The authors thank Tyra Witt, Cheri Mueske, Brant Newman and Dr. Peter J. Psaltis, MBBS, PhD for their valuable contributions. This study was financially supported by European Regional Development Fund &#8211; FNUSA-ICRC (No. CZ.1.05/1.100/02.0123), American Heart Association Scientist Development Grant (AHA #06-35185N), National Institutes of Health (T32HL007111) and The Grainger Innovation Fund &#8211; Grainger Foundation.

Tutti gli studi sugli animali sono stati approvati dalla cura degli animali e del Comitato Istituzionale Utilization (IACUC) presso la Mayo Clinic. 1. Progettazione e analisi di un 2205 in acciaio inossidabile Stent <ol><li> Progettare un stent di metallo nudo con CAD <ol><li> Effettuare un cilindro cavo estruso selezionando in funzione &#39;Estrusione / base&#39; con lo spessore di parete pari allo spessore stent puntone. </li><li> Progettare un modello di stent su un piano di schizzo diverso tangente al cilindro estruso. Effettuare la larghezza del modello piatto per abbinare la circonferenza del cilindro cavo estruso. </li><li> Trasferire sul cilindro cavo utilizzando la funzione di avvolgere il design modello piatto. </li><li> Salvare la parte nel suo formato nativo e anche in formato ACIS deve essere esportato per FEA. </li></ol></li><li> Analisi agli elementi finiti per i modelli stent <ol><li> Importare la geometria solida salvato in formato ACIS nel modulo parte del software FEA per ulteriori analysè. </li><li> Modello 2 cilindri analitici coassialmente allo stent nella parte modellatore del software FEA. Il cilindro esterno ha un diametro iniziale maggiore del diametro dello stent per simulare la pressa e il cilindro interno ha un diametro iniziale di 1 mm per simulare un pallone per gonfiaggio. </li><li> Fare doppio clic sulla voce di albero del modellatore assemblea &#39;casi&#39; di montare sopra dette parti in posizioni relative. </li><li> Utilizzare il modulo di maglia del software FEA, specificare il tipo di elemento come 20 nodi elemento esaedrica con l&#39;integrazione ridotta, specificare le dimensioni dell&#39;elemento, e maglie dello stent. </li><li> Specificare attrito coppie a contatto rigide tra lo stent e due cilindri, rispettivamente nelle &quot;Proprietà&quot; della interazione del modello. </li><li> Assegnare comportamento sforzo-deformazione elasto-plastico di acciaio inossidabile 2205 al modello dello stent. </li><li> Definire condizioni al contorno per crimpare primo luogo il cilindro esterno di 1 mm che simula il cRimping dello stent. Rimuovere il cilindro esterno per simulare il rilassamento dello stent crimpato. Espandere il cilindro interno di 3 mm per simulare l&#39;espansione e, infine, rimuovere il cilindro interno per simulare recoil dello stent. </li><li> Definire i parametri di simulazione, tra cui il numero di processori e quantità di RAM allocati nella voce albero modello di &#39;Analisi&#39; ed eseguire la simulazione. </li><li> Una volta che la simulazione, aprire il file risultato (filename.odb) e post-elaborare i risultati di studiare le deformazioni principali e migliorare iterativamente il disegno stent per ottenere una deformazione principale del 20%, che è inferiore al limite cedimento del materiale, . </li></ol></li></ol> 2. Stent fabbricazione e la sperimentazione per la piegatura e di espansione <ol><li> Stent fabbricazione <ol><li> Ottenere i tubi in acciaio inossidabile 2205 di pistola foratura e molatura di precisione barra magazzino materiale presso una società di lavorazioni meccaniche di precisione, come azione di precisione Prodotti in Pioneer,OH. </li><li> Trasferire i tubi a terra di precisione e il modello di progettazione dello stent piatta ad una società di stent taglio come Laserage Technology Corporation a Waukegan, IL per taglio laser e elettrolucidatura. </li><li> Passivare la superficie degli stent elettrolucidate da essi submersing in un acido forte (50% HCl) per 10 minuti seguiti da una base (10% NaHCO3) per altri 10 min. ATTENZIONE: maneggiare sostanze chimiche con i dispositivi di protezione adeguata e sotto una cappa aspirante. Infine, lavare gli stent con alcool etilico e acqua deionizzata. Questo processo è chiamato decapaggio acido. </li></ol></li><li> Prove di stent fabbricati per la piegatura e l&#39;espansione <ol><li> Crimp lo stent su un pallone a tre ante con una mano tesa strumento di piegatura. Tenere lo stent e il pallone a tre ante in pinza. Premere la maniglia per deformare radialmente lo stent da crimpare sul pallone. </li><li> Ispezionare lo stent a pressare utilizzando un microscopio per uniforme aggraffatura e qualsiasi segno di fallimento nella struttura a causaper deformazione plastica. </li><li> Espanderlo al diametro progettato 3 mm pressurizzando il palloncino a tre ante con acqua. Esaminare gli stent espanso per le fratture microscopiche e l&#39;espansione uniforme. </li></ol></li></ol> 3. Caratterizzazione di stent per Retained Campo Magnetico NOTA: magnete cilindrico del diametro di 2 pollici e 1 pollice di altezza è stato utilizzato in questo studio. I poli del magnete sono allineate lungo l&#39;asse. La densità di flusso magnetico superficiale del magnete è di circa 1 T. <ol><li> Magnetizzare gli stent diametralmente o assiale con un forte magnete al neodimio. Tenere il stent vicino al magnete forte per circa 1 min per la magnetizzazione. </li><li> Tenere il stent su una delle facce piane con il suo diametro lungo le linee del campo magnetico per essere magnetizzate diametralmente o tenere lo stent accanto alla superficie cilindrica con asse lungo le linee del campo magnetico per magnetizzare assialmente. Fiel magnetico a nuovod dello stent è risultato essere stabile per almeno 24 ore, ma utilizzare lo stent non appena possibile dopo magnetizzazione. </li><li> Montare gli stent individualmente su mandrini di vetro e poi montare i mandrini di vetro nel mandrino precisione dell&#39;apparecchio sondaggio magnetica. Sonda microsensore magnetica può essere posizionata con precisione vicino allo stent senza toccare la superficie con la XYZ fasi di montaggio del dispositivo di tastatura magnetica (Figura 4). </li><li> Misurare la lettura basale della microsensore magnetico lontano dallo stent e quindi misurare il campo magnetico trattenuto sulla superficie dello stent posizionando la sonda con le fasi XYZ del proiettore tastatura magnetica. </li></ol> 4. magnetici Studi cellulare Capture <ol><li> Ottenere cellule, l&#39;etichettatura con spion e colorazione con colorante fluorescente <ol><li> Derivare le cellule endoteliali escrescenza (EOC) da sangue periferico suino come descritto in 5,7. Cultura in un pallone T-75 until circa l&#39;80% confluenti (5x10 6 a 8x10 6 cellule). </li><li> Sintetizzare SPIONs come diametro di 10 nm magnetite (Fe 3 O 4) nucleo circondato da 50 poli di spessore nm (lattico-co-glicolico acido) (PLGA) Coperture come descritto in 8,9. </li><li> Incubare la EOC derivato con spion ad una concentrazione di 200 ug / ml di mezzo di coltura cellulare per 16 ore a 37 ° C. </li><li> Aspirare il mezzo di coltura cellulare delicatamente. Lavare delicatamente le cellule aggiungendo 10 ml di tampone fosfato salino (PBS) al pallone, dondolo, e aspirare il PBS. </li><li> Colorare le cellule con colorante fluorescente (CM-DII) per la visualizzazione durante gli esperimenti. Ciò avviene secondo le istruzioni del fabbricante aggiungendo il colorante a 10 ml di mezzo di coltura cellulare ad una concentrazione di 5 microlitri / ml e incubando le cellule con per 30 minuti a 37 ° C. </li><li> Lavare le cellule con PBS come al punto 4.1.4 e incubare con 3 ml di 0,25% soluzione di tripsina-EDTA per 5 min a 37 ° C persollevare le cellule dal pallone. </li><li> Trasferire la sospensione cellulare in una provetta da 15 ml, rabboccare con PBS e centrifugare a 500 xg per 5 min a formare un pellet di cellule. </li><li> Risospendere il pellet cellulare in PBS ad una concentrazione di 1-2x10 6 cellule / ml e miscelare accuratamente pipettando in e fuori del tubo conico più volte. </li></ol></li><li> Studi in vitro di cellule <ol><li> Progettare e fabbricare (ad esempio, stampa 3D) un semplice attrezzo per tenere lo stent appena sopra la superficie di un vetrino di vetro. </li><li> Smagnetizzare uno stent utilizzando un degausser elettromagnetica o magnetizzare uno stent diametralmente o assiale con un forte magnete al neodimio. </li><li> Pipettare il EOC spion marcato sospesa in PBS nel piatto contenente gli stent di controllo assiale magnetizzati o diametralmente magnetizzate o non magnetizzate. Immagine gli stent con EOC sospesa in PBS immediatamente per la fluorescenza usando un microscopio a fluorescenza invertito. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 5. in vivo Animal Studies <ol><li> Impianto di stent <ol><li> Disegna sangue periferico da 4 maiali Yorkshire sani - peso di circa 50 kg - tre settimane prima di impianto di stent, rispettivamente, e la cultura EOC come descritto in 5,7. </li><li> Somministrare farmaci anti-piastrinici di partenza 3 giorni prima dell&#39;intervento (aspirina 325 mg e clopidogrel 75 mg al giorno). </li><li> Il giorno impianto dello stent, anestetizzare i maiali con intramuscolare Telazol, Xilazina, e atropina (5 / 2-3 / 0,05 mg / kg, rispettivamente), come indicato nelle linee guida istituzionali applicabili cura degli animali e l&#39;uso. </li><li> Intubare e posizionare il maiale inalazione di 1-2,5% anestesia isoflurano. </li><li> Radere la regione ventrale del collo del maiale e condurre la procedura in condizioni sterili generali. </li><li> Implant 1 magnetizzato e 1 stent non magnetizzato nell&#39;arteria coronaria destra (RCA) usando la tecnica di cateterizzazione cardiaca standard. <ol><li> Catheterizzazione di animali deve essere effettuata da un cardiologo interventista addestrato. Accedere alla carotide destra con una guaina French 9. </li><li> Incannulare l&#39;obiettivo coronarica e iniettare mezzo di contrasto iodato per ottenere immagini fluoroscopiche. </li><li> Mettere un filo guida coronarico standard di 0.014 pollici nell&#39;arteria. Far avanzare il palloncino e stent utilizzando questo filo guida e distribuire il stent in un vaso di diametro 3-3,5 mm. </li></ol></li><li> Occludere il flusso di sangue all&#39;interno prossimale RCA agli stent impiantati utilizzando un over pallone filo e fornire circa 2x10 6 EOC autologo marcato con spion sospeso in 4 ml di PBS attraverso il catetere centrale su un periodo di 2 min. </li><li> Ripristinare il flusso di sangue al RCA dopo 2 min di occlusione aggiuntivo. </li><li> Trasferire l&#39;animale alla sala di risveglio e monitorare attentamente l&#39;animale fino a che non ha ripreso conoscenza. </li><li> Continuare a somministrare farmaci anti-piastrinici (aspirina 325 mg e clopidogrel 75 mg) inviare operativamente fino sacrificio. </li></ol></li><li> Espianto stent e istologia <ol><li> Euthanize gli animali 7 giorni dopo l&#39;intervento dapprima anestetizzare l&#39;animale come spiegato precedentemente e quindi somministrare per via endovenosa una dose letale di pentobarbital sodio (100 mg / kg) secondo le linee guida istituzionali applicabili cura degli animali e di uso. </li><li> Chirurgicamente raccogliere i segmenti arteriosi stent. Fissare le arterie espiantati in 10% formalina tampone per almeno 30 min. Lasciare i campioni in buffer di formalina per ulteriori analisi istologica. </li><li> Esternalizzare il campione fissato alle strutture in grado di svolgere l&#39;istologia con stent di metallo. Durante questo processo, i campioni vengono incorporare in metilmetacrilato, in sezione, e analizzati istologicamente usando la tecnica di colorazione di Mallory con Prussia macchia blu per particelle di ferro. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Abbiamo sviluppato uno stent magnetica che può funzionare come un stent di metallo nudo e in grado di attirare le cellule endoteliali spion marcato. In studi precedenti che coinvolgono stent magnetici, i ricercatori hanno usato nichel stent rivestiti commerciali e bobine o mesh in materiali magnetici a causa della indisponibilità di uno stent ferromagnetico 5,10-14. Altri gruppi hanno usato anche la natura paramagnetica attualmente in commercio stent in acciaio inox 304-grado per il targeting di nanopartice...

Patients implanted with vascular stents manufactured from thrombogenic materials like stainless steel, cobalt chromium, and platinum chromium &#8211; both bare metal stents (BMS) and drug eluting stents (DES) &#8211; need anti-platelet therapy to prevent thrombus formation. BMS heal rapidly, but are subject to late stage restenosis due to incomplete healing. DES require long term anti-platelet therapy due to delayed healing. Anti-platelet therapy administered to avoid thrombosis as a result of incomplete or delayed healing leads to increased bleeding risk and may not be suitable for certain patients1,2. An ideal stent will heal completely and quickly thus avoiding long-term anti-platelet therapy and late stage restenosis. This complete healing can only be achieved if the stent is rapidly coated with a monolayer of endothelial cells after implantation. Coating the stents with biocompatible materials such as gold or other biopolymers has been shown to improve thrombo-resistance, but none of these techniques achieved ideal blood compatibility as may be possible by coating with endothelial cells3,4.
A stent can be coated with endothelial cells post implantation by attracting circulating progenitor cells. This self-seeding technique can be achieved by utilizing ligands and antibodies. But this technique is limited by the low number of circulating endothelial progenitor cells. A promising strategy is to deliver cells directly to the stent immediately following implantation during a short period of blood flow occlusion3,5. This strategy requires a technique for rapidly capturing cells and retaining them on the stent even after restoring blood flow. We have developed a technique in which a magnetic stent is used to attract and retain magnetically-labeled endothelial cells delivered post implantation. To achieve this, a functional BMS with sufficient magnetic properties to capture and retain magnetically-labeled endothelial cells is required6.
In this paper, we discuss the methods for designing, manufacturing, and testing a 2205 stainless steel stent. The stents were designed using CAD and FEA. The manufactured stents were magnetized using a neodymium magnet and the retained magnetic field was measured using a magneto-resistance microsensor probe. We then tested the stents for magnetically-labeled cell capture in a culture dish during our in-vitro experiments. Finally, the stents were tested in-vivo by implanting magnetic and non-magnetic stents in 4 pigs and histologically analyzing the stented arteries.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>2205 Stainless steel</td>
			<td>Carpenter Technology Corporation</td>
			<td>N/A</td>
			<td>Round bar stock material</td>
		</tr>
		<tr>
			<td>Abaqus</td>
			<td>Dassault systems</td>
			<td>N/A</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td></td>
			<td></td>
			<td>Prescription drug.</td>
		</tr>
		<tr>
			<td>Clopidogrel</td>
			<td></td>
			<td></td>
			<td>Commercial name: Plavix. Prescription drug.</td>
		</tr>
		<tr>
			<td>CM-DiI</td>
			<td>Life Technologies</td>
			<td>V-22888</td>
			<td>Molecular Probes, Eugene, OR</td>
		</tr>
		<tr>
			<td>Endothelial growth medium-2</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand Held Crimping tool</td>
			<td>Blockwise engineering</td>
			<td>M1-RMC</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00011324</td>
			<td>CAUTION: wear proptective equipment and handle under fume hood</td>
		</tr>
		<tr>
			<td>Isoflurane anesthesia</td>
			<td>Piramal Critical Care, Inc.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00011674</td>
			<td></td>
		</tr>
		<tr>
			<td>NdFeB magnet 2&#34; Dia x 1&#34; thick</td>
			<td>Amazing magnets</td>
			<td>D1000P</td>
			<td>Axially magnetized disc magnet with poles on flat faces</td>
		</tr>
		<tr>
			<td>Over-The-Wire trifold balloon</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any commercially available OTW trifold balloon can be used</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td>Commonly known as PBS</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate (NaHCO3)</td>
			<td>Sigma Aldrich</td>
			<td>MFCD00003528</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>Zoetis</td>
			<td></td>
			<td>Commercial Name: Sleepaway (26%), FatalPlus, Beuthanasi.&#160; Controlled substance to be ordered only by licensed veternarian</td>
		</tr>
		<tr>
			<td>SolidWorks</td>
			<td>Dassault systems</td>
			<td>N/A</td>
			<td>Software</td>
		</tr>
		<tr>
			<td>SpinTJ-020 micro sensor</td>
			<td>MicroMagneitcs Sensible Solutions</td>
			<td>N/A</td>
			<td>Long probe STJ-020 microsensor</td>
		</tr>
		<tr>
			<td>SPION</td>
			<td>Mayo Clinic</td>
			<td>N/A</td>
			<td>Nanoparticles synthesized internally (Ref: Lee, S. J. et al. Nanoparticles of magnetic ferric oxides encapsulated with poly(D,L latide-co-glycolide) and their applications to magnetic resonance imaging contrast agent. J Magn Magn Mater 272, 2432-2433, doi:DOI 10.1016/j.jmmm.2003.12.416 (2004))</td>
		</tr>
		<tr>
			<td>Telazol</td>
			<td>Zoetis</td>
			<td></td>
			<td>Controlled substance to be ordered only by licensed veternarian</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td>Gibco, Grand Island, NY</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer Animal Health</td>
			<td></td>
			<td>Commercial name: Rompun. Controlled sunstance to be ordered only by a licensed veternarian</td>
		</tr>
	</tbody>
</table>

ferromagnetic bare metal stent for endothelial cell capture and retention

È necessario un rapido endotelizzazione di stent cardiovascolari per ridurre la trombosi dello stent e per evitare la terapia anti-piastrinica che può ridurre il rischio di sanguinamento. La possibilità di utilizzare forze magnetiche per catturare e trattenere le cellule endoteliali escrescenza (EOC) marcate con nanoparticelle di ossido di ferro super-paramagnetiche (spion) è stato indicato in precedenza. Ma questa tecnica richiede lo sviluppo di uno stent meccanicamente funzionale da un materiale magnetico e biocompatibile seguita da in-vitro e in vivo alcune prove endotelizzazione rapida. Abbiamo sviluppato uno stent debolmente ferromagnetico in acciaio inox 2205 duplex utilizzando computer aided design (CAD) e il suo design è stata ulteriormente raffinata con analisi agli elementi finiti (FEA). Il disegno finale dello stent mostrato una deformazione principale sotto del limite frattura del materiale durante crimpatura meccanica ed espansione. Cento stent sono stati fabbricati e un loro sottoinsieme è stato utilizzato per prove meccaniche, retained misure di campo magnetico, studi in vitro di cattura delle cellule, e in vivo studi di impianto. Dieci stent sono stati testati per la distribuzione per verificare se sono sostenuti aggraffatura e l&#39;espansione del ciclo senza guasti. Altri 10 sono stati magnetizzati con stent un forte magnete al neodimio e il loro campo magnetico conservato è stato misurato. Gli stent hanno mostrato che il magnetismo trattenuto era sufficiente a catturare spion marcato EOC nei nostri studi in vitro. Spion marcato EOC cattura e la conservazione sono stati verificati in modelli animali di grandi dimensioni mediante l&#39;impianto di stent 1 magnetizzato e 1 stent controllo non magnetizzato in ognuno dei 4 maiali. Le arterie stent sono stati espiantati dopo 7 giorni e analizzati istologicamente. Gli stent debolmente magnetici sviluppati in questo studio sono stati in grado di attrarre e trattenere le cellule endoteliali spion marcato che possono promuovere una rapida guarigione.

<html> Disegno stent iterativo basato su FEA (Figura 1) ha mostrato uno stent che può crimpare ed espandere con un ceppo principale 20%, che è inferiore al 30% deformazione ultima. Piegatura e di prova di espansione (Figura 2) non ha mostrato segni di frattura. Immagini di stent deformato mostrato buon accordo con deformazioni FEA calcolate e anche foto microscopia mostrato fratture (Figura 3). Come previsto dalle misure di campo magnetico a nuovo (figure 4 e 5...

Watch this Scientific Journal Video about Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention at JoVE.com

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

I nostri obiettivi erano di progettare, produrre e testare stent ferromagnetici per la cattura delle cellule endoteliali. Dieci stent sono stati testati per la frattura e altre 10 stent sono stati testati per magnetismo mantenuto. Infine, 10 stent sono stati testati in vitro e altre 8 stent sono stati impiantati in 4 maiali per mostrare la cattura e la conservazione delle cellule.

Bare Metal Stent ferromagnetico per Capture cellule endoteliali e conservazione

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

ferromagnetic-bare-metal-stent-for-endothelial-cell-capture

Research

JoVE Journal

Bioengineering

11.4K Views.  Mayo Clinic.  I nostri obiettivi erano di progettare, produrre e testare stent ferromagnetici per la cattura delle cellule endoteliali. Dieci stent sono stati testati per la frattura e altre 10 stent sono stati testati per magnetismo mantenuto. Infine, 10 stent sono stati testati in vitro e altre 8 stent sono stati impiantati in 4 maiali per mostrare la cattura e la conservazione delle cellule. 

Video: Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Autologo di cellule progenitrici endoteliali-Seeding Tecnologie e test di biocompatibilità per dispositivi cardiovascolari in un modello animale di grandi dimensioni

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Ricerca

Bioingegneria

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the arterial wall lead to tissue injury, which initiates restenosis2-7. This hypothesis needs further investigations including better quantifications of non-uniform strain distribution on the artery following stent implantation. A non-contact surface strain measurement method for the stented artery is presented in this work. ARAMIS stereo optical surface strain measurement system uses two optical high speed cameras to capture the motion of each reference point, and resolve three dimensional strains over the deforming surface8,9. As a mesh stent is deployed into a latex vessel with a random contrasting pattern sprayed or drawn on its outer surface, the surface strain is recorded at every instant of the deformation. The calculated strain distributions can then be used to understand the local lesion response, validate the computational models, and formulate hypotheses for further in vivo study.

Stent-induced arterial strain distributions are characterized using an optical surface strain measurement system. This visualization technique is used to gain insights into the impact of stent implantation on the host vessel.

Monitoraggio dei Mechanics parete durante la distribuzione stent in un recipiente

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Elettrico Cell-substrato Impedance Sensing per la quantificazione dei Endothelial proliferazione, Barriera funzione e motilità

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Fabbricazione di meccanicamente Tunable e Bioactive metallo Ponteggi per applicazioni biomediche

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

Co-trapianto di tessuto ovarico umano con le cellule endoteliali derivati dal: una combinazione di strategia basati su cellule accelerato aspersione con consegna diretta Paracrine

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Immobilizzazione degli individui Live Caenorhabditis elegans utilizzando un Chip microfluidico Polydimethylsiloxane di ultra-sottile con ritenzione idrica

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Crescita degli strati cellulari endoteliali umani e ovini sulle membrane biomateriali

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

Mitigazione dell'attaccamento delle cellule trasmesse dal sangue agli impianti metallici attraverso l'immobilizzazione peptidica derivata da CD47

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Segmentazione della crescita delle cellule endoteliali in piastre a 6 pozzi su uno shaker orbitale per studi meccanobiologici

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Un sistema microfisiologico per studiare l'interazione leucocitaria-cellula endoteliale durante l'infiammazione

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...