この作品は、NHMRCとスタッフォード・フォックス医療財団からの助成金によってサポートされていました。著者はスー・マッケイ、リリアナペペ、アンナDeftereosと実験的医療や外科ユニットのアマンダRixon、セントビンセント病院、メルボルンの外科的な支援を認めます。サポートはまた、ビクトリア朝の州政府のイノベーション省、産業と地域開発の運用インフラ支援プログラムによって提供されています。

ここで説明するプロトコルは、セントビンセント病院メルボルン、オーストラリアの動物倫理委員会によって承認されており、オーストラリア国立保健医療研究評議会のガイドラインに厳守下で行いました。 注：二室プロトコールを以下に記載します。 2つの異なるモデルおよびその特定のチャンバ設計は、 図1に示されている。チャンバは、（1）（ラット動静脈ループ室モデルの場合）、ポリカーボネートで作られています。これは、内径13ミリメートル、高さ4ミリメートルを有する円筒形です。壁内の1つのポイントでウィンドウが椎弓根のために妨げられることなくアクセスすることができます。 （ラットのフロースルー茎室モデルの）第2のモデルでは、チャンバは、アクリル製の矩形（10×4×8 mm 3で内部寸法）です。それは、チャンバを犯すように大腿動脈および静脈に適応するように両側に2つの1.5ミリメートルの開口部を有します。 1.ラット動静脈ループ商工モデル（Oneチャム動物当たりBER） 注：手術を開始する前に、すべての機器が適切に滅菌されていることを確認してください。同様に、滅菌タオル上の機器の残りを確保し、手続き中の汚染を避けるために、手術野から合理的な距離にあります。 <ol><li>手術のための動物の準備<ol><li>動静脈ループを作成するための血管のサイズが大きいために、少なくとも250gのラットを使用してください。 </li><li> 4％のイソフルラン吸入で動物をAnaesthetize。つま先ピンチに無応答性を評価することによって麻酔の十分な深さを裏付けます。麻酔した後、適切に2％イソフルランと手順を通して麻酔動物を維持します。 </li><li>温暖化パッド上に仰臥位で動物を置き、手術中の乾燥を防ぐために、目に無菌潤滑剤を適用します。 </li><li>電気かみそりを使用して、両方の股間を剃ると湿ったガーゼで髪を削除します。 </li><li>クロルヘキシジンと手術部位をPREP/ 70％エタノール溶液および無菌タオルで動物をドレープ。鎮痛剤としてカルプロフェン（5 mgの/ kgを皮下）の単回投与を管理します。 </li></ol></li><li>大腿静脈グラフトの収穫<ol><li> ＃15ブレードを使用して、鼠径靱帯に左鼠径部に平行に長さ4cmの皮膚切開を行います。これは、鼠径部脂肪パッドを露出させます。 </li><li>上腹部管に基づいて、その血管茎に取り付けられ、それを残してはさみで円周方向に脂肪パッドを通してカット。 </li><li>マイクロはさみを使用して、腹壁とその下にある大腿血管の間にフィルム状の結合組織の癒着を解放します。 </li><li>腹壁に開創器を置き、内側引き出します。これは、鼠径靱帯と大腿血管の全長を公開しています。 </li><li>マイクロ鉗子を使用し、湾曲したハサミを静かに引っ張り、切断によって上腹部静脈を分析し、その周囲の脂肪からそれを分離します。ループを構築するときに、この静脈は、テザーとして機能します。 </li><li> Usinグラムマイクロ鉗子湾曲マイクロハサミは大腿血管や神経の心窩部支店への分岐末端に鼠径靭帯からのすべての方法を含む血管周囲鞘を開きます。 </li><li>マイクロ鉗子を使用して、その外膜によって大腿静脈をピックアップし、静かに周囲の組織および添付動脈からそれを分離します。離れて組織を引っ張り、それを介して切断することにより、マイクロ鉗子湾曲ラウンド尖ったマイクロハサミでこれを行います。 注：これは血栓症の脆弱性が発生内膜への外傷を引き起こす可能性があるとして、静脈壁の全体の厚さをつかむことはありません。 </li><li>ライゲーション側枝は10/0ナイロン縫合糸で切開中に発見されたまたはバイポーラ凝固でそれらを凝固します。 </li><li>大腿静脈完全に無料で、4/0シルク縫合糸で、その近位端および遠位端を連結。少なくとも10ミリメートルの長さの静脈グラフトを取得することを確認しますと、男のロープのテザーとして使用する上腹部の枝の約0.5センチメートル長を含みますチャンバー内の開ループを保持します。 </li><li>マイクロ鉗子とストレートマイクロはさみを使用して、静かに引き出し、切断することにより、移植片の端部から外膜をトリミングします。これはまた、顕微吻合前に、後に行うことができます。 </li><li>ヘパリン添加生理食塩水（ヘパリンの10 U / ml）で静脈グラフトをフラッシュし、溶液中で休むようにそれを残します。連続走行4/0絹縫合糸と2つまたは3つの追加の簡単な中断されたステッチを使用して傷を閉じます。 </li></ol></li><li>商工会議所の動静脈ループと移植の作成<ol><li>繰り返しは、対側肢にまったく同じ方法で、1.2.4に1.2.1を繰り返します。 </li><li>マイクロ鉗子を使用して、上腹部動脈と静脈の両方を分析し、単離周囲の脂肪パッドを形成します。静かに血管から組織を引っ張ることによってこれを行います</li><li>マイクロ鉗子を使用して、その外膜により大腿動脈をピックアップし、周囲の組織からそれを解放します。マイクロ鉗子湾曲ラウンド尖ったマイクでこれを行います離れて組織を引っ張り、それを切断することにより、roのはさみ。その側枝を結紮または凝固。 </li><li> 4/0絹縫合糸を用いて、上腹部管の出現に大腿動脈と静脈の先端を連結。 </li><li>大腿動脈と静脈のそれぞれの近位に単一のクランプを置きます。鋭いストレートマイクロはさみを使用して、上腹部枝の出現に各容器の先端でクリーンな横カットを行います。船の下の滅菌プラスチックコントラストの背景を配置します。 </li><li>すべての血液が内腔から除去されるまで、ヘパリン化生理食塩水の寛大な量と激しく血管をフラッシュします。 </li><li>術野に静脈グラフトを持参し、必要に応じて、ステップ1.2.10に従って船 &#39;末端から任意の冗長な外膜を除去します。 </li><li> 10/0ナイロン縫合糸と顕微吻合の両方を実行します。大腿静脈と大腿動脈への遠位端部に静脈グラフトの近位端を吻合。これがf血液が流れるようになります静脈グラフトの内側バルブから抵抗なく静脈側への動脈をROM。 注：任意のねじれずに大腿血管とその自然な位置にある静脈グラフトの残りの部分を確認してください。 </li><li>両方の吻合部に漏れがないか確認してください。上に脂肪の小片を置き、静かに5〜10分間圧縮することにより、吻合部位から出てくる非脈動血のように見える小さな漏れを解決します。急速にフィールド全体をあふれさせるより大きな脈動漏れが追加ステッチが必要になります。 </li><li>動静脈ループの開存性を確認してください。大腿動脈の穏やかな閉塞は、大腿静脈内の同じがそれをガブガブ飲むべきである、それが縮小する必要があります。 </li><li>ねじれやよじれのないその自然な位置にある後者の休息と動静脈ループの下に組織工学室のベースを置きます。 </li><li> 6/0ナイロン縫合糸で鼠径靭帯およびその下の筋筋膜に、チャンバのベースを固定します。 </li><li>上に蓋を置きベース大腿血管は、ノッチ（室側の窓）を通ってチャンバに入るようにします。蓋を閉じたとき、それはテザーを所定の位置に動静脈ループを保持するように作用チャンバーベースと蓋との間で、心窩部枝をキャッチしていることを確認します。 </li><li>連続走行4/0絹縫合糸と2つまたは3つの追加の簡単な中断されたステッチを使用して傷を閉じます。 </li><li>動物は加温パッド上で麻酔から回復することを可能にします。 </li><li>それは胸骨横臥位を維持するのに十分な意識を取り戻したまで無人の動物を放置しないでください。同様に、完全に回復するまで、他の動物の会社に手術を受けた動物を返しません。 24時間後、鎮痛剤としてカルプロフェン（皮下5ミリグラム/キログラム）の別の単一用量を投与。 </li><li> 5日間のための局所抗生物質軟膏で傷口を扱います。傷口が開いている場合、1.1.5を介してステップ1.1.2のように動物を麻酔し、1.3のように傷口を閉じます0.14。毎日、動物の健康状態を監視します。動物はinacitivity、食欲不振、体重減少及び色の損失の複数の適度な兆候を示している場合（23 G針で0.25ミリリットルで163ミリグラム/ kg）を腹腔内lethabarb注入の致死量を用いて動物を安楽死させます。 </li></ol></li></ol> 2.フロースルー椎弓根商工会議所（動物ごとに2つのチェンバース） <ol><li>手術のための動物の準備<ol><li>繰り返しは、1.1.4を介して1.1.1を繰り返します。二つのチャンバは、単一のラットの双方の鼠径部領域に移植することができます。 </li></ol></li><li>大腿血管の単離と商工会議所の挿入<ol><li>繰り返しは、1.2.8を介して1.2.1を繰り返します。 </li><li>動脈と完全に周囲の組織から解放され、その枝を結紮静脈の両方で、術野にチャンバをもたらします。 </li><li>チャンバーベースがないねじれやよじれがないことを確認することの対応するスリットに無傷の大腿血管のそれぞれを置きます。 </li><li> attachiによってチャンバを閉じますベースに蓋をngの。連続走行4/0絹縫合糸と2つまたは3つの追加の簡単な中断されたステッチを使用して傷を閉じ、前述のように、動物が回復することを可能にします。 </li></ol></li></ol> 3.チェンバースの収穫と組織処理<ol><li>実験の時間点（4-6週間移植後）に到達したら、ステップ1.1.2のように動物を麻酔し、1.1.5まで手順1.1.3を繰り返します。 </li><li> ＃15ブレードを使用して傷を開き、チャンバが完全に露出するまでハサミで組織を切断。 </li><li>その後、遠位方向に血液を牛乳、静かに2マイクロピンセットで血管を閉塞し、最終的には近位鉗子を解放：血管開通のための構築物およびテストに大腿骨近位部の血管を公開します。容器は再び血液でいっぱいになると、これは、開通性を確認します。動静脈ループの場合には近位大腿血管を結紮し、両方の近位および遠位番目の場合には電子フロースルー設定、および一括含む組織でチャンバーを削除します。 </li><li>実験の最後に、（23 G針で0.25ミリリットルで163ミリグラム/ kg）を腹腔内lethobarb注入の致死量を用いて動物を安楽死させます。 </li><li> 24時間室温で4％パラホルムアルデヒド中で組織を修正しました。ディバイド複数の横断切片に組織（1-2 mm厚）、パラフィンワックスまたはそれぞれパラフィン切片のための最適な切削温度化合物（5ミクロン）または凍結切片（10μm）を、中に埋め込 ​​む。3,4,8,17,22、 24 </li><li>組織の一般的な形態を調べるために、このようなヘマトキシリン​​およびエオシンなどの日常的な組織学的染色を実行します。例えば心筋細胞のための心筋トロポニンTの免疫染色を関心、3,4,8,17,22,24,29の細胞型を同定するための特定の抗体を用いて免疫組織化学染色を行ってください。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+microcirculation.">Engineering the microcirculation.</a> Tissue Eng Part B Rev. 14 (1), 87-103 (2008).</li><li>Yap, K. K., 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=Enhanced+liver+progenitor+cell+survival+and+differentiation+in+vivo+by+spheroid+implantation+in+a+vascularized+tissue+engineering+chamber.">Enhanced liver progenitor cell survival and differentiation in vivo by spheroid implantation in a vascularized tissue engineering chamber.</a> Biomaterials. 34 (16), 3992-4001 (2013).</li><li>Findlay, M. W., 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=Tissue-engineered+breast+reconstruction:+Bridging+the+gap+toward+large-volume+tissue+engineering+in+humans.">Tissue-engineered breast reconstruction: Bridging the gap toward large-volume tissue engineering in humans.</a> Plast Reconstr Surg. 128 (6), 1206-1215 (2011).</li><li>Tee, R., Morrison, W. A., Dusting, G. J., Liu, G. S., Choi, Y. S., Hsiao, S. T., Dilley, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+engineered+cardiac+muscle+flaps+in+syngeneic+rats.">Transplantation of engineered cardiac muscle flaps in syngeneic rats.</a> Tissue Eng Part A. 18 (19-20), 1992-1999 (2012).</li><li>Dolderer, J. H., 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=Long-term+stability+of+adipose+tissue+generated+from+a+vascularized+pedicled+fat+flap+inside+a+chamber.">Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber.</a> Plast Reconstr Surg. 127 (6), 2283-2292 (2011).</li><li>Sekine, H., 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=Endothelial+cell+coculture+within+tissue-engineered+cardiomyocyte+sheets+enhances+neovascularization+and+improves+cardiac+function+of+ischemic+hearts.">Endothelial cell coculture within tissue-engineered cardiomyocyte sheets enhances neovascularization and improves cardiac function of ischemic hearts.</a> Circulation. 118, 145-152 (2008).</li><li>Ting, A. C., 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=The+adipogenic+potential+of+various+extracellular+matrices+under+the+influence+of+an+angiogenic+growth+factor+combination+in+a+mouse+tissue+engineering+chamber.">The adipogenic potential of various extracellular matrices under the influence of an angiogenic growth factor combination in a mouse tissue engineering chamber.</a> Acta Biomater. 10 (5), 1907-1918 (2014).</li><li>Zhan, W., 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=Self-synthesized+extracellular+matrix+contributes+to+mature+adipose+tissue+regeneration+in+a+tissue+engineering+chamber.">Self-synthesized extracellular matrix contributes to mature adipose tissue regeneration in a tissue engineering chamber.</a> Wound Repair Regen. 23 (3), 443-452 (2015).</li><li>Messina, A., Bortolotto, S. K., Cassell, O. C., Kelly, J., Abberton, K. M., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+vascularized+organoid+using+skeletal+muscle+as+the+inductive+source.">Generation of a vascularized organoid using skeletal muscle as the inductive source.</a> FASEB J. 19 (11), 1570-1572 (2005).</li><li>Lim, S. Y., Hern&#225;ndez, D., Dusting, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+vascularized+heart+tissue+from+stem+cells.">Growing vascularized heart tissue from stem cells.</a> J Cardiovasc Pharmacol. 62 (2), 122-129 (2013).</li><li>Poon, C. 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=Preparation+of+an+adipogenic+hydrogel+from+subcutaneous+adipose+tissue.">Preparation of an adipogenic hydrogel from subcutaneous adipose tissue.</a> Acta Biomater. 9 (3), 5609-5620 (2013).</li><li>Dilley, R. J., Morrison, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularisation+to+improve+translational+potential+of+tissue+engineering+systems+for+cardiac+repair.">Vascularisation to improve translational potential of tissue engineering systems for cardiac repair.</a> Int J Biochem Cell Biol. 56, 38-46 (2014).</li><li>Lesman, A., Koffler, J., Atlas, R., Blinder, Y. J., Kam, Z., Levenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vessel-like+networks+within+multicellular+fibrin-based+constructs.">Engineering vessel-like networks within multicellular fibrin-based constructs.</a> Biomaterials. 32 (31), 7856-7869 (2011).</li><li>Hussey, A. 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=Seeding+of+pancreatic+islets+into+prevascularized+tissue+engineering+chambers.">Seeding of pancreatic islets into prevascularized tissue engineering chambers.</a> Tissue Eng Part A. 15 (12), 3823-3833 (2009).</li><li>Chen, X., Aledia, A. S., Popson, S. A., Him, L., Hughes, C. C., George, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+anastomosis+of+endothelial+progenitor+cell-derived+vessels+with+host+vasculature+is+promoted+by+a+high+density+of+cotransplanted+fibroblasts.">Rapid anastomosis of endothelial progenitor cell-derived vessels with host vasculature is promoted by a high density of cotransplanted fibroblasts.</a> Tissue Eng Part A. 16 (2), 585-594 (2010).</li><li>Lin, R. Z., Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor-2+facilitates+rapid+anastomosis+formation+between+bioengineered+human+vascular+networks+and+living+vasculature.">Fibroblast growth factor-2 facilitates rapid anastomosis formation between bioengineered human vascular networks and living vasculature.</a> Methods. 56 (3), 440-451 (2012).</li><li>Dolderer, J. 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著者には競合する利益を宣言しません。

微小循環のエンジニアリングは、現在、二つのアプローチを通して、本質的に検討されています。最初は、移植されたときに、ホストの血管床からの毛細血管は、このように周囲に栄養素だけでなく、の配信を保証する、吻合と呼ばれるプロセスを通じて移植構築物のものと接続するようにin vitroでの構文内で高度に相互接続血管網を開発したが含ままた、コア。21,32,33にこれ?...

機能的な血管化組織を作製する組織工学アプローチを使用して再生医療における新興のパラダイムである。損傷した組織または欠陥のある臓器の置換のために新しく健康な組織を操作するために1,2の多くのアプローチが開発されている、3-6実験で小動物モデルで有望な臨床的可能性。7,8。しかし、血管新生は、臨床的に関連する大きさの組織を成長させる可能性を制限する組織工学のための大きな課題の一つである。9 組織は新しい血管が受信者血管床から成長外因性経路のいずれかに従うと、移植された組織は、10または脈管構造が成長し、新たに開発組織と一体となって展開する内因性血管新生経路を構築を通じて侵入する血管新生化するための現在のアプローチ。11外因性アプローチ伝統的な足場上に細胞を播種することを含みます血管の内部成長が遅すぎると非常に薄いインプラント（としてin vitroで 、以前に培養培地により供給される栄養素は、血液循環から供給されることを期待して生きている動物に完全な構築物を注入する。12,13コンセプトは&lt;単純化されて1〜2ミリメートルの厚さ）が生存したままになります。十分かつ迅速な血管新生によって栄養と酸素を提供することは、骨、筋肉、脂肪および固体臓器など、より複雑で、より大きな組織工学代用を成長させるための任意の成功した試みの心臓部である。14,15組み込み血管新生は、の可能性を提供していますより大きな構築物は、その拡大の血液供給に見合ったプログレッシブ組織の成長によって開発します。一つのデザインは、細胞播種足場を伴うまたは伴わない血管茎のチャンバ内へのin vivoでの移植である。5,6これは厚い本質的に血管新生した組織の生成のための新しい手順への道を開いた。16,17 &lt;/ P&gt; より最近では、戦略は、移植前に、事前血管を通す組織移植片に開発されてきました。これらの組み込まれた血管ネットワークは、移植厚い組織移植片のすべての部分の生存率を改善するための完全な血液供給の迅速な提供を可能にする注入でホスト血管と吻合することを目的としている。18 私たちは、灌流血管茎と細胞含有生体材料を含む皮下移植半硬質の密閉チャンバを伴う小動物におけるインビボ血管組織工学モデルを開拓してきました。室は、移植された血管からの血管新生発芽を刺激し、虚血性環境を作成します。3血管茎が再構成された動静脈ループまたは無傷のフロースルー動脈と静脈をいずれかであることができる。3-6,19この血管茎の芽機能と豊富な動脈を両方の芸術でリンク-capillary、静脈網erioleと静脈は血管茎で終わる。3,20また、周囲の中空の支持室が潜在的に機械的な力を変形させることから、発達中の組織を保護し、血管新生を高めるために、虚血性ドライブを延長する。3,21,22血管茎は単純に移植された場合室内の保護された空間内の正常組織ではなく、血管新生発芽は通常の創傷と同じタイムラインに沿って停止し、新しい組織が椎弓根の周りに蓄積されません。調べでは、支持的脈管構造とし、臨床的に関連する大きさの3次元機能に血管新生組織構築物を生成するために、このin vivoでの設定を使用しています。4,23さらに、その無傷の血管茎と工学血管組織構築物は、損傷部位でのその後の移植のために採取することができます。24,25より臨床的に実現可能なシナリオは、復興のための決定的なサイトでチャンバを作成することになります胸としてUCH。したがって、この新規組織工学アプローチは、再建手術のための機能的な標的組織の新たな供給源を提供するための臨床的可能性を有することができる。26-28 以下のプロトコルは、異なる動物モデルで適応および血管新生、マトリックス産生、および細胞遊走および分化の複雑な過程を調べるために使用することができるラットにおけるインビボ血管新生組織工学室を構築する一般的なガイドを提供します。

<table border="0" cellpadding="0" cellspacing="0" width="649">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:123px;">1 15 Blade Scalpel</td>
			<td style="width:187px;">Braun</td>
			<td style="width:163px;">BB515</td>
			<td style="width:176px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Toothed Adson Forceps</td>
			<td>Braun</td>
			<td>BD527R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Needle Holder</td>
			<td>Braun</td>
			<td>BM201R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Bipolar Coagulator&#160;</td>
			<td>Braun</td>
			<td>US335</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Needle Holder B-15-8.3</td>
			<td>S &#38; T</td>
			<td>00763</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Dilator Forceps D-5a.2</td>
			<td>S &#38; T</td>
			<td>00125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Jeweler&#39;s Forceps JF-5</td>
			<td>S &#38; T</td>
			<td>00108</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Straight SAS-11</td>
			<td>S &#38; T</td>
			<td>00098</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Micro Scissors - Curved SDC-11</td>
			<td>S &#38; T</td>
			<td>00090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 Single Clamps B-3</td>
			<td>S &#38; T</td>
			<td>00400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 10/0 nylon suture</td>
			<td>S &#38; T</td>
			<td>03199</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 6/0 nylon suture</td>
			<td>Braun</td>
			<td>G2095469</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 4/0 Silk Sutures</td>
			<td>Braun</td>
			<td>C0760145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xilocaine 1%</td>
			<td>Dealmed</td>
			<td>150733</td>
			<td>10 mg/ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heparin Sodium</td>
			<td>Dealmed</td>
			<td>272301</td>
			<td>5000 UI / ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ringer Lactate</td>
			<td>Baxter</td>
			<td>JB2323</td>
			<td>500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 dome-shaped tissue engineering chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 flow-through chamber</td>
			<td>custom made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lectin I, Griffonia Simplicifolia&#160;</td>
			<td>Vector Laboratories</td>
			<td>B-1105</td>
			<td>1.67 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Troponin T antibody</td>
			<td>Abcam</td>
			<td>Ab8295</td>
			<td>4 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human-specific Ku80 antibody</td>
			<td>Abcam</td>
			<td>Ab80592</td>
			<td style="width:176px;">0.06 &#956;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Desmin antibody</td>
			<td>Dako</td>
			<td>M0760</td>
			<td style="width:176px;">2.55 &#956;g/mL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cell Tracker CM-DiI dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C-7000</td>
			<td>3 mg/106 cells</td>
		</tr>
	</tbody>
</table>

tissue engineering by intrinsic vascularization in an in vivo tissue engineering chamber

再建手術では、複雑で自家復興の現在の方法に代わる、コスト高と貿易別のものの欠陥のための臨床的な必要性があります。組織工学は、この需要の増加に対処するための約束を保持しています。しかし、ほとんどの組織工学戦略が悪いため、血管新生の安定した機能的組織代替物を生成することができません。本論文では、灌流動脈と静脈のいずれかの動静脈ループまたはフロースルー椎弓根構成としては、保護された中空室の内部に向けられている固有の血管新生のin vivo組織工学室モデルに焦点を当てています。この室ベースのシステムでは、血管新生発芽は、動静脈の血管から発生し、このシステムは徐々に線維血管組織でチャンバ空間を埋め駆動虚血性および炎症性の内因性の細胞遊走を魅了しています。外因性の細胞/チャンバーの構築時に行列注入は、セルシュールを強化しますvivalと開発工学組織の特異性を決定します。我々の研究は、この室モデルがうまくそのような脂肪、心筋、肝臓などのような様々な組織を生成することができることを示しています。しかしながら、修正及び改良は、標的組織の形成を一貫して再現可能であることを確認する必要があります。この記事では、生体内で二つの異なる血管組織工学室モデルの製造のための標準化されたプロトコルについて説明します。

上記のプロトコールに記載されるように、組織工学室の顕微作成を行いました。チャンバ内部で発生組織3.種々の組織タイプの正常インビボ血管新生室（ 図2）を使用して操作されたプロトコルのステップで説明するように組織学的に調べることができます。これらは、新生児ラット心筋細胞（ 図2A）、脂肪組織の細胞外マトリックス?...

Watch this Scientific Journal Video about Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber at JoVE.com

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

中に組み込み血管新生による組織工学<em&gt;インビボ</em&gt;ティッシュ・エンジニアリング商工会議所

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

tissue-engineering-intrinsic-vascularization-an-vivo-tissue

Research

JoVE Journal

Bioengineering

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

8.8K ビュー.  St Vincent's Institute of Medical Research. This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling. 

ビデオ: Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心筋組織工学のための生物由来注射用材料の作製

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

生物工学

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

動脈組織工学におけるエラストマーのPGSの足場

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

マウスモデルにおける腸のティッシュエンジニアリング

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

心臓組織工学のためのキャピラリーフォースリソグラフィ

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

人間の3次元の組織工学<em&gt; in vitroで</em&gt;腫瘍テストシステム

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三次元の全層皮膚等価物と自動傷つけ処理の発生

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

エンジニアリング3D細胞化血管組織再生のためのコラーゲンゲル

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

サンドイッチ状の微小環境は、Cell /材料の相互作用を活用します

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

組織工学のための熱応答性ナノ構造表面の調製

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

組織工学のための血管新生単位としてマウスの脂肪組織由来の微小血管フラグメントの単離

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...