著者は、テキサスA＆M大学のエンジニアリングに感謝し、本研究の財政支援のためのステーション（TEES）を実験します。リンジーネイルは感謝して少数の参加のためのテキサスA＆M大学のルイ・ストークス・アライアンス（LSAMP）と国立科学財団（NSF）大学院研究フェローシッププログラム（GRFP）からの支援を認めています。偉張のおかげで、テキサスA＆M大学論文フェローシップ。

1. PCL-DAマクロマーの合成<ol><li>アクリルの反応を実行します。 <ol><li>テフロンで覆われた磁気撹拌棒を備えた250mlの丸底フラスコにPCLジオール（N = M〜10,000グラム/モル）を20gを秤量します。 </li><li> DCM中のPCLジオールを溶解します。 <ol><li>フラスコ（濃度= 0.17グラム/ミリリットル）に120 mLlof DCMを追加します。 </li><li>圧力上昇を避けるためにも、DCMの蒸発を防止しながら、フラスコの首に緩くゴム隔膜を配置します。 </li><li> 〜250rpmで30分間〜攪拌溶液がポリマーを完全に溶解しました。 </li></ol></li><li>溶液に4-ジメチルアミノピリジン（DMAP）の〜6.6ミリグラムを加え、撹拌しながら溶解します。 </li><li>フラスコの首にしっかりとゴム隔膜を配置します。ソリューションは、攪拌を継続することを許可します。 </li><li>ゴム隔膜を介して、優しく、正N 2圧力針入口とオープン針を使用して〜3分間N 2でフラスコをパージアウトレットとして。 </li><li> N 2入口と出口を削除します。 </li><li>ゴム製セプタムを通して挿入された針を備えたガラス製シリンジを介して滴下し、トリエチルアミン（Et 3 Nを）の0.56ミリリットル（4.0ミリモル）を追加します。 </li><li>ゴム製セプタムを通して挿入された針を備えたガラス製シリンジを介して塩化アクリロイルを滴下の0.65ミリリットル（8.0ミリモル）を追加します。 </li><li>フラスコに、N 2注入口を返し、その内容は〜のために、正N 2圧下で30分間攪拌することができます。 </li><li> 55ºCにオイルバスを事前に加熱します。 </li><li>割り当てられた〜30分後、N 2注入口を削除し、凝縮器をセプタムを交換してください。 </li><li>予備加熱した油浴中にフラスコを浸し。 </li><li>フラスコの内容物を20時間撹拌します。 </li><li>割り当てられた20時間後、油浴からフラスコを除去し、内容物を室温に冷却します。 </li><li>ロータリーエバポレーターを使用して、フラスコからDCMの溶媒を除去します。 </li></ol></li><li> P粗製のPCL-DA製品をurify。 <ol><li>フラスコに、酢酸エチル〜135ミリリットルを加え、粗PCL-DAを溶解します。 </li><li>重力はきれいな250ミリリットル丸底フラスコにろ紙を通して溶液をフィルタリングします。 （注：ソリューションを容易に通過しない、ろ紙上に厚くすることができるので、慎重に加熱銃で穏やかな熱を適用する場合。。） </li><li>ロータリーエバポレーターを使用して、フラスコから酢酸エチルの溶媒を除去します。 </li><li>フラスコに、DCMの〜140ミリリットルを加え、粗PCL-DAを溶解します。 </li><li> 500ミリリットル分液漏斗にコンテンツを転送します。 </li><li>ロートに、2 M炭酸カリウム（K 2 CO 3）の13.5ミリリットルを追加します。 </li><li>ファンネルキャップ。ゆっくりコックを介して圧力を解放するように注意しながら、漏斗を反転し、穏やかに一度か二度旋回することにより、2層を混合。 3回繰り返します。 </li><li>パラフィルムの層でキャップを交換し、混合物は、O / N（〜12時間）を分離することができます。 </li><li>底を収集し、または250ミリリットルの三角フラスコにganic層。 </li><li>フラスコに無水硫酸マグネシウム（MgSO 4）の〜5グラムを加え、穏やかに渦巻きます。 </li><li>重力は丸底フラスコ定性濾紙とクリーン250ml中に混合物をフィルタリングします。 </li><li>ロータリーエバポレーターを使用して、フラスコからDCMの溶媒を除去します。 </li><li>残留DCMを除去し、高真空下で乾燥。 （注：PCL-DAは光から離して保管する必要があります。） </li><li> 1 H NMR 22,23とアクリルを確認してください。 </li></ol></li></ol> SMP足場（図1）を準備する。2. <ol><li>溶融塩のテンプレートを準備します。 <ol><li>直径〜460±70μmの塩化ナトリウム（NaCl）で粒子を得るために425ミクロンのふるいを使用してください。 （注：平均粒径は、ImageJソフトウェアを走査型電子顕微鏡[SEM]像から確認することができる。）14 </li><li> 3ミリリットルのガラスバイアル（ID = 12.9ミリメートル）には、以前にふるいにかけNaClを1.8グラムを追加します。 </li><li>ゆっくりとバイアルに（塩の重量に基づく）、4回に分けて、DI水（0.146グラム）を7.5重量％を追加します。水の各部分を加えた後、金属へらで混ぜます。 </li><li>バイアルに蓋を、組織内にラップし、遠心分離管に垂直に配置します。 3,220×gで15分間遠心。 </li><li>キャップを外し、空気乾燥O / N（〜12時間）をしましょう​​。 </li></ol></li><li>新しいガラスバイアルに、DCM 1ml当たりPCL-DA 0.15gのを組み合わせることにより、「マクロマー溶液」を調製します。 （注：1の足場のために、〜1mlの溶液を準備する必要があります）キャップとは〜1分間ボルテックスミキサー上で高速で溶液を混合。 </li><li>新しい3 mLのガラスバイアル中、1-ビニル-2-ピロリドン（NVP）10重量％の2,2-ジメトキシ-2-フェニルアセトフェノン（DMP）に基づいて、「開始剤溶液」を調製します。 NVP 1mlにDMPの0.115グラムを組み合わせます。 （注：1の足場のために、〜180μLが必要です。）キャップ、（光を遮断する）アルミホイルで包み、ボルテックスミキサーで高速で溶液を混合〜1分間。 （注：上級で製造した場合、溶液は冷蔵し、光から保護されるべきです。） </li><li>アルミホイルで（キャップ​​除く）マクロマー溶液を含むバイアルをラップ（光を遮断するために）、およびピペットで15体積％を追加の光開始剤溶液（マクロマー溶液の全体積に基づいて）。キャップと〜1分間ボルテックスミキサー上で高速で溶液を混合。 </li><li> （光を遮断する）アルミホイルで（キャップ​​を除く）溶融塩テンプレートを含むバイアルをラップし、ピペットで先に調製したマクロマー/光開始剤溶液（〜0.6ミリリットルまたはテンプレートが完全に覆われるまで）を追加します。 </li><li>バイアルに蓋を、組織内にラップし、遠心分離管に垂直に配置します。テンプレート全体マクロマー溶液を配布する1,260×gで10分間遠心。 </li><li> 、アルミホイルを外し、バイアルをキャップを取ると3分間UV光（365nmの、25 W）に公開します。空気乾燥O / N。 </li><li> 「塩含有scaffolを削除ガラスバイアルの上部を得点し、破砕した後、ピンセットでバイアルからD」。 </li><li> （：1容量：容量1）400ミリリットルビーカーに、水/エタノールの溶媒〜200ミリリットルを準備します。 </li><li>毎日の溶媒変更で4日間水/エタノール溶媒中に沈め足場を維持します。 </li><li>溶剤や空気乾燥O / Nから足場を取り外します。 </li></ol> 3. SMP骨格にポリドーパミンコーティングを塗布する（図1） <ol><li>テフロン被覆された撹拌棒を備えた400ミリリットルのビーカーでは、ドパミン塩酸塩溶液の〜200ミリリットル（10 mMトリス緩衝液中の2 mg / mlの、pH値= 8.5、25ºC）を準備します。 〜150rpmで攪拌​​します。 </li><li>距離の半分足場を介して足場に、〜;使い捨て針（ゲージ= 20の長さ= 40 mm）を配置します。針ハブの周りにワイヤーをラップします。 </li><li>ビーカーの縁にワイヤーを固定することにより、攪拌ドーパミン溶液に（溶液表面上の針ハブ付き）足場を沈めます。 </li><li>針ハブにシリンジを配置し、足場から空気を引くためにそれを使用して足場を脱気。 （注：より多くの空気が除去できない場合に脱ガスが完了し、溶液を完全に足場に浸透しています。） </li><li> 16時間攪拌したドーパミン溶液中に沈め足場を維持します。 </li><li>溶液から足場を外し、針を取り除きます。 24時間室温で真空オーブン中でDI水とドライで洗い流してください。 </li><li> 1時間85ºCオーブンに足場を置きます。 </li><li>足場を室温に冷却します。最終的な円筒状の足場は〜直径6mm X〜5ミリメ​​ートルの高さになります。 </li></ol> 4.「セルフフィット」行動を評価します<ol><li>厚さ〜5mmである硬質プラスチックのシートを使用して、「不規則なCMF欠損モデル」を用意します。 図2Aに示されたように、〜6ミリメートルよりもわずかに小さい平均直径を有するプラスチックシート内の空隙を作成するためにドリルを使用してください。 </li><li> ABで（生理食塩水の臨床使用を表す）エーカー、熱DI水〜60ºCの温度。 </li><li> 〜60ºC、水の入ったビーカーの中に足場を置きます。水にすべての領域を露出させ、水面下の足場をプッシュするためにピンセットを使用してください。 〜2分間、または足場が著しく（ 図2B）を軟化するまで続けます。 </li><li>ビーカーから足場を取り外し、すぐにモデルの欠損部に（手で）を押します。 </li><li> RT（〜5〜10分間）（ 図2C）に冷却してください。 </li><li>新しい、固定の一時的な形状と比較的より剛性の状態（ 図2D）のリターンを観察する欠陥から削除します。 </li></ol> 5.テスト形状記憶挙動<ol><li>動的機械的分析器の使用（DMA;本明細書中で使用される、例えば 、TA InstrumentsのQ800）を、2サイクル（N）の形状固定性（ 式中、R f）を決定すると、足場の上に歪み制御環式熱機械的圧縮試験を実行します形状回復（R rの）（ 図3）。 <ol><li> 5分間60℃（ 高 T）に平衡化。 </li><li> 50％/分で最大ひずみ（εM = 50％）に圧縮します。 </li><li> εmを （5分）でホールド。 </li><li> 25℃（ 低 T）に冷却し、一時的な形状を固定するための10分間維持します。 </li><li>負荷を削除します。 </li><li>ストレスのない状態での究極のひずみ（εU）を測定します。 </li><li> 60°C（T 高 ）に再加熱し、10分には永続的形状を回復するために維持します。 </li><li>回収されたひずみ（εpを ）を測定します。 </li><li>まだ（T 高 ）、60℃で、2 番目のサイクルを開始する（N = 2）1 回目のサイクルの後に回収され、高さ（N = 1）の50％に足場を圧縮しています。 </li><li> 5.1を繰り返します.3-5.1.8、N = 2のため。 </li><li>以下の式を使用して、N = 1および2のR fおよびR rを計算します。 式中、R f（N）= [U（N）/εmのε]のR R（N）= [εmを - εP（N）] / [εmを - εpを（N-1）] </li></ol></li></ol> 6.可視化細孔サイズおよび細孔相互接続性<ol><li>走査型電子顕微鏡の使用（SEM;本明細書中で使用される例えば FEI QuantaがSEM）を、孔径および孔相互接続性を観察します。 <ol><li>ピンセットを使用して、SMPの足場を保持1分間液体N 2中に沈めました。 </li><li>きれいなカミソリの刃で足場の中央に沿って液体N 2と骨折から削除します。 </li><li>カーボンテープを用いて、試料上にSMP足場半分の一方を固定破断面を上に​​向けてステージ。 </li><li>金 - 白金（〜4 nm）のでコートをスパッタ。 </li><li> 10〜15 kVの（ 図4A）の推奨加速電圧でのSEM画像をキャプチャします。 </li></ol></li></ol> インビトロ生物活性の7テスト<ol><li> 50mlの遠心分離管に、〜1×SBF 24の30ミリリットルを追加します。 </li><li>元の、円筒状に成形された永続的形状に足場を取得します。きれいなブレードを用いて（円形縁全体に）半分に足場をカットします。 </li><li>準備された遠心管とキャップ内に個々の足場半分を置きます。 </li><li>無SBF変更を静的条件下での水浴中で37℃でチューブを維持します。 </li><li> 14日後、24時間SBF、空気乾燥から足場を取り外します。 </li><li>カーボンテープを使用して、破断面を上に​​向けて試料ステージ上に足場を固定。 </li><li>金 - 白金（〜4 nm）のでコートをスパッタ。 </li><li>再でSEM画像をキャプチャ10〜15 kVの（ 図4B）の加速電圧を賞賛しました。 </li></ol>

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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=Polydopamine-mediated+immobilization+of+multiple+bioactive+molecules+for+the+development+of+functional+vascular+graft+materials.">Polydopamine-mediated immobilization of multiple bioactive molecules for the development of functional vascular graft materials.</a> Biomaterials. 33, 8343-8352 (2012).</li><li>Wu, C., Fan, W., Chang, J., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mussel-inspired+porous+SiO+2+scaffolds+with+improved+mineralization+and+cytocompatibility+for+drug+delivery+and+bone+tissue+engineering.">Mussel-inspired porous SiO 2 scaffolds with improved mineralization and cytocompatibility for drug delivery and bone tissue engineering.</a> J Mater Chem. 21, 18300-18307 (2011).</li><li>Ku, S. H., Ryu, J., Hong, S. K., Lee, H., Park, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+functionalization+route+for+cell+adhesion+on+non-wetting+surfaces.">General functionalization route for cell adhesion on non-wetting surfaces.</a> Biomaterials. 31, 2535-2541 (2010).</li><li>Schoener, C. A., Weyand, C. B., Murthy, R., Grunlan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+memory+polymers+with+silicon-containing+segments.">Shape memory polymers with silicon-containing segments.</a> J Mater Chem. 20, 1787-1793 (2010).</li><li>Zhang, D., Giese, M. L., Prukop, S. L., Grunlan, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(&#949;-caprolactone)-based+shape+memory+polymers+with+variable+polydimethylsiloxane+soft+segment+lengths.">Poly(&#949;-caprolactone)-based shape memory polymers with variable polydimethylsiloxane soft segment lengths.</a> J Polym Sci Pol Chem. 49, 754-761 (2011).</li><li>Kokubo, T., Takadama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+useful+is+SBF+in+predicting+in+vivo+bone+bioactivity.">How useful is SBF in predicting in vivo bone bioactivity.</a> Biomaterials. 27, 2907-2915 (2006).</li><li>Murphy, W. L., Dennis, R. G., Kileny, J. L., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+fusion:+an+approach+to+improve+pore+interconnectivity+within+tissue+engineering+scaffolds.">Salt fusion: an approach to improve pore interconnectivity within tissue engineering scaffolds.</a> Tissue Eng. 8, 43-52 (2002).</li><li>Yang, Q., Chen, L., Shen, X., Tan, Z. <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+polycaprolactone+tissue+engineering+scaffolds+by+improved+solvent+casting/particulate+leaching+method.">Preparation of polycaprolactone tissue engineering scaffolds by improved solvent casting/particulate leaching method.</a> J Macromol Sci Phys. 45, 1171-1181 (2006).</li><li>Madbouly, S. A., Kratz, K., Klein, F., L&#252;zow, K., Lendlein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermomechanical+behaviour+of+biodegradable+shape-memory+polymer+foams.">Thermomechanical behaviour of biodegradable shape-memory polymer foams.</a> 1190, (2009).</li><li>Luo, X., Mather, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+shape+memory+elastomeric+composites.">Preparation and characterization of shape memory elastomeric composites.</a> Macromolecules. 42, 7251-7253 (2009).</li><li>Lendlein, A., Schmidt, A. M., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AB-polymer+networks+based+on+oligo(&#603;-caprolactone)+segments+showing+shape-memory+properties.">AB-polymer networks based on oligo(&#603;-caprolactone) segments showing shape-memory properties.</a> Proc Natl Acad Sci. 98, 842-847 (2001).</li></ol>

The authors have nothing to disclose.

このプロトコルは、自己フィッティング行動だけでなく、骨誘導性および生物活性ポリドーパミン被覆された、PCLベースの足場の製造を記載し​​、不規則なCMFの骨欠損の治療に関心のそれを作ります。プロトコルの態様は、様々な骨格の機能を変更するために変更することができます。 プロトコルは、UV硬化を可能にするために、PCLジオールのアクリルから始まります...

現在、頭蓋顎顔面（CMF）骨欠損治療のゴールドスタンダードと考えられ、収穫自家移植片の移植は複雑な移植手順、ドナー部位の罹患率と限られた可用性1によって妨げられています。特定の困難は、オッセオインテグレーションを得るために、グラフトの再吸収を防止するために、欠陥にしっかり剛性自家移植片を成形し、固定されています。組織工学は、自家移植の代替戦略と合成骨代替物（ 例えば、骨セメント）-2,3-として研究されてきました。組織工学的アプローチの成功に重要なプロパティの特定のセットとの足場です。まず、オッセオインテグレーションを得るために、足場は、隣接する骨組織4との密接な接触を形成しなければなりません。足場はまた、細胞移動、栄養拡散とneotissue堆積4,5を可能にして、骨伝導性でなければなりません。この動作は、一般的に、生分解性、SCAで達成されます高度に相互接続孔の形態を呈するffolds。骨組織5を周囲との統合との結合を促進するために、最後に、足場は、生物活性であるべきです。 ここでは、これらの特性を持つ組織工学の足場を準備するためのプロトコルを提示します。重要なことには、この足場は、形状記憶挙動6による不規則なCMF欠陥に「自己適合」する能力を示します。温度応答性形状記憶ポリマー（SMPS）は、7,8の熱への暴露時に形状変化を起こすことが知られています。 SMPSが永続的形状および一時的形状を維持し、永続的形状を回復する「スイッチングセグメントを「決定」netpoints」（ すなわち 、化学的または物理的架橋）から構成されています。スイッチングセグメントはガラス転移（T g）のいずれかに相当する熱転移温度（T トランス）を示すか、ポリマーの転移（T mを）溶かします。として結果は、SMPSが順次T &lt;T トランスで一時的な形状に固定し、T&gt; T トランスでの一時的な形状に変形し、T&gt; T トランスで永続的形状に回復することができます。 6を次のようにこのように、SMP足場は、CMFの欠損内に「自己フィッティング」を達成できました。生理食塩水（T&gt; T トランス）を温めるに暴露した後、SMP足場は、形状回復が不良境界に足場の拡大を推進して、凹凸欠陥に手で押したように一般的に準備された円筒状の足場を許可する、可鍛性になります。 （T &lt;T トランス）を冷却すると、足場は形状固定性が欠損内に、新しい一時的な形状を維持しながら、その比較的より剛性の状態に戻ることになります。このプロトコルでは、SMPの足場は、生分解性ポリマーは、組織再生、および他の生物医学的用途のために広く研究9-11、ポリカプロラクトン（PCL）から調製されます。形状記憶のために、目PCLのEのT mは、T トランスとして機能し、PCL 12の分子量に応じて、43〜60ºCの間で変化します。このプロトコルでは、足場のT トランス （ すなわち T m）は56.6±0.3ºC6です。 骨伝導性を達成するために、プロトコルは、溶媒キャスティング微粒子浸出（SCPL）法6,13,14に基づいて高度に相互接続された細孔を有するPCLベースSMP足場を作るために開発されました。ポリカプロラクトンジアクリレート（PCL-DA）（M = N〜10,000グラム/モル）を急速な、光化学的な架橋を可能にするために利用された塩のテンプレート上溶剤キャストを可能にするために、ジクロロメタン（DCM）に溶解しました。光化学硬化、溶媒の蒸発の後、塩のテンプレートを水に浸出することによって除去しました。平均塩サイズは足場の細孔サイズを調節します。重要なことには、塩テンプレート前細孔interconnectiviを達成するために、キャスティング溶媒を水と融合しましたTY。 生物活性は、細孔壁6上にポリドーパミンコーティングのその場形成のことで、SMPの足場に付与しました。生物活性は、多くの場合、ガラスまたはガラス-セラミック充填材15を含めることによって足場に導入されます。しかし、これらは、望ましくない脆性、機械的特性を生じ得ます。ドーパミンは、基板16〜19の様々な上に付着し、薄いポリドーパミン層を形成することが示されています。このプロトコルでは、SMPの足場は、すべての細孔壁面6にポリドーパミンのnanothickコーティングを形成するためにドーパミンのわずかに塩基性液（pH = 8.5）に供しました。改善された細胞接着のための表面の親水性を向上させ、拡散に加えて、ポリドーパミンは、擬似体液（SBF）18,20,21への曝露の際に、ハイドロキシアパタイト（HAP）を形成するという点で生物活性であることが示されています。最後のステップでは、コートされた足場は、85ºC（T&gt; T トランス）WHで熱処理を露出させますICHは、足場の高密度化につながります。熱処理は、以前おそらく近接14に再編成するPCLの結晶ドメインに、足場形状記憶動作のために必須であることが認められました。 我々はさらに、孔の形態を、不規則なモデル欠陥内のセルフフィット挙動を特徴づけるための方法を記載するという点でひずみ制御環状の熱機械的圧縮試験を形状記憶挙動（ すなわち形状回復と定着性を形作る）、 および in vitro生物活性に 。足場の特性を調整するための戦略も提供されます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Polycaprolactone-diol (Mn ~ 10,000 g/mol)</td>
			<td>Sigma-Aldrich</td>
			<td>440752</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>D65100</td>
			<td>Dried over 4A molecular sieves</td>
		</tr>
		<tr>
			<td>4-dimethylaminopyridine (DMAP)</td>
			<td>Sigma-Aldrich</td>
			<td>D5640</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine (Et3N)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryloyl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A24109</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Carbonate (K2CO3)</td>
			<td>Sigma-Aldrich</td>
			<td>209619</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous magnesium sulfate (MgSO4)</td>
			<td>Fisher</td>
			<td>M65</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2-dimethoxy-2-phenyl acetophenone (DMP)</td>
			<td>Sigma-Aldrich</td>
			<td>196118</td>
			<td></td>
		</tr>
		<tr>
			<td>1-vinyl-2-pyrrolidinone (NVP)</td>
			<td>Sigma-Aldrich</td>
			<td>V3409</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>459844</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopamine Hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H8502</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris buffer (2mol/L)</td>
			<td>Fisher</td>
			<td>BP1759</td>
			<td>Used at 10 mM concentration, pH = 8.5</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>VWR</td>
			<td>47729-972</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Transilluminator (365 nm, 25 W)</td>
			<td>UVP</td>
			<td>95-0426-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynamic Mechanical Analyzer (DMA)</td>
			<td>TA Instruments</td>
			<td>Q800</td>
			<td></td>
		</tr>
		<tr>
			<td>High Resolution Sputter Coater</td>
			<td>Cressington</td>
			<td>208HR</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope (SEM)</td>
			<td>FEI</td>
			<td>Quanta 600</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a bioactive, pcl-based "self-fitting" shape memory polymer scaffold

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to the success of this approach is a scaffold that is able to conformally fit within an irregular defect while also having the requisite biodegradability, pore interconnectivity and bioactivity. By nature of their shape recovery and fixity properties, shape memory polymer (SMP) scaffolds could achieve defect &#8220;self-fitting.&#8221; In this way, following exposure to warm saline (~60 &#186;C), the SMP scaffold would become malleable, permitting it to be hand-pressed into an irregular defect. Subsequent cooling (~37 &#186;C) would return the scaffold to its relatively rigid state within the defect. To meet these requirements, this protocol describes the preparation of SMP scaffolds prepared via the photochemical cure of biodegradable polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method. A fused salt template is utilized to achieve pore interconnectivity. To realize bioactivity, a polydopamine coating is applied to the surface of the scaffold pore walls. Characterization of self-fitting and shape memory behaviors, pore interconnectivity and in vitro bioactivity are also described.

得られたPCLベースSMP足場は自己フィッティングモデルCMF欠陥に（ 図2）が可能です。短時間の露光後の生理食塩水（〜60℃）を暖めるために、円筒状の足場は、足場が手動に押され、モデルの欠陥内で膨張することができるように柔らかく。室温まで冷却した後、足場が欠陥から除去する際に保持され、新しい一時的な形状に固定されています。 SMP足場の形?...

Watch this Scientific Journal Video about Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold at JoVE.com

Fabrication of a Bioactive, PCL-based &quot;Self-fitting&quot; Shape Memory Polymer Scaffold

Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

生理活性、PCLベースの「セルフフィット」形状記憶ポリマー足場の作製

Tissue engineering has been explored as an alternative strategy for the treatment of critical-sized cranio-maxillofacial (CMF) bone defects. Essential to ...

fabrication-bioactive-pcl-based-self-fitting-shape-memory-polymer

Research

JoVE Journal

Bioengineering

Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

12.5K ビュー.  Texas A&M University. Scaffolds capable of fitting within cranio-maxillofacial (CMF) bone defects while exhibiting osteoconductivity and bioactivity are of interest. This protocol describes the preparation of a shape memory scaffold based on polycaprolactone diacrylate (PCL-DA) using a solvent-casting particulate-leaching (SCPL) method employing a fused salt template and application of a bioactive polydopamine coating.

ビデオ: Fabrication of a Bioactive, PCL-based "Self-fitting" Shape Memory Polymer Scaffold

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

アクティブな細胞培養のための形状記憶ポリマー

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

生物工学

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, 'SurgiLux': Preparation and Demonstration

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures can act as a nidus for infection and in many procedures are unable to effect wound repair or interfere with functional tissue regeneration.1 Surgical glues and adhesives, such as those based on fibrin and cyanoacrylates, have been developed as alternatives to sutures for the repair of such wounds. However, current commercial adhesives also have significant disadvantages, ranging from viral and prion transfer and a lack of repair strength as with the fibrin glues, to tissue toxicity and a lack of biocompatibility for the cyanoacrylate based adhesives. Furthermore, currently available surgical adhesives tend to be gel-based and can have extended curing times which limit their application.2 Similarly, the use of UV lasers to facilitate cross-linking mechanisms in protein-based or albumin 'solders' can lead to DNA damage while laser tissue welding (LTW) predisposes thermal damage to tissues.3 Despite their disadvantages, adhesives and LTW have captured approximately 30% of the wound closure market reported to be in excess of US $5 billion per annum, a significant testament to the need for sutureless technology.4
In the pursuit of sutureless technology we have utilized chitosan as a biomaterial for the development of a flexible, thin film, laser-activated surgical adhesive termed 'SurgiLux'. This novel bioadhesive uses a unique combination of biomaterials and photonics that are FDA approved and successfully used in a variety of biomedical applications and products. SurgiLux overcomes all the disadvantages associated with sutures and current surgical adhesives (see Table 1).
In this presentation we report the relatively simple protocol for the fabrication of SurgiLux and demonstrate its laser activation and tissue weld strength. SurgiLux films adhere to collagenous tissue without chemical modification such as cross-linking and through irradiation using a comparatively low-powered (120 mW) infrared laser instead of UV light. Chitosan films have a natural but weak adhesive attraction to collagen (~3 KPa), laser activation of the chitosan based SurgiLux films emphasizes the strength of this adhesion through polymer chain interactions as a consequence of transient thermal expansion.5 Without this 'activation' process, SurgiLux films are readily removed.6-9 SurgiLux has been tested both in vitro and in vivo on a variety of tissues including nerve, intestine, dura mater and cornea. In all cases it demonstrated good biocompatibility and negligible thermal damage as a consequence of irradiation.6-10

A Chitosan Based, Laser Activated Thin Film Surgical Adhesive, &#039;SurgiLux&#039;: Preparation and Demonstration

The fabrication of a novel, flexible thin film surgical adhesive from FDA approved ingredients, chitosan and indocyanine green is described. Bonding of this adhesive to collagenous tissue through a simple activation process with a low-powered infra-red laser is demonstrated.

キトサンベース、レーザー活性薄膜外科用接着剤、 &#39;SurgiLux&#39;：準備とデモ

Sutures are a 4,000 year old technology that remain the 'gold-standard' for wound closure by virtue of their repair strength (~100 KPa). However, sutures ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

機械的に適応型ポリマーナノコンポジットの環境制御のMicrotensileテスト<em&gt; ex vivoで</em&gt;キャラ

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

あらかじめ設計されたサイズと形状を用いるポリマー繊維およびバイオハイブリッド繊維のマイクロ流体製造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

バインダーとしてのバクテリアセルロースを利用堅牢な天然繊維プリフォームの製造

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

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 ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

肝組織工学のための三次元細胞培養プラットフォーム：逆コロイド結晶ポリ（エチレングリコール）足場の作製

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

マクロを含む光の組織ファントムの作製と評価

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

腎組織のプライマリ セグメントと新規スキャフォールド フリー構造技術と定款の

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

伸縮性、デュアル チャネル、オルガン マイクロ流体チップのスケーラブルな作製

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...