicabile. </li></ol></li></ol> 2. Operazione Chirurgica <ol><li> Indurre pecore per anestesia con diazepam (0,5 mg / kg) e ketamina (4 mg / kg) per via endovenosa (IV). In alternativa, utilizzare Telazol (4 mg / kg) IV. <ol><li> Eseguire intubazione oro-tracheale con diametro 8,5-10,0 mm interno ammanettato endotracheale tubo 7. </li><li> Somministrare anestesia inalatoria attraverso un circuito rirespirazione con un ventilatore marea di volume (7-10 ml / kg) o di un ventilatore regolata pressione (15-20 cm H 2 O). Utilizzare un vaporizzatore di precisione per amministrare isoflurano o sevoflurano ad una quantità adeguata (3% -4% inizialmente) per raggiungere un mezzo di profonda fase anestetico chirurgico. La concentrazione alveolare minima perpecora è di 1,4% o 1,9%, rispettivamente, 8. </li><li> Valutare profondità dell&#39;anestesia osservando risposta motoria agli stimoli, riflesso palpebrale, posizione degli occhi, e la frequenza cardiaca. Monitorare la saturazione di ossigeno nel sangue (95% -100%) con pulsossimetria, CO 2 concentrazione (45-55 mmHg) in gas espiratorio utilizzando capnografia e mantenere la temperatura corporea durante la procedura (38,5-39,5 ° C) con un auto regolata riscaldamento coperta. </li></ol></li><li> Shave lana tutto il collo della pecora, e più di un vena cefalica, con una lama # 40 su Clippers standard. Preparare la pelle di entrambi i siti per la chirurgia con il 70% di alcool isopropilico e il 7,5% betadine scrub garza saturi. Inizia con l&#39;alcol garza per facilitare la rimozione di oli per la pelle. Si alternano tra betadine garza e alcol garza tre volte. </li><li> Posizionare pecore sul tavolo operatorio in decubito dorsale sulla cima di una coperta riscaldamento. Passare un tubo orogastrico di medie dimensioni per consentire l&#39;espulsione passiva del contenuto dello stomaco. Estendere lacollo di pecora e utilizzare ammortizzazione di supporto secondo necessità per mantenere il posizionamento. <ol><li> Eseguire uno scrub asettica finale con il 7,5% betadine garza imbevuta e lasciar riposare per 5 minuti prima di un intervento chirurgico. </li></ol></li><li> Somministrare fluidi IV (Ringer lattato o 0,9% Saline) a 10 ml / kg / ora attraverso un catetere venoso posizionato nella vena cefalica. Somministrare antibiotici intraoperatorie e analgesia: la penicillina G procaina 6.600 U / kg per via intramuscolare (IM), gentamicina 1,6 mg / kg IM, e buprenorfina 0,005-0,01 mg IV o IM. </li><li> Fare un cm un&#39;incisione ~ 12 longitudinalmente sul collo linea mediana ventrale con cauterizzazione electro. Isolare sinistra e destra carotide arterie (~ 6 cm), rimuovendo il tessuto connettivo con una tecnica dissezione smussa. Legare e cauterizzare microvasi diramano dalle arterie carotidi per ridurre al minimo il sanguinamento. </li><li> Mantenere la sterilità utilizzando (un non-sterile) infermiera chirurgica per assistere con scavare tutto il cablaggio e tubazioni (sonda di mandata, fili di cristallo ad ultrasuonie il tubo del catetere) nello strato sottocutaneo della pelle. Utilizzare un trocar smussata, che esce attraverso l&#39;incisione chirurgica preparata al collo dorsolaterale. <ol><li> Reach sotto il telo sterile e girare la testa della pecora modo che il lato del collo può essere visualizzata sotto il telo. </li><li> Utilizzare un emostatico curvato da 8 cm a tunnel attraverso lo spazio sottocutaneo tra la linea mediana ventrale incisione del collo e il lato del collo. Aprire e chiudere il emostatico di sezionare senza mezzi termini uno spazio per i tubi ~ 1,5 centimetri di larghezza. Le punte della pinza emostatica dovrebbero risiedere a metà strada tra la testa e le spalle, circa 10 cm caudale verso destra o di sinistra. Girare il hemostat modo che le punte siano rivolte verso la pelle superficiale. </li><li> Reach sotto il telo sterile e fare un 1,5 centimetri incisione attraverso la pelle, sulle punte del emostato con una sterile # 11 lama. Visualizza le punte della pinza emostatica per confermare una chiara uscita attraverso la pelle. </li><li> Passare il tubo Tygon contenente unll cablaggio e il tubo attraverso il tunnel sottocutaneo. Tenere i cavi e tubi sopra il telo sterile. </li><li> Reach sotto il telo per rimuovere il tubo esterno Tygon dal collo, esponendo il cablaggio impiantato quando lascia il collo della pecora. Estrarre singole linee fuori per minimizzare il gioco nello spazio sottocutaneo. Lasciare una distanza sufficiente per fissare correttamente la strumentazione per l&#39;arteria. </li></ol></li><li> Inserire 4 millimetri sonde flusso Doppler su entrambe le arterie carotidi e ottenere una lettura iniziale (Figura 1). Somministrare 100 U / kg di eparina IV 30 min prima bloccaggio dell&#39;arteria. </li><li> Continuare somministrazione di eparina a 100 U / kg / ora fino alla fine dell&#39;intervento. Bloccare la carotide utilizzando non schiacciamento morsetti vascolari e asportare una porzione (circa 4 cm di lunghezza). La portata carotideo controlaterale aumenterà del 50% -100% per mantenere il flusso di sangue al cervello. NOTA: È possibile limitare tratto longitudinale rinculo del vaso nativo rimuovendosegmenti più brevi rispetto sostituiti e / o stiramento dell&#39;arteria nativa con vascolare morsetti per accorciare gap fino procedura anastomotica completo è completato. Questo aiuterà limitare la tensione sui singoli suture holding e l&#39;innesto impiantato. </li><li> Suturare la TEV in luogo utilizzando semplici punti interrotti con 7-0 prolina ethalloy doppia armato monofilamento sutura. Se necessario, applicare miorilassanti lisce vascolari quali papaverina (15 mg / ml) o nicardipina (1,25 mg / ml) per via topica alla vascolarizzazione nativa per evitare vasocostrizione che ostacolerebbe sutura anastomotica. NOTA: Iniziare suture con circa 1 mm di distanza. Questo può variare notevolmente da caso a caso. La composizione e lo spessore TEV influenzerà la distanza effettiva tra suture. Poiché lo spessore del tessuto nativo o TEV diminuisce, può essere necessario collocare le suture più vicini. <ol><li> Prima di ancoraggio quattro punti del TEV alla arteria nativa mettendo due s oppostetitches su entrambe estremità prossimale e distale (Figura 2 -Di). Tenere ogni ancoraggio insegnato usando emostatiche. NOTA: descrizioni prossimale e distale si riferiscono alla direzione del flusso di sangue attraverso il foglio. </li><li> Aggiungere 5-6 più suture sul lato superficiale sia l&#39;estremità prossimale e distale per iniziare l&#39;anastomosi. (Figura 2 -Pr). Contemporaneamente ruotare vascolare morsetti 180 gradi. </li><li> Ristabilire tensione sulle suture di ancoraggio. Aggiungi supplementare (5-6) interrotto suture di estremità prossimale e distale sul lato ruotato del TEV. </li></ol></li><li> Una volta che il TEV viene suturata saldamente in posizione, ruotarlo indietro alla posizione iniziale e rimuovere vascolare morsetti uno alla volta, morsetto distale. Lieve sanguinamento nei siti anastomosi è comune. Questo può naturalmente risolvere dopo alcuni minuti di rilascio morsetto e reclamping o richiedere il posizionamento di suture supplementari. Posizionare la sonda Doppler (Figura 3-FL) Sul nativa dell&#39;arteria prossimale al flusso di sangue che entra nel TEV e portata monitor. NOTA: Aspettatevi le portate della carotide sinistra e destra per equilibrare dopo circa 15 min. Se la portata sulla carotide con impiantato TEV costante gocce, è possibile che il TEV è coagulazione. Altre anomalie possibili per quanto riguarda il flusso possono essere attribuiti alla costrizione della prossimale arterie nativo o distale al TEV. In questo caso, l&#39;uso di ulteriori rilassante della muscolatura liscia vascolare può essere applicato, e il vaso nativo deve ritornare ad un tono basale dopo 30-60 min a seguito della chiusura del tessuto sul sito dell&#39;innesto. <ol><li> Se lo si desidera, asportare la carotide controlaterale e suturare nuovamente in sede come controllo &quot;Sham&quot;. Questo è più clinicamente rilevante che lasciare la carotide destra solo e soltanto di collegare la sonda flusso, cristalli ultrasonici, e il catetere. Se un controllo sham è voluto, fare questo prima di passare al punto 2.11. </li></ol></li><li> Suturare 1 millimetro cristalli ultrasuoni (Figura 3 -cr1 CR2) a lati opposti del TEV con 7-0 Proline. Filo di sutura attraverso la testa cristallo ultrasuoni e cucite solo allo strato superficiale del TEV. </li><li> Cateterizzare l&#39;arteria con una versione modificata 18 G catetere con una abbottonatura Teflon tessuto (Figura 3- Ca &amp; Figure 4A). Posizionare il distale del catetere per il TEV in tessuto arterioso nativo. <ol><li> Suturare la abbottonatura alla parete arteriosa con 5/0 Ethibond per controllare qualsiasi emorragia. Utilizzare cicloesanone aderire il tubo microbore al catetere che è stata lavata con soluzione salina. Utilizzare il tubo come una linea di estensione. </li><li> Utilizzare un G Luer stub adattatore 20 con una spina iniezione Surflo per sigillare l&#39;estremità del tubo esteriorizzato (Figura 4B). Per mantenere la pervietà del catetere, ottenere il volume di adescamento della linea e lavare con 10 ml di soluzione salina e poi 5.000 U / ml di eparina sodica iniettabile ogni 2-3 giorni. </li></ol></li><li> Registrare la distanza tra la sonda di flusso ei cristalli ultrasonici, così come la distanza tra la sonda di flusso e il catetere. Questa velocità dell&#39;onda di polso permetterà di calcolare in combinazione con il software. Se non sono necessari calcoli, non impiantare un catetere. </li><li> Ottenere una lettura intraoperatoria se lo si desidera per garantire tutto l&#39;hardware è impiantato funzionale (vedi sezione 3). </li><li> Fissare le linee impiantati e fili alla vicina muscolatura con 2/0 seta e un ago cono (Figura 3). <ol><li> Posizionare la sonda di flusso vascolare filo parallelamente alla nave, con la caudale sonda e il filo che si estende cranialmente, e poi fare una &quot;U-Turn&quot; verso la muscolatura laterale. Fissare il filo muscolatura adiacente, utilizzando 2-0 seta su un ago conico a due posizioni, in modo che la sonda filo o flusso non è in grado di provocare sollecitazioni sulla nave. Assicurarsi suture sono aderente ma non stringere troppo e strangulate musculature (Figura 3). </li><li> Suturare la linea di fili di cristallo e arteriosa catetere alla muscolatura laterale, consentendo ~ 1,5 cm slack, simile a passaggi precedenti per il fissaggio della sonda di flusso (Figura 3). </li><li> Gruppo tutti i cavi e le linee insieme e ancorare al muscolatura poco prima di uscire fuori attraverso tunnel sottocutaneo, simile a fasi precedenti. </li></ol></li><li> Chiudere il sito chirurgico con un Vicryl sutura 2-0 in livelli utilizzando un modello di sutura in esecuzione su plancia e sottocutaneo, in esecuzione materasso punto sulla pelle (plancia, ago non tagliare, la pelle, il taglio ago). Chiudere l&#39;1,5 centimetri un&#39;incisione al collo dorsale attorno i fili esteriorizzato e le linee con 2/0 Vicryl e un ago di taglio. </li><li> Inserire fili sonda flusso, linee, cateteri e fili di cristallo ultrasuoni in un sacchetto (10 cm x 10 cm) che viene suturata saldamente alla pelle della pecora (Figura 5 - dopo il recupero). </li><li> Gradualmente svezzare anestesia pecore offe il volume del ventilatore di marea quindi estubare le pecore quando respiro spontaneo è ripreso. Rimuovere il catetere venoso inserito nella vena cefalica e benda, se necessario. </li><li> Fasciatura al collo con tripla pomata antibiotica sulle incisioni, un pad Telfa, tratto rotolo di garza, e elasticon. </li><li> Amministrare analgesia postoperatoria: meglumina flunixin 2,2 mg / kg IM una volta durante il recupero, poi 1,1 mg / kg IM una volta al giorno per due giorni, buprenorfina 0,005-0,01 mg IV o IM due volte al giorno per un giorno. </li></ol> 3. In Vivo Monitoring <ol><li> Mettere le pecore nel carrello mobile per garantire una corretta moderazione. In questo modo le pecore a mantenere la calma e consapevole, senza compromettere l&#39;hardware. Potrebbe essere necessario per ambientarsi pecore al carrello 2 o 3 volte per 30 minuti prima di ottenere registrazioni di strumentazione. </li><li> Rimuovere tutti i cavi e le linee dalla busta e la connessione a dispositivi di monitoraggio. Collegare la sonda del flusso di un misuratore di portata, cristalli 1 millimetro ultrasuoni collegato a TRBBox -USB, e cateteri linee a trasduttori di pressione. Un diagramma di flusso di questa configurazione è fornito (Figura 6). </li><li> Calibrare le sonde di flusso e trasduttori di pressione prima di acquisizione dati. NOTA: A causa della variabilità potenziale tra le versioni del software e le differenze nelle apparecchiature utilizzate, le tarature e le impostazioni varieranno da caso a caso. </li><li> Utilizzare un oscilloscopio per mettere a punto la misura di cristallo Sonometrics, secondo il protocollo del produttore. </li><li> Registrare i dati utilizzando il software del computer (Figura 7). Tracce nella metà superiore della figura 7 in colore bianco corrisponde al TEV impiantato, mentre tracce nella metà inferiore di colore rosso corrisponde al Sham / nativo. Sia per il TEV e Sham la portata (ml / min), la pressione sanguigna arteriosa (mmHg) e il diametro (mm) vengono registrate vivo. </li><li> Record per almeno 1 minuto, senza disturbi. Esportare questi dati per un&#39;analisi più dettagliata. Dopo la registrazione, disconnect tutti i cavi e posto nuovamente dentro il sacchetto suturato sul collo della pecora. </li></ol>

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	<li>Goldman, 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=Long-term+patency+of+saphenous+vein+and+left+internal+mammary+artery+grafts+after+coronary+artery+bypass+surgery:+Results+from+a+Department+of+Veterans+Affairs+Cooperative+Study.">Long-term patency of saphenous vein and left internal mammary artery grafts after coronary artery bypass surgery: Results from a Department of Veterans Affairs Cooperative Study.</a> Journal of the American College of Cardiology. 44, 2149-2156 (2004).</li><li>Achouh, 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=Long-term+(5-+to+20-year)+patency+of+the+radial+artery+for+coronary+bypass+grafting.">Long-term (5- to 20-year) patency of the radial artery for coronary bypass grafting.</a> The Journal of Thoracic And Cardiovascular Surgery. 140, 73-79 (2010).</li><li>Conklin, B. S., Richter, E. R., Kreutziger, K. L., Zhong, D. S., Chen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+novel+decellularized+vascular+xenograft.">Development and evaluation of a novel decellularized vascular xenograft.</a> Medical Engineering &amp; Physics. 24, 173-183 (2002).</li><li>Zhu, C., 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=Development+of+anti-atherosclerotic+tissue-engineered+blood+vessel+by+A20-regulated+endothelial+progenitor+cells+seeding+decellularized+vascular+matrix.">Development of anti-atherosclerotic tissue-engineered blood vessel by A20-regulated endothelial progenitor cells seeding decellularized vascular matrix.</a> Biomaterials. 29, 2628-2636 (2008).</li><li>Quint, C., 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=Decellularized+tissue-engineered+blood+vessel+as+an+arterial+conduit.">Decellularized tissue-engineered blood vessel as an arterial conduit.</a> Proceedings of the National Academy of Sciences. 108, 9214-9219 (2011).</li><li>Kaushal, 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=Functional+small-diameter+neovessels+created+using+endothelial+progenitor+cells+expanded+ex+vivo.">Functional small-diameter neovessels created using endothelial progenitor cells expanded ex vivo.</a> Nat Med. 7, 1035-1040 (2001).</li><li>Galatos, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+Analgesia+in+Sheep+and+Goats.">Anesthesia and Analgesia in Sheep and Goats.</a> Veterinary Clinics of North America: Food Animal Practice. 27, 47-59 (2011).</li><li>Okutomi, T., Whittington, R. A., Stein, D. J., Morishima, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effects+of+sevoflurane+and+isoflurane+anesthesia+on+the+maternal-fetal+unit+in+sheep.">Comparison of the effects of sevoflurane and isoflurane anesthesia on the maternal-fetal unit in sheep.</a> J Anesth. 23, 392-398 (2009).</li><li>Swartz, D. D., Russell, J. A., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+fibrin-based+functional+and+implantable+small-diameter+blood+vessels.">Engineering of fibrin-based functional and implantable small-diameter blood vessels.</a> American Journal of Physiology - Heart and Circulatory Physiology. 288, H1451-H1460 (2005).</li><li>Niklason, L. 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=Functional+arteries+grown+in+vitro.">Functional arteries grown in vitro.</a> Science. 284, 489-493 (1999).</li><li>Dahl, S. L. 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=Readily+Available+Tissue-Engineered+Vascular+Grafts.">Readily Available Tissue-Engineered Vascular Grafts.</a> Science Translational Medicine. 3, 68ra69 (2011).</li><li>Wu, W., Allen, R. A., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast-degrading+elastomer+enables+rapid+remodeling+of+a+cell-free+synthetic+graft+into+a+neoartery.">Fast-degrading elastomer enables rapid remodeling of a cell-free synthetic graft into a neoartery.</a> Nature Medicine. 18, 1148-1153 (2012).</li><li>Saami, K. Y., Bryan, W. T., Joel, L. B., Shay, S., Randolph, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+an+endothelium+layer+after+preconditioning.">The fate of an endothelium layer after preconditioning.</a> Journal of vascular surgery : Official Publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter. 51, 174-183 (2010).</li><li>Ueberrueck, T., 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=Comparison+of+the+ovine+and+porcine+animal+models+for+biocompatibility+testing+of+vascular+prostheses.">Comparison of the ovine and porcine animal models for biocompatibility testing of vascular prostheses.</a> Journal of Surgical Research. 124, 305-311 (2005).</li><li>Labb&#233;, R., Germain, L., Auger, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+completely+biological+tissue-engineered+human+blood+vessel.">A completely biological tissue-engineered human blood vessel.</a> The FASEB Journal. 12, 47-56 (1998).</li><li>Samaha, F. J., Oliva, A., Buncke, G. M., Buncke, H. J., Siko, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinical+study+of+end-to-end+versus+end-to-side+techniques+for+microvascular+anastomosis.">A clinical study of end-to-end versus end-to-side techniques for microvascular anastomosis.</a> Plastic and Reconstructive Surgery. 99, 1109-1111 (1997).</li><li>Huang, 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=A+novel+end-to-side+anastomosis+technique+for+hepatic+rearterialization+in+rat+orthotopic+liver+transplantation+to+accommodate+size+mismatches+between+vessels.">A novel end-to-side anastomosis technique for hepatic rearterialization in rat orthotopic liver transplantation to accommodate size mismatches between vessels.</a> European Surgical Research. 47, 53-62 (2011).</li><li>Peng, H., Schlaich, E. M., Row, S., Andreadis, S. T., Swartz, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Ovine+ex+vivo+Arteriovenous+Shunt+Model+to+Test+Vascular+Implantability.">A Novel Ovine ex vivo Arteriovenous Shunt Model to Test Vascular Implantability.</a> Cells, Tissues, Organs. 195, 108 (2011).</li><li>Zilla, P., Bezuidenhout, D., Human, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prosthetic+vascular+grafts:+Wrong+models,+wrong+questions+and+no+healing.">Prosthetic vascular grafts: Wrong models, wrong questions and no healing.</a> Biomaterials. 28, 5009-5027 (2007).</li><li>Berger, K., Sauvage, L. R., Rao, A. M., Wood, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Healing+of+Arterial+Prostheses+in+Man:+Its+Incompleteness.">Healing of Arterial Prostheses in Man: Its Incompleteness.</a> Annals of Surgery. 175, 118-127 (1972).</li><li>Byrom, M. J., Bannon, P. G., White, G. H., Ng, M. K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+the+assessment+of+novel+vascular+conduits.">Animal models for the assessment of novel vascular conduits.</a> Journal of Vascular Surgery : Official Publication, the Society for Vascular Surgery [and] International Society for Cardiovascular Surgery, North American Chapter. 52, 176-195 (2010).</li><li>Swartz, D. D., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+vascular+tissue-engineering.">Animal models for vascular tissue-engineering.</a> Current Opinion in Biotechnology. 24, 916-925 (2013).</li><li>Liang, M. -. S., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+fibrin-binding+TGF-&#946;1+for+sustained+signaling+and+contractile+function+of+MSC+based+vascular+constructs.">Engineering fibrin-binding TGF-&#946;1 for sustained signaling and contractile function of MSC based vascular constructs.</a> Biomaterials. 32, 8684-8693 (2011).</li><li>Han, J., Liu, J. Y., Swartz, D. D., Andreadis, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+functional+effects+of+organismal+ageing+on+smooth+muscle+cells+derived+from+bone+marrow+mesenchymal+stem+cells.">Molecular and functional effects of organismal ageing on smooth muscle cells derived from bone marrow mesenchymal stem cells.</a> Cardiovascular Research. 87, 147-155 (2010).</li></ol>

There are no disclosures to report.

Lo scopo di questo rapporto è quello di fornire una procedura affidabile e riproducibile di TEV impianto di interesse nella carotide ovine. Le arterie carotidi native degli animali utilizzati in questo modello sono stati 0,5-0,75 mm di spessore e 4,5-5 mm di diametro esterno. La tecnica chirurgica qui descritta ha avuto successo per l&#39;impianto di TEV di varie geometrie di misura 0,25-1 mm di spessore, 4-5 mm di diametro esterno e 4 cm di lunghezza con grande successo dimostrando efficace fino a 3 mesi di durata, il...

L&#39;obiettivo principale per lo sviluppo di TEV è stato quello di fornire un sostituto per la sostituzione del trapianto autologo quando vasi autologhi non sono disponibili e limitare donatori di vista morbilità. Ad esempio, il numero di interventi di bypass coronarico all&#39;anno ha superato 350.000 negli Stati Uniti, e la fonte ideale di opportuni innesti rimane fianco arteria mammaria interna, discendente anteriore dell&#39;arteria coronaria e safena 1. Dal momento che molte persone che soffrono di malattie vascolari non possono avere le arterie e le vene adatte per la sostituzione del trapianto autologo, lo sviluppo di TEV è così diventato un campo di intensa ricerca da decenni 1-6. Mentre la progettazione e l&#39;ottimizzazione di nuovi TEV hanno subito molti progressi, relazioni sulle tecniche chirurgiche impiegate per impiantare i TEV stessi non è stato un argomento di tale discussione intensa. Piuttosto, i protocolli per quanto riguarda l&#39;impianto di TEV in modelli animali sono in gran parte lasciatifino alla ricerca investigatori. Il seguente manoscritto illustrato come impiantare TEV utilizzando un approccio anastomosi end-to-end. Questa procedura è stata ottimizzata utilizzando uno specifico modello di sutura anastomotica, stabilizzante tecnica di sutura, ottimizzando tensione longitudinale e l&#39;aggiunta di monitoraggio in vivo strumentazione. Questo metodo è in contrasto con alcune delle molte varianti che sono state usate in precedenza. Inoltre, questa procedura viene descritto come acquisire i parametri come la pressione arteriosa, TEV diametro / conformità e portata attraverso il TEV dopo l&#39;intervento fino espianto. Questa raccolta di dati fornisce un&#39;analisi indispensabile del TEV mentre è in fase di rimodellamento.

<table border="0" cellpadding="0" cellspacing="0" width="1505">
	<tbody>
		<tr height="43">
			<td dir="LTR" height="43" style="height:43px;width:335px;">Equipment</td>
			<td dir="LTR" style="width:208px;">Manufacturer</td>
			<td dir="LTR" style="width:311px;">Serial/Catalog #</td>
			<td dir="LTR" style="width:363px;">Notes</td>
		</tr>
		<tr height="56">
			<td dir="LTR" height="94" rowspan="2" style="height:94px;width:335px;">Pressure Transducer</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Becton Dickinson</td>
			<td dir="LTR" rowspan="2" style="width:311px;">P23XL-1</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each artery) 
			&#160;Used with water-filled diaphragm domes</td>
		</tr>
		<tr height="172">
			<td dir="LTR" height="172" style="height:172px;width:335px;">Amplifier and transducer box</td>
			<td dir="LTR" style="width:208px;">Gould</td>
			<td dir="LTR" style="width:311px;">5900 Signal Conditioner Cage</td>
			<td dir="LTR" style="width:291px;">1 
			Two transducers and amplifiers should be included in cage. While this specific unit may be discontinued, other commercially available pressure transducers with a BNC/analog output will communicate with the Sonometrics equipment</td>
		</tr>
		<tr height="121">
			<td dir="LTR" height="121" style="height:121px;width:335px;">T403 Console with TS420 perivascular flowmeter module(x2)&#160;</td>
			<td dir="LTR" style="width:208px;">Transonic Systems</td>
			<td dir="LTR" style="width:311px;">T403 module and TS420 (x2)</td>
			<td dir="LTR" style="width:291px;">1 
			Flow probes measuring flow through each of the carotid arteries will connect to each of the TS420 units.&#160;&#160;</td>
		</tr>
		<tr height="126">
			<td dir="LTR" height="126" style="height:126px;width:335px;">Digital ultrasonic measurement unit</td>
			<td dir="LTR" style="width:208px;">Sonometrics</td>
			<td dir="LTR" style="width:311px;">TR-USB</td>
			<td dir="LTR" style="width:291px;">1</td>
		</tr>
		<tr height="48">
			<td dir="LTR" height="48" style="height:48px;width:335px;">Flow Probe Precision S-Series 4mm</td>
			<td dir="LTR" rowspan="4" style="width:208px;">Transonic Systems Inc.</td>
			<td dir="LTR" rowspan="4" style="width:311px;">MC4PSS-LS-WC100-CM4B-GA</td>
			<td dir="LTR" rowspan="4" style="width:291px;">2</td>
			<td dir="LTR" rowspan="4" style="width:363px;"></td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="49" rowspan="2" style="height:49px;width:335px;">1mm&#160; Sonometrics Crystals</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Sonometrics Systems</td>
			<td dir="LTR" rowspan="2" style="width:311px;">1R-38S-20-NC-SH</td>
			<td dir="LTR" style="width:291px;">&#160;2-4 (2 for each artery)</td>
		</tr>
		<tr height="81">
			<td dir="LTR" height="134" rowspan="2" style="height:134px;width:335px;">Catheter for implantation</td>
			<td dir="LTR" style="width:208px;">BD (Becton Dickinson)</td>
			<td dir="LTR" rowspan="2" style="width:311px;">&#160;381447</td>
			<td dir="LTR" style="width:291px;">&#160;1+ (1 for each artery) 
			Catheter is cut and secured to microbore tubing, stylette is utilized for insertion&#160;</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="73" rowspan="2" style="height:73px;width:335px;">Tygon Microbore Tubing</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Norton Performance Plastics</td>
			<td dir="LTR" style="width:311px;">(AAQ04127) Formulation S-54-HL</td>
			<td dir="LTR" style="width:291px;">NA (cut to length for an extension set)</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="49" rowspan="2" style="height:49px;width:335px;">Luer Stub Adapter</td>
			<td dir="LTR" style="width:208px;">BD (Becton Dickinson)</td>
			<td dir="LTR" style="width:311px;">427564 (20 gauge)</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each arterial catheter)</td>
		</tr>
		<tr height="24">
			<td dir="LTR" height="82" rowspan="2" style="height:82px;width:335px;">Surflo Injection Plug</td>
			<td dir="LTR" rowspan="2" style="width:208px;">Terumo</td>
			<td dir="LTR" rowspan="2" style="width:311px;">SR-IP2</td>
			<td dir="LTR" style="width:291px;">1+ (1 for each arterial catheter)</td>
		</tr>
		<tr height="39">
			<td dir="LTR" height="128" rowspan="2" style="height:128px;width:335px;">Meadox</td>
			<td dir="LTR" rowspan="2" style="width:208px;">PTFE (Teflon) Felt</td>
			<td dir="LTR" rowspan="2" style="width:311px;">19306</td>
			<td dir="LTR" style="width:291px;">NA (cut to size) 
			The PTFE felt used in our studies was discontinued. However, comparable companies such as &#8220;Surgical Mesh&#8221; offer products which are equivalent.&#160;</td>
		</tr>
	</tbody>
</table>

surgical technique for the implantation of tissue engineered vascular grafts and subsequent in vivo monitoring

Lo sviluppo di ingegneria tessutale Vessels (TEV) è avanzata dalla capacità di di routine ed efficace TEV impianto (4-5 mm di diametro) in un grande modello animale. Un protocollo passo-passo per il posizionamento tra posizionale della valutazione TEV digitale in tempo reale del TEV e arterie carotidi native è descritto qui. In vivo monitoraggio è reso possibile dal impiantazione di sonde di flusso, cateteri e cristalli ultrasonici (grado di registrare i cambiamenti di diametro dinamici di TEV impiantati e delle arterie carotidi native) al momento della chirurgia. Una volta impiantato, i ricercatori possono calcolare i modelli flusso di sangue arterioso, invasiva della pressione arteriosa e del diametro dell&#39;arteria che producono parametri come la velocità dell&#39;onda di polso, indice di aumento, le pressioni di impulsi e la conformità. L&#39;acquisizione dei dati viene eseguita utilizzando un singolo programma per computer per l&#39;analisi per tutta la durata dell&#39;esperimento. Tali dati inestimabile permette di comprendere meglio TEV rimodellamento della matrice, la sua resemblanCé a controlli nativi / sham e prestazioni TEV generale in vivo.

Più di 30 pecore sono stati sottoposti alla tecnica chirurgica descritta nella presente relazione per l&#39;impianto di TEV (in corso di stampa) 9. Una tabella che riassume le più recenti operazioni di ovini dopo l&#39;ottimizzazione del protocollo sono riportati nella tabella 2. Tutte le pecore recuperati dopo TEV l&#39;impianto senza pericolo di vita complicazioni. In alcuni animali, la fibrosi è stata osservata in arteria nativa vicino alla punta del catetere arterioso. Non è stato oss...

Watch this Scientific Journal Video about Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring at JoVE.com

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

Un protocollo step-by-step per il collocamento inter-posizionale di ingegneria tessutale Vessels (TEV) nella carotide di una pecora con anastomosi end-to-end e di valutazione digitale in tempo reale in vivo fino al sacrificio degli animali.

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 ...

surgical-technique-for-implantation-tissue-engineered-vascular-grafts

Research

JoVE Journal

Bioengineering

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

11.6K Views.  State University of New York Buffalo School of Medicine.  Un protocollo step-by-step per il collocamento inter-posizionale di ingegneria tessutale Vessels (TEV) nella carotide di una pecora con anastomosi end-to-end e di valutazione digitale in tempo reale in vivo fino al sacrificio degli animali. 

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

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Imaging bioluminescenza per la valutazione delle risposte immunitarie A seguito di impianto di tessuto cardiaco Composti (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Ricerca

Bioingegneria

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Ingegneria biologica basata innesti vascolari Utilizzando un bioreattore pulsatile

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Senza contatto, Label-free monitoraggio delle cellule e matrice extracellulare mediante Spettroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Produzione, caratterizzazione e possibilità di utilizzo di un 3D tissutale umano esofageo mucose Modello

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

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 ...

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. ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

applicazioni di<em&gt; In Vivo</em&gt; Test funzionale del Topo tibiale anteriore per la valutazione di ingegneria tessutale muscolo scheletrico di riparazione

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Scaling di Engineered vascolari innesti Usando 3D Stampato Guide e il metodo di impilamento Anello

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Una nuova tecnica di sutura Tenorrhaphy con innesto di collagene ingegnerizzato per riparare grandi difetti tendinei

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.