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.

Tüm hayvan çalışmaları Mayo Clinic Kurumsal Hayvan Bakım ve Kullanım Komitesi (IACUC) tarafından onaylanmıştır. Bir 2205 Paslanmaz Çelik Stent 1. Tasarımı ve Analizi <ol><li> CAD kullanarak çıplak metal stent Tasarımı <ol><li> Stent payanda kalınlığına eşit et kalınlığına sahip &#39;ekstrüde patron / baz&#39; özelliği seçerek haddelenmiş oyuk bir silindir yapın. </li><li> Haddelenmiş silindir teğet farklı bir skeç düzlemde bir stent desen tasarlayın. Haddelenmiş oyuk silindirin çevresini maç düz desen genişliğini olun. </li><li> Şal özelliğini kullanarak boş silindirin üzerine düz desen tasarımı aktarın. </li><li> Kendi özgün biçiminde ve aynı zamanda FEA için ihraç edilecek ACIS biçiminde bir bölümünü kaydedebilirsiniz. </li></ol></li><li> Stent modelleri için sonlu elemanlar analizi <ol><li> Daha fazla analizi kullanılmıştır için FEA yazılımının bir parçası modülüne ACIS formatında kaydedilen katı geometri İthalatolduğunu. </li><li> FEA yazılımının bir parçası modelleyici içinde stent koaksiyal 2 analitik silindir Model. Dış silindir kıvırma simüle etmek için stent çapından bir başlangıç ​​çapı daha büyük olan ve iç silindir enflasyonu bir balon simüle etmek için 1 mm&#39;lik bir başlangıç ​​çapına sahiptir. </li><li> Above monte montaj modelleyicinin &#39;örneklerini&#39; ağaç öğesi üzerine çift tıklayın göreli konumları parçaları söyledi. </li><li> Indirgenmiş entegrasyonu ile 20 düğüm hexahedral elemanı olarak eleman türünü belirtin, FEA yazılımı örgü modülünü kullanın eleman boyutunu belirtin ve stent örgü. </li><li> Stent ve model ağacı &#39;etkileşim özelliklerinin&#39; sırasıyla iki silindir arasındaki sürtünmesiz sert kontakt çiftleri belirleyin. </li><li> Stent modeline 2205 paslanmaz çelikten elasto-plastik gerilme-deformasyon davranışını atayın. </li><li> Öncelikle c taklit 1 mm dış silindir kıvrım için sınır şartları tanımlayınstentin Rimping. Kıvrılmış stentin gevşeme simüle etmek için dış silindiri çıkartın. Genişleme simüle etmek için 3 mm iç silindir genişletin ve nihayet, stent geri tepme simüle etmek için iç silindir çıkarın. </li><li> &#39;Analiz&#39; modeli ağaç kaleminde ayrılan işlemci ve RAM miktarı sayısı dahil simülasyon parametreleri tanımlayın ve simülasyon çalıştırın. </li><li> Simülasyon tamamlandıktan sonra, ana suşları incelemek için sonuçlar Sonuç dosyası (filename.odb) ve post-process açın ve iteratif malzeme hatası sınırından daha az% 20 anapara gerginlik elde etmek için stent tasarımını geliştirmek . </li></ol></li></ol> Sıkma ve Genişleme 2. Stent İmalatı ve Test <ol><li> Stent fabrikasyon <ol><li> Böyle Pioneer Eylem Hassas Ürünler olarak bir hassas işleme şirketi de silah sondaj ve hassas taşlama çubuk stok malzeme ile 2205 paslanmaz çelik borular elde etmek,OH. </li><li> Lazer kesim ve elektro böyle Waukegan, IL Laserage Technology Corporation olarak stent kesme şirkete hassas taşlanmış tüpleri ve düz model stent tasarımı aktarın. </li><li> Başka bir 10 dakika boyunca, bir baz (% 10 NaHCO 3), ardından 10 dakika boyunca kuvvetli bir asit (50% HCI) bunları batırmak suretiyle elektro-stent yüzey pasifleştirilmesi. DİKKAT: Uygun koruyucu ekipman ve davlumbaz altında kimyasal anlaştım. Son olarak, bir etil alkol ve deiyonize su ile stent yıkayın. Bu işlem, asit temizleme adlandırılır. </li></ol></li><li> Kıvırma ve genişleme için imal stent Testi <ol><li> Aracı Sıkma bir el kullanarak üç katlı balonu üzerine stenti kıvırın. Stent ve sıkma aracı üç katlı balon tutun. Radyal balon kıvrılır edilecek stent deforme için kolu basın. </li><li> Üniforma sıkma için bir mikroskop ve bağlı yapıda başarısızlık herhangi bir işaret kullanılarak kıvrımlı stent inceleyinplastik deformasyona. </li><li> Su ile üç katlı balon basınç ile 3 mm tasarlanmış çapa genişletin. Mikroskobik kırıklar ve uniform genişleme için genişletilmiş stentler inceleyin. </li></ol></li></ol> Tutulan Manyetik Alan için Stent 3. Karakterizasyonu Not: 2 inç çapında ve 1 inç yüksekliğinde plastik bir silindir mıknatıs bu çalışmada kullanılmıştır. Mıknatıs kutupları ekseni boyunca hizalanır. Mıknatısın yüzey manyetik akı yoğunluğu yaklaşık T. 1 <ol><li> Güçlü neodimyum mıknatıs kullanarak taban tabana veya eksenel stent çekmek. Mıknatıslanma yaklaşık 1 dakika boyunca güçlü mıknatıs yakın stent tutun. </li><li> Taban tabana manyetize veya eksenel ve mıknatısla manyetik alan çizgileri boyunca kendi ekseni ile silindirik yüzeye sonraki stent tutun edilecek manyetik alan çizgileri boyunca çapı düz yüzeylerinden birine stent tutun. Tutulan manyetik fielstentin d, en az 24 saat süre ile stabil olabilir, ama manyetizasyon sonra mümkün olan en kısa sürede stent bulunmuştur. </li><li> Cam mandreller üzerine tek tek stent monte edin ve daha sonra manyetik sondalama fikstür hassas aynasının cam millerini monte edin. Manyetik mikrosensor prob tam XYZ manyetik sondalama fikstür montajını aşamaları kullanarak yüzeyi (Şekil 4) dokunmadan stent yakın yerleştirilmiş olabilir. </li><li> Uzakta stent manyetik mikrosensor bazal okuma ölçün ve sonra manyetik sondalama fikstür XYZ aşamalarını kullanarak prob konumlandırarak stent yüzeyine muhafaza manyetik alan ölçer. </li></ol> 4. Manyetik Hücre Yakalama Çalışmaları <ol><li> Elde edilmesi hücreleri, floresan boya ile spion ve boyama ile markalama <ol><li> 5,7 anlatıldığı gibi domuz periferik kandan endotel hücreleri akıbet (EOC) türevi. Unti T-75 balonuna Kültürl yaklaşık% 80 konfluent (5x10 6 6 8x10 için hücre). </li><li> 10 nm çapında manyetit (Fe 3 O 4) 8,9 de tarif edildiği gibi 50 nm kalınlığında bir poli (laktik-ko-glikolik asit) (PLGA) Kabuk ile çevrili temel olarak SPIONs sentezleme. </li><li> 37 o C&#39;de 16 saat süre ile hücre kültür ortamında 200 ug / ml bir konsantrasyonda spion türetilen EOC inkübe </li><li> Yavaşça hücre kültür ortamı aspire. Yavaşça, şişeye, fosfat tamponlu salin (PBS), 10 ml ekleyerek sallanan ve PBS aspire hücreleri yıkayın. </li><li> Deneyler sırasında görselleştirmek için floresan boya (CM-DII) olan hücreleri leke. Bu 5 ul / ml arasında bir konsantrasyonda, hücre kültür ortamı 10 ml boya eklenmesi ve 37 ° C sıcaklıkta 30 dakika için hücreler ile inkübe edilerek, üretici talimatlarına göre yapılır. </li><li> Aşama 4.1.4 gibi hücrelerin PBS ile yıkayın ve 37 ° C&#39;de 5 dakika süreyle% 0.25 tripsin-EDTA çözeltisi 3 ml inkübeşişeden hücreler kaldırın. </li><li> 5 dakika bir hücre tanesinin meydana getirmek üzere, 500 xg PBS ile bir 15 mL konik tüp üst hücre süspansiyonu kapalı transferi ve santrifüjlenir. </li><li> 1-2x10 6 hücre / ml&#39;lik bir konsantrasyonda PBS içinde hücre pelletini yeniden askıya ve birkaç kez ve konik tüp üzerinden pipetleme ile iyice karıştırın. </li></ol></li><li> In vitro hücre çalışmaları <ol><li> Tasarım ve fabricate (örneğin, 3D baskı) sadece cam lamel yüzeyi üzerinde stenti tutmak için basit bir fikstür. </li><li> Elektromanyetik degausser kullanarak bir stent manyetikliğini ya da güçlü bir neodimyum mıknatıs kullanarak taban tabana veya eksenel bir stent çekmek. </li><li> Eksenel manyetize ya taban tabana manyetize ya da olmayan manyetize kontrol stentler içeren çanak içine PBS içinde asılı spion etiketli EOC pipetle. Resim ters bir floresan mikroskobu kullanılarak hemen floresan PBS içinde süspansiyon haline getirilmiş EOC olan stent. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 5. In-vivo Hayvan Araştırmaları <ol><li> Stent implantasyonu <ol><li> 4 sağlıklı Yorkshire domuz periferik kan Draw - 5,7 açıklandığı gibi 3 hafta önce sırasıyla implantasyonu stent ve kültür EOC için - yaklaşık 50 kg ağırlığındaki. </li><li> (Günlük aspirin 325 mg ve klopidogrel 75 mg) ameliyat 3 gün önce başlayan anti-trombosit ilaç yönetmek. </li><li> Stent yerleştirme gününde, kas içi Telazol, Xylazine ve Atropin ile domuz uyutmak (5 / 2-3 / 0,05 mg / kg sırasıyla) uygulanabilir kurumsal hayvan bakımı ve kullanımı kurallarda belirtildiği gibi. </li><li> Entübe ve 1-2,5% İsofluranın anestezi inhalasyon üzerine domuz yerleştirin. </li><li> Domuz ventral boyun bölgesini Tıraş ve genel steril şartlarda prosedürü yürütmek. </li><li> İmplant manyetize 1 ve standart kalp kateterizasyonu tekniği kullanılarak sağ koroner artere (RCA) içine 1 olmayan manyetize stent. <ol><li> CatheteHayvanların haklara ait eğitimli girişimsel kardiyoloji tarafından yapılmalıdır. 9 Fransız kılıf ile sağ karotis arter erişin. </li><li> Hedef koroner arterin cannulate ve skopi görüntüler elde etmek için iyotlu kontrast boya enjekte edilir. </li><li> Arterdeki bir 0.014 inçlik standart koroner kılavuz teli yerleştirin. Balon Advance ve bu kılavuz teli kullanarak stent ve 3-3.5 mm çapında bir kap içinde stent dağıtın. </li></ol></li><li> Tel balonu üzerinde kullanılarak implante stent RCA proksimal içindeki kan akışını tıkamak ve 2 dakikalık bir süre boyunca merkezi bir kateter yardımı ile PBS 4 ml süspansiyon haline spion ile etiketlenmiş yaklaşık 2x10 6 otolog EOC sağlar. </li><li> Ek tıkanıklığı 2 dakika sonra RCA kan akışını geri yükleyin. </li><li> Ayılma odasına hayvan aktarın ve bilinci yerine kadar yakından hayvan izleyin. </li><li> Anti-trombosit ilaç yönetmek devam (aspirin 325 mg ve klopidogrel 75 mg) Kurban kadar ameliyat sonrası. </li></ol></li><li> Stent eksplant ve histoloji <ol><li> Daha önce açıklandığı gibi ilk hayvan anestezi ile hayvanlar ameliyat sonrası 7 gün Euthanize ve daha sonra damardan uygulanan kurumsal hayvan bakımı ve kullanım yönergelerine uygun olarak sodyum pentobarbital (100 mg / kg) bir öldürücü dozunu. </li><li> Cerrahi stent arter segmentleri hasat. 30 dakika en az% 10 formalin tamponu içinde eksplante arterleri düzeltildi. Daha histolojik analiz için formalinle tampon örnekleri bırakın. </li><li> Metal stent ile histoloji yapabilen tesislere sabit numune Outsource. Bu işlem sırasında, numune, metilmetakrilat katıştırmak çapraz kesit ve demir parçacıkları Prusya mavisi boyası ile Mallory&#39;nin boyama tekniği kullanılarak histolojik olarak değerlendirildi. </li></ol></li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+imaging+of+metallic+implants+and+materials:+a+compilation+of+the+literature.">MR imaging of metallic implants and materials: a compilation of the literature.</a> AJR Am J Roentgenol. 151 (4), 811-814 (1988).</li><li>Lopic, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+determination+of+magnetic+force+on+a+coronary+stent+in+MRI.">Quantitative determination of magnetic force on a coronary stent in MRI.</a> J Magn Reson Imaging. 37 (2), 391-397 (2013).</li></ol>

The authors declare that they have no competing financial interests.

Biz çıplak metal stent olarak işlev görebilir ve casus etiketli endotel hücrelerini çekebilecek bir manyetik stent geliştirdi. Manyetik stent kapsayan önceki çalışmalarda, araştırmacılar nedeniyle ferromanyetik stent 5,10-14 olmayışı manyetik malzemelerden yapılmış nikel kaplamalı ticari stent ve bobinler veya kafesleri kullandık. Diğer gruplar da nanoparçacık yüklü endotel hücrelerini 3 hedefleme için piyasada mevcut 304 kalite paslanmaz çelik stent paramanyetik doğa...

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

Kardiyovasküler stentlerin hızlı endotelizasyonunun stent trombozu azaltmak ve kanama riskini azaltabilir, anti-trombosit tedavisi önlemek için gereklidir. Süper paramanyetik demir oksit nanopartiküller (casus) ile işaretlenmiş endotel hücreleri akıbet (EOC) yakalamak ve korumak için manyetik kuvvetler kullanarak fizibilite önceden gösterilmiştir. Ancak bu teknik, hızlı endotelizasyon kanıtlamak için in vitro ve in vivo test ardından manyetik ve biyo-uyumlu bir malzemeden, mekanik olarak işlevsel bir stent geliştirilmesini gerektirmektedir. Biz bilgisayar destekli tasarım (CAD) kullanarak 2205 dubleks paslanmaz çelikten zayıf ferromanyetik stent geliştirdi ve tasarımı daha da sonlu elemanlar analizi (FEA) kullanılarak rafine edilmiştir. Stentin nihai tasarım, mekanik kıvırma ve genişleme sırasında malzemenin kırılma sınırının altında bir ana zorlanma sergiledi. Yüz stent imal edildi ve bunların bir alt kümesi mekanik test, ret kullanılmıştırained manyetik alan ölçümleri, in-vitro hücre yakalama çalışmaları ve in vivo implantasyon çalışmaları. On stent onlar arızasız döngüsünü sıkma ve genişleme sürdürülebilir olmadığını doğrulamak için dağıtım için test edildi. Başka bir 10 stentler, güçlü bir neodimyum mıknatıs kullanarak mıknatıslanmamış ve bunların muhafaza manyetik alan ölçüldü. Stentler muhafaza manyetizma eden in vitro çalışmalarda spion etiketli EOC yakalamak için yeterli olduğunu göstermiştir. Spion etiketli EOC yakalama ve tutma 1 manyetize stent ve 4 domuz her 1 olmayan manyetize edilmiş kontrol stent implante büyük hayvan modellerinde doğrulanmıştır. Stentli arterler 7 gün sonra eksplante ve histolojik olarak incelendi. Bu çalışmada geliştirilen zayıf manyetik stentler çeken ve hızlı iyileşmeyi teşvik edebilir spion etiketli endotel hücrelerini tutabilen idi.

<html> FEA (Şekil 1) dayalı iteratif stent tasarımı kıvrım ve% 30 nihai gerginlik daha azdır% 20 anapara zorlanma ile genişletebilirsiniz stent gösterdi. Sıkma ve genişleme testi (Şekil 2) kırık bulgusuna rastlanmadı. Deforme stentin Resimleri FEA hesaplanan deformasyonlar iyi anlaşmayı gösterdi ve aynı zamanda mikroskopi resimler hiçbir kırıklarına (Şekil 3) gösterdi. Muhafaza manyetik alan ölçümleri (Şekil 4 ve 5) beklendi?...

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

Hedeflerimiz, tasarlamak, üretmek ve endotel hücre yakalama için ferromanyetik stent test edildi. On stent kırığı için test edildi ve 10 daha fazla stent muhafaza manyetik için test edildi. Nihayet, 10 stent in vitro test edildi ve 8 daha stent hücre yakalama ve tutma göstermek için 4 domuzlarda implante edildi.

Endotel Hücre yakalama ve Saklanmasına İlişkin ferromanyetik Bare Metal Stent

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 Görüntüleme.  Mayo Clinic.  Hedeflerimiz, tasarlamak, üretmek ve endotel hücre yakalama için ferromanyetik stent test edildi. On stent kırığı için test edildi ve 10 daha fazla stent muhafaza manyetik için test edildi. Nihayet, 10 stent in vitro test edildi ve 8 daha stent hücre yakalama ve tutma göstermek için 4 domuzlarda implante edildi. 

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.

Otolog Endotelyal progenitör Hücre-Yayımlama Teknoloji ve Büyük Hayvan Modeli Kardiyovasküler Cihazlar için Biyouyumluluk Testleri

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

Biyomühendislik

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.

Bir Gemi Stent Dağıtım sırasında Duvar Mekaniği izlenmesi

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.

Elektrik Hücre-substrat Empedans endotel Yayılmasının, Bariyer Fonksiyonu Nicelik için Algılama ve Motilite

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.

Biyomedikal Uygulamalar için Mekanik ayarlanabilir ve Biyoaktif Metal iskelelerinin Fabrikasyon

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.

İnsan yumurtalık dokusu mühendislik endotel hücreleri ile ortak nakli: bir hücre tabanlı strateji birleştirerek perfüzyon doğrudan parakrin teslim ile hızlandırılmış

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.

Bir Ultra-thin Polydimethylsiloxane mikrosıvısal çip su tutma ile kullanarak canlı Caenorhabditis elegans bireylerin immobilizasyon

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.

Biyomalzeme Membranlar Üzerinde İnsan ve Koyun Korneal Endotel Hücre Tabakalarının Büyümesi

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.

CD47-Türetilmiş Peptid İmmobilizasyonu Ile Metal İmplantlara Kan Yoluyla Bulaşan Hücre Bağlılığının Azaltılması

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.

Mechanobiyolojik Çalışmalar için Orbital Çalkalayıcıda Endotel Hücrelerinin 6 Kuyu Plakalarında Parçalanması

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.

Enflamasyon Sırasında Lökosit-Endotelyal Hücre Etkileşimini İnceleyen Mikrofizyolojik Bir Sistem

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