Questo lavoro è stato sostenuto, in parte, da una sovvenzione del NIH (RO1 HL098228) per CKB.

NOTA: Tutte le procedure sugli animali sono state approvate dalla Hospital Institutional Animal Care ed uso Comitato dei Bambini di Nationwide. 1. Graft Manufacturing <ol><li> Rendere la soluzione ε-caprolattone e L-lattide copolimero P (LA / CL) aggiungendo 100mg P (LA / CL) in 2 ml di diossano sotto una cappa aspirante. Mettere la soluzione in un vortice e mescolare continuamente per 1-1,5 ore per sciogliere completamente. </li><li> Nel frattempo, rimuovere un foglio di acido poliglicolico (PGA) feltro dal congelatore e tagliare diverse sezioni 5 x 8 mm. Anche tagliare la punta di una pipetta microlitri 0,1-10 appena sopra il filtro. </li><li> Inserire un ago G 19 (1,5 di lunghezza) nella estremità distale di un puntale e avvolgere il PGA sentire in tutto il ago utilizzando micro pinze. </li><li> Attentamente spingere il feltro all&#39;estremità distale della punta della pipetta, dove il lume è dritto di parte prossimale, utilizzando un ottuso ago 18 G, mentre il ago G 19 è inserito in esso. </li><li> PipettaSoluzione al 40 microlitri P (CL / LA) nella punta della pipetta dall&#39;alto. Saturare il PGA feltro con la soluzione. Poi spingere le bolle d&#39;aria utilizzando un dispenser pipetta. Ripetere questo processo, se necessario. </li><li> Luogo innesti in un tubo da 50 ml e collocarlo in una -80 ° C freezer per 20 minuti. Assicurarsi che la testa degli aghi faccia verso il basso. </li><li> Trasferire la provetta in un liofilizzatore e vuoto per 24 ore. Assicurarsi di aprire il coperchio del tubo di avere il flusso d&#39;aria. </li><li> Estrarre gli innesti e rimuoverli dagli aghi. Tagliare entrambe le estremità dell&#39;innesto lasciando una sezione ~ 5 mm e riporli sugli aghi per mantenere la loro forma. Mantenere gli innesti in un essiccatore. </li><li> Posizionare il patibolo sotto la luce UV in una cappa di biosicurezza O / N prima della semina delle cellule. </li></ol> 2. Midollo osseo Mononuclear raccolta di cellule e di isolamento <ol><li> Euthanize i topi con overdose di ketamina / xylazina (ketamina, 200 mg / kg e xylazina, 20 mg / kg). </li><li> Rimuovere le ossa (femore di und tibie) da 3 topi per 10 impiantazioni innesto e posto in una capsula di Petri con 10 cc RPMI. Tagliare le due estremità delle ossa e filo midollo osseo utilizzando una siringa con un ago 25 G in una nuova piastra di Petri con 3 cc RPMI. Raccogliere midollo osseo e soluzione RPMI in una provetta da 15 cc e lavare la capsula di Petri con ulteriori RPMI 2 cc per raccogliere la restante BM. </li><li> Prendere campione (5-10 microlitri) e conta delle cellule utilizzando un contatore di cellule automatico o emocitometro. Registrare il risultato. </li><li> Mettere 5 cc Ficoll in una provetta da 15 cc centrifuga e aggiungere midollo osseo e soluzione RPMI. Aggiungere la soluzione molto delicatamente per evitare che la miscelazione con Ficoll. </li><li> Centrifugare a 528 xg per 30 minuti con &quot;NO FRENO&quot; a 24 ° C. </li><li> Rimuovere lo strato superiore di colore rosa. Raccogliere lo strato centrale chiara Figura 1, che è lo strato MNC, e diluire con PBS 1:1. </li><li> Centrifugare la soluzione diluita MNC a 528 xg per 10 min a 24 ° C. </li><li> Rimuovere il surnatante e diliuto il pellet con 5 ml di PBS. </li><li> Centrifugare pellet a 528 xg per 10 min a 24 ° C. </li><li> Rimuovere il surnatante. Diluire il pellet con il volume appropriato di RPMI (~ 200 microlitri). </li><li> Prelevare un campione microlitri 5-10 e contare le cellule utilizzando un contatore di cellule automatico o emocitometro. Registrare il risultato. Ripetere la cella contando ancora una volta e calcolare il numero medio di cellule. </li><li> Diluire la concentrazione cellulare a 1.000.000 cells/10 microlitri utilizzando RPMI. </li></ol> 3. Semina cellulare <ol><li> Scaffold Prewet con l&#39;aggiunta di 5 ml RPMI luminally per 5 minuti, quindi rimuovere RPMI. </li><li> Aggiungere 10 ml di midollo osseo derivate mono cellule nucleari in RPMI dal punto 2.12 al lume ponteggio e attendere 10 minuti per consentire alle cellule di attaccare sul patibolo. </li><li> Tagliare un ago 19 G per 1 cm di lunghezza e mettere l&#39;ago nel lume del ponteggio per mantenere la forma del ponteggio. Trasferire il campione in una piastra da 24 pozzetti. &lt;/ Li&gt; <li> Aggiungere 1,000 ml RPMI a ciascun pozzetto e incubare O / N in un incubatore. </li></ol> 4. Graft impianto <ol><li> Sterilizzare in autoclave tutti gli strumenti chirurgici prima della chirurgia: 1x forbici sottili, 3x micro pinze, pinze vascolari micro 2x, 1x morsetto pinze applicano, porta-aghi micro 1x, forbici primavera 1x, 1x divaricatore. </li><li> 6-8 settimane vecchia femmina C57BL / 6 sono usati come ingegneria tessutale destinatari innesto vascolare. Rimuovere il mouse dalla gabbia e si pesa, poi anestetizzare attraverso un&#39;iniezione intraperitoneale nel quadrante inferiore destro dell&#39;addome con un cocktail di ketamina / xilazina (ketamina, 100 mg / kg e xilazina, 10 mg / kg). Ketoprofene (5 mg / kg, IP) è usato come un analgesico pre-anestesia. </li><li> Controllare il livello di sedazione da pizzicare racconto, poi ritagliare i capelli addominale. Lubrificare gli occhi con pomata oftalmica sterile, e posizionare il mouse in una posizione prona dorsale su un taccuino. Disinfettare l&#39;addome con Betadine e alcool pastiglie. Cover il mouse con un telino sterile ed esporre solo la zona di incisione. </li><li> Effettuare una laparotomia mediana incisione da sotto la xifoidea alla regione sovrapubica, e inserire un divaricatore di auto-conservazione. Avvolgere l&#39;intestino in soluzione salina inumidito garza. Senza mezzi termini definire l&#39;aorta sottorenale e la vena cava. </li><li> Inserire due micro pinze vascolari su entrambi i lati prossimale e distale della aorta e vena cava poi senza mezzi termini separare l&#39;aorta dalla vena cava Transect la vena cava. Se necessario, legare i rami dell&#39;aorta addominale con un 10-0 monofilamento sugli aghi conici. </li><li> Impiantare una vena cava inferiore interposizione di innesto con l&#39;estremità prossimale e distale di anastomosi terminare con una sterile 10-0 sutura. Tagliare il trapianto, di solito 1-2 mm, a seconda dell&#39;anatomia del mouse. Fissare l&#39;innesto con un punto su entrambi estremità prossimale e distale e inizia a suturare continuamente con 4-5 stiches dall&#39;altro lato dell&#39;innesto. Dopo aver terminato la parte anteriore, capovolgere i morsetti e innestiverso l&#39;altro lato e suturare il lato posteriore dell&#39;innesto. Durante l&#39;impianto, lavare l&#39;innesto con la soluzione di eparina frequentemente per evitare la trombosi acuta. </li><li> Rimuovere il morsetto prossimale e controllare l&#39;emorragia applicando una topica agente emostatico sterile riassorbibile. Quando l&#39;emorragia si arresta completamente, rimuovere il morsetto distale e controllare l&#39;emorragia allo stesso modo. Assicurarsi che il sangue scorre attraverso l&#39;innesto. </li><li> Chiudere la muscolatura addominale e la pelle in due strati con un 6-0 nera in poliammide monofilamento di sutura con ago filettato incluso. </li><li> Iniettare 0,5 ml di soluzione fisiologica per via sottocutanea e posizionare il mouse in una gabbia di recupero su un rilievo di riscaldamento fino a quando il mouse è completamente mobile. Al momento del recupero, ritorno il mouse in una nuova gabbia con biancheria da letto di carta. Dare farmaci antidolorifici (ibuprofene, 30 mg / kg, acqua potabile) per 48 ore. </li></ol>

<ol><li>Heart Association, A. merican <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heart+Disease+and+Stroke+Statistics%E2%80%942012+Update%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Heart Disease and Stroke Statistics—2012 Update.</a> Circulation. 125, (2012).</li>
<li>Shinoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creation+Of+Viable+Pulmonary+Artery+Autografts+Through+Tissue+Engineering%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Creation Of Viable Pulmonary Artery Autografts Through Tissue Engineering.</a> The Journal of Thoracic and Cardiovascular Surgery. 115, 536-546 (1998).</li>
<li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Late-term+results+of+tissue-engineered+vascular+grafts+in+humans%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Late-term results of tissue-engineered vascular grafts in humans.</a> The Journal of Thoracic and Cardiovascular Surgery. 139, 431-436 (2010).</li>
<li>Shin'oka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Midterm+clinical+result+of+tissue-engineered+vascular+autografts+seeded+with+autologous+bone+marrow+cells%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Midterm clinical result of tissue-engineered vascular autografts seeded with autologous bone marrow cells.</a> The Journal of Thoracic and Cardiovascular Surgery. 129, 1330-1338 (2005).</li>
<li>Brennan, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+vascular+grafts+demonstrate+evidence+of+growth+and+development+when+implanted+in+a+juvenile+animal+model%5BTitle%5D)+AND+%22Ann+Surg%22%5BJournal%5D)">Tissue-engineered vascular grafts demonstrate evidence of growth and development when implanted in a juvenile animal model.</a> Ann Surg. 248, 370-377 (2008).</li>
<li>Roh, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Construction+of+an+autologous+tissue-engineered+venous+conduit+from+bone+marrow-derived+vascular+cells%3A+optimization+of+cell+harvest+and+seeding+techniques%5BTitle%5D)+AND+%22Journal+of+Pediatric+Surgery%22%5BJournal%5D)">Construction of an autologous tissue-engineered venous conduit from bone marrow-derived vascular cells: optimization of cell harvest and seeding techniques.</a> Journal of Pediatric Surgery. 42, 198-202 (2007).</li>
<li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+vascular+grafts+form+neovessels+that+arise+from+regeneration+of+the+adjacent+blood+vessel%5BTitle%5D)+AND+%22The+FASEB+Journal%22%5BJournal%5D)">Tissue-engineered vascular grafts form neovessels that arise from regeneration of the adjacent blood vessel.</a> The FASEB Journal. 25, 2731-2739 (2011).</li>
<li>Hibino, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+critical+role+for+macrophages+in+neovessel+formation+and+the+development+of+stenosis+in+tissue-engineered+vascular+grafts%5BTitle%5D)+AND+%22The+FASEB+Journal%22%5BJournal%5D)">A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts.</a> The FASEB Journal. 25, 4253-4263 (2011).</li>
<li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+the+Natural+History+of+Extracellular+Matrix+Production+in+Tissue-Engineered+Vascular+Grafts+during+Neovessel+Formation%5BTitle%5D)+AND+%22Cells+Tissues+Organs%22%5BJournal%5D)">Characterization of the Natural History of Extracellular Matrix Production in Tissue-Engineered Vascular Grafts during Neovessel Formation.</a> Cells Tissues Organs. 195, 60-72 (2012).</li>
<li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Beyond+Burst+Pressure%3A+Initial+Evaluation+of+the+Natural+History+of+the+Biaxial+Mechanical+Properties+of+Tissue+Engineered+Vascular+Grafts+in+the+Venous+Circulation+Using+a+Murine+Model%5BTitle%5D)+AND+%22Tissue+Eng.+Part+A%22%5BJournal%5D)">Beyond Burst Pressure: Initial Evaluation of the Natural History of the Biaxial Mechanical Properties of Tissue Engineered Vascular Grafts in the Venous Circulation Using a Murine Model.</a> Tissue Eng. Part A. 20, (2013).</li>
<li>Mirensky, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+vascular+grafts%3A+does+cell+seeding+matter%5BTitle%5D)+AND+%22Journal+of+Pediatric+Surgery%22%5BJournal%5D)">Tissue-engineered vascular grafts: does cell seeding matter.</a> Journal of Pediatric Surgery. 45, 1299-1305 (2010).</li>
<li>Roh, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+vascular+grafts+transform+into+mature+blood+vessels+via+an+inflammation-mediated+process+of+vascular+remodeling%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences%22%5BJournal%5D)">Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling.</a> Proceedings of the National Academy of Sciences. 107, 4669-4674 (2010).</li>
<li>Mirensky, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue-engineered+arterial+grafts%3A+long-term+results+after+implantation+in+a+small+animal+model%5BTitle%5D)+AND+%22Journal+of+Pediatric+Surgery%22%5BJournal%5D)">Tissue-engineered arterial grafts: long-term results after implantation in a small animal model.</a> Journal of Pediatric Surgery. 44, 1127-1133 (2009).</li>
<li>Lee, Y. U., Naito, Y., Kurobe, H., Breuer, C. K., Humphrey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biaxial+mechanical+properties+of+the+inferior+vena+cava+in+C57BL%2F6+and+CB-17+SCID%2Fbg+mice%5BTitle%5D)+AND+%22Journal+of+Biomechanics%22%5BJournal%5D)">Biaxial mechanical properties of the inferior vena cava in C57BL/6 and CB-17 SCID/bg mice.</a> Journal of Biomechanics. 46, 2277-2282 (2013).</li>
</ol>

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Il modello murino di TEVG è uno strumento prezioso per studiare i meccanismi cellulari e molecolari della formazione neotissue e lo sviluppo di stenosi. La testa di serie BM-MNC stato mostrato in entrambe le immagini istologiche e SEM delle cellule inseminate sulla marza 11. Efficienza semina delle cellule è stata anche dimostrata utilizzando un test DNA 7. Usando questo sistema modello abbiamo dimostrato che semina delle cellule riduce l&#39;incidenza dello sviluppo di TEVG stenosi, che era la p...

Difetti cardiaci congeniti sono condizioni gravi che colpiscono circa l&#39;8% dei nati vivi negli Stati Uniti. Circa il 25% di questi neonati con difetti cardiaci congeniti o 2,4 per 1.000 nati vivi, richiedono un trattamento invasivo nel primo anno della loro vita 1. Il trattamento più efficace per la malattia cardiaca congenita è la chirurgia ricostruttiva. Purtroppo, complicazioni derivanti dall&#39;uso di condotti vascolari attualmente disponibili sono la causa più importante di morbilità e mortalità postoperatoria. Per risolvere questo problema, abbiamo sviluppato i primi di ingegneria tessutale innesti vascolari (TEVGs) per uso clinico 2. TEVGs sono stati costruiti con tubi in poliestere biodegradabile seminati con autologo di midollo osseo cellule derivate-mononucleari (BM-MNC) e impiantati come condotti venosi per la chirurgia cardiaca congenita. I risultati hanno mostrato ottimi tassi di pervietà a 1-3 anni di follow-up, ma con una significativa incidenza di stenosi <sup&gt; 3,4. Era chiaro che era necessaria una migliore comprensione della formazione neotissue vascolare e il meccanismo alla base dello sviluppo di TEVG stenosi. Per comprendere meglio lo sviluppo di TEVGs e il meccanismo di sviluppo stenosi, un modello ovino emo 5,6. In questo modello, i TEVGs trasformata con successo in navi viventi e erano simili in entrambi morfologia e la funzione delle vene native. Questo uso di un grande modello animale era un buon primo passo per fornire importanti informazioni pre-clinico che ha aiutato l&#39;uso clinico di TEVGs. Tuttavia, la piena comprensione dei meccanismi cellulari e molecolari della formazione neotissue vascolare in TEVGs utilizzando modelli animali di grandi dimensioni è limitata a causa di limitazioni nella caratterizzazione molecolare dei fenotipi di cellule vascolari a causa della mancanza di strumenti di specie molecolari specifici. Per superare queste carenze, un modello murino di TEVGs è stato sviluppato a causa del rapido progresso nel campo della genetica mouse e la loro vasta moleculaCaratterizzazione r con il vantaggio aggiunto di una scala temporale ridotta. Il murino IVC modello interposizione fedelmente ricapitolato il processo di neovessel di formazione che si verifica in grandi animali e nell&#39;uomo, ma nel corso di un corso di tempo molto più breve 6-9. Qui, un protocollo dettagliato per la produzione innesto su piccola scala utilizzando scaffold biodegradabili, BM-MNC raccolta e l&#39;isolamento, BM-MNC semina sull&#39;impalcatura, e l&#39;impianto innesto in un modello murino sono stati descritti.

<table><tbody><tr><td>Polyglycolic acid (PGA) felt</td><td>Biomedical Structures</td><td></td><td>Custome ordered</td></tr><tr><td>Pipet tip, 0.1-10 &amp;mu;l&amp;nbsp;</td><td>Fisher Sientific</td><td>02-707-456</td><td></td></tr><tr><td>Lyophilizer&amp;nbsp;</td><td>Labconco</td><td>7070020</td><td></td></tr><tr><td>RPMI medium 1604</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Petri dish</td><td>BD</td><td>353003</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>15 cc tube&amp;nbsp;</td><td>BD</td><td>352096</td><td></td></tr><tr><td>Ficoll</td><td>Sigma</td><td>10831-100ml</td><td>Also called &#039;Histopaque&#039;</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Littauer bone cutter 4.5&quot; Straight</td><td>Roboz</td><td>RS-8480</td><td>For BM harvesting</td></tr><tr><td>Forceps 4.5&quot;</td><td>Roboz</td><td>RS-8120</td><td>For BM harvesting</td></tr><tr><td>Scissors 4.5&quot;</td><td>Roboz</td><td>RS-5912</td><td>For BM harvesting</td></tr><tr><td>Microscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>C57BL/6J (H-2b), Female</td><td>Jackson Laboratories</td><td>664</td><td>8-12 weeks</td></tr><tr><td>Ketamine hydrochloride injection</td><td>Hospira Inc.</td><td>NDC 0409-2053</td><td></td></tr><tr><td>Xylazine sterile solution</td><td>Akorn Inc.</td><td>NADA# 139-236</td><td></td></tr><tr><td>Ketoprofen</td><td>Fort Dodge Animal Health</td><td>NDC 0856-4396-01</td><td></td></tr><tr><td>Ibuprofen</td><td>PrecisionDose</td><td>NDC 68094-494-59</td><td></td></tr><tr><td>Heparin sodium</td><td>Sagent Pharmaceticals</td><td>NDC 25021-400</td><td></td></tr><tr><td>Saline solution (sterile 0.9% sodium chloride)</td><td>Hospira Inc.</td><td>NDC 0409-0138-22</td><td></td></tr><tr><td>0.9% Sodium chloride injection</td><td>Hospira Inc.</td><td>NDC 0409-4888-10</td><td></td></tr><tr><td>Petrolatum ophthalmic ointment</td><td>Dechra Veterinary Products</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Iodine prep pads</td><td>Triad Disposables, Inc.</td><td>NDC 50730-3201-1</td><td></td></tr><tr><td>Alcohol prep pads</td><td>McKesson Corp.</td><td>NDC 68599-5805-1</td><td></td></tr><tr><td>Cotton tipped applicators</td><td>Fisher Scientific</td><td>23-400-118</td><td></td></tr><tr><td>Fine scissor</td><td>FST</td><td>14028-10</td><td></td></tr><tr><td>Micro-adson forcep</td><td>FST</td><td>11018-12</td><td></td></tr><tr><td>Clamp applying forcep</td><td>FST</td><td>00072-14</td><td></td></tr><tr><td>S&amp;amp;T Vascular clamp</td><td>FST</td><td>00396-01</td><td></td></tr><tr><td>Spring scissors</td><td>FST</td><td>15008-08</td><td></td></tr><tr><td>Colibri retractors</td><td>FST</td><td>17000-04</td><td></td></tr><tr><td>Dumont #5 forcep</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>Dumont #7 - fine forceps</td><td>FST</td><td>11274-20</td><td></td></tr><tr><td>Dumont #5/45 forceps</td><td>FST</td><td>11251-35</td><td></td></tr><tr><td>Tish needle holder/forceps</td><td>Micrins</td><td>MI1540</td><td></td></tr><tr><td>Black polyamide monofilament suture, 10-0</td><td>AROSurgical Instruments Corporation</td><td>TI638402</td><td>For suturing the graft</td></tr><tr><td>Black polyamide monofilament suture, 6-0</td><td>AROSurgical Instruments</td><td>SN-1956</td><td>For musculature and skin closure</td></tr><tr><td>Non-woven sponges</td><td>McKesson Corp.</td><td>94442000</td><td></td></tr><tr><td>Absorbable hemostat</td><td>Ethicon</td><td>1961</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>18 G 1.5 in, Needle</td><td>BD</td><td>305190</td><td></td></tr><tr><td>25 G 1 in, Needle</td><td>BD</td><td>305125</td><td></td></tr><tr><td>30 G 1 in, Needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-700</td><td></td></tr><tr><td>Warming pad</td><td>Gaymar</td><td>TP-22G</td><td></td></tr><tr><td>Trimmer</td><td>Wahl</td><td>9854-500</td><td></td></tr></tbody></table>

implantation of inferior vena cava interposition graft in mouse model

Scaffold biodegradabili seminati con cellule mononucleari del midollo osseo (BMC) sono spesso utilizzati per la chirurgia ricostruttiva per il trattamento di anomalie cardiache congenite. I risultati clinici a lungo termine hanno mostrato ottimi tassi di pervietà, tuttavia, con una significativa incidenza di stenosi. Per studiare i meccanismi cellulari e molecolari della formazione neotissue vascolare e prevenire lo sviluppo di stenosi in ingegneria tessutale innesti vascolari (TEVGs), abbiamo sviluppato un modello murino dell&#39;innesto con circa 1 mm di diametro interno. In primo luogo, le TEVGs stati assemblati da impalcature tubolari biodegradabili fabbricate da un tessuto non tessuto di acido poliglicolico feltro maglia rivestito con ε-caprolattone e L-lattide copolimero. I ponteggi sono stati poi collocati in un liofilizzatore, aspirati per 24 ore, e conservati in un essiccatore fino semina delle cellule. In secondo luogo, midollo osseo è stato prelevato da topi donatori e cellule mononucleari sono stati isolati da centrifugazione in gradiente di densità. Terzo, circa un milione di cellule eranoseminate su un ponteggio e incubati O / N. Infine, i ponteggi seminati stati impiantati come infrarenali innesti vena cava interposizione di C57BL / 6 topi. Gli innesti impiantati dimostrato eccellente pervietà (&gt; 90%), senza evidenza di complicanze tromboemboliche o di formazione aneurismatica. Questo modello murino ci aiuterà a comprendere e quantificare i meccanismi cellulari e molecolari della formazione neotissue nel TEVG.

Uno schema di TEVG impianto è mostrato in Figura 1. Midollo osseo è stato raccolto da un topo donatore e mono cellule nucleari sono stati isolati mediante centrifugazione densità e quindi seminate su uno scaffold biodegradabile. Gli scaffold seminati sono stati incubati O / N e impiantati a un mouse destinatario come la vena cava inferiore interposizione innesto. La Figura 2 illustra la microscopia elettronica a scansione del ponteggio PGA-P (CL / LA). Il...

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Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Per migliorare la nostra conoscenza della formazione neotissue cellulare e molecolare, un modello murino della TEVG è stata sviluppata di recente. Gli innesti sono stati impiantati come infrarenali innesti vena cava interposizione di C57BL / 6 topi. Questo modello raggiunge risultati simili a quelli ottenuti nella nostra ricerca clinica, ma nel corso di un time-course di gran lunga ridotto.

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

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

implantation-of-inferior-vena-cava-interposition-graft-in-mouse-model

Research

JoVE Journal

Medicine

11.8K Views.  Nationwide Children's Hospital.  Per migliorare la nostra conoscenza della formazione neotissue cellulare e molecolare, un modello murino della TEVG è stata sviluppata di recente. Gli innesti sono stati impiantati come infrarenali innesti vena cava interposizione di C57BL / 6 topi. Questo modello raggiunge risultati simili a quelli ottenuti nella nostra ricerca clinica, ma nel corso di un time-course di gran lunga ridotto. 

Video: Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

Il trapianto cardiaco eterotopico nel Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

Ricerca

Biologia

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

Il trapianto di valvola polmonare Utilizzando un modello murino di trapianto cardiaco eterotopico

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Medicina

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

Eterotopico cervicale trapianto cardiaco nei topi

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Stenosi della Vena Cava inferiore: un modello murino di trombosi venosa profonda

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Immunologia e infezioni

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

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

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

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

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

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

Trapianto cardiaco cervicale eterotopico di topo utilizzando bracciali vascolari

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Un modello di interposizione venosa di coniglio che imita la chirurgia di rivascolarizzazione usando grafts di venina per valutare l'iperplasia intima sotto la pressione sanguigna arteriosa

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

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

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

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

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

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

Bioingegneria

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

Ricostruzione del circuito sanguigno in un modello di trapianto di cuore di topo addominale

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

Una tecnica di bracciale modificata per il modello di trapianto di cuore eterotopico cervicale del topo

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...

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

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