著者は、このプロジェクトの初期のイテレーションへの技術的な貢献のためにガブリエルアロムとマイケルYeranosianに感謝したいと思います。このプロジェクトはOREF臨床医科学トレーニンググラント（NL）によって資金を供給された、HHリー外科研究助成（NL）、退役軍人局BLR＆Dメリットレビュー1 I01 BX00012601（DM）と筋骨格移植財団若手研究賞（FP）。

注：すべての動物の手術は、実験を開始する前に、地元の獣医スタッフと動物使用委員会によって承認された。 エレクトロポリカプロラクトン足場の調製<ol><li>医療グレードのエステルを秤量し、溶解PCLポリマーの溶液を10％w / wを作成する1,1,1,3,3,3-ヘキサフルオロ-2-プロパノールで顆粒状にポリ（εカプロラクトン）を終了する。均質な溶液を確保するために、少なくとも3時間、攪拌プレートを用いて溶液攪拌をしましょう​​。 </li><li>足場の製作のための高度に整列PCL繊維のカフを作成するためにPCLソリューションをエレクトロスピニング。 <ol><li>すべての回でのファンとの化学ドラフト内でエレクトロスピニングセットアップを準備します。これは、エレクトロスピニングプロセスの単離された真空媒体として機能する大型アクリルボックスで構成され、電圧源により駆動源のPCL溶液を、モーター駆動の収集マンドレル、及び真空ポートのエントリポイントを有している。 acryliを清掃してください完全にエタノールでCボックスとエレクトロ製品の品質を損なう可能性のある不純物を除去するためにパラフィルムシートのすべての表面を覆う。 </li><li>負荷を約3ミリリットル18 G、1 1/2インチの針を平滑末端で10mlシリンジに上記溶液の。注射器を上に押して、すべての気泡を除去。プログラム可能なシリンジポンプにソリューションをロックします。電圧源線の取り付けのためのアクリルボックスの外側針の1/2インチを約残しながらアクリルボックスに小さな穴を通して針を挿入します。 </li><li>高度に整列PCL繊維のための集電体としての旋盤マンドレルを回転さ30ミリメートルを使用します。アルミ箔の薄いストリップでしっかりとマンドレルをカバーしています。約15cm離れた注射針からボックスの反対側のモーターにマンドレルをロックします。 </li><li>真空ポートにプラスチック製のホースを挿入し、ヒュームフードの真空源に接続します。真空源をオンにして、蓋付きのアクリルボックスをカバーしています。 </li><li> Sら2.5ミリリットル/時プログラム可能なシリンジポンプの注入速度。 3,450 rpmでマンドレルを動作させるためにモーターの電源を入れ、ワニ口クリップを使用して、箱の外側針の先端に電圧源の正極リードを添付してください。 </li><li> PCL溶​​液の注入が開始されると、電源をオンにし、20kVの動作電圧に設定する。 </li><li> PCL溶​​液0.5mlから均質なカフを作成するために12分間この溶液を注入する。 注：平均して、それぞれのカフは、3~4層の足場の合計を作成するために使用できる2つの6ストリップシートを作成するのに十分な電界紡糸材料を有する。 </li></ol></li><li>レーザーは、低真空の設定、10.0 keVの着陸電圧、6.4ミリメートル作動距離、および3.0のプローブ径で運転VersaLaserカッター2.3上の複数の小さなシートを形成するPCLカフをカット。 注：この例では、コンピュータ支援設計が1.5mmの複数のシートを得るために、カッターを指示するために使用された×35ミリメートル×150ミクロンと足場均等に15％の孔面積で150μmの直径の穴を配布した。 </li><li>プラズマは、イオン加速をPCL表面の親水性を誘導するためにプラズマクリーナーを使用して、PCL足場をエッチングする。 450トルに真空を設定し、ハイパワー（29.6 W）で30秒間足場を扱う。 </li><li>無菌環境での70％エタノール中で足場を浸し。 </li><li>コートコラーゲンとの個々の足場は、in vivoでの細胞接着および増殖を促進する。 <ol><li> 4℃での1×PBS中で9：1のPurecolコラーゲン3 mg / mlの標準溶液、10×PBS、2.5滅菌溶液と0.1 N NaOHで1：8に希釈することによって、コラーゲンコーティング溶液を作成する。溶液の均一性を確保するために十分に混合する。 </li><li>コー​​ト個々1.5ミリメートル×35ミリメートル×上記のコラーゲン溶液の薄層フィルムと150μmの足場。無菌環境下で24時間乾燥することができます。 </li></ol></li><li> 5-0ビクリル縫合糸を使用して、スタックおよび4つの個々の1.5ミリメートル×35ミリメートル×150μmの足場を固定Krackowは、移植の準備ができている最終的な厚さ0.6mm、多層、コラーゲンコーティングされた足場を作成するためにステッチ使用。 </li></ol> 2.ラット手術プロトコル<ol><li>吸入チャンバーにラットを配置し、2リットル/分の酸素と2％のイソフルランを送達することにより麻酔を誘導する。確認してラットが十分に足を後肢に圧力をかけ、あらゆる応答を評価することにより麻酔している。 </li><li>非滅菌バックテーブルの上には、皮下に25mg / kgのアンピシリン及び0.03 mg / kgをブプレノルフィンを注入する。 </li><li>目に眼軟膏を適用します。手術後肢から毛皮をクリップし、3クロルヘキシジンの交互のスクラブと70％エタノールで手術部位を分取</li><li>低体温症を防ぐために加熱されたパッドの上に、手術台にラットを転送します。セキュアなノーズコーン、および2リットル/分の酸素の2％イソフルランで手続きを麻酔を維持は、ノーズコーンを提供しました。露出された作動肢を残して、無菌状態でドレープ。 </li><li&gt;手術を開始する前に、最初の足圧を後肢への応答の欠如によって十分な麻酔を確認。 </li><li>膝蓋骨のレベルを中心と膝に2cmの長さの垂直切開内側を、確認します。切開が膝の上にセンタリングされるまで横方向に皮膚を撤回。 </li><li>ちょうど膝蓋骨に外側から内側に切断し、大腿四頭筋の筋腱接合部のレベルに遠位脛骨結節に膝蓋腱挿入のレベルに近位に拡張することで、内側傍関節切開を作るためにメスを使用してください。膝蓋骨や大腿四頭筋の腱をカットしないように注意してください。 </li><li>膝蓋腱のすぐ横膝カプセルを通じて1センチメートル垂直切開することにより横方向にリリース膝蓋骨。 </li><li>膝が延長されることを確認してください。細かいハサミを取り、横方向から内側に膝蓋骨の下を通過。伸筋機構はどちらの側に変換することができるようにハサミを数回を広げた。 </li><li>トンを曲げながら彼の膝、膝関節の内部を露出させるために横方向に膝蓋骨を翻訳。顆間ノッチと大腿骨顆の明確な可視化を確認してください。メスを用いて、ノッチにACLとPCLを横断する。 </li><li> 1.6ミリメートルのKワイヤと負荷電力ドリル。顆間でACL原点にKワイヤ先端を置きます。 superolaterallyドリルとメスで、必要に応じて、任意の軟組織を除去し、大腿骨の外側面に出口点を視覚化。移植のための明確なの通路を確保するために数回内外Kワイヤを渡します。 </li><li>脛骨プラトー上のACLフットプリント上のKワイヤを配置します。前外側ドリルと前外側脛骨近位端上の出口点を視覚化。 Kワイヤが脛骨を出るポイントが完全に可視化されるように、必要に応じて、軟組織をクリアするためにメスを使用してください。 </li><li>大腿骨のトンネルを通って（理想的にはこれ以上の2よりインチ長）を短縮キース針を渡します。 Kの目を通して移植片の一端から二縫合糸末端を通すeith針。大腿骨トンネルを通じて移植片の一端を引っ張って針を使用してください。 </li><li>脛骨トンネルを通って移植片のもう一方の端を渡す前に、手順を繰り返します。 </li><li> 8の字ステッチで骨膜またはその他の周囲の軟組織への移植片の大腿骨の端を固定する4-0ビクリル縫合糸を使用してください。手動張力拡張子で膝移植片。 8の字ステッチで骨膜またはその他の周囲の軟組織への移植片の脛骨の端を固定します。 </li><li> 8の字ステッチ過去両端に1〜2ミリメートルを残して、両端の余分な移植片を切断するはさみを使用してください。 </li><li>膝を拡張し、膝蓋骨を減らす。 4-0ビクリルを使用して、膝蓋骨の横方向の亜脱臼を防止、内側関節包を閉じるには、シングル8の字ステッチを配置。 </li><li>下の筋肉を縫合や皮膚がクローズされると、目に見える縫合糸を持ってさせないように注意しながら、実行中の表皮下5-0モノクリルまたはビクリル縫合糸で皮膚を閉じます。 </li><li>麟蹄皮下にブプレノルフィンとCTラット術後3日間の合計ごとに12時間。注入時の任意の排水や縫合不全のために手術部位を確認してください。跛行といくつかの腫れ、手術後の最初の数日で正常であるが、速やかに獣医スタッフと一緒にすべての術後の懸念に対処する。外科的切開が完全に治癒したとき動物は、術後2週間後に社会住宅に戻ることができる。 </li></ol> 3.データ収集プロトコル<ol><li>屠殺時に、開胸続いクローズCO 2チャンバー内で個別にラットを窒息させる。 </li><li>股関節で分離することにより、外科的に再構築され、反対側の手足の両方を収穫。 <ol><li>再構成された手足のために、外科的に後十字靭帯および残骸を含むすべての軟組織を除去するだけで、大腿骨、脛骨、および移植片を分離するために微細な切開により、天然のACLを破壊した。 </li> &lt;李は&gt;対側肢の場合は、ネイティブACLだけでなく、細かい解剖による大腿骨と脛骨を除くすべての軟組織を取り除く。 </li><li>大腿骨 - グラフト - 脛骨複合体の各端部から骨から1cmに¾が、すべてを削除するには、そのようなドレメルなどの回転工具を使用してください。 </li><li>このプロセスの間、および生体力学的試験を通して、定期的かつ頻繁には誤った結果を変更することが収穫膝の乾燥を防ぐために、生理食塩水で靱帯領域をスプレー。 </li></ol></li><li>組織学的分析のために、48時間、25℃で4％パラホルムアルデヒド溶液中で個別に膝を固定する。次に、完全な脱灰用Immunocal試薬の溶液中で膝を沈める。このプロセスは、サンプルの石灰の内容に依存しており、5日ほどかかる場合があります。サンプルの品質を下げることができ、不完全な脱灰などの進捗状況を評価するために毎日の個々のサンプルを確認してください。切片を実行、完了したら、必要に応じて取り付け、及び染色スライドさせます。 </li><li> Perfor組織工学靱帯の機能的能力を評価するための生体力学的試験をメートル。 <ol><li>個別に各骨の骨端の周りに28 G亜鉛めっき鋼線を包むことにより、大腿骨と脛骨を固定します。これは骨ではなく、興味のある靭帯でサンプルの早期引張障害から不正確な生体力学的試験データを防ぐためです。 </li><li>ポットメタクリレート（PMMA）骨セメントの混合物中に大腿骨。これを行うには、2セメント成分を混合し、すぐに完全に自由に突出した骨端及び添付靱帯との接合鍋に骨の骨幹を包む、金属中に大腿骨を固定するために、粘性混合物を使用しています。徐々に締まりのない物質に、最終的には、固体硬化マトリックス中に混合粘性のコンポーネントを変換するための自​​発的なフリーラジカル重合を許可します。 注：このプロセスは数分かかり、手作業で作らボーラスの温度を評価することによってモニターすることができる残りのセメント。温度は一過発熱重合反応の間に増加し、材料が固化した後、室温に治まる必要があります。 </li><li>理想的な機械的試験のために20°屈曲時に膝の靭帯を維持しながら除き、脛骨を接合するため、上記と同じプロセスを繰り返します。 </li><li>引張試験装置の上に接合、大腿骨 - グラフト - 脛骨複合体をマウントして、障害に張力の開始からの時間の関数としての負荷及び変位を記録するために準備する。この例では、1 kNのロードセルをインストロンモデル5564を使用した。 </li><li>プリテンションが0.5N /分のランプ速度にて2 Nに移植した後は、0.5mm /秒の歪速度での故障までの移植片をテスト。プロセスの間、靱帯半ば物質で失敗していることと、骨の大腿骨と脛骨が安全であることと早まって不正確にテストさ靱帯の生体力学的特性を評価することができるもの、失敗しないことを確認してください。 </li><li>生成された荷重 - 変位を使用してください試験された靱帯の破壊荷重および剛性を計算する曲線。 </li></ol></li></ol>

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L., 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+role+of+macrophages+in+early+healing+of+a+tendon+graft+in+a+bone+tunnel.">The role of macrophages in early healing of a tendon graft in a bone tunnel.</a> J Bone Joint Surg Am. 90 (3), 565-579 (2008).</li><li>Dagher, E., 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=Immobilization+modulates+macrophage+accumulation+in+tendon-bone+healing.">Immobilization modulates macrophage accumulation in tendon-bone healing.</a> Clin Orthop Relat Res. 467 (1), 281-287 (2009).</li><li>Bedi, 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=Effect+of+early+and+delayed+mechanical+loading+on+tendon-to-bone+healing+after+anterior+cruciate+ligament+reconstruction.">Effect of early and delayed mechanical loading on tendon-to-bone healing after anterior cruciate ligament reconstruction.</a> J Bone Joint Surg Am. 92 (14), 2387-2401 (2010).</li><li>Bedi, A., Kawamura, S., Ying, L., Rodeo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+tendon+graft+healing+between+the+intra-articular+and+extra-articular+ends+of+a+bone+tunnel.">Differences in tendon graft healing between the intra-articular and extra-articular ends of a bone tunnel.</a> HSS J. 5 (1), 51-57 (2009).</li><li>Fu, S. 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=Effect+of+graft+tensioning+on+mechanical+restoration+in+a+rat+model+of+anterior+cruciate+ligament+reconstruction+using+free+tendon+graft.">Effect of graft tensioning on mechanical restoration in a rat model of anterior cruciate ligament reconstruction using free tendon graft.</a> Knee Surg Sports Traumatol Arthrosc. 21 (5), 1226-1233 (2013).</li><li>Fan, H., Liu, H., Wong, E. J., Toh, S. L., Goh, J. 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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=Increase+in+cell+migration+and+angiogenesis+in+a+composite+silk+scaffold+for+tissue-engineered+ligaments.">Increase in cell migration and angiogenesis in a composite silk scaffold for tissue-engineered ligaments.</a> J Orthop Res. 27 (4), 495-503 (2009).</li><li>Freeman, J. W., Woods, M. D., Laurencin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+of+the+anterior+cruciate+ligament+using+a+braid-twist+scaffold+design.">Tissue engineering of the anterior cruciate ligament using a braid-twist scaffold design.</a> J Biomech. 40 (9), 2029-2036 (2007).</li><li>Bashur, C. A., Shaffer, R. D., Dahlgren, L. A., Guelcher, S. A., Goldstein, A. 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The authors declare that they have no competing financial interests.

ACL損傷は現時点での再構築のための限られた選択肢で、整形外科スポーツ手術に共通する条件である。 インビボでの再生を可能にするACLのための適切な組織工学代替を開発するために、適切な動物モデルが必要である。無胸腺ラットにACL再建の再現可能なモデルを使用して、in vivoでの移植のように、本 ​​研究では、生分解性の操作された移植片の製造は、記載されている。...

前十字靱帯（ACL）の破裂は、膝1の最も一般的な靱帯損傷の一つである。破裂したACLのは、外科的介入なしに治癒することができませんので、日常生活だけでなく、スポーツへの参加の活動には限界が毎年10億ドル3の推定コストで、毎年2手術を受けるために175000以上の患者をドライブ。現在、自家移植または同種移植腱のいずれかが靭帯再建のために使用されている。高い成功率は、自家移植および同種移植の交換の両方を達成することができるものの、深刻な合併症は、これらの再構成オプション4に関連している。自家移植片組織は、特に再破裂またはマルチ靭帯損傷の事例では、ドナー部位の病的状態と関連しており、供給が限られている。一方、同種移植組織は、遅延移植片統合、有害な炎症反応、理論的な感染のリスク、および限られたサップとリンクされているLY 5。合成非分解性移植片は、1970年代と1980年代に開発されたが、時期尚早グラフト破裂、異物反応、骨溶解、及び滑膜炎6によって妨げられた。これらの深刻な懸念の結果として、米国での臨床使用のために利用可能な合成移植片はまだありません。 原因既存の移植オプションと、生物学、工学、再生医療における最近の進展にこれらの制限のために、ACL移植のための組織工学的ソリューションに大きな関心が集まっている。現在の組織工学戦略が永久合成材料注入7に関連する制限を回避しながら宿主組織の内部成長を可能にするために、生物分解性合成材料を用いる。 ポリカプロラクトン（PCL）は、FDAがuのあった8ドレッシング癒着バリア、創傷などの医療用途の数、のために承認された生分解性ポリマーである血管、骨、軟骨、神経、皮膚、食道組織工学5,9-16を含む多種多様な用途でのsed。良好な生体適合性、比較的長い生体内半減期は、十分な機械的強度、高弾性、組織工学におけるこのポリマーの普及に貢献する。創傷治癒のげっ歯類モデルにおいて、移植エレクトロスピンPCLは、非免疫原性であること、および副作用13ずに局所組織に統合することが示された。エレクトロPCLのSEM像を図1に示されている。 現在のFDAの規制基準では、小規模および大規模の両方の動物モデルにおける有効性と安全性は、PCLまたは米国での臨床試験に移行するために、他の設計のACL移植のために必要とされるであろう。さらに、in vivoでの条件は、多くの場合、in vitroでの組織工学のACLグラフトのプロパティを増大させることができます。屈筋digitorと自家ACL再建のラットモデルumの伸筋腱が以前ネイティブACLが切断された、説明したが、大腿骨と脛骨のトンネルを掘削し、移植片は、縫合糸17〜22との代わりに渡され、固定した。本稿では、設計のACL置換の評価のためではなく、自家ベースの復興のために、このモデルの修正（ 図2）を説明します。 多くの動物モデルは、靱帯組織工学のために存在するが、ラットは、多くの理由のために、より大きなモデルに比べて有利である。これらの利点は、より簡単に飼育と取り扱い、より少ない倫理的配慮、そして低コスト17,23が含まれています。また、ラットモデルは、軟骨、腱、および骨組織工学24を含む整形外科組織再生のためのモデルとして広く使用されている。具体的には、無胸腺ヌードラットを最終的な移植oを可能にする、細胞媒介性免疫応答25の欠如に起因して選択したこのモデルにおけるfの異種ドナー細胞は、さらに、将来的に操作された移植片を強化する。このメソッドの論文では、ACL再建の無胸腺ラットモデルにおける製造および無細胞の外科的移植、生分解性ポリマーのグラフトを記述する。

<table border="0" cellpadding="0" cellspacing="0" width="888">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:77px;">Catalog Number</td>
			<td style="width:317px;">Comments/Description</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Medical grade ester terminated poly (&#949;-caprolactone), granule form (MW = 110,000)</td>
			<td style="width:147px;">Lactel Absorbable Polymers</td>
			<td style="width:77px;"></td>
			<td style="width:317px;">Custom synthesized polymer to desired molecular weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>105228</td>
			<td style="width:317px;">Solvent for PCL polymer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">18G x 1 1/2&#34;bevel needle</td>
			<td style="width:147px;">BD Medical</td>
			<td>305196</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Remote Infuse/Withdraw Programmable Syringe Pump</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td style="width:77px;">702101</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">VersaLaser VLS2.30 Laser Engraver</td>
			<td style="width:147px;">Microgeo USA</td>
			<td style="width:77px;">VLS2.30</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Expanded Plasma Cleaner 115V</td>
			<td style="width:147px;">Harrick Plasma</td>
			<td style="width:77px;">PDC-001</td>
			<td style="width:317px;">Plasma etch just prior to collagen coating for surface modification</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">PureCol&#160; Collagen Standard Solution, 3 mg/ml</td>
			<td style="width:147px;">Advanced Biomatrix</td>
			<td style="width:77px;">5015-A</td>
			<td style="width:317px;">Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1N NaOH 1:9 in 1x PBS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Suture, 5-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1086471</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture, 4-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">6540072</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Sharp-pointed Dissecting Scissors (Straight; 4.5 inch)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:77px;">8940</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Buphrenorphine hydrochloride</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:77px;">B9275</td>
			<td style="width:317px;">Use 0.03 mg/kg for both intra- and post-operatively for pain control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Ampicillin, injectable</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1185678</td>
			<td style="width:317px;">Use 25 mg/kg subcutaneously during the procedure</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">K-wire, 1.6 mm</td>
			<td style="width:147px;">Spectrum Surgical</td>
			<td style="width:77px;">SI040062</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Keith Needle, Straight 1 1/2&#34;</td>
			<td style="width:147px;">Delasco Dermatology Lab &#38; Supply</td>
			<td style="width:77px;">KE-112</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Immunocal Decalcifying Solution</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>NC9491030</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td>
			<td style="width:147px;">US Dental Depot</td>
			<td style="width:77px;">OPTICRYL 100410&#160;</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:347px;">Instron Model 5564 Tensile Testing Machine</td>
			<td style="width:147px;">Instron</td>
			<td style="width:77px;">5564</td>
			<td style="width:317px;">Any comparable tensile testing apparatus is suitable</td>
		</tr>
	</tbody>
</table>

athymic rat model for evaluation of engineered anterior cruciate ligament grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

シングル外科医による92ラットの手術の我々の経験では、切開から傷の完了までの平均手術時間は4.7分の標準偏差は、16.9分であった。屠殺時に、ラットは356±23グラムの重さであった。すべてのラットはよく手術を許容し、合併症を経験していない。すぐに手術後、ラットを手術四肢に体重を支えることが認めたが、わずかにぐったりを示した。手術後1週間では、すべてのラットはありませ?...

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Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

エンジニアード前十字靭帯移植片の評価のための無胸腺ラットモデル

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

athymic-rat-model-for-evaluation-engineered-anterior-cruciate

Research

JoVE Journal

Bioengineering

10.3K ビュー.  University of California Los Angeles. Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

ビデオ: Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

ティッシュエンジニアーコンストラクトの成長評価のための磁気共鳴エラストグラフィの方法論

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

生物工学

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

外科用回収、単離と<em&gt;インビトロ</em&gt;組織工学アプリケーションのためのヒト前十字靭帯由来細胞の拡大

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

組織工学血管移植片とその後の移植のために手術手技<em&gt;インビボ</em&gt;モニタリング

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

マウスモデルにおける組織工学血管の生成とグラフト

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

生産、特性評価および3D組織工学ヒト食道粘膜モデルの使用可能性

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

偏神経組織のエンジニア3Dシルクコラーゲンベースモデル

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

の応用<em&gt;インビボ</em組織工学骨格筋の修復を評価するためのラット前脛骨の&gt;機能テスト

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

3Dプリントガイドとリングスタッキング法を用いたエンジニア血管移植片のスケーリング

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

運動調節血清による靭帯構築物の治療：翻訳組織工学モデル

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

高スループットの自動モデル IgG1 CHO 細胞での生産のための Microbioreactor システム

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...