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.

Alle tierexperimentellen Studien wurden von der Institutional Animal Care and Utilization Committee (IACUC) an der Mayo Klinik genehmigt. 1. Entwurf und Analyse von einem 2205 Edelstahl-Stent <ol><li> Entwerfen einer Bare-Metal-Stent unter Verwendung von CAD- <ol><li> Stellen Sie eine extrudierte Hohlzylinder, indem Sie auf &#39;extrudierten Chef / base &quot;-Funktion mit der Wanddicke gleich der Stentstrebendicke. </li><li> Entwerfen Sie ein Stentmuster auf einer anderen Skizze Ebene, die den Strangpresszylinder. Stellen Sie die Breite der flachen Muster, um den Umfang des extrudierten Hohlzylinder entsprechen. </li><li> Übertragen Sie das flache Design-Muster auf den Hohlzylinder mit dem Wrap-Funktion. </li><li> Speichern Sie das Teil in ihrem nativen Format sowie in ACIS Format für FEA exportiert werden. </li></ol></li><li> Finite-Elemente-Analyse für die Stent-Modelle <ol><li> Importieren Sie die Volumengeometrie in ACIS-Format gespeichert in den Teil-Modul des FEA-Software für weitere Analytikist. </li><li> Modell 2 analytischen Zylinder koaxial mit dem Stent in dem Teil-Modellierer des FEA-Software. Der äußere Zylinder hat einen Anfangsdurchmesser größer als der Durchmesser des Stents der Kreppvorrichtung zu simulieren, und der innere Zylinder weist einen Anfangsdurchmesser von 1 mm, um einen Ballon zum Aufblasen simulieren. </li><li> Doppelklicken Sie auf das Strukturelement der Baugruppe Modeler &#39;Fälle&#39;, um die oben genannten Teile zusammenzubauen in relativen Positionen. </li><li> Verwenden Sie die Mesh-Modul des FEA-Software, geben Sie den Elementtyp als 20 Knoten Hexaeder-Element mit reduzierten Integration, geben Sie die Elementgröße und Netz den Stent. </li><li> Geben reibungs starren Kontaktpaare zwischen dem Stent und der beiden Zylinder jeweils in den &quot;Interaktionseigenschaften&quot; der Modellbaum. </li><li> Weisen Sie elasto-plastische Spannungs-Dehnungs-Verhalten von 2205 aus Edelstahl mit dem Stent-Modell. </li><li> Randbedingungen zu definieren, um den Außenzylinder bis 1 mm, die den c simuliert zunächst CrimpRimping des Stents. Entfernen des äußeren Zylinders, um die Entspannung des gequetschten Stents simulieren. Erweitern Sie den inneren Zylinder bis 3 mm auf Expansion zu simulieren und schließlich, entfernen Sie den inneren Zylinder, um Rückstoß des Stents zu simulieren. </li><li> Definieren Sie die Simulationsparameter einschließlich der Anzahl der Prozessoren und Größe des Arbeitsspeichers im &quot;Analysis&quot; Modellbaum Posten zugeordnet und führen Sie die Simulation. </li><li> Sobald die Simulation abgeschlossen ist, öffnen Sie die Ergebnisdatei (filename.odb) und Post-Prozess die Ergebnisse an den Hauptdehnungen studieren und iterativ zu verbessern, den Stent-Design, um eine Hauptdehnung von 20%, der kleiner ist als der Ausfallgrenze des Materials zu erreichen . </li></ol></li></ol> 2. Stent Herstellung und Testing für Press- und Expansion <ol><li> Stent Herstellungs <ol><li> Besorgen Sie sich die 2205 Edelstahlrohre von gun Bohren und Präzisionsschleifen Stangenmaterial bei einer Präzisionsbearbeitung Unternehmen wie Aktions Precision Products in Pioneer,OH. </li><li> Übertragen Sie die präzisionsgeschliffen Röhren und die Abwicklung Stent-Design auf einen Stent Schneid Unternehmen wie Laserage Technology Corporation in Waukegan, IL zum Laserschneiden und Elektropolieren. </li><li> Passivieren der Oberfläche der elektro Stents durch Eintauchen davon in einer starken Säure (50% HCl) 10 min lang, gefolgt von einer Basis (10% NaHCO 3) für weitere 10 min. VORSICHT: Gehen Sie mit Chemikalien geeignete Schutzausrüstung und unter einer Abzugshaube. Schließlich, waschen Sie die Stents mit Ethylalkohol und entionisiertem Wasser. Dieser Vorgang wird als Säurebeizen. </li></ol></li><li> Testen von hergestellten Stent zum Crimpen und Expansion <ol><li> Crimp den Stent auf eine dreifach gefaltete Ballons mit einem Handpresswerkzeug. Halten Sie den Stent und den Dreifach-Ballon in die Crimp-Werkzeug. Drücken Sie den Griff, um radial zu verformen, um den Stent auf dem Ballon gequetscht werden. </li><li> Untersuchen Sie den gecrimpten Stent unter Verwendung eines Mikroskops für eine gleichmäßige Kräuselung und keine Anzeichen einer Störung in der Struktur aufgrundim plastischen Bereich. </li><li> Erweitern Sie es an die entworfen Durchmesser von 3 mm durch Unterdrucksetzen des trifold Ballon mit Wasser. Untersuchen Sie die erweiterten Stents für die mikroskopische Brüche und gleichmäßige Expansion. </li></ol></li></ol> 3. Charakterisierung der Stent für Rück Magnetic Field HINWEIS: Zylindermagneten von 2 Zoll Durchmesser und 1 Zoll Höhe wurde in dieser Studie verwendet. Die Pole des Magneten sind entlang der Achse ausgerichtet. Die Oberflächenmagnetflussdichte des Magneten ungefähr 1 T. <ol><li> Magnetisieren die Stents diametral oder axial unter Verwendung einer starken Neodym-Magneten. Halten Sie den Stent in der Nähe des starken Magneten für ca. 1 min für die Magnetisierung. </li><li> Halten des Stents an einer der ebenen Flächen mit einem Durchmesser entlang der magnetischen Feldlinien an diametral magnetisiert sein oder halten den Stent neben der zylindrischen Fläche, deren Achse entlang der magnetischen Feldlinien, um sie axial zu magnetisieren. Einbehaltene Magnet field des Stents wurde gefunden, daß für mindestens 24 Stunden stabil sein, sondern müssen über die Stents so bald wie möglich nach der Magnetisierung. </li><li> Montieren Sie die stents einzeln auf Glasdorne und hängen Sie dann das Glas Dorne in der Präzisionsspannfutter des magnetischen Sondenbefestigung. Magnetische Mikrosonde präzise ohne Berührung der Oberfläche mit der XYZ-Tische Anordnung der magnetischen Sondenvorrichtung (4) nahe dem Stent werden. </li><li> Messung der Basislinie Lesen der magnetischen Mikro weit weg vom Stent und dann den zurückgehalten Magnetfeld an der Stentoberfläche durch Positionierung der Sonde unter Verwendung der XYZ-Tische des magnetischen Sondenbefestigung. </li></ol> 4. Magnetzellaufnahmestudien <ol><li> Gewinnung von Zellen, die Markierung mit SPION und Färbung mit Fluoreszenzfarbstoff <ol><li> Leiten Sie die endothelialen Zellen Auswuchs (EOC) aus Schweine peripheren Blut wie in 5,7 beschrieben. Kultur in einem T-75-Kolben until etwa 80% konfluent (5x10 6 bis 8x10 6 Zellen). </li><li> Synthetisieren SPIONs als 10 nm Durchmesser Magnetit (Fe 3 O 4) Kern, umgeben von 50 nm dicke Poly (milch-co-Glykolsäure) (PLGA) Schale nach 8,9 beschrieben. </li><li> Inkubieren des abgeleiteten EOC mit SPION bei einer Konzentration von 200 ug / ml Zellkulturmedium für 16 Stunden bei 37 o C. </li><li> Saugen Sie das Zellkulturmedium sanft. Sanft die Zellen werden durch Zugabe von 10 ml Phosphat-gepufferter Salzlösung (PBS) in den Kolben, schaukeln, und Absaugen des PBS. </li><li> Färben die Zellen, die mit Fluoreszenzfarbstoff (CM-Dil) zur Visualisierung während der Experimente. Dies wird nach den Angaben des Herstellers durch Zugabe des Farbstoffs zu 10 ml Zellkulturmedium in einer Konzentration von 5 &amp; mgr; l / ml und Inkubation mit den Zellen für 30 min bei 37 ° C durchgeführt. </li><li> Waschen der Zellen mit PBS wie in Schritt 4.1.4 und Inkubation mit 3 ml 0,25% Trypsin-EDTA-Lösung für 5 min bei 37 ° C aufheben die Zellen aus dem Kolben. </li><li> Übertragen der Zellsuspension in ein 15 ml konisches Röhrchen, oben mit PBS gewaschen und bei 500 xg für 5 Minuten, um ein Zellpellet zu bilden. </li><li> Re-suspendieren des Zellpellets in PBS bei einer Konzentration von 1-2x10 &amp; sup6; Zellen / ml und gründlich durch Pipettieren in die und aus der konischen Röhrchen mehrere Male. </li></ol></li><li> In-vitro-Zellstudien <ol><li> Design und zu verarbeiten (zum Beispiel 3D-Drucken) eine einfache Befestigung des Stents direkt über der Oberfläche eines Deckglases zu halten. </li><li> Entmagnetisieren eines Stents unter Verwendung einer elektromagnetischen Degausser oder magnetisieren einen Stent diametral oder axial unter Verwendung einer starken Neodym-Magneten. </li><li> Pipettieren Sie die SPION-markierten EOC in PBS in die Schüssel mit den axial magnetisiert oder diametral magnetisierte oder nicht-magnetisierten Kontrollstents suspendiert. Bild die Stents mit EOC in PBS unmittelbar für die Fluoreszenz suspendiert mit einem inversen Fluoreszenzmikroskop. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 5. In-vivo-Tierstudien <ol><li> Stentimplantation <ol><li> Zeichnen peripheres Blut von 4 gesunden Yorkshire Schweine - einem Gewicht von etwa 50 kg - 3 Wochen vor der Implantation bzw. Stent und Kultur EOC nach 5,7 beschrieben. </li><li> Verwalten Anti-Thrombozyten-Medikament, ab 3 Tage vor der Operation (Aspirin 325 mg und 75 mg Clopidogrel täglich). </li><li> Auf der Stentimplantation Tag, betäuben die Schweine mit intramuskulären Telazol, Xylazin und Atropin (5 / 2-3 / 0,05 mg / kg), wie in den geltenden institutionellen Tierpflege und Leitlinien für die Verwendung angegeben. </li><li> Intubieren und legen Sie das Schwein auf Einatmen von 1-2,5% Isofluran-Narkose. </li><li> Rasieren Sie die ventralen Halsbereich des Schweins und führen das Verfahren im allgemeinen sterilen Bedingungen. </li><li> Implantat 1 magnetisiert und 1 nichtmagnetisierten Stents in der rechten Koronararterie (RCA) unter Verwendung von Standardherzkathetertechnik. <ol><li> Katheterisierung von Tieren sollte durch eine ausgebildete interventioneller Kardiologe durchgeführt werden. Besuchen Sie das rechte Halsschlagader mit einem 9 Französisch Scheide. </li><li> Kanülieren das Ziel der Herzkranzgefäße und injizieren jodhaltigen Kontrastfarbstoff zur Durchleuchtungsbilder zu erhalten. </li><li> Legen Sie eine 0,014 Inch Standard koronaren Führungsdraht in die Arterie. Schieben Sie den Ballon und Stent mit diesen Führungsdraht und den Stent in einer 3-3,5 mm Durchmesser Schiffes. </li></ol></li><li> Verstopfen den Blutfluss innerhalb der RCA proximal der implantierten Stents unter Verwendung eines über dem Draht Ballon und liefern etwa 2x10 6 autologe EOC mit SPION in 4 ml PBS über die Zentralkatheter über eine 2-minütige Zeitspanne ausgesetzt markiert. </li><li> Wiederherstellung des Blutflusses zum RCA nach 2 min zusätzlicher Okklusion. </li><li> Übertragen Sie das Tier in den Aufwachraum und das Tier genau zu überwachen, bis er wieder zu sich kam. </li><li> Weiter zum Anti-Thrombozyten-Medikament zu verabreichen (325 mg Aspirin und Clopidogrel 75 mg) nach der Operation bis zum Opfer. </li></ol></li><li> Stent Explantation und Histologie <ol><li> Euthanize der Tiere 7 Tage nach der Operation durch erste Betäubung des Tieres wie zuvor erläutert und dann intravenös verabreichen eine tödliche Dosis Natriumpentobarbital (100 mg / kg) nach geltenden institutionellen Animal Care und Verwenden Richtlinien. </li><li> Chirurgisch Ernte der Stent arteriellen Segmenten. Fixierung der explantierten Arterien in 10% Formalin-Puffer für ein Minimum von 30 min. Lassen Sie die Proben in Formalin-Puffer für weitere histologische Analyse. </li><li> Outsource die feste Probe zu Einrichtungen der Lage ist, die Histologie mit Metall-Stents. Während dieser Verarbeitung werden die Proben in Methylmethacrylat eingebettet werden, im Querschnitt, und mit Mallorys Färbetechnik mit Berliner Blau Färbung für Eisenpartikel histologisch analysiert. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Wir entwickelten eine magnetische Stent, der als ein Bare-Metal-Stent funktionieren kann und SPION-markierten Endothelzellen zu gewinnen. In früheren Studien, bei denen magnetische Stents haben Forscher vernickelt kommerziellen Stents und Spulen oder Maschen aus magnetischen Materialien bestehen aufgrund der Nichtverfügbarkeit eines ferromagnetischen Stent 5,10-14 verwendet. Andere Gruppen haben auch für die Ausrichtung von Nanopartikeln beladen Endothelzellen 3 verwendet die paramagnetischen Ch...

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

Schnelle Endothelialisierung kardiovaskuläre Stents erforderlich ist, um Stent-Thrombose zu verringern und die Anti-Thrombozyten-Therapie, die das Blutungsrisiko zu verringern, zu vermeiden. Die Durchführbarkeit der Verwendung von magnetischen Kräfte zu erfassen und behalten endothelialen Zellen Auswuchs (EOC) mit Super-paramagnetische Eisenoxid-Nanopartikel (SPION) markiert wurde zuvor gezeigt. Aber diese Technik erfordert die Entwicklung eines mechanisch funktionale Stent aus einem magnetischen und biokompatiblen Material, gefolgt von in-vitro und in-vivo-Tests, um eine schnelle Endothelisierung zu beweisen. Wir entwickelten ein schwach ferromagnetischen Stent aus 2205 Duplex-Edelstahl mit Computer Aided Design (CAD) und sein Design wurde weiter unter Verwendung von Finite-Elemente-Analyse (FEA) verfeinert. Das endgültige Design des Stents zeigten eine Hauptdehnung unterhalb der Bruchgrenze des Materials bei der mechanischen Crimpen und Expansion. Einhundert Stents wurden hergestellt und eine Teilmenge davon wurde für die mechanische Prüfung, ret verwendetained Magnetfeldmessungen, in-vitro Zelleinfang Studien und in-vivo-Implantation Studien. Zehn Stents wurden für den Einsatz getestet, um zu überprüfen, ob sie anhalt Crimpen und Expansionszyklus ohne Ausfall. Weitere 10 Stents wurden unter Verwendung einer starken Neodym-Magneten magnetisiert und ihre beibehalten Magnetfeld gemessen. Die Stents zeigten, dass die zurückgehalten Magnetismus war ausreichend, um SPION-markierten EOC in unserer In-vitro-Studien zu erfassen. SPION-markierten EOC-Abscheidung und Retention wurde in Großtiermodellen durch Implantation von 1 magnetisierten Stent und 1 nicht-magnetisierten Kontrollstent in jedem der 4 Schweinen nachgewiesen. Stent versehenen Arterien wurden nach 7 Tagen explantiert und histologisch ausgewertet. Die in dieser Studie entwickelt schwach magnetischen Stents waren in der Lage, die Gewinnung und Bindung SPION-markierten Endothelzellen, die schnelle Heilung fördern können.

<html> Iterative Stentdesign basierend auf FEA (Abbildung 1) zeigte einen Stent, der Kräuselung und erweitern mit einer Hauptdehnung von 20%, die weniger als 30% der Bruchdehnung ist kann. Press- und Expansionstest (Abbildung 2) zeigte keine Anzeichen von Bruch. Fotos von der verformten Stent zeigte eine gute Übereinstimmung mit FEM berechneten Verformungen und auch Mikroskopie Bilder zeigten keine Frakturen (Abbildung 3). Wie aus dem Bilanzmagnetfeldmessungen ...

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

Unser Ziel war es, entwickeln, produzieren und testen ferro Stents für Endothelzellen-Capture. Zehn Stents wurden zur Fraktur getestet und 10 weitere Stents wurden für einbehaltene Magnetismus getestet. Schließlich wurden 10 Stents in vitro getestet und 8 weitere Stents wurden in 4 Schweinen implantiert, um die Zellaufnahme und Retention zu zeigen.

Ferromagnetische Bare Metal Stent für Endothelzellen-Abscheidung und Retention

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 Aufrufe.  Mayo Clinic.  Unser Ziel war es, entwickeln, produzieren und testen ferro Stents für Endothelzellen-Capture. Zehn Stents wurden zur Fraktur getestet und 10 weitere Stents wurden für einbehaltene Magnetismus getestet. Schließlich wurden 10 Stents in vitro getestet und 8 weitere Stents wurden in 4 Schweinen implantiert, um die Zellaufnahme und Retention zu zeigen. 

Serie: 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.

Autologe endotheliale Vorläuferzellen Cell-Sätechnik und Biokompatibilitätstestung Für Herz-Kreislauf-Geräte in Großtiermodell

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

Forschung

Biotechnik

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.

Die Überwachung der Wand Mechanik Während der Entfaltung des Stents in einem Gefäß

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.

Elektro-Zell-Substrat-Impedance Sensing zur Quantifizierung der endothelialen Proliferation, Barrierefunktion und Beweglichkeit

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.

Herstellung von mechanisch Tunable und Bioactive Metallgerüste für biomedizinische Anwendungen

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 Transplantation von menschlichen Eierstockgewebe mit veränderter Endothelzellen: eine Zell-basierte Strategie-Kombination beschleunigt Perfusion mit direkten Parakrine Lieferung

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.

Immobilisierung von Live Caenorhabditis Elegans Einzelpersonen, die eine ultraflache Polydimethylsiloxan Mikrofluidik-Chip mit Wassereinlagerungen

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.

Wachstum von Human- und Schafhornhaut-Endothelzellschichten auf Biomaterialmembranen

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.

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.

Segmentierung des Wachstums von Endothelzellen in 6-Well-Platten auf einem Orbitalschüttler für mechanobiologische Studien

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.

Ein mikrophysiologisches System zur Untersuchung der Leukozyten-Endothelzell-Interaktion während einer Entzündung

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