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.

Toutes les études sur les animaux ont été approuvées par le Comité d&#39;utilisation institutionnel de protection des animaux et (IACUC) à la Clinique Mayo. 1. Conception et analyse d&#39;un stent en acier inoxydable 2,205 <ol><li> Conception d&#39;un stent en métal nu en utilisant CAD <ol><li> Faites un cylindre creux extrudé en sélectionnant sur option &#39;extrudé patron / de base »avec l&#39;épaisseur de paroi égale à l&#39;épaisseur de l&#39;entretoise stent. </li><li> Concevoir un modèle de stent sur un plan d&#39;esquisse différents tangent au cylindre extrudé. Faire de la largeur du motif plat en fonction de la circonférence du cylindre creux extrudé. </li><li> Transférer la conception de modèle à plat sur le cylindre creux en utilisant la fonction de rembobinage. </li><li> Enregistrez la pièce dans son format natif et aussi en format ACIS être exportés pour être FEA. </li></ol></li><li> Analyse par éléments finis pour les modèles de stent <ol><li> Importez la géométrie solide enregistrée au format ACIS dans le module de partie du logiciel de FEA pour d&#39;autres analysest. </li><li> Modèle 2 cylindres analytiques coaxiale au stent dans la partie modeleur du logiciel FEA. Le cylindre extérieur a un diamètre initial supérieur au diamètre du stent pour simuler la pince et le cylindre intérieur a un diamètre initial de 1 mm pour simuler un ballonnet de gonflage. </li><li> Double-cliquez sur l&#39;élément du modeleur de montage de l&#39;arbre «instances» pour assembler le dessus dudit pièces dans des positions relatives. </li><li> Utilisez le module de maillage du logiciel FEA, spécifier le type d&#39;élément comme élément hexaédrique 20 nœuds avec intégration réduite, spécifier la taille de l&#39;élément, et la maille du stent. </li><li> Spécifiez paires de contact rigides friction entre le stent et les deux cylindres respectivement dans les «propriétés d&#39;interaction» de l&#39;arbre du modèle. </li><li> Attribuer contrainte-déformation comportement élasto-plastique de l&#39;acier inoxydable 2,205 pour le modèle de stent. </li><li> Définir les conditions aux limites pour le sertissage d&#39;une part le cylindre extérieur à 1 mm qui simule le cRimping du stent. Retirer le cylindre extérieur pour simuler la relaxation du stent serti. Développez le cylindre intérieur à 3 mm pour simuler l&#39;expansion et enfin, retirer le cylindre intérieur pour simuler le recul du stent. </li><li> Définir les paramètres de simulation, y compris le nombre de processeurs et la quantité de RAM alloués dans le &#39;Analyse&#39; élément de modèle de l&#39;arbre et exécuter la simulation. </li><li> Une fois la simulation terminée, ouvrez le fichier de résultat (filename.odb) et post-traitement des résultats pour étudier les principales souches et itérative améliorer la conception de stent pour obtenir une souche principale de 20%, ce qui est inférieur à la limite de l&#39;échec de la matière . </li></ol></li></ol> 2. Stent fabrication et les essais pour le sertissage et expansion <ol><li> Stent fabrication <ol><li> Obtenir les 2205 tubes en acier inoxydable par le forage des armes à feu et la précision bar à broyer la matière à une entreprise d&#39;usinage de précision tels que l&#39;action Precision Products dans Pioneer,OH. </li><li> Transférer les tubes au sol de précision et la conception modèle de stent à plat pour une entreprise de stent coupe comme Laserage Technology Corporation à Waukegan, IL pour la coupe et le polissage électrolytique laser. </li><li> Passiver la surface des stents en les immergeant électropoli dans un acide fort (HCl 50%) pendant 10 min, suivie par une base (10% de NaHCO 3) pour un autre 10 min. ATTENTION: manipuler des produits chimiques avec un équipement de protection approprié et sous une hotte. Enfin, lavez les stents à l&#39;alcool éthylique et de l&#39;eau déminéralisée. Ce processus est appelé décapage à l&#39;acide. </li></ol></li><li> Essai de stent fabriqué pour le sertissage et l&#39;expansion <ol><li> Sertir le stent sur un ballon à trois volets en utilisant une main tendue outil de sertissage. Tenez le stent et le ballon à trois volets dans l&#39;outil de sertissage. Appuyez sur la poignée pour déformer radialement le stent à être sertie sur le ballon. </li><li> Inspectez le stent serti en utilisant un microscope pour uniforme sertissage et aucun signe de défaillance de la structure en raisonà déformation plastique. </li><li> Développer pour le diamètre de 3 mm conçu en pressurisant le ballon à trois volets avec de l&#39;eau. Examiner les stents élargis pour les fractures microscopiques et l&#39;expansion uniforme. </li></ol></li></ol> 3. Caractérisation de stent pour le champ magnétique conservée REMARQUE: aimant cylindrique de 2 pouces de diamètre et 1 pouce de hauteur a été utilisé dans cette étude. Les pôles de l&#39;aimant sont alignés le long de l&#39;axe. La densité de surface de flux magnétique de l&#39;aimant est d&#39;environ 1 T. <ol><li> Magnétiser les stents diamétralement ou axialement l&#39;aide d&#39;un aimant néodyme forte. Tenez le stent à proximité de l&#39;aimant puissant pour environ 1 min pour la magnétisation. </li><li> Maintenir l&#39;extenseur sur l&#39;une des faces plates avec son diamètre le long des lignes de champ magnétique d&#39;être magnétisés diamétralement ou tenir le stent à côté de la surface cylindrique avec son axe le long des lignes de champ magnétique pour magnétiser axialement. Fiel magnétique retenuD du stent a été jugée stable pendant au moins 24 heures, mais utiliser le stent dès que possible après aimantation. </li><li> Montez les stents individuellement sur des mandrins de verre et puis montez les mandrins de verre dans le mandrin de précision de l&#39;appareil de sondage magnétique. Sonde de microcapteurs magnétiques peut être positionné précisément à proximité du stent sans toucher la surface en utilisant la scène XYZ ensemble de l&#39;appareil de sondage magnétique (Figure 4). </li><li> Mesurer la lecture de référence du micro magnétique loin du stent, puis mesurer le champ magnétique retenu à la surface de l&#39;endoprothèse en positionnant la sonde en utilisant les stades XYZ de l&#39;appareil de sondage magnétique. </li></ol> 4. Études cellule de capture magnétiques <ol><li> Obtention de cellules, l&#39;étiquetage avec SPION et coloration avec un colorant fluorescent <ol><li> Dériver les cellules endothéliales excroissance (COE) du sang périphérique porcine comme décrit dans 5,7. Culture dans un flacon T-75 until environ 80% de confluence (5x10 6 à 8x10 6 cellules). </li><li> Synthétiser SPIONs que le diamètre de 10 nm de la magnétite (Fe 3 O 4) de noyau entouré par 50 nm d&#39;épaisseur de poly (lactique-co-acide glycolique) (PLGA) comme décrit dans l&#39;enveloppe 8,9. </li><li> Incuber les COE dérivé avec SPION à une concentration de 200 ug / ml de milieu de culture cellulaire de 16 h à 37 o C. </li><li> Aspirer le milieu de culture cellulaire doucement. Laver avec précaution les cellules en ajoutant 10 ml d&#39;une solution saline tamponnée au phosphate (PBS) dans le ballon, à bascule, et en aspirant le PBS. </li><li> Colorer les cellules avec un colorant fluorescent (CM-Dil) pour visualiser lors des expériences. Ceci est réalisé selon les instructions du fabricant en ajoutant le colorant à 10 ml de milieu de culture cellulaire à une concentration de 5 ul / ml et l&#39;incubation avec les cellules pendant 30 min à 37 ° C. </li><li> Laver les cellules avec du PBS comme à l&#39;étape 4.1.4 et incuber avec 3 ml de solution de trypsine-EDTA 0,25% pendant 5 minutes à 37 ° C etlever les cellules de la fiole. </li><li> Transférer la suspension cellulaire à un tube conique de 15 ml, de haut avec du PBS, et centrifuger à 500 g pendant 5 min pour former un culot cellulaire. </li><li> Remettre en suspension le culot cellulaire dans du PBS à une concentration de 1-2x10 6 cellules / ml et bien mélanger par pipetage dans et hors du tube conique à plusieurs reprises. </li></ol></li><li> Des études in vitro de cellules <ol><li> Concevoir et fabriquer (par exemple, l&#39;impression 3D), un appareil simple pour tenir le stent juste au-dessus de la surface d&#39;une lamelle de verre. </li><li> Démagnétisez un stent à l&#39;aide d&#39;un démagnétiseur électromagnétique ou magnétiser un stent diamétralement ou axialement l&#39;aide d&#39;un aimant néodyme forte. </li><li> Pipeter l&#39;EOC de SPION marqué en suspension dans PBS dans le plat contenant les stents de contrôle axialement magnétisés ou diamétralement magnétisés ou non magnétisées. Image les stents avec EOC en suspension dans PBS immédiatement pour fluorescence à l&#39;aide d&#39;un microscope inversé à fluorescence. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 5. Des études in vivo animaux <ol><li> Implantation d&#39;un stent <ol><li> Dessinez le sang périphérique à partir de 4 porcs Yorkshire sains - pesant environ 50 kg - 3 semaines avant l&#39;implantation du stent, respectivement, et la culture COE comme décrit dans 5,7. </li><li> Administrer un médicament anti-plaquettaire à partir de 3 jours avant la chirurgie (aspirine 325 mg et clopidogrel 75 mg par jour). </li><li> Le jour stent implantation, anesthésier les porcs avec intramusculaire Telazol, xylazine, et de l&#39;atropine (5 / 2-3 / 0,05 mg / kg, respectivement) comme indiqué dans les soins des animaux et de l&#39;utilisation des directives institutionnelles applicables. </li><li> Intuber et placer le porc sur l&#39;inhalation de 1-2,5% isoflurane. </li><li> Rasez la région du cou ventrale du porc et de mener la procédure dans des conditions stériles générales. </li><li> Implant 1 magnétisé et 1 stent non-aimanté dans l&#39;artère coronaire droite (RCA) en utilisant la technique de cathétérisme cardiaque standard. <ol><li> Catheterisation des animaux doit être réalisée par un cardiologue interventionnel formés. Accéder à l&#39;artère carotide droite avec une gaine 9 français. </li><li> Cathétériser l&#39;artère coronaire cible et d&#39;injecter un colorant de contraste iodé pour obtenir des images de radioscopie. </li><li> Placer un fil de guidage coronaire norme 0,014 pouce dans l&#39;artère. Avancer le ballon et le stent en utilisant ce fil de guidage et de déployer le stent dans un vaisseau 3-3,5 mm de diamètre. </li></ol></li><li> Occlure le flux sanguin à l&#39;intérieur de l&#39;extrémité proximale de RCA les stents implantés au moyen d&#39;un ballonnet sur ​​fil et d&#39;environ 2x10 6 fournir EOC autologues marquées avec SPION en suspension dans 4 ml de PBS par l&#39;intermédiaire du cathéter central sur une période de 2 min. </li><li> Restaurer le flux sanguin vers le RCA après 2 min d&#39;occlusion supplémentaire. </li><li> Transférer l&#39;animal à la salle de récupération et de suivre de près l&#39;animal jusqu&#39;à ce qu&#39;il a repris conscience. </li><li> Continuer à administrer des médicaments anti-plaquettaire (aspirine 325 mg et clopidogrel 75 mg) après l&#39;opération jusqu&#39;au sacrifice. </li></ol></li><li> Expiant stent et l&#39;histologie <ol><li> Euthanasier les animaux 7 jours après la chirurgie par abord anesthésier l&#39;animal comme expliqué précédemment, puis administrer par voie intraveineuse une dose létale de pentobarbital de sodium (100 mg / kg) que par le soin des animaux et de l&#39;utilisation des directives institutionnelles applicables. </li><li> Récolter chirurgicalement les segments artériels stentées. Fixer les artères explantées dans un tampon de formol à 10% pour un minimum de 30 min. Laissez les échantillons dans un tampon de formol pour une analyse ultérieure histologique. </li><li> Externaliser l&#39;échantillon fixe d&#39;installations capables d&#39;effectuer l&#39;histologie avec les stents métalliques. Au cours de ce traitement, les échantillons sont incorporer dans le méthacrylate de méthyle, en coupe transversale, et analysés histologiquement en utilisant la technique de coloration de Mallory avec taches bleu de Prusse pour les particules de fer. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

Nous avons développé un stent magnétique qui peut fonctionner comme un stent en métal nu et peut attirer les cellules endothéliales Spion marqué. Dans des études précédentes impliquant des stents magnétiques, les chercheurs ont utilisé nickel stents actifs commerciaux et des bobines ou des mailles en matériaux magnétiques en raison de l&#39;indisponibilité d&#39;un stent ferromagnétique 5,10-14. D&#39;autres groupes ont également utilisé la nature paramagnétique disponibles dans le commerce ...

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

Endothélialisation rapide des endoprothèses vasculaires est nécessaire pour réduire la thrombose de stent et d&#39;éviter la thérapie anti-plaquettaire qui peut réduire le risque de saignement. La faisabilité de l&#39;utilisation des forces magnétiques pour capturer et conserver les cellules endothéliales excroissance (COE) marqués avec des nanoparticules d&#39;oxyde de fer super-paramagnétiques (SPION) a été montré précédemment. Mais cette technique nécessite la mise au point d&#39;un stent mécaniquement fonctionnelle à partir d&#39;un matériau magnétique et biocompatible suivi par in-vitro et in-vivo des tests pour prouver endothélialisation rapide. Nous avons développé un stent faiblement ferromagnétique acier inoxydable duplex 2205 en utilisant conception assistée par ordinateur (CAD) et sa conception a été affinée en utilisant une analyse par éléments finis (FEA). La conception finale du stent présenté une déformation principale sous la limite de rupture du matériau pendant le sertissage mécanique et d&#39;expansion. Une centaine de stents ont été fabriqués et un sous-ensemble d&#39;entre eux a été utilisé pour les essais mécaniques, retained mesures de champ magnétique, des études in vitro de capture de cellules, in vivo et des études d&#39;implantation. Dix stents ont été testés pour déploiement pour vérifier si elles soutenue sertissage et l&#39;expansion du cycle sans défaillance. 10 autres ont été magnétisés stents utilisant un aimant de néodyme forte et leur champ magnétique retenu a été mesurée. Les stents ont montré que le magnétisme retenu était suffisante pour capturer SPION marqué EOC dans nos études de in vitro. Capture et la conservation des COE SPION marqué a été vérifiée dans les grands modèles animaux par l&#39;implantation d&#39;une endoprothèse magnétisé et 1 non-aimanté stent de contrôle dans chacun des 4 porcs. Les artères ont été explantées stentées après 7 jours et analysés histologiquement. Les stents faiblement magnétiques développés dans cette étude étaient capables d&#39;attirer et de retenir les cellules endothéliales Spion marqué qui peut favoriser la guérison rapide.

<html> Conception de stent itératif basé sur FEA (Figure 1) a montré un stent qui peut sertir et étendre avec une souche principale de 20%, ce qui est moins que la déformation à la rupture de 30%. Sertissage et l&#39;essai d&#39;expansion (figure 2) ne présentaient aucun signe de fracture. Photos du stent déformé ont montré une bonne entente avec déformations FEA calculés et aussi des photos microscopie montré aucune fracture (Figure 3). Comme on s&#39;y att...

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

Nos objectifs étaient de concevoir, fabriquer et tester stents ferromagnétiques pour la capture des cellules endothéliales. Dix stents ont été testés pour la fracture et 10 autres stents ont été testés pour le magnétisme retenu. Enfin, 10 stents ont été testés in vitro et 8 autres stents ont été implantés dans 4 porcs pour montrer capture et la conservation cellule.

Ferromagnétique Bare Metal Stent pour endothéliale capture de cellules et de la conservation

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 vues.  Mayo Clinic.  Nos objectifs étaient de concevoir, fabriquer et tester stents ferromagnétiques pour la capture des cellules endothéliales. Dix stents ont été testés pour la fracture et 10 autres stents ont été testés pour le magnétisme retenu. Enfin, 10 stents ont été testés in vitro et 8 autres stents ont été implantés dans 4 porcs pour montrer capture et la conservation cellule. 

Vidéo: 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.

Autologue de cellules progénitrices endothéliales-ensemencement de la technologie et les tests de biocompatibilité pour les appareils de cardio-vasculaires dans le modèle des grands animaux

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

Recherche

Bio-ingénierie

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.

Suivi des Mécanique mur pendant le déploiement du stent dans un vaisseau

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.

Électrique Cell-substrat Impedance Sensing pour la quantification de la prolifération endothéliale, de la fonction barrière, et la motilité

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.

Fabrication de mécanique accordable et bioactifs Métal échafaudages pour des applications biomédicales

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 de tissu ovarien humain avec des cellules endothéliales ingénieries : une combinaison de stratégie basés sur les cellules accéléré la Perfusion avec livraison directe 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.

Immobilisation des individus vivants Caenorhabditis elegans en utilisant une puce de microfluidique de polydiméthylsiloxane ultra-mince avec rétention d’eau

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.

Croissance des couches cellulaires endothéliales coréennes humaines et brebis sur les membranes biomatériaux

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.

Atténuation de l’attachement cellulaire transmissible par le sang aux implants métalliques grâce à l’immobilisation du peptide dérivé du 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.

Segmentation de la croissance des cellules endothéliales dans des plaques à 6 puits sur un agitateur orbital pour les études mécanobiologiques

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 système microphysiologique pour étudier l’interaction leucocytaire-endothéliale pendant l’inflammation

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