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.

すべての動物実験は、メイヨークリニックでの動物実験及び利用委員会（IACUC）によって承認されました。 2205ステンレス鋼ステントの1設計と解析<ol><li> CADを使用して、ベアメタルステントの設計<ol><li>ステントストラットの厚さに等しい壁厚に「押し出されたボス/ベース」機能を選択することにより、押し出された中空円筒を行います。 </li><li>押し出された円筒に対して接線方向に異なるスケッチ平面上のステントパターンを設計します。押し出された中空円筒の円周に合わせて、フラットパターンの幅を確認します。 </li><li>ラップ機能を使用して中空シリンダー上にフラットパターン設計を転送します。 </li><li> FEAのためにエクスポートするために、そのネイティブフォーマットでも、ACISフォーマットの一部を保存します。 </li></ol></li><li>ステントモデルの有限要素解析<ol><li>さらにanalysためFEAソフトウェアの一部のモジュールにACIS形式で保存されたソリッドジオメトリをインポートです。 </li><li> FEAソフトウェアの一部モデラーでステントにモデル2の分析シリンダ同軸。外筒は、クリンパと内筒の膨張バルーンをシミュレートするために1mmの初期直径を有するシミュレートするためにステントの直径よりも大きい初期直径を有しています。 </li><li>上記を組み立てる組立モデラーの「インスタンス」ツリー項目をダブルクリックして、相対的な位置で部品を述べました。 </li><li> FEAソフトウェアのメッシュモジュールを使用し、低減積分で20ノードの六面体エレメントとしての要素型を指定し、要素のサイズを指定し、ステントメッシュ。 </li><li>ステントとモデルツリーの「相互作用のプロパティ」で、それぞれ2つの気筒間の摩擦の剛性接点ペアを指定します。 </li><li>ステントモデルに2205ステンレス鋼の弾塑性応力 - ひずみ挙動を割り当てます。 </li><li>まずCをシミュレートする1​​ミリメートルに外筒を圧着するための境界条件を定義します。ステントのリンピン。クリンプステントの緩和をシミュレートするために、外筒を取り外します。展開をシミュレートし、最終的には、ステントの反動をシミュレートするために、内筒を除去するために、3mmの内筒を展開します。 </li><li> 「分析」モデルツリーの項目に割り当てられたプロセッサとRAMの量の数を含む、シミュレーションパラメータを定義し、シミュレーションを実行します。 </li><li>シミュレーションが完了すると、結果は主要な系統を研究し、反復的物質の故障限界未満である20％の主ひずみを達成するために、ステントの設計を改善するために、結果ファイル（filename.odb）及び後処理を開き。 </li></ol></li></ol>圧着および拡張のための2ステントの作製とテスト<ol><li>ステントの製造<ol><li>このようなパイオニアでアクション精密製品等の精密加工会社に銃の掘削および精密研削バーストック材料によって2205ステンレス鋼管を取得し、OHです。 </li><li>レーザー切断と電解研磨のためにこのようなウォーキーガン、イリノイ州のLaserageテクノロジー社としてステント切断会社に精密研磨管とフラットパターンのステント設計を転送します。 </li><li>さらに10分間塩基（10％のNaHCO 3）、続いて10分間、強酸（50％塩酸）でそれらを浸すことにより、電解ステントの表面を不動態化します。注意：適切な保護具を持つとヒュームフードの下に化学物質を扱います。最終的には、エチルアルコールと脱イオン水を用いてステントを洗浄します。このプロセスは、酸洗いと呼ばれています。 </li></ol></li><li>圧着および拡張のために製造のステントのテスト<ol><li>圧着工具、手持ちを使用してつ折りバルーン上にステントを圧着します。ステントと圧着工具でつ折りバルーンを保持します。放射状にバルーン上に圧着されるように、ステントを変形させるためにハンドルを押します。 </li><li>均一な圧着用顕微鏡とによる構造の故障の兆候を使用してクリンプステントを検査塑性変形します。 </li><li>水でつ折りバルーンを加圧して3mmの設計直径にそれを展開します。微細な骨折および均一な拡張のための拡張したステントを調べます。 </li></ol></li></ol>留保磁場用ステントの3キャラクタリゼーション注：2インチ、直径1インチの高さの円筒磁石は、この研究において使用しました。磁石の極は、軸に沿って整列されています。磁石の表面磁束密度は約1であるT. <ol><li>強力なネオジム磁石を使用して、直径又は軸方向にステントを磁化。磁化のため、約1分間の強力な磁石に近いステントを保持します。 </li><li>直径磁化又は軸方向にそれを磁化する磁力線に沿ってその軸と円筒面の隣にステントを保持する磁力線に沿ってその直径を平らな面の一つにステントを保持します。留保磁気金融商品取引法ステントの開発は、少なくとも24時間安定であるが、磁化後できるだけ早くステントを使用することが見出されました。 </li><li>ガラスマンドレル上に個別にステントをマウントして、磁気探査治具の精度チャックにガラスマンドレルをマウントします。磁気マイクロプローブは、正確にXYZを用いて表面に触れることなく、ステントの近くに配置することが可能な磁気探査固定具（ 図4）の組み立てをステージ。 </li><li>遠く離れたステントからの磁気マイクロのベースラインの読みを測定した後、磁気探査治具のXYZステージを使用してプローブを配置することによって、ステントの表面に保持する磁界を測定します。 </li></ol> 4.磁気細胞キャプチャー研究<ol><li>入手細胞、蛍光色素でSPIONおよび染色による標識<ol><li> 5,7で説明したように、ブタの末梢血から内皮成長細胞（EOC）を求めよ。 unti T-75フラスコで培養リットル約80％コンフルエント（5×10 6 6 8×10個の細胞）。 </li><li> 10 nmの直径のマグネタイト （Fe 3 O 4）8,9に記載のように厚さ50nmのポリ（乳酸-コ-グリコール酸）（PLGA）シェルによって囲まれたコアとしてSPIONsを合成します。 </li><li> 37 O℃で16時間、細胞培養培地に200μg/ mlの濃度でSPIONと導出EOCインキュベート</li><li>静かに細胞培養培地を吸引除去します。優しく、フラスコにリン酸緩衝生理食塩水（PBS）を10mlを加える揺動し、PBSを吸引することにより細胞を洗浄します。 </li><li>実験中に可視化するための蛍光色素（CM-DiIを）で細胞を染色します。これは、5μL/ mlの濃度で細胞培養培地10mlに染料を添加し、37℃で30分間細胞をインキュベートすることにより、製造業者の指示に従って行われます。 </li><li>ステップ4.1.4のようにPBSで細胞を洗浄し、37℃で5分間、0.25％トリプシン-EDTA溶液3mlでインキュベートフラスコから細胞を持ち上げます。 </li><li>細胞ペレットを形成するために、5分間500×gで15ミリリットルコニカルチューブ、PBSでトップオフ、および遠心分離機に細胞懸濁液を転送します。 </li><li> 1-2x10 6細胞/ mlの濃度でPBS中に細胞ペレットを再懸濁し、数回にし、円錐管からピペッティングにより完全に混和します。 </li></ol></li><li> インビトロ細胞試験<ol><li> （例えば、3D印刷）だけカバーガラスの表面上にステントを保持するための簡単な治具を設計・製作してください。 </li><li>電磁消磁装置を使用してステントを消磁や強いネオジム磁石を使用して、直径又は軸方向にステントを磁化。 </li><li>軸方向に磁化さや直径磁化または非磁化対照ステントを含む皿にPBSに懸濁SPION標識EOCをピペット。画像倒立蛍光顕微鏡を用いて蛍光についてすぐにPBSに懸濁EOCを有するステント。 </li></ol></li></ol><pクラス= &quot;jove_title&quot;&gt; 5。 生体内で動物研究<ol><li>ステント移植<ol><li> 4健康ヨークシャーブタからの末梢血を描く- 5,7で説明したように3週間前にそれぞれ移植し、ステントと文化EOCする-約50キロで計量。 </li><li> （毎日アスピリン325mgのクロピドグレル75 mg）を、手術の3日前に開始する抗血小板薬を投与します。 </li><li>該当機関の動物の管理と使用に関するガイドラインに記載されているようにステント移植の日には、筋肉内テラゾール、キシラジン、およびアトロピン（それぞれ5 / 2-3 / 0.05ミリグラム/ kg）で豚を麻酔。 </li><li>挿管と1から2.5パーセントイソフルラン麻酔の吸入に豚を配置します。 </li><li>豚の腹側ネック領域を剃ると、一般的な無菌状態で手続きを行っています。 </li><li>インプラント1は、標準的な心臓カテーテル技術を使用して右冠動脈（RCA）に着磁1非磁性ステント。 <ol><li> Cathete動物のrizationは訓練されたインターベンション心臓病専門医が行ってください。 9フランスの鞘で右頚動脈にアクセスします。 </li><li>ターゲット冠動脈にカニューレを挿入し、透視画像を取得するためにヨード造影色素を注入します。 </li><li>動脈内の0.014インチの標準冠状動脈ガイドワイヤを配置します。このガイドワイヤーを使用して、バルーンやステントを進め、3〜3.5ミリメートルの直径の容器内にステントを展開。 </li></ol></li><li>ワイヤーバルーン上で使用して移植したステントへのRCA近位内の血流を閉塞し、2分間かけて、中央カテーテルを介して4mlのPBSに懸濁SPIONで標識された約2×10 6自家EOCを提供します。 </li><li>追加の閉塞の2分後のRCAへの血流を復元します。 </li><li>回復室に​​動物を移し、それは意識を取り戻したまで密接に動物を監視します。 </li><li>抗血小板薬（アスピリン325mgのクロピドグレル75メートルの管理を継続しますg）を犠牲にするまで、術後。 </li></ol></li><li>ステント植片および組織学<ol><li>該当機関の動物の管理と使用のガイドラインに従って静脈内ペントバルビタールナトリウム（100mg / kg）の致死量を投与し、その後、以前に説明したように、第1の動物を麻酔により7日手術後、動物を安楽死させます。 </li><li>外科的ステント付動脈セグメントを収穫。最低30分間​​10％ホルマリン緩衝液中に外植動脈を修正しました。さらなる組織学的分析のためにホルマリンバッファ内のサンプルを残します。 </li><li>金属ステントと組織学を行うことのできる施設に固定された試料を外部委託しています。この処理の間、サンプルは、メチルメタクリレートに埋め込んされる断面に、鉄粒子のためのプルシアンブルー染色でマロリーの染色技術を使用して組織学的に分析しました。 </li></ol></li></ol>

<ol>
	<li>Garg, S., Serruys, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+stents:+current+status.">Coronary stents: current status.</a> J Am Coll Cardiol. 56, 1-42 (2010).</li><li>Austin, D., 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=Drug-eluting+stents+versus+bare-metal+stents+for+off-label+indications:+a+propensity+score-matched+outcome+study.">Drug-eluting stents versus bare-metal stents for off-label indications: a propensity score-matched outcome study.</a> Circ Cardiovasc Interv. 1 (1), 45-52 (2008).</li><li>Polyak, B., 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=High+field+gradient+targeting+of+magnetic+nanoparticle-loaded+endothelial+cells+to+the+surfaces+of+steel+stents.">High field gradient targeting of magnetic nanoparticle-loaded endothelial cells to the surfaces of steel stents.</a> P Natl Acad Sci USA. 105 (2), 698-703 (2008).</li><li>Tassiopoulos, A. K., Greisler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+mechanisms+of+endothelialization+of+cardiovascular+implants:+a+review+of+recent+investigative+strategies.">Angiogenic mechanisms of endothelialization of cardiovascular implants: a review of recent investigative strategies.</a> J Biomat Sci-Polym E. 11 (11), 1275-1284 (2000).</li><li>Pislaru, S. V., 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=Magnetic+forces+enable+rapid+endothelialization+of+synthetic+vascular+grafts.">Magnetic forces enable rapid endothelialization of synthetic vascular grafts.</a> Circulation. 114, I314-I318 (2006).</li><li>Uthamaraj, S., 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=Design+and+validation+of+a+novel+ferromagnetic+bare+metal+stent+capable+of+capturing+and+retaining+endothelial+cells).">Design and validation of a novel ferromagnetic bare metal stent capable of capturing and retaining endothelial cells).</a> Ann Biomed Eng. 42 (12), 2416-2424 (2014).</li><li>Gulati, R., 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=Diverse+origin+and+function+of+cells+with+endothelial+phenotype+obtained+from+adult+human+blood.">Diverse origin and function of cells with endothelial phenotype obtained from adult human blood.</a> Circ Res. 93 (11), 1023-1025 (2003).</li><li>Lee, S. J., 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=Nanoparticles+of+magnetic+ferric+oxides+encapsulated+with+poly(D,L+latide-co-glycolide)+and+their+applications+to+magnetic+resonance+imaging+contrast+agent.">Nanoparticles of magnetic ferric oxides encapsulated with poly(D,L latide-co-glycolide) and their applications to magnetic resonance imaging contrast agent.</a> J Magn Magn Mater. 272 (3 Special Issue), 2432-2433 (2004).</li><li>Lee, S. J., 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=Magnetic+enhancement+of+iron+oxide+nanoparticles+encapsulated+with+poly(D,L-latide-co-glycolide).">Magnetic enhancement of iron oxide nanoparticles encapsulated with poly(D,L-latide-co-glycolide).</a> Colloid Surface A. (1-3), 255-251 (1016).</li><li>Forbes, Z. G., 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=Locally+targeted+drug+delivery+to+magnetic+stents+for+therapeutic+applications.">Locally targeted drug delivery to magnetic stents for therapeutic applications.</a> Computer Architectures for Machine Perception, 2003 IEEE International Workshop on. , 1-6 (2003).</li><li>Rathel, T., 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=Magnetic+Stents+Retain+Nanoparticle-Bound+Antirestenotic+Drugs+Transported+by+Lipid+Microbubbles.">Magnetic Stents Retain Nanoparticle-Bound Antirestenotic Drugs Transported by Lipid Microbubbles.</a> Pharm Res-Dordr. 29 (5), 1295-1307 (2012).</li><li>Gunn, J., Cumberland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stent+coatings+and+local+drug+delivery+-+state+of+the+art.">Stent coatings and local drug delivery - state of the art.</a> Eur Heart J. 20 (23), 1693-1700 (1999).</li><li>Lu, A., Jia, G., Gao, G., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+magnetic+stent+on+coronary+restenosis+after+percutaneous+transluminal+coronary+angioplasty+in+dogs.">The effect of magnetic stent on coronary restenosis after percutaneous transluminal coronary angioplasty in dogs.</a> Chin Med J (Engl. 114 (8), 821-823 (2001).</li><li>Kempe, H., Kempe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+magnetite+nanoparticles+for+implant-assisted+magnetic+drug+targeting+in+thrombolytic+therapy.">The use of magnetite nanoparticles for implant-assisted magnetic drug targeting in thrombolytic therapy.</a> Biomaterials. 31 (36), 9499-9510 (2010).</li><li>Chorny, M., 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=Targeting+stents+with+local+delivery+of+paclitaxel-loaded+magnetic+nanoparticles+using+uniform+fields.">Targeting stents with local delivery of paclitaxel-loaded magnetic nanoparticles using uniform fields.</a> P Natl Acad Sci USA. 107 (18), 8346-8351 (2010).</li><li>Polyak, B., Friedman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+targeting+for+site-specific+drug+delivery:+applications+and+clinical+potential.">Magnetic targeting for site-specific drug delivery: applications and clinical potential.</a> Expert Opin Drug Del. 6 (1), 53-70 (2009).</li><li>Liu, J. Y., 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=Magnetic+stent+hyperthermia+for+esophageal+cancer:+an+in+vitro+investigation+in+the+ECA-109+cell+line.">Magnetic stent hyperthermia for esophageal cancer: an in vitro investigation in the ECA-109 cell line.</a> Oncol Rep. 27 (3), 791-797 (2012).</li><li>Gunn, J., Cumberland, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+stent+design+influence+restenosis.">Does stent design influence restenosis.</a> Eur Heart J. 20 (14), 1009-1013 (1999).</li><li>Aviles, M. O., 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=In+vitro+study+of+ferromagnetic+stents+for+implant+assisted-magnetic+drug+targeting.">In vitro study of ferromagnetic stents for implant assisted-magnetic drug targeting.</a> J Magn Magn Mater. 311 (1), 306-311 (2007).</li><li>Mardinoglu, A., 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=Theoretical+modelling+of+physiologically+stretched+vessel+in+magnetisable+stent+assisted+magnetic+drug+targeting+application.">Theoretical modelling of physiologically stretched vessel in magnetisable stent assisted magnetic drug targeting application.</a> J Magn Magn Mater. 323 (3-4), 324-329 (2011).</li><li>Liu, Z. Y., 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=Stress+corrosion+cracking+of+2205+duplex+stainless+steel+in+H2S-CO2+environment.">Stress corrosion cracking of 2205 duplex stainless steel in H2S-CO2 environment.</a> J Mater Sci. 44 (16), 4228-4234 (2009).</li><li>Alverez-Armas, I., Degallaix-Moreuill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Duplex stainless steels. , (2009).</li><li>Tefft, B. J., 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=Magnetizable+Duplex+Steel+Stents+Enable+Endothelial+Cell+Capture.">Magnetizable Duplex Steel Stents Enable Endothelial Cell Capture.</a> Ieee T Magn. 49 (1), 463-466 (2013).</li><li>Pelton, A. R., 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=Fatigue+and+durability+of+Nitinol+stents.">Fatigue and durability of Nitinol stents.</a> J Mech Behav Biomed Mater. 1 (2), 153-164 (2008).</li><li>Knowles, M., 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=Finite+element+analysis+of+a+balloon-expandable+stent+and+superior+mesenteric+arterial+wall+interaction.">Finite element analysis of a balloon-expandable stent and superior mesenteric arterial wall interaction.</a> J Vasc Surg. 60 (6), 1722-1723 (2014).</li><li>Veeram Reddy, S. R., 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=A+novel+biodegradable+stent+applicable+for+use+in+congenital+heart+disease:+bench+testing+and+feasibility+results+in+a+rabbit+model.">A novel biodegradable stent applicable for use in congenital heart disease: bench testing and feasibility results in a rabbit model.</a> Catheter Cardiovasc Interv. 83 (3), 448-456 (2014).</li><li>Shellock, F. 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.

私たちは、ベアメタルステントとして機能することが可能とSPION標識内皮細胞を引き付けることができる磁気ステントを開発しました。磁気ステントを伴う以前の研究では、研究者が原因で、強磁性ステント5,10-14の使用不能に磁性材料で作られたニッケル被覆商業ステントおよびコイルまたはメッシュを使用しています。他のグループはまた、ナノ粒子ロード内皮細胞3を標?...

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

心血管ステントの急速な内皮化は、ステント血栓症を低減し、出血のリスクを減らすことができる抗血小板療法を回避するために必要とされます。超常磁性酸化鉄ナノ粒子（SPION）で標識された内皮細胞の伸長（EOC）を取得し、保持するために磁力を使用することの実現可能性は、以前に示されています。しかし、この技術は、迅速な内皮化を証明するために、in vitroおよびin vivo試験に続いて、磁性および生体適合性の材料から機械的に機能的なステントの開発が必要です。我々は、コンピュータ支援設計（CAD）を使用して、2205二相ステンレス鋼から弱い強磁性ステントを開発し、その設計は、さらに、有限要素解析（FEA）を用いて精製しました。ステントの最終的な設計は、機械的圧着し、拡張中に材料の破壊限界以下の主ひずみを示しました。百ステントを作製し、それらのサブセットは、機械的試験のために使用した、RETained磁場測定、 インビトロ細胞取り込み研究及びインビボ移植実験。十ステントは、それらが故障せずに圧着し、膨張サイクルを持続かどうかを確認するために展開するために試験しました。別のステント10は、強力なネオジム磁石を用いて着磁し、その保持磁界を測定しました。ステントは、保持磁力が我々のインビトロ研究においてSPION標識EOCを捕捉するのに十分であったことを示しました。 SPION標識EOCを捕捉および保持を4匹のブタのそれぞれに1着磁ステント1の非磁化対照ステントを移植することにより、大型動物モデルにおいて確認されました。ステント動脈を7日後に外植し、組織学的に分析しました。本研究で開発した弱磁性のステントは、誘致し、迅速な治癒を促進することができるSPION標識内皮細胞を保持することができました。

<html> FEA（ 図1）に基づく反復ステント設計は、圧着し、30％の最終的な株よりも小さい20％の主ひずみを拡張することができ、ステントを示しました。圧着及び拡張試験（ 図2）は 、破壊の徴候を示さありませんでした。変形したステントの写真は、FEA計算された変形と良い一致を示し、また、顕微鏡写真には、骨折に（ 図3）を示さありませんでした。?...

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

私たちの目標は、内皮細胞の捕捉のための強磁性体のステントを設計、製造、テストしました。十ステントは、骨折のために試験し、10以上のステント保持磁力を試験しました。最後に、10のステントは、 インビトロで試験したところ、8複数のステントは、細胞捕獲および保持を示すために、4匹のブタに移 ​​植しました。

内皮細胞捕捉および保持のための強磁性ベアメタルステント

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 ビュー.  Mayo Clinic. 私たちの目標は、内皮細胞の捕捉のための強磁性体のステントを設計、製造、テストしました。十ステントは、骨折のために試験し、10以上のステント保持磁力を試験しました。最後に、10のステントは、 インビトロで試験したところ、8複数のステントは、細胞捕獲および保持を示すために、4匹のブタに移 ​​植しました。 

ビデオ: 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.

大動物モデルにおける心血管デバイス用自家血管内皮前駆細胞播種技術と生体適合性試験

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

生物工学

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.

容器内ステントの展開中の壁の力学を監視する

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.

内皮増殖、バリアー機能、運動性の定量のための電気細胞 - 基質インピーダンスセンシング

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.

バイオメディカル応用のための機械的に調整可能な生物活性金属スキャフォールドの作製

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.

設計の内皮細胞とひと卵巣組織の共同移植: セル ベースの戦略の組み合わせは直接パラクリン配信による血液灌流を加速

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.

極薄ポリジメチルシロキサン マイクロ流体チップを用いた保水ライブ線虫個人の固定化

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.

生体物質膜上におけるヒトおよび羊角膜内皮細胞層の増殖

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由来ペプチド固定化による金属インプラントへの血液媒介性細胞の取り付けの軽減

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.

メカノ生物学的研究のための軌道シェーカー上の6ウェルプレートにおける内皮細胞の成長のセグメント化

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.

炎症時の白血球-内皮細胞相互作用を研究するための微生理学的システム

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